Abstract: The tumor suppressor p53 controls a broad range of cellular responses. Induction of a transient (cell cycle arrest) or a permanent (senescence) block of cell proliferation, or the activation of cell death pathways in response to genotoxic stress comprise the major arms of the survival-death axis governed by p53. Due to these biological properties, inactivation of p53 is a crucial step in tumor development and progression, reflected by the high incidence of TP53 mutations in different types of human cancers. The remarkable potency of p53 in suppressing tumorigenic outgrowth has promoted the expectation that tumor cells expressing wild-type p53 (wtp53) should be more prone to elimination by cytotoxic treatments than tumor cells expressing mutant p53 (mutp53) with defunct wtp53 activities. However, recent findings yielded somewhat unexpected insights concerning the preponderance of the survival-promoting effects of wtp53 in cancer cells, a rather undesired property from the therapeutic point of view. In this commentary we will discuss the possibility that the developmentally established distinct patterns of wtp53 mediated responses in different tissues are an important factor in determining the ultimate outcome of cellular responses mediated by wtp53 in different types of tumor cells, with a particular focus on the divergent impact of wtp53 in malignant tumors of the central nervous system. We infer that a selective gain of pro-survival functions of wtp53 in cancer cells will confer a survival advantage that counteracts tumor therapy.

Abstract: Missense point mutations in the TP53 gene are frequent genetic alterations in human tumor tissue and cell lines derived thereof. Mutant p53 (mutp53) proteins have lost sequence-specific DNA binding, but have retained the ability to interact in a structure-selective manner with non-B DNA and to act as regulators of transcription. To identify functional binding sites of mutp53, we established a small library of genomic sequences bound by p53(R273H) in U251 human glioblastoma cells using chromatin immunoprecipitation (ChIP). Mutp53 binding to isolated DNA fragments confirmed the specificity of the ChIP. The mutp53 bound DNA sequences are rich in repetitive DNA elements, which are dispersed over non-coding DNA regions. Stable down-regulation of mutp53 expression strongly suggested that mutp53 binding to genomic DNA is functional. We identified the PPARGC1A and FRMD5 genes as p53(R273H) targets regulated by binding to intronic and intra-genic sequences. We propose a model that attributes the oncogenic functions of mutp53 to its ability to interact with intronic and intergenic non-B DNA sequences and modulate gene transcription via re-organization of chromatin.

Abstract: Tumors that acquire resistance against death stimuli constitute a severe problem in the context of cancer therapy. To determine genetic alterations that favor the development of stress-resistant tumors in vivo, we took advantage of polyclonal tumors generated after retroviral infection of newborn Elambda-MYC mice, in which the retroviral integration acts as a mutagen to enhance tumor progression. Tumor cells were cultivated ex vivo and exposed to gamma-irradiation prior to their transplantation into syngenic recipients, thereby providing a strong selective pressure for pro-survival mutations. Secondary tumors developing from stress-resistant tumor stem cells were analysed for retroviral integration sites to reveal candidate genes whose dysregulation confer survival. In addition to the gene encoding the antiapoptotic Bcl-x(L) protein, we identified the gadd45b locus to be a novel common integration site in these tumors, leading to enhanced expression. In accord with a thus far undocumented role of Gadd45beta in tumorigenesis, we showed that NIH3T3 cells overexpressing Gadd45beta form tumors in NOD/SCID mice. Interestingly and differently to other known 'classical' antiapoptotic factors, high Gadd45beta levels did not protect against MYC-, UV- or gamma-irradiation-induced apoptosis, but conferred a strong and specific survival advantage to serum withdrawal.

Abstract: Signaling mediated by activation of the transmembrane receptor Notch influences cell-fate decisions, differentiation, proliferation, and cell survival. Activated Notch reduces proliferation by altering cell-cycle kinetics and promotes differentiation in hematopoietic progenitor cells. Here, we investigated if the G(1) arrest and differentiation induced by activated mNotch1 are dependent on tumor suppressor p53, a critical mediator of cellular growth arrest. Multipotent wild-type p53-expressing (p53(wt)) and p53-deficient (p53(null)) hematopoietic progenitor cell lines (FDCP-mix) carrying an inducible mNotch1 system were used to investigate the effects of proliferation and differentiation upon mNotch1 signaling. While activated Notch reduced proliferation of p53(wt)-cells, no change was observed in p53(null)-cells. Activated Notch upregulated the p53 target p21(cip/waf) in p53(wt)-cells, but not in p53(null)-cells. Induction of the p21(cip/waf) gene by activated Notch was mediated by increased binding of p53 to p53-binding sites in the p21(cip/waf) promoter and was independent of the canonical RBP-J binding site. Re-expression of p53(wt) in p53(null) cells restored the inhibition of proliferation by activated Notch. Thus, activated Notch inhibits proliferation of multipotent hematopoietic progenitor cells via a p53-dependent pathway. In contrast, myeloid and erythroid differentiation was similarly induced in p53(wt) and p53(null) cells. These data suggest that Notch signaling triggers two distinct pathways, a p53-dependent one leading to a block in proliferation and a p53-independent one promoting differentiation.

Abstract:

Abstract: In human breast cancer, mutations in the p53 gene are associated with poor prognosis. However, analysis of patient data so far did not clarify, whether missense point mutations in the p53 gene, in addition to causing loss of wild-type p53 function, also confer a gain of function phenotype to the encoded mutant p53. As heterogeneity of patient material and data might obscure a clear answer, we studied the effects of a coexpressed mutant p53(R270H) in transgenic mice in which SV40 early proteins initiate the development of mammary adenocarcinoma (WAP-T mice). In such tumors the endogenous wild-type p53 is functionally compromised by complex formation with SV40 T-antigen, thereby constituting a loss of wild-type p53 function situation that allowed analysis of the postulated gain of function effects of mutant p53(R270H). We found that mutant p53(R270H) in bi-transgenic mice enhanced the transition from intraepithelial neoplasia to invasive carcinoma, resulting in a higher frequency of invasive carcinoma per gland and per mouse, a more severe tumor phenotype, and more frequent pulmonary metastasis. Surprisingly, mutant p53(R270H) in this system does not increase genomic instability. Therefore, other postulated gain of function activities of mutant p53 must be responsible for the effects described here.

Abstract:

Abstract: Since the very early days of p53 research, the gain of oncogenic activities by some mutant p53 proteins had been suspected as an important factor contributing to cancer progression. Considerable progress towards understanding the biology of mutant p53 has been made during the last years, the quintessence being the realization that the impact of mutant p53 proteins on the transcriptome of a tumor cell is much more global than previously thought. The emerging role of mutant p53 proteins in coordinating oncogenic signaling and chromatin modifying activities reveals an until now unsuspected function of these proteins as important modifiers of the oncogenic transcriptional response. Notwithstanding the fact that the sequence-specific DNA binding activity of mutant p53 proteins is impaired, they are still able to associate with specific loci on DNA by utilizing different mechanisms. The ability to associate with DNA appears to be crucial for the master role of mutant p53 proteins in coordinating oncogenic transcriptional responses.

Abstract:

Abstract: In this report we analyzed the impact of interleukin-4 (IL-4) on tumor-associated simian virus 40 (SV40) large T-antigen (TAg)-specific CD8+ cytotoxic T cells during rejection of syngeneic SV40 transformed mKSA tumor cells in BALB/c mice. Strikingly, challenge of naïve mice with low doses of mKSA tumor cells revealed a CD8+ T cell-dependent prolonged survival time of naïve IL-4-/- mice. In mice immunized with SV40 TAg we observed in IL-4-/- mice, or in wild type mice treated with neutralizing anti-IL-4 monoclonal antibody, a strongly enhanced TAg-specific cytotoxicity of tumor associated CD8+ T cells. The enhanced cytotoxicity in IL-4-/- mice was accompanied by a significant increase in the fraction of CD8+ tumor associated T-cells expressing the cytotoxic effector molecules granzyme A and B and in granzyme B-specific enzymatic activity. The data suggest that endogenous IL-4 can suppress the generation of CD8+ CTL expressing cytotoxic effector molecules especially when the antigen induces only a very weak CTL response.

Abstract: To determine pathways cooperating with p53 in cellular senescence when the retinoblastoma protein (pRb)/p16INK4a pathway is defunct, we stably transfected the p16INK4a-negative C6 rat glioma cell line with a temperature-sensitive mutant p53. Activation of p53(Val-135) induces a switch in pocket protein expression from pRb and p107 to p130(Rb2) and stalls the cells in late G1, early S-phase at high levels of cyclin E. Maintenance of the arrest depends on the functions of p130(Rb2) repressing cyclin A. Inactivation of p53 in senescent cultures restores the pocket proteins to initial levels and initiates progression into S-phase, but the cells fail to resume proliferation, likely due to DNA damage becoming apparent in the arrest and activating apoptosis subsequent to the release from p53-dependent growth suppression. The data indicate that p53 can cooperate selectively with p130(Rb2) to induce cellular senescence, a pathway that may be relevant when the pRb/p16INK4a pathway is defunct.

Abstract:

Abstract: Following genotoxic stress, p53 either rescues a damaged cell or promotes its elimination. The parameters determining a specific outcome of the p53 response are largely unknown. In mouse fibroblasts treated with different irradiation schemes, we monitored transcriptional and non-transcriptional p53 activities and identified determinants that initiate an anti- or a pro-apoptotic p53 response within the context of p53-independent stress signaling. The primary, transcription-mediated p53 response in these cells is anti-apoptotic, while induction of p53-dependent apoptosis requires an additional, transcription-independent p53 activity, provided by high intracellular levels of activated p53. High intracellular levels of p53 were selectively generated after apoptosis-inducing high-dose UV-irradiation, and correlated with a strongly delayed upregulation of Mdm2. Following high-dose UV-irradiation, p53 accumulated in the cytoplasm and led to activation of the pro-apoptotic protein Bax. As p53-dependent Bax-activation is transcription-independent, we postulated that certain transcription-deficient mutant p53 proteins might also exert this activity. Indeed we found an endogenous, transcription-inactive mutant p53 that upon genotoxic stress induced Bax-activation in vivo. Our results demonstrate the impact and in vivo relevance of non-transcriptional mechanisms for wild-type and mutant p53-mediated apoptosis.

Abstract: BACKGROUND: The tumor suppressor gene p53 (TP53) controls numerous signaling pathways and is frequently mutated in human cancers. Novel p53 isoforms suggest alternative splicing as a regulatory feature of p53 activity. RESULTS: In this study we have analyzed mRNA expression of both wild-type and mutated p53 and its respective Deltap53 isoform in 88 tumor samples from breast cancer in relation to clinical parameters and molecular subgroups. Three-dimensional structure differences for the novel internally deleted p53 isoform Deltap53 have been predicted. We confirmed the expression of Deltap53 mRNA in tumors using quantitative real-time PCR technique. The mRNA expression levels of the two isoforms were strongly correlated in both wild-type and p53-mutated tumors, with the level of the Deltap53 isoform being approximately 1/3 of that of the full-length p53 mRNA. Patients expressing mutated full-length p53 and non-mutated (wild-type) Deltap53, "mutational hybrids", showed a slightly higher frequency of patients with distant metastasis at time of diagnosis compared to other patients with p53 mutations, but otherwise did not differ significantly in any other clinical parameter. Interestingly, the p53 wild-type tumors showed a wide range of mRNA expression of both p53 isoforms. Tumors with mRNA expression levels in the upper or lower quartile were significantly associated with grade and molecular subtypes. In tumors with missense or in frame mutations the mRNA expression levels of both isoforms were significantly elevated, and in tumors with nonsense, frame shift or splice mutations the mRNA levels were significantly reduced compared to those expressing wild-type p53. CONCLUSION: Expression of p53 is accompanied by the functionally different isoform Deltap53 at the mRNA level in cell lines and human breast tumors. Investigations of "mutational hybrid" patients highlighted that wild-type Deltap53 does not compensates for mutated p53, but rather may be associated with a worse prognosis. In tumors, both isoforms show strong correlations in different mutation-dependent mRNA expression patterns.

Abstract:

Abstract: Growth regulatory functions of Rb2/p130, which aim at a sustained arrest such as in quiescent or differentiated cells, qualify the protein also to act as a central regulator of growth arrest in cellular senescence. In this respect, Rb2/p130 functions are connected to signaling pathways induced by p53, which is a master regulator in cellular senescence. Here, we summarize the pathways, which specify pRb2/p130 to control this arrest program and distinguish its functions from those of pRb/p105.

Abstract: Telomerase activity is repressed in most human somatic tissues during differentiation processes but strongly up-regulated in most human tumors. Regulation of human telomerase activity primarily occurs at the level of transcriptional initiation of the TERT gene, which encodes the catalytic subunit of telomerase. We have generated a novel transgenic mouse model to study the regulation of the human TERT gene promoter in an in vivo system. For this purpose, we have cloned an 8.0-kbp human TERT promoter fragment in front of the bacterial lacZ reporter gene (hTERTp-lacZ), which encodes the beta-galactosidase enzyme. Expression of the reporter gene was monitored by reverse transcription-PCR analysis, 5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside staining of whole mount preparations, and histologic sections. We find that the activity of the human TERT promoter in most normal mouse tissues recapitulates the expression of the hTERT gene in normal human tissues and is under tighter control when compared with the endogenous mouse TERT gene expression. In testis, where highest lacZ expression was observed, the expression of the reporter gene was restricted to the spermatogonial stem cells and the spermatocytes. Intriguingly, we find increased levels of lacZ expression in mammary tumors of hTERTp-lacZ x p53(+/-) bitransgenic mouse mammary tumor model. Thus, this transgenic mouse model provides a suitable in vivo system to analyze the expression of the human TERT gene under physiologic conditions and during tumorigenesis.

Abstract: Despite the loss of sequence-specific DNA binding, mutant p53 (mutp53) proteins can induce or repress transcription of mutp53-specific target genes. To date, the molecular basis for transcriptional modulation by mutp53 is not understood, but increasing evidence points to the possibility that specific interactions of mutp53 with DNA play an important role. So far, the lack of a common denominator for mutp53 DNA binding, i.e. the existence of common sequence elements, has hampered further characterization of mutp53 DNA binding. Emanating from our previous discovery that DNA structure is an important determinant of wild-type p53 (wtp53) DNA binding, we analyzed the binding of various mutp53 proteins to oligonucleotides mimicking non-B DNA structures. Using various DNA-binding assays we show that mutp53 proteins bind selectively and with high affinity to non-B DNA. In contrast to sequence-specific and DNA structure-dependent binding of wtp53, mutp53 DNA binding to non-B DNA is solely dependent on the stereo-specific configuration of the DNA, and not on DNA sequence. We propose that DNA structure-selective binding of mutp53 proteins is the basis for the well-documented interaction of mutp53 with MAR elements and for transcriptional activities mediates by mutp53.

Abstract: Sequence-specific DNA binding is a major activity of the tumor suppressor p53 and a prerequisite for the transactivating potential of the protein. p53 interaction with target DNA is tightly regulated by various mechanisms, including binding of different components of the transcription machinery, post-translational modifications, and interactions with other factors that modulate p53 transactivation in a cell context- and promoter-specific manner. The bi-functional redox factor 1 (Ref-1/APE1) has been identified as one of the factors, which can stimulate p53 DNA binding by redox-dependent as well as redox-independent mechanisms. Whereas stimulation of p53 DNA binding by the redox activities of Ref-1 is understood quite well, little is known about mechanisms that underlie the redox-independent effects of Ref-1. We report in this study a previously unknown activity of Ref-1 as a factor promoting tetramerization of p53. We demonstrate that Ref-1 promotes association of dimers into tetramers, and de-stacking of higher oligomeric forms into the tetrameric form in vitro, thereby enhancing p53 binding to target DNA.

Abstract: BACKGROUND: p53 is the most frequently mutated gene in human cancers and its functional integrity is an important predictor of treatment response and clinical outcome. The majority of mutations found in different types of cancer cluster within the DNA binding domain encoded by exons 5-8. In clinical specimens the functional status of p53 is, therefore, often evaluated by direct mutation analysis of exons 5-8 or indirectly by immunostaining and evaluation of the subcellular localization pattern or protein accumulation. MATERIALS AND METHODS: In a panel of glioma cell lines, the status of the P53 gene was analyzed by temperature gradient gel electrophoresis (TGGE) of exons 5-8 and direct sequencing of all p53 exons. The nuclear accumulation of p53 in unstressed cells was assessed by immunostaining. These data were correlated with stress induction of the p53 protein, nuclear translocation and a direct determination of the transcriptional activity of endogenous p53 protein and induction of p53 target genes. RESULTS: Our analysis demonstrated that a p53 gene mutation analysis limited to exons 5-8 and analysis of immunostaining patterns can not serve as reliable predictors of functional p53 in tumor cells. Conversely, in some presumably rare cases, the transcriptional activity of p53 may be retained in tumor cells in the presence of a mutation and a pathological immunostaining pattern. In our analysis, the constitutive dephosphorylation at Ser 376 correlated with the nuclear accumulation of p53, but not with the transcriptional activity of the protein. This suggests that constitutive dephosphorylation at Ser376 may be one of the factors determining stabilization of mutant and wild-type p53, which is frequently observed in glial tumors. CONCLUSION: The incidence of a dysfunctional p53 protein in gliomas may be higher than expected, based on a single parameter evaluation by mutation analysis of exons 5-8 or assessment of p53 accumulation and subcellular localization by immunostaining.

Abstract: We describe the construction and phenotypic characterization of 23 whey acidic protein (WAP)-mutp53 transgenic mouse lines. The mutp53-expressing lines showed a mosaic expression pattern for the transgenes, leading to a heterogeneous yet mouse line-specific expression pattern for mutp53 upon induction. Only few lines were obtained, in which the majority of the induced mammary epithelial cells expressed the mutp53 transgene, most of the transgenic lines did not express mutp53, or expressed the transgene in less than 2% of the induced mammary epithelial cells. Hormone requirements for mutp53 transgene expression from the WAP-promoter differed in high and low expressing lines, being low in high expressing lines, and even lower in multiparous mutp53 mice, where persistent expression of the transgene occurred. Repeated induction of mutp53 expression through repeated parturition resulted in the formation of expanding mutp53-expressing foci within the mammary alveolar epithelium. The data suggest that epigenetic mechanisms play a role in modulating the expression of the mutp53 transgene. To support this idea, we crossed a nonexpressing WAP-mutp53 line with a strongly SV40 T-antigen-expressing WAP-T mouse line. In the bitransgenic mice, T-antigen-induced chromatin remodeling led to re-expression of epigenetically silenced mutp53 transgene(s). In these mice, mutp53 expression was much more variable compared to SV40 T-antigen expression, and seemed to depend on the coexpression of SV40 T-antigen. Mutp53 expression in this system thus resembles the situation in many human tumors, where one can observe a heterogeneous expression of mutp53, despite a homogeneous distribution of the p53 mutation in the tumor cells.

Abstract: DNA binding is central to the ability of p53 to function as a tumor suppressor. In line with the remarkable functional versatility of p53, which can act on DNA as a transcription, repair, recombination, replication, and chromatin accessibility factor, the modes of p53 interaction with DNA are also versatile. One feature common to all modes of p53-DNA interaction is the extraordinary sensitivity of p53 to the topology of its target DNA. Whereas the strong impact of DNA topology has been demonstrated for p53 binding to sequence-specific sites or to DNA lesions, the possibility that DNA structure-dependent recognition may underlie p53 interaction with other types of DNA has not been addressed until now. We demonstrate for the first time that conformationally flexible CTG.CAG trinucleotide repeats comprise a novel class of p53-binding sites targeted by p53 in a DNA structure-dependent mode in vitro and in vivo. Our major finding is that p53 binds to CTG.CAG tracts by different modes depending on the conformation of DNA. Although p53 binds preferentially to hairpins formed by either CTG or CAG strands, it can also bind to linear forms of CTG.CAG tracts such as canonic B DNA or mismatched duplex. Intriguingly, by binding to a mismatched duplex p53 can induce further topological alterations in DNA, indicating that p53 may act as a DNA topology-modulating factor.

Abstract: The serine/threonine-specific casein kinase I delta (CKIdelta) is ubiquitously expressed in all tissues, is p53 dependently induced in stress situations and plays an important role in various cellular processes. Our immunohistochemical analysis of the human placenta revealed strongest expression of CKIdelta in extravillous trophoblast cells and in choriocarcinomas. Investigation of the functional role of CKIdelta in an extravillous trophoblast hybrid cell line revealed that CKIdelta was constitutively localized at the centrosomes and the mitotic spindle. Inhibition of CKIdelta with the CKI-specific inhibitor IC261 led to structural alterations of the centrosomes, the formation of multipolar spindles, the inhibition of mitosis and, in contrast to other cell lines, the induction of apoptosis. Our findings indicate that CKIdelta plays an important role in the mitotic progression and in the survival of cells of trophoblast origin. Therefore, IC261 could provide a new tool in treating choriocarcinomas.

Abstract: Induction of apoptosis is one of the central activities by which p53 exerts its tumor-suppressing function. Aside from its primary function as a transcription factor, it can promote apoptosis independent of transcription. Recent studies have started to define the mechanisms of non-transcriptional pro-apoptotic p53 activities operating within the intrinsic mitochondria-mediated pathway of apoptosis. So far, two different mechanisms have been described, each of which was assigned to a specific localization of the p53 protein, either in the cytosol or directly at the mitochondria. Although mechanistically different, both transcription-independent modes of apoptosis induction converge, as they both initiate permeabilization of the outer mitochondrial membrane via activation of the pro-apoptotic Bcl-2 family members Bax or Bak.

Abstract: The archetypal human tumor suppressor p53 is considered to have unique transactivation properties. The assumption is based on the fact that additionally identified human p53 isoforms lack transcriptional activity. However, we provide evidence for the existence of an alternatively spliced p53 isoform (Deltap53) that exerts its transcriptional activity independent from p53. In contrast to p53, Deltap53 transactivates the endogenous p21 and 14-3-3sigma but not the mdm2, bax, and PIG3 promoter. Cell cycle studies showed that Deltap53 displays its differential transcriptional activity only in damaged S phase cells. Upon activation of the ATR-intra-S phase checkpoint, Deltap53, but not p53, transactivates the Cdk inhibitor p21. Induction of p21 results in downregulation of cyclin A-Cdk activity and accordingly attenuation of S phase progression. Data demonstrate that the Deltap53-p21-cyclin A-Cdk pathway is crucial to facilitate uncoupling of repair and replication events, indicating that Deltap53 is an essential element of the ATR-intra-S phase checkpoint.

Abstract: The Japanese-German Workshops "Molecular and Cellular Aspects of Carcinogenesis," held biennially since 1987, have been organized traditionally at the University of Essen Medical School and West German Cancer Center Essen in Germany. This 9th Workshop was held on September 18-20, 2003. It was generously supported by the Cancer Research Program of the Ministry of Education, Culture, Sports, Science and Technology and the International Cooperation Program for the 2nd Term Comprehensive 10-Year Strategy for Cancer Control of the Ministry of Health, Labour and Welfare on the Japanese side, and by the Deutsche Forschungsgemeinschaft (DFG) on the German side. Additional support from many other sponsors in Germany and Japan is gratefully acknowledged. The Workshop participants are listed at the end of this Report.

Abstract: The dominant oncogenic properties of mutant p53 have been recognized as a phenomenon associated with tumor progression a long time ago, even before it was realized that the major function of wild type p53 is that of a tumor suppressor. Recent advances in this fascinating area in tumor cell biology reveal that the community of mutant p53 proteins is comprised of proteins that are extremely diverse both structurally and functionally, and elicit a multitude of cellular responses that not only are entirely distinct from those mediated by wild type p53, but also vary among different mutant p53 proteins. Aberrant regulation of transcription is one of the mechanisms underlying the ability of some mutant p53 proteins to act as oncogenic factors. Systematic analyses of the transcriptional activities of mutant p53 suggest that not the loss of transcriptional activity as such, but alterations of target DNA selectivity may be the driving force of mutant p53 specific transcription underlying the growth-promoting effects of mutant p53. This article focuses on mechanistic aspects of mutp53 "gain-of-function" with the emphasis on possible mechanisms underlying transcriptional activation by mutp53.

Abstract: Activation of the tumor suppressor p53 after genotoxic insults may result in two different responses: growth arrest or apoptosis. In this study, we analysed how mitogenic stimulation of primary mouse lymphocytes influences p53 signaling upon gamma-irradiation. We found that G(0) lymphocytes rapidly went into p53-dependent apoptosis, whereas stimulated lymphocytes went into a p53-dependent, p21-mediated growth arrest. The switch in p53 response upon stimulation did neither result from a switch in transcriptional activation of major p53 target genes, nor from the high level of p21 expressed in stimulated, irradiated cells. Growth stimulation, however, led to the upregulation of the antiapoptotic factors Bcl-x(L) and Bfl-1. In resting cells, p53 induced apoptosis after gamma-irradiation was accompanied by a breakdown of the mitochondrial membrane potential (psi(m)) that was counteracted by growth stimulation. We propose that growth stimulation intercepted p53 proapoptotic signaling at the level of mitochondrial integrity, most likely by upregulating the antiapoptotic factors Bcl-x(L) and Bfl-1. Upregulation of Bcl-x(L) and of Bfl-1 upon growth stimulation was mediated by the PKC-dependent activation of NF-kappaB. Consequently, blocking PKC activity restored apoptosis in stimulated, irradiated splenocytes. The inherent coupling of growth stimulation with antiapoptotic signaling in primary lymphocytes might provide hints as to how precancerous lymphocytes bypass the need for mutational inactivation of p53. Thus, our findings might explain the relatively low frequency of p53 mutations in lymphomas in comparison to other tumor entities.

Abstract: The most import biological function of the tumor suppressor p53 is that of a sequence-specific transactivator. In response to a variety of cellular stress stimuli, p53 induces the transcription of an ever-increasing number of target genes, leading to growth arrest and repair, or to apoptosis. Long considered as a "latent" DNA binder that requires prior activation by C-terminal modification, recent data provide strong evidence that the DNA binding activity of p53 is strongly dependent on structural features within the target DNA and is latent only if the target DNA lacks a certain structural signal code. In this review we discuss evidence for complex interactions of p53 with DNA, which are strongly dependent on the dynamics of DNA structure, especially in the context of chromatin. We provide a model of how this complexity may serve to achieve selectivity of target gene regulation by p53 and how DNA structure in the context of chromatin may serve to modulate p53 functions.

Abstract: The concept that the tumor suppressor p53 is a latent DNA-binding protein that must become activated for sequence-specific DNA binding recently has been challenged, although the "activation" phenomenon has been well established in in vitro DNA binding assays. Using electrophoretic mobility shift assays and fluorescence correlation spectroscopy, we analyzed the binding of "latent" and "activated" p53 to double-stranded DNA oligonucleotides containing or not containing a p53 consensus binding site (DNAspec or DNAunspec, respectively). In the absence of competitor DNA, latent p53 bound DNAspec and DNAunspec with high affinity in a sequence-independent manner. Activation of p53 by the addition of the C-terminal antibody PAb421 significantly decreased the binding affinity for DNAunspec and concomitantly increased the binding affinity for DNAspec. The net result of this dual effect is a significant difference in the affinity of activated p53 for DNAspec and DNAunspec, which explains the activation of p53. High affinity nonspecific DNA binding of latent p53 required both the p53 core domain and the p53 C terminus, whereas high affinity sequence-specific DNA binding of activated p53 was mediated by the p53 core domain alone. The data suggest that high affinity nonspecific DNA binding of latent and high affinity sequence-specific binding of activated p53 to double-stranded DNA differ in their requirement for the C terminus and involve different structural features of the core domain. Because high affinity nonspecific DNA binding of latent p53 is restricted to wild type p53, we propose that it relates to its tumor suppressor functions.

Abstract: The casein kinase I isoform delta (CKIdelta) plays an important role in vesicular trafficking, chromosome segregation, cell cycle progression, cytokinesis, developmental processes, and circadian rhythm. In this study we examined the distribution pattern of CKIdelta and quantified its kinase activity in various tissues of BALB/c mice. Whereas CKIdelta is ubiquitously expressed, differences in the kinase activity were detected in organs with comparable CKIdelta protein levels. To elucidate the role of CKIdelta in splenocytes, which displayed the highest kinase activity, the cell type-specific distribution of CKIdelta within the spleen was investigated. Immunohistochemical analysis revealed a strong CKIdelta immunolabeling in lymphoid cells of the white pulp, while in the red pulp CKIdelta immunoreactivity was found in cells of various haematopoietic lineages. Furthermore, high CKIdelta kinase acitivity was observed in isolated lymphocytes and granulocytes of young BALB/c mice. In lymphocytes the CKIdelta activity increased upon mitogenic stimulation, whereas upon gamma-irradiation CKIdelta protein and activity levels were diminished. Interestingly, the comparison of CKIdelta activity in p53+/+ and p53-/- lymphocytes revealed a higher activity in p53+/+ lymphocytes. In addition, we observed an increased immunostaining in cells of hyperplastic B follicles and advanced B-cell lymphomas in p53-deficient mice. Thus, our results indicate that CKIdelta plays several roles in lymphocyte physiology.

Abstract: Cancer formation and progression is a complex process determined by several mechanisms that promote cell growth, invasiveness, neo-angiogenesis, and render neoplastic cells resistant to apoptosis. The tumor suppressor p53 and the proto-oncogenic factor ets-1 are important regulators of such mechanisms. While it is well established that p53 and ets-1 influence various aspects of cell behavior by regulating the transcription of specific genes, little is known about the functional relationship between these transcription factors. We found that the gene encoding thromboxane synthase (TXSA), which we recently identified as a factor promoting invasion and resistance to apoptosis in gliomas, is a novel target gene for both p53 and ets-1. We demonstrate that p53 and ets-1 coregulate TXSA in an antagonistic and inter-related manner, with ets-1 being a potent transcriptional activator and p53 inhibiting ets-1-dependent transcription. Negative interference with ets-1 transcription requires functional p53 and is lost in mutant p53 proteins. We show that ets-1 and p53 associate physically in vitro and in vivo and that their interaction, rather than a direct binding of p53 to the TXSA promoter, is required for transcriptional repression of TXSA by wild-type p53. An important implication of our findings is that the loss of p53-mediated negative control over ets-1-dependent transcription may lead to the acquisition of an invasive phenotype in tumor cells.

Abstract: Transcriptional activation of p53-regulated genes is initiated by sequence-specific DNA binding of p53 to target binding sites. Regulation of sequence-specific DNA binding is complex and occurs at various levels. We demonstrate that DNA topology is an important parameter for regulating the selective and highly specific interaction of p53 with its target binding sites. Specific binding of wild-type p53 is greatly enhanced when cognate binding sites are present in a non-linear stem-loop conformation. The C-terminal domain plays a key role in regulating the specific interactions of p53 with target binding sites in a DNA conformation-dependent manner. The C-terminal domain is required for binding to target sites in a non-linear DNA conformation in contrast to the strong inhibitory effects of the C terminus on p53 interaction with linear DNA. We propose that selective binding of p53 to various promoters may be determined by the DNA conformation within p53 cognate sites.

Abstract: Cytoplasmic accumulation of wild-type p53 in tumor cells indicates that the tumor suppressor is inactive with regard to growth suppressive functions. Whether this occurs randomly during tumor development or characterizes a certain tumor cell subset is not known. Here we assayed primary glioblastomas for expression and subcellular localization of p53 and determined a correlation with expression of intermediate filament proteins characterizing glial cell development. Sixty-nine percent of the tumors were p53 positive in immunohistochemistry. A significant number of tumors (23%) accumulated wild-type p53 in the cytoplasm, which correlated with the presence of vimentin and glial fibrillary acidic protein, except for 1 case. Tumors with exclusive nuclear p53 contained none or only one of these intermediate filament proteins. In an alternative approach, tumors positive for glial fibrillary acidic protein were screened for expression of p53 and vimentin. Thirty-eight percent of these tumors showed cytoplasmic p53, and all of those also expressed vimentin. Tumors with only nuclear p53 were vimentin negative, except for 1 case. No mutation was detected in p53 exons 5 to 8 in tumors with cytoplasmic p53, suggesting that they express wild-type p53. The data indicate that a cytoplasmic accumulation of wild-type p53 in human primary glioblastomas correlates with a certain intermediate filament protein expression, suggesting that it identifies a certain subset of tumors.

Abstract: This study describes a tumor progression model for ductal pancreatic cancer in mice overexpressing TGF-alpha. Activation of Ras and Erk causes induction of cyclin D1-Cdk4 without increase of cyclin E or PCNA in ductal lesions. Thus, TGF-alpha is able to promote progression throughout G1, but not S phase. Crossbreeding with p53 null mice accelerates tumor development in TGF-alpha transgenic mice dramatically. In tumors developing in these mice, biallelic deletion of Ink4a/Arf or LOH of the Smad4 locus is found suggesting that loci in addition to p53 are involved in antitumor activities. We conclude that these genetic events are critical for pancreatic tumor formation in mice. This model recapitulates pathomorphological features and genetic alterations of the human disease.

Abstract: Metabolic labeling of primate cells revealed the existence of phosphorylated and hypophosphorylated DNA polymerase alpha-primase (Pol-Prim) populations that are distinguishable by monoclonal antibodies. Cell cycle studies showed that the hypophosphorylated form was found in a complex with PP2A and cyclin E-Cdk2 in G1, whereas the phosphorylated enzyme was associated with a cyclin A kinase in S and G2. Modification of Pol-Prim by PP2A and Cdks regulated the interaction with the simian virus 40 origin-binding protein large T antigen and thus initiation of DNA replication. Confocal microscopy demonstrated nuclear colocalization of hypophosphorylated Pol-Prim with MCM2 in S phase nuclei, but its presence preceded 5-bromo-2'-deoxyuridine (BrdU) incorporation. The phosphorylated replicase exclusively colocalized with the BrdU signal, but not with MCM2. Immunoprecipitation experiments proved that only hypophosphorylated Pol-Prim associated with MCM2. The data indicate that the hypophosphorylated enzyme initiates DNA replication at origins, and the phosphorylated form synthesizes the primers for the lagging strand of the replication fork.

Abstract: Protective immunity of BALB/c mice immunized with simian virus 40 (SV40) large T antigen (TAg) against SV40-transformed, TAg-expressing mKSA tumor cells is critically dependent on both CD8(+) and CD4(+) T lymphocytes. By depleting mice of T-cell subsets at different times before and after tumor challenge, we found that at all times, CD4(+) and CD8(+) cells both were equally important in establishing and maintaining a protective immune response. CD4(+) cells do not contribute to tumor eradication by directly lysing mKSA cells. However, CD4(+) lymphocytes provide help to CD8(+) cells to proliferate and to mature into fully active cytotoxic T lymphocytes (CTL). Depletion of CD4(+) cells by a single injection of CD4-specific monoclonal antibody at any time from directly before injection of the vaccinating antigen to up to 7 days after tumor challenge inhibited the generation of cytolytic CD8(+) lymphocytes. T helper cells in this system secrete the typical Th-1 cytokines interleukin 2 (IL-2) and gamma interferon. Because in this system TAg-specific CD8(+) cells secrete only minute amounts of IL-2, it appears that T helper cells provide these cytokines for CD8(+) T cells. Moreover, this helper effect of CD4(+) T cells in mKSA tumor rejection in BALB/c mice does not simply improve the activity of TAg-specific CD8(+) CTL but actually enables them to mature into cytolytic effector cells. Beyond this activity, the presence of T helper cells is necessary even in the late phase of tumor cell rejection in order to maintain protective immunity. However, despite the support of CD4(+) T helper cells, the tumor-specific CTL response is so weak that only at the site of tumor cell inoculation and not in the spleen or in the regional lymph nodes can TAg-specific CTL be detected.

Abstract: As the key integrator of nuclear structure and function, the nuclear matrix is likely to be an important target for structural and functional alterations during the process of neoplastic transformation. Here I summarize and discuss data demonstrating that the major transforming protein of the small DNA tumor virus simian virus 40 (SV40), the SV40 large tumor antigen (large T), specifically targets the chromatin and the nuclear matrix during viral transformation. I then turn to recent evidence endorsing the concept that mutant p53--the most commonly expressed oncogene in human cancer--might exert its oncogenic activities by specifically interacting with the nuclear matrix. The data suggest that SV40 large T and mutant p53 might be members of a new family of oncogenes that exert their oncogenic functions by directly modulating nuclear structure and function.

Abstract: In cultured human keratinocytes, the tumor suppressor p53 acts as a control element in the protective response to UVB radiation and is affected by a variety of factors linked to cellular adhesion and differentiation. Because keratinocytes within the epidermis are not a homogeneous population but differ in their proliferative capacity and differentiation status, we compared the UVB responsiveness of primary keratinocyte populations isolated from various skin biopsies using p53 expression as a marker for their sensitivity to UVB. Besides keratinocytes exhibiting a UVB dose- and time-dependent upregulation of p53, keratinocyte populations were detected with high p53 expression levels even without irradiation. Such keratinocytes did not regulate p53 expression in response to UVB. Furthermore their p53-mediated UVB response was influenced by cocultivation with human dermal fibroblasts (HDF) but not with cell cycle-arrested human normal keratinocytes or HaCaT keratinocytes. When these cells were cultivated together with arrested HDF, they did not only reveal increased p53 expression levels after UVB treatment but also a more pronounced transcriptional activation of the p53 downstream target gene p21. These findings indicate that the UVB response of keratinocytes, specifically the activation of the tumor suppressor p53, is heterogeneous and can be affected by growth conditions.

Abstract: The ductal carcinoma in situ (DCIS) of the mammary gland represents an early, pre-invasive stage in the development of invasive breast carcinoma and is increasingly diagnosed since the introduction of high-quality mammography screening. Uncertainties in the prognosis for patients with DCIS have caused a controversial discussion about adequate treatment, and it is suspected that most patients undergoing mastectomy may be overtreated. In order to improve treatment and treatment decision, it therefore is highly desirable to identify prognostic markers and therapeutic targets for DCIS. We here introduce a set of transgenic mice (WAP-T and WAP-T-NP lines) presenting with various morphological forms of DCIS-like lesions. In these mice the SV40 large tumor antigen is specifically induced in epithelial cells of the terminal duct lobular units (TDLU). As a consequence of continuous expression of the oncogene, the animals develop multifocal DCIS and consequently invasive carcinoma within strain specific periods of latency. DCIS lesions in transgenic mice exhibit distinct architectural and cytological features which closely resemble those commonly present in humans. We therefore propose these transgenic lines as an experimental model to study the underlying molecular events leading to DCIS and its progression to invasive disease.

Abstract: Members of the casein kinase 1 family of serine/threonine kinases are highly conserved from yeast to mammals and seem to play an important role in vesicular trafficking, DNA repair, cell cycle progression and cytokinesis. We here report that in interphase cells of various mammalian species casein kinase 1 delta (CK1delta) specifically interacts with the trans Golgi network and cytoplasmic, granular particles that associate with microtubules. Furthermore, at mitosis CK1delta is recruited to the spindle apparatus and the centrosomes in cells, which have been exposed to DNA-damaging agents like etoposide or gammairradiation. In addition, determination of the affinity of CK1delta to different tubulin isoforms in immunoprecipitation-Western analysis revealed a dramatically enhanced complex formation between CK1delta and tubulins from mitotic extracts after introducing DNA damage. The high affinity of CK1delta to the spindle apparatus in DNA-damaged cells and its ability to phosphorylate several microtubule-associated proteins points to a regulatory role of CK1delta at mitosis.

Abstract: Our previous work (Dudenhöffer et al., 1999) unveiled a link between the capacity of p53 to regulate homologous recombination processes and to specifically bind to heteroduplex junction DNAs. Here, we show that p53 participates in ternary complex formation after preassembly of nucleoproteins, consisting of the human recombinase hRad51 and junction DNA. The cancer-related mutant p53(273H), which is defective in inhibiting recombination processes, displays a reduced capacity to associate with hRad51-DNA complexes, even under conditions which support DNA-binding. This suggests that hRad51-p53 contacts play a role in targeting p53 to heteroduplex joints and indicates an involvement in recombination immediately following hRad51-mediated strand transfer. To study the initial phase of strand exchange, when heteroduplex joints arise, we applied oligonucleotide based strand transfer assays. We observed that hRad51 stimulates exonucleolytic DNA degradation by p53, when it generates strand transfer intermediates. In agreement with this observation, artificial 3-stranded junction DNAs, designed to mimic nascent recombination intermediates, were found to represent preferred exonuclease substrates, especially when comprising a mismatch within the heteroduplex part. From our data, we propose a model according to which, p53-dependent correction of DNA exchange events is triggered by high-affinity binding to joint molecules and by stabilizing contacts with hRad51 oligomers. Oncogene (2000) 19, 4500 - 4512.

Abstract: Mutant p53 proteins were shown to exert complex DNA-interactions in vitro, like binding to MAR-DNA, but so far it is unknown whether such interactions also occur in vivo. Therefore we analysed the binding of mutant (mut) p53 (Gly245-->Ser) in Onda 11 glioma cells to cellular DNA in vivo, using p53-specific chromatin immunoprecipitation (CHIP) after in vivo cross-linking of mut p53 to genomic DNA with cisplatin. We identified genomic DNA fragments to which mut p53 (Gly245-->Ser) could be cross-linked in vivo. Purified recombinant mut p53 (Gly245-->Ser) was able to bind specifically to such elements in PCR-EMSA in vitro, supporting the idea that this mut p53 protein interacts with genomic DNA in vivo. The genomic DNA fragments identified are vastly different in sequence, but display as a common feature a high likelihood to adopt a non B-DNA conformation. Therefore we propose that structural determinants within these DNA elements are important for their interaction with mut p53 (Gly245-->Ser) in vivo. Oncogene (2000) 19, 4178 - 4183

Abstract: The p53-targeted kinases casein kinase 1delta (CK1delta) and casein kinase 1epsilon (CK1epsilon) have been proposed to be involved in regulating DNA repair and chromosomal segregation. Recently, we showed that CK1delta localizes to the spindle apparatus and the centrosomes in cells with mitotic failure caused by DNA-damage prior to mitotic entry. We provide here evidence that 3-[(2,4,6-trimethoxyphenyl)methylidenyl]-indolin-2-one (IC261), a novel inhibitor of CK1delta and CK1epsilon, triggers the mitotic checkpoint control. At low micromolar concentrations IC261 inhibits cytokinesis causing a transient mitotic arrest. Cells containing active p53 arrest in the postmitotic G1 phase by blockage of entry into the S phase. Cells with non-functional p53 undergo postmitotic replication developing an 8N DNA content. The increase of DNA content is accompanied by a high amount of micronucleated and apoptotic cells. Immunfluorescence images show that at low concentrations IC261 leads to centrosome amplification causing multipolar mitosis. Our data are consistent with a role for CK1delta and CK1epsilon isoforms in regulating key aspects of cell division, possibly through the regulation of centrosome or spindle function during mitosis.

Abstract: Mutant p53 not simply is an inactivated tumor suppressor, as at least some mutant p53 proteins exhibit oncogenic properties. Mutant p53 thus is the most commonly expressed oncogene in human cancer. Accordingly, the expression of mutant p53 in tumors often correlates with bad prognosis, and expression of mutant p53 in p53-negative tumor cells enhances their transformed phenotype. The molecular basis for this "gain of function" is not yet understood. However, the finding that mutant p53 tightly associates with the nuclear matrix in vivo, and with high affinity binds to nuclear matrix attachment region (MAR) DNA in vitro, suggests that these activities are connected and may result in perturbation of nuclear structure and function in tumor cells. MAR-binding of mutant p53 most likely is due to conformation-selective DNA binding by mutant p53, i.e. the specific interaction of a given mutant p53 protein with regulatory or structural genomic DNA elements that are able to adopt specific non-B-DNA conformations. In support to this assumption, human mutant p53 (Gly(245)-->Ser) was shown to bind to repetitive DNA elements in vivo that might be part of MAR elements. This further supports a model according to which mutant p53, by interacting with key structural components of the nucleus, exerts its oncogenic activities through perturbation of nuclear structure and function. J. Cell. Biochem. Suppl. 35:115-122, 2000.

Abstract: In this study we further characterized the 3'-5' exonuclease activity intrinsic to wild-type p53. We showed that this activity, like sequence-specific DNA binding, is mediated by the p53 core domain. Truncation of the C-terminal 30 amino acids of the p53 molecule enhanced the p53 exonuclease activity by at least 10-fold, indicating that this activity, like sequence-specific DNA binding, is negatively regulated by the C-terminal basic regulatory domain of p53. However, treatments which activated sequence-specific DNA binding of p53, like binding of the monoclonal antibody PAb421, which recognizes a C-terminal epitope on p53, or a higher phosphorylation status, strongly inhibited the p53 exonuclease activity. This suggests that at least on full-length p53, sequence-specific DNA binding and exonuclease activities are subject to different and seemingly opposing regulatory mechanisms. Following up the recent discovery in our laboratory that p53 recognizes and binds with high affinity to three-stranded DNA substrates mimicking early recombination intermediates (C. Dudenhoeffer, G. Rohaly, K. Will, W. Deppert, and L. Wiesmueller, Mol. Cell. Biol. 18:5332-5342), we asked whether such substrates might be degraded by the p53 exonuclease. Addition of Mg2+ ions to the binding assay indeed started the p53 exonuclease and promoted rapid degradation of the bound, but not of the unbound, substrate, indicating that specifically recognized targets can be subjected to exonucleolytic degradation by p53 under defined conditions.

Abstract: It is generally assumed that TGFbeta induces cell cycle arrest through the cooperative action of cell cycle inhibitors p15, p27 and p21. Here, we found that several pancreatic carcinoma cell lines exert TGFbeta-induced negative growth control in spite of the loss of p15 and p16 expression. In these cell lines, TGFbeta-induced growth control correlates with the upregulation of the p21 protein and active pRb expression. Conversely, cells without p21 and/or pRb expression are resistant to TGFbeta -induced growth inhibition. Moreover, overexpression of p21 in the p21-deficient cell line Panc Tu1 leads to growth arrest. Thus, TGFbeta-induced growth control correlates with p21 expression and pRb status independent of p15 and/or p16 expression.

Abstract: The tumour suppressor p53 is a potent mediator of cellular responses against genotoxic insults. In this review we describe the multiple functions of p53 in response to DNA damage, with an emphasis on p53's role in DNA repair. We summarize data demonstrating that p53 actively participates in various processes of DNA repair and DNA recombination via its ability to interact with components of the repair and recombination machinery, and by its various biochemical activities. An important aspect in evaluating p53 functions is provided by the finding that the core domain of p53 harbours two mutually exclusive biochemical activities, sequence-specific DNA binding required for its transactivation function, and 3'-5' exonuclease activity, possibly involved in aspects of DNA repair. Based on the finding that modifications of p53 which lead to activation of its sequence-specific DNA-binding activity result in inactivation of its 3'-5' exonuclease activity, we propose that p53 exerts its functions as a 'guardian of the genome' at various levels: in its noninduced state, p53 should not be regarded as a 'dead' protein but, for example, via its exonuclease activity might be actively involved in prevention and repair of endogenous DNA damage. Upon induction through exogenous DNA damage, p53 will exert its well-documented functions as a superior response element in various types of cellular stress. This dual role model for p53 in maintaining genomic integrity significantly enhances p53's possibilities as a guardian of the genome.

Abstract: Surface plasmon resonance measurements were used for detecting and quantifying protein-protein interactions between the tumor suppressor protein p53, the SV40 large T antigen (T-ag), the cellular DNA polymerase alpha-primase complex (pol-prim), and the cellular single-strand DNA binding protein RPA. Highly purified p53 protein bound to immobilized T-ag with an apparent binding constant of 2 x 10(8) M(-1). Binding of p53 to RPA was in the same order of magnitude with a binding constant of 4 x 10(8) M(-1), when RPA was coupled to the sensor chip via its smallest subunit, and 1 x 10(8) M(-1), when RPA was coupled via its p70 subunit. Furthermore, p53 bound human DNA polymerase alpha-primase complex (pol-prim) with a K(A) value of 1 x 10(10) m(-1). Both the p68 subunit and the p180 subunit of pol-prim could interact with p53 displaying binding constants of 2 x 10(10) m1(-1) and 5 X 10(9) M(-1), respectively. Complex formation was also observed with a p180/p68 heterodimer, and again with a binding constant similar. Hence, there was no synergistic effect when p53 bound to higher order complexes of pol-prim. A truncated form of p53, consisting of amino acids 1-320, bound pol-prim by four orders of magnitude less efficiently. Therefore, an intact C-terminus of p53 seems to be important for efficient binding to pol-prim. It was also tried to measure complex formation between p53, pol-prim, and T-ag. However there was no evidence for the existence of a ternary complex consisting of T-ag, pol-prim, and p53.

Abstract: Transcriptional activation by the tumor suppressor p53 is regulated at multiple levels, including posttranslational modifications of the p53 protein, interaction of p53 with various regulatory proteins, or at the level of sequence-specific DNA binding to the response elements in p53's target genes. We here propose as an additional regulatory mechanism that the DNA topology of p53-responsive promoters may determine the interaction of p53 with its target genes. We demonstrate that sequence-specific DNA binding (SSDB) and transcriptional activation by p53 of the mdm2 promoter is inhibited when this promoter is present in supercoiled DNA, where it forms a non-B-DNA structure which spans the p53-responsive elements. Relaxation of the supercoiled DNA in vitro resulted in conversion of the non-B-DNA to a B-DNA conformation within the mdm2 promoter, and correlated with an enhanced SSDB of p53 and an elevated expression of a reporter gene. In contrast, sequence specific DNA binding and transcriptional activation of the p21 promoter were not inhibited by DNA supercoiling. We propose that conformational alterations within p53-responsive sites, which either promote or prohibit sequence specific DNA binding of p53, are an important feature in orchestrating the activation of different p53 responsive promoters.

Abstract:

Abstract: In this review we describe the multiple functions of p53 in response to DNA damage, with an emphasis on p53's role in DNA repair. We summarize data demonstrating that p53, through its various biochemical activities and via its ability to interact with components of the repair and recombination machinery, actively participates in various processes of DNA repair and DNA recombination. An important aspect in evaluating p53 functions arises from the finding that the p53 core domain harbors two mutually exclusive biochemical activities, sequence-specific DNA binding, required for its transactivation function, and 3'->5' exonuclease activity, possibly involved in various aspects of DNA repair. As modifications of p53 that lead to activation of its sequence-specific DNA-binding activity result in inactivation of its 3'-> 5' exonuclease activity, we propose that p53 exerts its functions as a 'guardian of the genome' at various levels: in its non-induced state, p53 should not be regarded as a non-functional protein, but might be actively involved in prevention and repair of endogenous DNA damage, for example via its exonuclease activity. Upon induction through exogenous DNA damage, p53 will exert its well-documented functions as a superior response element in various types of cellular stress. The dual role model for p53 in maintaining genomic integrity significantly enhances p53's possibilities as a guardian of the genome.

Abstract: Recently, we described a new biological function of p53 in inhibiting recombination processes when encountering mismatches in heteroduplexes (Dudenhöffer et al., 1998). Here, we characterized protein domains of p53 participating in this process by in vitro analysis of mutated p53 proteins, and by applying our SV40-based assay system on monkey cells, which express different p53 variants. We present evidence that both binding of artificial recombination intermediates and p53-dependent recombination control require an intact p53 core and the oligomerization domain, strongly suggesting that the recognition of DNA undergoing recombination represents an essential step of this genomic surveillance mechanism. Further analyses indicated a role of the C-terminus in negatively regulating recombination control, an effect which can be neutralized by concurrent mismatch recognition. p53 lacking the oligomerization domain totally lost its ability to suppress homologous recombination. The cancer-related mutant p53(273H) was also significantly defective in this function, although we observed only twofold reductions in the corresponding transactivation activities on p53-response elements in episomal constructs. HDM2, an inhibitor of p53's transcriptional and growth regulatory activities, interfered with the inhibition of DNA exchange processes by p53 only weakly. Thus, functions of p53 in recombination control can be structurally dissociated from p53-dependent transcriptional transactivation.

Abstract: We recently reported that murine MethA mutant but not wild-type p53 specifically binds to MAR-DNA elements (MARs) with high affinity. Here we show that this DNA binding activity is exerted not only by MethA mutant p53 but also by other murine mutant p53 proteins isolated from the transformed murine BALB/c cell lines 3T3tx and T3T3 and differing in their conformational status. High affinity MAR-DNA binding was not restricted to the Xbal-IgE-MAR-DNA fragment from the murine immunoglobulin heavy chain gene enhancer locus [Cockerill et al. (1987): J Biol Chem 262:5394-5397] used in previous studies, as MethA p53 also specifically interacted with other A/T-rich bona fide MARs. Not only murine but also human mutant p53 proteins carrying the mutational hot spot amino acid exchanges 175Arg-->His, 273Arg-->Pro, or 273Arg-->His bound to the Xbal-IgE-MAR-DNA fragment. We therefore conclude that high affinity MAR-DNA binding is a property common to a variety of mutant p53 proteins.

Abstract: The primary CTL response of BALB/c mice infected with the lymphocytic choriomeningitis (LCM) virus strain WE is directed exclusively against one major epitope, n118, whereas a viral variant, ESC, that does not express n118 induces CTL against minor epitopes. We identified one minor epitope, g283, that induces primary lytic activity in ESC-infected mice. Infections of mice with WE and ESC were used to study the hierarchical control of a T cell response. Presentation of minor epitopes is not reduced in WE-infected cells. Generation of CTL against n118 does not suppress the generation of minor epitope-specific CTL systemically, as mice coinfected with WE and ESC developed CTL against n118 and g283. However, elimination of ESC and development of minor epitope-specific CTL in ESC infection were slower than elimination of WE and development of CTL against n118. CD8+ T cells against the minor epitope were activated in ESC and WE infection, but did not expand in the latter to show lytic activity in a primary response. We explain the absence of minor epitope-specific lytic activity in WE infection by the fast reduction of virus load due to the early developing n118-specific CTL. Immunodominance of CTL epitopes in primary virus infections thus can be explained as a kinetic phenomenon composed of 1) expansion of CD8+ T cells specific for individual epitopes, 2) stimulatory effect of virus load, and 3) negative feedback control on virus load by the fastest CTL population.

Abstract: The novel early response gene p22/PRG1 is linked to cell cycle entry and the induction of proliferation in various cell types although its exact function is still unknown. The p22/PRG1 promoter region contains a 20 bp sequence matching the consensus binding motif for the tumor suppressor protein p53. Gel shift assays demonstrated that p53 specifically binds to an oligonucleotide derived from the p53 binding site of the p22/PRG1 promoter. Chloramphenicol acetyltransferase (CAT) reporter gene assays confirmed that this site confers p53-dependent transcriptional activity to the p22/PRG1 promoter. In Hela cells, p22/PRG1 promoter constructs induced CAT expression only when cotransfected with an expression plasmid for wild-type, but not for mutant p53. Similarly, CAT expression was inducible at the permissive (31 degrees C) but not at the non-permissive temperature (39 degrees C) in the rat embryo fibroblast-derived cell line clone-6 that expresses a temperature-sensitive mutant p53. Conversion of this mutant p53 to a functional p53 at the permissive temperature was accompanied by a significant increase of endogenous p22/PRG1 mRNA level in this cell line. Gamma-irradiation of rat splenocytes or doxorubicin-treatment of Hela cells increased p53 levels followed by transcriptional activation of p22/PRG1 and p21/Waf1 in parallel. Our data demonstrate that p22/PRG1 transcription is induced by p53 during p53-dependent cell cycle arrest and apoptosis. Therefore, p22/PRG1 represents a novel target for transcriptional activation by p53.

Abstract: Mutant, but not wild-type p53 binds with high affinity to a variety of MAR-DNA elements (MARs), suggesting that MAR-binding of mutant p53 relates to the dominant-oncogenic activities proposed for mutant p53. MARs recognized by mutant p53 share AT richness and contain variations of an AATATATTT "DNA-unwinding motif," which enhances the structural dynamics of chromatin and promotes regional DNA base-unpairing. Mutant p53 specifically interacted with MAR-derived oligonucleotides carrying such unwinding motifs, catalyzing DNA strand separation when this motif was located within a structurally labile sequence environment. Addition of GC-clamps to the respective MAR-oligonucleotides or introducing mutations into the unwinding motif strongly reduced DNA strand separation, but supported the formation of tight complexes between mutant p53 and such oligonucleotides. We conclude that the specific interaction of mutant p53 with regions of MAR-DNA with a high potential for base-unpairing provides the basis for the high-affinity binding of mutant p53 to MAR-DNA.

Abstract: In this study we describe a novel putative p53-responsive gene, designated p22/PACAP response gene 1 (PRG1), recently identified as a proliferation-associated early-response gene in rats. By means of electrophoretic mobility shift assay and CAT-reporter gene assay, we could demonstrate that the p53 binding site residing in the promoter of p22/PRG1 is functional in vitro. Furthermore, in clone 6 cells expression of p22/PRG1 is induced in parallel to p21/Waf1 under conditions permitting mutant p53 to adopt wild-type configuration. An increase of p22/PRG1 transcription was also observed in gamma-irradiated rat splenocytes, which undergo p53-dependent apoptosis. Our findings demonstrate that p22/PRG1 fulfills all essential criteria as a p53 target gene and might be implicated in p53-dependent apoptosis.

Abstract: We demonstrate that wild-type p53 inhibits homologous recombination. To analyze DNA substrate specificities in this process, we designed recombination experiments such that coinfection of simian virus 40 mutant pairs generated heteroduplexes with distinctly unpaired regions. DNA exchanges producing single C-T and A-G mismatches were inhibited four- to sixfold more effectively than DNA exchanges producing G-T and A-C single-base mispairings or unpaired regions of three base pairs comprising G-T/A-C mismatches. p53 bound specifically to three-stranded DNA substrates, mimicking early recombination intermediates. The KD values for the interactions of p53 with three-stranded substrates displaying differently paired and unpaired regions reflected the mismatch base specificities observed in recombination assays in a qualitative and quantitative manner. On the basis of these results, we would like to advance the hypothesis that p53, like classical mismatch repair factors, checks the fidelity of homologous recombination processes by specific mismatch recognition.

Abstract: The temperature-sensitive mutant tsp53val135 accumulates in the cytoplasm of cells kept at the non-permissive temperature (39 degrees C), but is rapidly transported into the cell nucleus at the permissive temperature (30 degrees C). tsp53 thus may serve as a model for analysing cellular parameters influencing the subcellular location of p53. Here we provide evidence that retention of tsp53 in the cytoplasm at the non-permissive temperature is due to cytoskeletal anchorage of the p53 protein. Two sublines of C6 rat glioma cells differing in their expression of the intermediate filament protein vimentin (vimentin expressing or vimentin negative cells) were stably transfected with a vector encoding tsp53. Whereas cells of vimentin expressing C6 subclones retained tsp53 in the cytoplasm at the non-permissive temperature, cells of vimentin negative subclones exclusively harbored the tsp53 within their nuclei. Intermediate filament deficient cells that had been reconstituted with a full length vimentin protein again showed a cytoplasmic localization of tsp53, whereas in cells expressing a C-terminally truncated (tail-less) vimentin tsp53 localized to the nucleus. We conclude that cytoplasmic sequestration of tsp53 requires an intact intermediate filament system.

Abstract: Overexpression of p53 correlates with neoplasia in many cytological specimens. To test the specificity of overexpressed p53 as a tumour marker for the detection of pancreatic cancer, we analysed cytological specimens of pancreatic juice samples from patients with pancreatitis or pancreatic carcinoma (n = 42) for p53 protein overexpression. p53 protein overexpression was found in 59% of patients with pancreatitis and 67% of patients with pancreatic carcinoma. Thus, the assessment of p53 protein overexpression is not useful in the diagnosis of pancreatic cancer. Overexpressed p53 during pancreatitis appears to be wild-type p53. Overexpression of p53 may result from DNA damage occurring during chronic inflammation. It is well established that p53 can induce apoptosis upon DNA damage. Consequently, we found apoptotic cell death in five out of five tested cytological preparations from patients with pancreatitis as well as in one out of one pancreatic carcinoma specimen.

Abstract: Phosphopeptide analyses of the simian virus 40 (SV40) large tumor antigen (LT) in SV40-transformed rat cells, as well as in SV40 lytically infected monkey cells, showed that gel-purified LT that was not complexed to p53 (free LT) and p53-complexed LT differed substantially in their phosphorylation patterns. Most significantly, p53-complexed LT contained phosphopeptides not found in free LT. We show that these additional phosphopeptides were derived from MDM2, a cellular antagonist of p53, which coprecipitated with the p53-LT complexes, probably in a trimeric LT-p53-MDM2 complex. MDM2 also quantitatively bound the free p53 in SV40-transformed cells. Free LT, in contrast, was not found in complex with MDM2, indicating a specific targeting of the MDM2 protein by SV40. This specificity is underscored by significantly different phosphorylation patterns of the MDM2 proteins in normal and SV40-transformed cells. Furthermore, the MDM2 protein, like p53, becomes metabolically stabilized in SV40-transformed cells. This suggests the possibility that the specific targeting of MDM2 by SV40 is aimed at preventing MDM2-directed proteasomal degradation of p53 in SV40-infected and -transformed cells, thereby leading to metabolic stabilization of p53 in these cells.

Abstract: Sequence-specific transactivation of target genes is one of the most important molecular properties of the tumor suppressor p53. Binding of p53 to its target DNAs is tightly regulated, with modifications in the carboxy-terminal regulatory domain of the p53 protein playing an important role. In this study we examined the possible influence of DNA structure on sequence-specific DNA binding by p53, by analysing its binding to p53 consensus elements adopting different conformations. We found that p53 has the ability to bind to consensus elements which are present in a double-helical form, as well as to consensus elements which are located within alternative non-B-DNA structures. The ability of a consensus element to adopt either one of these conformations is dependent on its sequence symmetry, and is strongly influenced by its sequence environment. Our data suggest a model according to which the conformational status of the target DNA is an important determinant for sequence-specific DNA binding by p53. Modifications in the carboxy-terminal regulatory region of p53 possibly determine the preference of p53 for a given DNA conformation.

Abstract: We used clone 6 cells (rat embryo fibroblasts transformed by the temperature sensitive mutant p53val135 and an activated H-ras-gene (Michalovitz et al., 1990)), growth arrested at 32 degrees C, as a model to analyse whether and how transformed cells, growth-arrested by an overexpressed wild-type p53, might overcome p53-mediated growth inhibition. When clone 6 cells were kept at 32 degrees C for about 2 weeks, foci of cells appeared which grew temperature-independent. Analysis of individual clones of such cell demonstrated that the ectopically expressed tsp53-gene had not been altered by an additional mutation, but that the tsp53 in these cells at 32 degrees C had lost its ability to upregulate expression of the p53 target genes waf1 and mdm2. This loss of p53-specific transactivation correlated with nuclear exclusion of the tsp53 at 32 degrees C, which was most likely mediated by cytoplasmic retention of the tsp53 protein via short-lived anchor proteins. Cytoplasmic retention of the tsp53 at 32 degrees C was also observed in PC12 pheochromocytoma cells ectopically expressing tsp53val135, there occurring without specific selection. Also in these cells nuclear exclusion of the tsp53 correlated with loss of p53 mediated growth inhibition. Nuclear exclusion of p53 thus might serve as an epigenetic mechanism to eliminate the growth-inhibitory function of p53.

Abstract: To investigate a possible role of p53 in DNA exchange mechanisms, we have developed a model system which allows us to quantify homologous recombination rates in eukaryotic cells. We generated two types of simian virus 40 (SV40) whose genomes were mutated in such a way that upon double infection of monkey cells, virus particles can be released only after interchromosomal exchange of genetic material. This test system allowed us to determine recombination rates in the order of 10(-4) to 10(-6) for chromatin-associated SV40 genomes. To study the role of p53-T-antigen (T-Ag) complexes in this process, we designed viral test genomes with an additional mutation leading to a single amino acid exchange in T-Ag (D402H) and specifically blocking T-Ag-p53 interactions. Analysis of primary rhesus monkey cells endogenously expressing wild-type p53 showed a decreased recombination rate upon loss of efficient T-Ag-p53 complex formation. However, cells expressing mutant p53 (LLC-MK2 cells), the introduction of mutant T-Ag did not affect the DNA exchange rates. Our data are interpreted to indicate an inhibitory role of wild-type p53 in recombination. In agreement with this hypothesis, p53-T-Ag complex formation alleviates the inhibitory effect of wild-type p53.

Abstract: BALB/c mice are often considered "low responders" or even "nonresponders" with regard to cytolytic CD8+ T lymphocytes and SV40 large T Ag (TAg). Large TAg and fragments thereof were produced by recombinant technology and injected into BALB/c mice that were subsequently challenged by i.p. injection of syngeneic TAg-expressing mKSA tumor cells. Two portions of the TAg were found to induce protective immunity, one stretching from amino acid residues 1-272 and the other from amino acid residues 683-708. In mice thus protected, the spleens were virtually free of cytotoxic T cells but CD8+ T lymphocytes obtained from the peritoneal cavity during rejection of the mKSA cells were directly lytic for TAg-expressing target cells. Depleting immune mice of CD4+ or CD8+ T lymphocytes by treatment with mAb abolished their ability to resist tumor development. We conclude that immunity against SV40 TAg-expressing tumor cells in BALB/c mice is dependent on both CD4+ and CD8+ T lymphocytes.

Abstract: Inactivation of the tumor suppressor p53 by single missense point mutations characterizes a large number of human tumors. At least some mutant p53 proteins not only have lost the tumor suppressor function, but at the same time reveal a variety of dominant oncogenic properties. The molecular basis of this 'gain of function' is still unknown. In this report we describe a new biochemical activity of mutant p53, the specific high-affinity interaction with MAR/SAR DNA-elements (nuclear matrix/scaffold attachment regions). This DNA-binding activity can be distinguished from the previously reported DNA-binding activities of p53 by its specificity for mutant p53, the high binding affinity, and the domains of the mutant p53 molecule involved in MAR/SAR DNA-binding. The MAR/SAR-binding region of mutant p53 maps to a bipartite domain consisting of the mutated core region and the C-terminal 60 amino acids, carrying the unspecific DNA-binding domain and the oligomerization motif. MAR/SAR elements are considered as important regulatory elements in a variety of nuclear processes. We propose a model according to which the specific interaction of mutant p53 with MAR/SAR elements might interfere with these processes, thereby exerting pleiotropic oncogenic effects.

Abstract: The tumor suppressor p53 is a multifunctional protein whose main duty is to preserve the integrity of the genome. This function of wild-type p53 as "guardian of the genome" is achieved at different levels, as a cell cycle checkpoint protein, halting the cell cycle upon DNA damage, and via a direct involvement in processes of DNA repair. Alternatively, p53 can induce apoptosis. Mutations in the p53 gene occur in about 50% of all human tumors and eliminate the tumor suppressor functions of p53. However, many mutant p53 proteins have not simply lost tumor suppressor functions but have gained oncogenic properties which contribute to the progression of tumor cells to a more malignant phenotype. The molecular basis for this gain of function of mutant p53 is still unknown. However, mutant (mut) p53 specifically binds to nuclear matrix attachment region (MAR) DNA elements. MAR elements constitute important higher order regulatory elements of chromatin structure and function. By binding to these elements, mut p53 could modulate important cellular processes, like gene expression, replication, and recombination, resulting in phenotypic alterations of the tumor cells. Mut p53 thus could be the first representative of a new class of oncogenes, which exert their functions via long-range alterations or perturbation of chromatin structure and function.

Abstract: Expression of the simian virus 40 large T antigen (large T) in F111 rat fibroblasts generated only minimal transformants (e.g., F5 cells). Interestingly, F111-derived cells expressing only an amino-terminal fragment of large T spanning amino acids 1 to 147 (e.g., FR3 cells), revealed the same minimal transformed phenotype as F111 cells expressing full-length large T. This suggested that in F5 cells the transforming domain of large T contained within the C-terminal half of the large T molecule, and spanning the p53 binding domain, was not active. Progression to a more transformed phenotype by coexpression of small t antigen (small t) could be achieved in F5 cells but not in FR3 cells. Small-t-induced progression of F5 cells correlated with metabolic stabilization of p53 in complex with large T: whereas in F5 cells the half-life of p53 in complex with large T was only slightly elevated compared with that of (uncomplexed) p53 in parental F111 cells or that in FR3 cells, coexpression of small t in F5 cells led to metabolic stabilization and to high-level accumulation of p53 complexed to large T. In contrast, coexpression of small t had no effect on p53 stabilization or accumulation in FR3 cells. This finding strongly supports the assumption that the mere physical interaction of large T with p53, and thus p53 inactivation, in F5 cells expressing large T only does not reflect the main transforming activity of the C-terminal transforming domain of large T. In contrast, we assume that the transforming potential of this domain requires activation by a cellular function(s) which is mediated by small t and correlates with metabolic stabilization of p53.

Abstract: Highly purified p53 protein from different sources was able to degrade DNA with a 3'-to-5' polarity, yielding deoxynucleoside monophosphates as reaction products. This exonuclease activity was dependent on Mg2+ and inhibited by addition of 5 mM nucleoside monophosphates. This exonuclease activity is intrinsic to the wild-type p53 protein: it copurified with p53 during p53 preparation; only purified wild-type p53, but not identically purified mutant p53 proteins displayed exonuclease activity; the exonuclease activity could be reconstituted from SDS gel-purified and urea-renatured p53 protein and mapped to the core domain of the p53 molecule; and finally, purified p53 protein could be UV-cross-linked to GMP. A p53-intrinsic exonuclease activity should substantially extend our view on the role of p53 as a "guardian of the genome."

Abstract: Injection into mice of chimeric proteins consisting of a portion of either the simian virus 40 large tumor antigen or nonstructural protein 1 of influenza A virus or of the murine tumor suppressor p53 on one hand and T-cell epitopes of lymphocytic choriomeningitis virus on the other resulted in antiviral protective immunity, which was independent of the epitopes' position in the protein and the same whether the latter was immunologically nonself or self. Mice of different haplotypes were protected when the corresponding class I molecule-restricted epitopes had been inserted close to each other in one carrier protein.

Abstract: A number of candidate tumor suppressor genes located on the human chromosome 17 are thought to have a role to play in the development of breast cancer. In addition to the p53 gene on 17p13.1 and the BRCA1 gene mapped to 17q12-21, other chromosomal regions for tumor suppressor genes have been suggested to exist on 17p13.3 and both the central and the distal parts of 17q, although definitive functional proof of their involvement in breast cancer tumorigenesis is still lacking. In this report we show that microcell transfer of a human chromosome 17 into wild-type p53 breast cancer cells CAL51 results in loss of tumorigenicity and anchorage-independent growth, changes in cell morphology and a reduction of cell growth rates of the neo-selected microcell hybrids. In the hybrid cells, which express the p53 wild-type protein, only the p- and the distal parts of the q arm of donor chromosome 17 are transferred. Thus, our results provide functional evidence for the presence of one or more tumor suppressor gene(s) on chromosome 17, which are distinct from the p53 and the BRCA1 genes.

Abstract: Replication of the small DNA tumor virus, simian virus 40 (SV40), is largely dependent on host cell functions, because SV40, in addition to virion proteins, codes only for a few regulatory proteins, the most important one being the SV40 large tumor antigen (T-antigen). This renders SV40 an excellent tool for studying complex cellular and viral processes. In this review we summarize and discuss data providing evidence for virtually all major viral processes during the life cycle of SV40 from viral DNA replication to virion formation, being performed at or within structural systems of the nucleus, in particular the chromatin and the nuclear matrix. These data further support the concept that viral replication in the nucleus is structurally organized and demonstrate that viruses are excellent tools for analyzing the underlying cellular processes. The analysis of viral replication at nuclear structures might also provide a means for specifically interfering with viral processes without interfering with the corresponding cellular functions.

Abstract: The temperature-sensitive mutant p53 tsp53val135 (tsp53) displays a mutant phenotype at 38 degrees C, but assumes properties of a wild-type (wt) p53 at 32 degrees C. We analysed the cellular responses of two cell lines which ectopically overexpress tsp53, and dramatically differ in their responses to tsp53 expressed at 32 degrees C. Clone 6 (cl6) cells [precrisis rat embryo fibroblasts transformed by tsp53val135 and an activated ras oncogene at 38 degrees C (Michalovitz et al., 1990. Cell 62, 671-680) stop to grow and arrest mainly in the G1 phase of the cell cycle, whereas MethAp53ts cells [BALB/c mouse MethA tumor cells, transfected with the same tsp53 encoding vector as cl6 cells (Otto and Deppert, 1993. Oncogene 8, 2591-2603)] do not growth arrest at 32 degrees C. Both cell lines expressed similar amounts of tsp53, which was mainly cytoplasmic at 38 degrees C and mainly nuclear at 32 degrees C. At 32 degrees C, both cell lines contained similar amounts of waf1/cip1 mRNA. However, the amount of mdm2 mRNA in MethAp53ts cells was considerably higher compared to that in cl6 cells. The different transcriptional regulation of the mdm2-gene in cl6 and MethAp53ts cells at 32 degrees C indicated that the tsp53 proteins in these cells were functionally different. This assumption was supported by our finding that at 32 degrees C phosphorylation of the tsp53 in these cells was markedly different. We conclude that the cellular environment is an important determinant of p53 function.

Abstract: BALB/c and C57BL/6J mice were immunized with recombinant vaccines consisting of lymphocytic choriomeningitis virus CD8+ T-lymphocyte epitopes and a carrier protein. During challenge infection with WE strain lymphocytic choriomeningitis virus, mutants with alterations in distinct amino acid residues of the epitopic nonapeptides appeared and multiplied. Splenocytes from WE-infected BALB/c mice lysed cells coated with the WE-type epitope; lysis was considerably less effective when the epitopic nonapeptide with which the syngeneic cells had been sensitized was the mutated form. Neither target was lysed by splenocytes from BALB/c mice infected with the variant virus. Mutants were not detected in F1 hybrid mice immunized with two viral epitopes that were restricted by class I molecules of both parents.

Abstract: When growth-arrested mouse fibroblasts re-entered the cell-cycle, the rise in tumour suppressor p53 mRNA level markedly preceded the rise in expression of the p53 protein. Furthermore, gamma-irradiation of such cells led to a rapid increase in p53 protein biosynthesis even in the presence of the transcription inhibitor actinomycin D. Both findings strongly suggest that p53 biosynthesis in these cells is regulated at the translational level. We present evidence for an autoregulatory control of p53 expression by a negative feed-back loop: p53 mRNA has a predicted tendency to form a stable stem-loop structure that involves the 5'-untranslated region (5'-UTR) plus some 280 nucleotides of the coding sequence. p53 binds tightly to the 5'-UTR region and inhibits the translation of its own mRNA, most likely mediated by the p53-intrinsic RNA re-annealing activity. The inhibition of p53 biosynthesis requires wild-type p53, as it is not observed with MethA mutant p53, p53-catalysed translational inhibition is selective; it might be restricted to p53 mRNA and a few other mRNAs that are able to form extensive stem-loop structures. Release from negative feed-back regulation of p53 biosynthesis, e.g. after damage-induced nuclear transport of p53, might provide a means for rapidly increasing p53 protein levels when p53 is required to act as a cell-cycle checkpoint determinant after DNA damage.

Abstract: Alternative splicing of the p53 transcript which so far has been demonstrated only in the murine system has been proposed as a general regulatory mechanism for the generation of functionally different p53 proteins. We analyzed by RT-PCR the pattern of p53 mRNAs within the region spanning exons 10 and 11 of the p53 gene in 13 different tissues from two independent mouse strains, in 10 different rat tissues and in six different human tissues. PCR products of the expected sizes, corresponding to the normally spliced and the alternatively spliced p53 mRNAs, were detected in mice. Alternatively spliced mRNA was found at approximately 25-20% the level of the normally spliced p53 mRNA in most tissues analyzed. In spleen and kidney the proportion of alternatively spliced p53 mRNA was much lower. Surprisingly, examination of p53 mRNAs isolated from 10 different rat tissues and six human tissues within the same region of the p53 gene showed only products of normal size. Although a potential homologous alternative 3' splice site within intron 10 of the human p53 gene is present in the genomic sequence of human p53, the expected corresponding alternatively spliced p53 mRNA was undetectable. These findings imply that the generation of functionally different forms of p53 by alternative splicing of p53 transcripts is a species-specific event, possibly indicating species-specific mechanisms for regulating p53 activities.

Abstract: Metabolic stabilization of the tumor suppressor p53 is a key event in cellular transformation by simian virus 40 (SV40). Expression of the SV40 large tumor antigen (large T) is necessary but not sufficient for this process, as metabolic stabilization of p53 complexed to large T in abortively SV40-infected cells strictly depends on the cellular systems analyzed (F. Tiemann and W. Deppert, J. Virol. 68:2869-2878, 1994). Comparative analyses of various cells differing in metabolic stabilization of p53 upon abortive infection with SV40 revealed that metabolic stabilization of p53 closely correlated with expression of the SV40 small t antigen (small t) in these cells: 3T3 cells do not express small t and do not stabilize p53 upon infection with wild-type SV40. However, ectopic expression of small t in 3T3 cells provided these cells with the capacity to stabilize p53 upon SV40 infection. Conversely, precrisis mouse embryo cells express small t and mediate metabolic stabilization of p53 upon infection with wild-type SV40. Infection of these cells with an SV40 small-t deletion mutant did not lead to metabolic stabilization of p53. Small-t expression and metabolic stabilization of p53 correlated with an enhanced transformation efficiency by SV40, supporting the conclusion that at least part of the documented helper effect of small t in SV40 transformation is its ability to promote metabolic stabilization of p53 complexed to large T.

Abstract: The tumor suppressor p53 is a key element in preserving the stability of the genetic information of vertebrates. In response to DNA damage, PSS induces a growth arrest thus allowing time for DNA repair to occur. However, p53 seems to exert additional functions in cellular proliferation, amongst them a so far unidentified role in the progression of cells through the cell cycle and in differentiation processes. Modulation of p53 activities, by regulating p53 interactions with different target genes and cellular protein partners through the cellular environment might explain the diversity of p53 functions in cellular growth control.

Abstract: To understand the process and biological significance of metabolic stabilization of p53 during simian virus 40 (SV40)-induced cellular transformation, we analyzed cellular and viral parameters involved in this process. We demonstrate that neither large T expression as such nor the cellular phenotype (normal versus transformed) markedly influence the stability of p53 complexed to large T in SV40 abortively infected BALB/c mouse fibroblasts. In contrast, metabolic stabilization of p53 is an active cellular event, specifically induced by SV40. The ability of SV40 to induce a cellular response leading to stabilization of p53 complexed to large T is independent from the cellular phenotype and greatly varies between different cells. However, metabolic stability was conferred only to p53 in complex with large T, whereas the free p53 in these cells remained metabolically unstable. Comparative analyses of cellular transformation in various cells differing in stability of p53 complexed to large T upon abortive infection with SV40 revealed a strong correlation between the ability of SV40 to induce metabolic stabilization and its transformation efficiency. Our data suggest that metabolic stabilization and the ensuing enhanced levels of p53 are important for initiation and/or maintenance of SV40 transformation.

Abstract: A major portion of the nucleoprotein (amino acids 67 through 300) and the glycoprotein-2 of lymphocytic choriomeningitis (LCM) virus were synthesized by using recombinant technology and were injected together with SDS twice in portions of 5 micrograms into BALB/c mice. As evidenced by diminished replication of LCM challenge virus, both proteins induced antiviral immunity, which was comparable in extent with the immunity caused by infection with LCM vaccinia recombinant viruses. Primed LCM-viral CTLs could not be demonstrated in these mice by culturing splenocytes in the presence of LCM virus, and Abs appeared slowly and in low quantities; but, after injection of large infectious doses, CTLs appeared faster and in higher numbers than in mice not previously treated with viral proteins. Depletion of CD8+ cells, but not of CD4+ cells, by treatment of mice with mAb abolished the antiviral immunity, demonstrating that protection was mediated by CD8+ T lymphocytes. Absence of CD4+ T lymphocytes before and during the period of immunization did not measurably affect the animals' antiviral immune status, indicating that activation of the CD8+ T lymphocytes was not dependent on help by CD4+ cells.

Abstract: We analysed p53 expression during proliferation of serum stimulated Swiss mouse 3T3 cells and of concanavalin A stimulated mouse spleen lymphocytes and correlated it to rate of DNA synthesis and to expression of PCNA. We also analysed mdm2 gene expression, as rising p53 levels during proliferation might require MDM2 protein expression to functionally antagonize p53 mediated growth inhibition. p53 protein synthesis closely paralleled DNA synthesis and PCNA expression, suggesting a direct involvement of p53 in cellular DNA synthesis. mdm2 expression in 3T3 cells could not be correlated with p53 expression and DNA synthesis and was not detected at all in stimulated lymphocytes. We conclude that p53 and mdm2 expression during proliferation are not functionally related and that mdm2 expression is not required for proliferation.

Abstract: In order to analyse the immortalizing and transforming potential of simian virus 40 (SV40), we compared the transformation efficiencies of SV40 in primary and in established BALB/c mouse fibroblasts. Five independently isolated clones of freshly immortalized normal fibroblasts (FTE cells) were established from precrisis BALB/c mouse embryo fibroblasts (pMEF cells) according to the protocol for establishing 3T3 cells (Todaro & Green, 1963). These cells expressed a wild-type p53 and were indistinguishable in all parameters analysed from original 3T3 cells kept in our laboratory. Using abortive infection to control gene dosage, followed by selection of transformed cells by cloning in soft agar, SV40 was able to transform primary cells with a much higher efficiency than 3T3 or FTE cells. Analysis of this unexpected result revealed that the different transformation efficiencies of SV40 in primary and established cells correlated with an altered cellular response to SV40 infection regarding metabolic stabilization of p53 complexed to large T during abortive infection. Whereas p53 in pMEF cells became stabilized upon abortive infection with SV40, p53 in 3T3 and FTE cells remained unstable. Our results strongly favour the hypothesis that metabolic stabilization and the ensuing higher levels of p53 in abortively infected cells enhance the transforming competence of large T.

Abstract: Minimal transformants of rat F111 fibroblasts were established after infection with the large T antigen (large T)-encoding retroviral expression vector pZIPTEX (M. Brown, M. McCormack, K. Zinn, M. Farrell, I. Bikel, and D. Livingston, J. Virol. 60:290-293, 1986). Coexpression of small t antigen (small t) in these cells efficiently led to their progression toward a significantly enhanced transformed phenotype. Small t forms a complex with phosphatase 2A and thereby might influence cellular phosphorylation processes, including the phosphorylation of large T. Since phosphorylation can modulate the transforming activity of large T, we asked whether the phosphorylation status of large T in minimally transformed cells might differ from that of large T in maximally transformed FR(wt648) cells and whether it might be altered by coexpression of small t. We found the phosphate turnover on large T in minimally transformed cells significantly different from that in fully transformed cells. This resulted in underphosphorylation of large T in minimally transformed cells at phosphorylation sites previously shown to be involved in the regulation of the transforming activity of large T. However, coexpression of small t in the minimally transformed cells did not alter the phosphate turnover on large T during progression; i.e., it did not induce a change in the steady-state phosphorylation of large T. This suggests that the helper function of small t during the progression of these cells was not mediated by modulating phosphatase 2A activity toward large T.

Abstract: The nuclear matrix plays an important role in simian virus 40 (SV40) DNA replication in vivo, since functional replication complexes containing large T and replicating SV40 minichromosomes are anchored to this structure (R. Schirmbeck and W. Deppert, J. Virol. 65:2578-2588, 1991). In the present study, we have analyzed the course of events leading from nuclear matrix-associated replicating SV40 minichromosomes to fully replicated minichromosomes and, further, to their encapsidation into mature SV40 virions. Pulse-chase experiments revealed that newly replicated SV40 minichromosomes accumulated at the nuclear matrix and were directly encapsidated into DNase-resistant SV40 virions at this nuclear structure. Alternatively, a small fraction of newly replicated minichromosomes left the nuclear matrix to associate with the cellular chromatin. During the course of infection, progeny virions continuously were released from the nuclear matrix to the cellular chromatin and into the cytoplasm-nucleoplasm. The bulk of SV40 progeny virions, however, remained at the nuclear matrix until virus-induced cell lysis.

Abstract: Immunization of C57BL/6 (B6) mice (H-2b) with the "large tumor antigen" (T-Ag) of simian virus 40 (SV40) in its soluble form without adjuvants primed CD8+ cytotoxic T lymphocytes (CTL) in vivo. CD8+ CTL primed in vivo by this non-structural 708-amino acid (aa) viral protein, and specifically restimulated in vitro, lysed H-2b target cells, either transfected with an SV40 T-Ag-encoding vector, or transformed by SV40 infection. H-2b RMA-S transfectants expressing the complete 708 aa T-Ag (which fail to transport peptides through the endoplasmic reticulum membranes) were not lysed. CTL were also efficiently primed in vivo by injection of the N-terminal 272 aa fragment of the T-Ag. Hence, this fragment contains the structure(s) required for a soluble protein to enter the "endogenous" class I-restricted antigen processing and presentation pathway for CD8+ CTL activation. In soluble form, the complete T-Ag or the N-terminal T-Ag fragment sensitized in vitro RBL5 cells for lysis by T-Ag-specific CTL lines and clones. This in vitro sensitization was blocked by brefeldin A. In contrast, specific recognition of RBL5 cells pulsed in vitro with synthetic, immunogenic nonapeptides (derived from N-terminal T-Ag epitopes) by CTL lines was insensitive to brefeldin A. Hence, T-Ag and its 272-aa N-terminal fragment can enter the "endogenous" processing pathway and prime CD8+ CTL in vivo and in vitro.

Abstract: To analyze the proposed growth-inhibitory function of wild-type p53, we compared simian virus 40 (SV40) DNA replication in primary rhesus monkey kidney (PRK) cells, which express wild-type p53, and in the established rhesus monkey kidney cell line LLC-MK2, which expresses a mutated p53 that does not complex with large T antigen. SV40 DNA replication proceeded identically in both cell types during the course of infection. Endogenously expressed wild-type p53 thus does not negatively modulate SV40 DNA replication in vivo. We suggest that inhibition of SV40 DNA replication by wild-type p53 in in vitro replication assays is due to grossly elevated ratios of p53 to large T antigen, thus depleting the replication-competent free large T antigen in the assay mixtures by complex formation. In contrast, the ratio of p53 to large T antigen in in vivo replication is low, leaving the majority of large T antigen in a free, replication-competent state.

Abstract: Injection of the 708-amino-acid (aa) viral protein "large tumor antigen" (T-Ag) of simian virus 40 (SV40) or its N-terminal 272-aa fragment into C57BL/6 (B6) mice (H-2b) primed CD8+ cytotoxic T lymphocytes (CTL) in vivo. Surprisingly, injection of this nonstructural viral protein (or its N-terminal fragment) in soluble form (without adjuvants) was as efficient in priming CD8+ CTL in vivo as the infection of B6 mice with the virus SV40. CTL activated in vivo by immunization with T-Ag proteins or SV40 infection specifically lysed syngeneic RBL5 cells transfected with a T-Ag-encoding vector; these RBL5/M7 transfectants efficiently presented N- and C-terminal T-Ag epitopes in association with H-2 class I restriction elements. N- and C-terminal T-Ag epitopes were recognized by CTL primed in vivo by immunization with the complete T-Ag protein or by infection with SV40, and (as expected) only N-terminal T-Ag epitopes were recognized by CTL primed in vivo by the soluble N-terminal T-Ag fragment. In CD8+ CTL populations primed in vivo by immunization with the complete T-Ag protein or by SV40 infection and restimulated in vitro with RBL5/M7 transfectants in a mixed tumor cell-lymphocyte culture (MTLC), CTL with specificity for C-terminal T-Ag epitopes were selectively expanded in vitro for months. Hence, the in vitro expansion of CTL population with heterogenous recognition specificities can dramatically distort the picture of its specific recognition repertoire primed in vivo.

Abstract: We found that simian virus 40 (SV40), in addition to the SV40 early proteins large T antigen (large T) and small antigen (small t), codes for a third early protein with a molecular weight of 17 kDa. This protein (17kT) is expressed from an alternatively spliced third SV40 early mRNA, using a splice donor site at position 4425 and a splice acceptor site at position 3679 of the SV40 genome. The 17kT protein consists of 135 amino acids. Of these, 131 correspond to the amino-terminus of large T, while the four carboxy-terminal amino acids are unique and encoded by a different reading frame. 17kT mRNA, and the corresponding protein, were found in all SV40 transformed cells analyzed, as well as in SV40 infected cells. Transfection of a cDNA expression vector encoding the 17kT protein into rat F111 fibroblasts induced phenotypic transformation of these cells. The expression of the transforming amino-terminal domain of large T as an independent 17kT protein might provide a means for individually regulating the various functions associated with this domain.

Abstract: Overexpression of mouse wild-type p53 (wt p53) in mouse Meth A tumor cells after transfection of wt p53 encoding vectors induced a strong growth-inhibitory response. Cells of only few of randomly selected surviving colonies contained and expressed the transfected wt p53 specific DNA. Despite expressing authentic wt p53, such cells (MethAp53wt) exhibited a similar phenotype as the parental Meth A cells. These cells overexpressed the mdm-2 (mouse double minute-2) gene, both at the RNA and at the protein level. Recently, the MDM-2 protein has been identified as a cellular target of p53, which can abolish its tumor suppressor activity. We, therefore, suggest that MDM-2 has mitigated the growth-inhibitory effect of wt p53 in MethAp53wt cells. Upregulation of mdm-2 expression in MethAp53wt cells was mediated by wt p53, as analysis of Meth A cells carrying a tsp53 (p53Val135) revealed a strict dependence of mdm-2 upregulation upon wt p53 expression. Our results propose that a balanced ratio of MDM-2 and p53 will allow cells to tolerate a limited expression of wt p53. This tolerance is not mediated by a direct inactivation of wt p53 via complex formation with MDM-2, as the majority of both MDM-2 and wt p53 in MethAp53wt cells was not complexed to each other.

Abstract: We report that p53 in resting and concanavalin A-stimulated Balb/c mouse lymphocytes cannot be distinguished on the basis of different reactivity with various epitope-specific monoclonal antibodies, regardless of whether the lymphocytes are stimulated with concanavalin A in the presence or absence of serum. Our results thus question the 'conformational hypothesis' put forward by Milner [Milner, J. (1991). Curr. Op. Cell Biol., 3, 282-286], according to which wild-type p53, depending on its conformational status, can act as a negative or a positive growth regulator.

Abstract: C57BL/6 (B6) mice (H-2b) were immunized with the large tumor antigen (T Ag) of simian virus 40 (SV40). Intraperitoneal or subcutaneous sensitization with soluble T Ag specifically primed cytotoxic lymphocyte precursors (CTLp). T Ag-specific cytotoxic T lymphocytes (CTL) were detected in a cytotoxicity assay after specific in vitro restimulation of effector cell populations from mice immunized with 2-10 micrograms purified, soluble T Ag and boosted with an injection of 2 micrograms T Ag 2-4 weeks after priming. Cells used for in vitro restimulation and as targets in cytotoxicity assays were syngeneic (B6-derived) RBL5 lymphoma cells expressing SV40 T Ag after transfection with a T Ag-encoding expression vector. Effector cells of this response were H-2 class I-restricted CD3+ CD4-CD8+ CTL. The magnitude of the anti-T Ag CTL response of B6 mice stimulated by soluble virus protein was comparable to the anti-T Ag CTL response of SV40-infected B6 mice. Injections of denatured or native T Ag protein primed CTLp equally well, but immunization with an equal dose of antigen emulsified in incomplete Freund's adjuvants inefficiently stimulated CTLp.

Abstract: We have analyzed in detail the phosphorylation of p53 from normal (3T3) and simian virus 40 (SV40)-transformed (SV3T3) BALB/c mouse cells and from normal (F111) and SV40-transformed [FR(wt648)] rat cells by two-dimensional tryptic peptide mapping and phosphoamino acid analyses. To accommodate the different half-lives of p53 in normal (half-life, 15 min) and transformed (half-life, 20 h) cells and possible differences in the rates of turnover of phosphate at specific sites, cells were labeled for 2 h (short-term labeling) or 18 h (long-term labeling). Depending on the labeling conditions, either close similarities or marked differences were observed in the phosphorylation patterns of p53 from normal and transformed cells. After the 2-h labeling, the phosphorylation patterns of p53 from normal and transformed mouse cells were quite similar. In contrast, p53 from normal and transformed rat cells exhibited dramatic quantitative and qualitative differences under these labeling conditions. The reverse was found after an 18-h label leading to steady-state phosphorylation of p53 in transformed cells: while p53 in transformed mouse cells revealed a marked quantitative increase in phosphorylation compared with p53 from normal cells, the corresponding patterns of p53 from normal and transformed rat cells were similar. Our data thus indicate species-specific differences in the phosphorylation of mouse and rat p53 in SV40-transformed cells, reflected by (i) different turnover rates at specific sites in mouse and rat p53 and (ii) phosphorylation of nonhomologous serine and threonine residues in rat p53, as revealed by indirect assignment of phosphorylation sites to the phosphopeptides of rat p53. Analyses of p53 from the SV40 tsA58 mutant-transformed F111 cell lines FR(tsA58)A (N type) and FR(tsA58)57 (A type) yielded no conclusive evidence for a direct correlation between phosphorylation of p53, the metabolic stabilization of p53, and expression of the transformed phenotype.

Abstract: In order to obtain insight into the parameters determining the subcellular localization of mutant and wild-type forms of p53, we analysed the subcellular distribution of p53 in four Balb/c mouse-derived cell lines ranging in their cellular phenotypes from normal (3T3), via minimal transformant (T3T3), to maximally transformed (3T3tx, Meth A). Epitope mapping showed the p53 proteins in 3T3 and in T3T3 cells to be in a wild-type conformation, as they reacted with PAb246, whereas p53 in 3T3tx and in Meth A cells were PAb246 negative and thus displayed a mutant conformation. Despite its reactivity with PAb246, p53 in T3T3 cells had an extended half-life and accumulated to abnormally high levels. We show that the conformationally wild-type p53 in 3T3 and T3T3 cells predominantly localized to the cell nucleus, with about half of it being tightly associated with nuclear structures. In contrast, approximately 60% of mutant p53 in 3T3tx and Meth A cells localized to the cytoplasm, the rest residing in the cell nucleus; all the nuclear p53 in these cells appeared to be structurally bound. The cytoplasmic location of mutant p53 in 3T3tx and Meth A cells was not seen by immunofluorescence microscopic analysis, and required cell fractionation for its detection. Both cytoplasmic and nuclear p53 of the mutant phenotype bound to hsc proteins with a similar stoichiometry, suggesting that hsc binding is not directly related to the subcellular distribution of these proteins. We suggest that the conformational phenotype of p53 is a major determinant of its subcellular location.

Abstract: Biologically active mutant p53 from Balb/c mouse tumor cells (Meth A) was analysed for its specific interaction with DNA. Restricted phage lambda DNA, representing DNA of high complexity with regard to sequence and secondary structure, was used to probe for such an activity in a target-bound DNA-binding assay, using doubly immunopurified p53. A single lambda DNA fragment was specifically retained with very high affinity (KD = 10(-10) M). Specific DNA binding was shown to be an intrinsic property of p53, as it could be blocked with p53-specific monoclonal antibodies PAb122 and PAb421. The characteristics of the DNA binding of p53 to this lambda DNA fragment, as well as the structural properties of this fragment, suggested the possibility that p53 might be able to interact with nuclear matrix attachment region (MAR) DNA. Indeed, established genomic MAR elements were specifically bound by Meth A p53, whereas no binding was observed to an AT-rich control DNA. The interaction of p53 with MAR elements in vitro is compatible with the idea that p53 in vivo is involved in the regulation of replication and/or expression of cellular DNA. Complex DNA interactions were not restricted to mutant p53 from Meth A cells. Mutant p53 of a different conformational phenotype (PAb246+ 'wild-type' as opposed to PAb246- 'mutant' for p53 from Meth A cells) from minimally transformed T3T3 cells, as well as genotypic wild-type p53 expressed by a recombinant baculovirus in insect cells, exhibited similar DNA-binding properties.

Abstract: Large T-antigen (T-Ag) in hamster cells transformed by the lymphotropic papova virus (LPV) exhibits similar properties as the T-Ag of simian virus 40 (SV40) with regard to its interaction with cellular targets. However, in contrast to SV40-transformed cells, LPV-transformed cells in cell culture select against high expression of LPV T-Ag. Southern analysis revealed that this selection process was accompanied by drastic changes at the DNA level, involving the loss of most of the integrated viral DNA copies. These changes probably were responsible for an approximately 100-fold downregulation of LPV T-Ag transcription. To elucidate the biological significance of this phenomenon, we studied the effects of the expression of LPV and SV40 T-Ag, respectively, in a variety of cells. Our data suggest that LPV T-Ag, like SV40 T-Ag, acts as an immortalizing and transforming protein. However, in contrast to SV40 T-Ag, high-level expression of LPV T-Ag seems to be detrimental to the establishment and maintenance of LPV-transformed cells in vitro.

Abstract: In an attempt to analyze the persistent infection of rhesus monkey cells with Simian virus 40 (SV40) in vitro, as described previously (reviewed in L. C. Norkin, Microbiol. Rev. 46, 384-425, 1982), we infected primary rhesus cell cultures (PRK), derived from a SV40-free monkey colony with SV40. Surprisingly, SV40 infected PRK cell cultures released as much infectious virus as cultures of the permissive African green monkey kidney cell line TC7. Infected PRK cells exhibited typical symptoms of a lytic infection, and the bulk of infected PRK cells died within 8 days postinfection (p.i.). A considerable proportion of infected PRK cells exhibited distinct SV40-caused cytopathic effects (CPE), similar to CPE in infected TC7 cells. We conclude that the in vivo persistence of SV40 in rhesus monkeys is not determined by cellular host factors, but by the immune system of the infected animals.

Abstract: To identify molecular differences between simian virus 40 (SV40) tsA58 mutant large tumor antigen (large T) in cells of tsA58 N-type transformants [FR(tsA58)A cells], which revert to the normal phenotype after the cells are shifted to the nonpermissive growth temperature, and mutant large T in tsA58 A-type transformants [FR(tsA58)57 cells], which maintain their transformed phenotype after the temperature shift, we asked whether the biological activity of these mutant large T antigens at the nonpermissive growth temperature might correlate with phosphorylation at specific sites. At the permissive growth temperature, the phosphorylation patterns of the mutant large T proteins in FR(tsA58)A (N-type) cells and in FR(tsA58)57 (A-type) cells were largely indistinguishable from that of wild-type large T in FR(wt648) cells. After a shift to the nonpermissive growth temperature, no significant changes in the phosphorylation patterns of wild-type large T in FR(wt648) or of mutant large T in FR(tsA58)57 (A-type) cells were observed. In contrast, the phosphorylation pattern of mutant large T in FR(tsA58)A (N-type) cells changed in a characteristic manner, leading to an apparent underphosphorylation at specific sites. Phosphorylation of the cellular protein p53 was analyzed in parallel. Characteristic differences in the phosphorylation pattern of p53 were observed when cells of N-type and A-type transformants were kept at 39 degrees C as opposed to 32 degrees C. However, these differences did not relate to the different phenotypes of FR(tsA58)A (N-type) and FR(tsA58)57 (A-type) cells at the nonpermissive growth temperature. Our results, therefore, suggest that phosphorylation of large T at specific sites correlates with the transforming activity of tsA mutant large T in SV40 N-type and A-type transformants. This conclusion was substantiated by demonstrating that the biological properties as well as the phosphorylation patterns of SV40 tsA28 mutant large T in cells of SV40 tsA28 N-type and A-type transformants were similar to those in FR(tsA58)A (N-type) and in FR(tsA58)57 (A-type) cells, respectively. The phenotype-specific phosphorylation of tsA mutant large T in tsA A-type transformants probably is a cellular process induced during establishment of SV40 tsA A-type transformants, since tsA28 A-type transformant cells could be obtained by a large-T-dependent in vitro progression of cells of the tsA28 N-type transformant tsA28.3 (M. Osborn and K. Weber, J. Virol. 15:636-644, 1975).

Abstract: Applying an in situ cell fractionation procedure, we analyzed structural systems of the cell nucleus for the presence of mature and replicating simian virus 40 (SV40) DNA. Replicating SV40 DNA intermediates were tightly and quantitatively associated with the nuclear matrix, indicating that elongation processes of SV40 DNA replication proceed at this structure. Isolated nuclei as well as nuclear matrices were able to continue SV40 DNA elongation under replication conditions in situ, arguing for a coordinated and functional association of SV40 DNA and large T molecules at nuclear structures. SV40 DNA replication also was terminated at the nuclear matrix. While the bulk of newly synthesized, mature SV40 DNA molecules then remained at this structure, some left the nuclear matrix and accumulated at the chromatin.

Abstract: The Rev2 cell line is a cellular revertant of the SV40 wild-type transformed rat cell line SV-52 [Bauer, M., Guhl, E., Graessmann, M. & Graessmann, A. (1987). J. Virol., 61, 1821-1827]. To characterize the level of cellular interference with the SV40 large T antigen (large T)-induced transformation pathway in Rev2 cells, we analysed the biological and biochemical properties of large T expressed in Rev2 cells. We found that Rev2 cells encoded an authentic wild-type large T, with regard to its sequence and its transforming functions. No differences were found in the metabolic stability of large T, or in complex formation with the cellular p53 protein, or in p53 metabolic stabilization. In contrast to SV-52 cells, Rev2 cells showed no association of large T with the chromatin fraction of isolated nuclei. This difference correlated with a reduced affinity of the Rev2 large T to SV40 DNA in vitro. The T proteins from both cell lines were phosphorylated at the same multiple sites. However, in Rev2 cells the phosphorylation of large T at specific serine -residues was significantly reduced. Thus the revertant phenotype of Rev2 cells may be due to an altered phosphorylation state of its large T protein, leading to altered nuclear localization and reduced transforming activity. The alterations of Rev2 large T properties and phosphorylation were very similar to the changes observed with mutant large T in FR(tsA58)A cells, an SV40 tsA58 N-type transformant, when the cells had reverted to the normal phenotype at the non-permissive growth temperature. Thus altered phosphorylation might provide a common structural basis for the biological inactivation of the large T proteins in these cells.

Abstract: To identify cellular targets of simian virus 40 large T antigen (SV40 large T) important for the maintenance of cellular transformation, we have compared biological properties of SV40 tsA58 mutant large T antigens expressed in cells of a matched pair of SV40 tsA58 N-type (temperature-sensitive) and A-type (temperature-insensitive) transformants of the normal rat fibroblast line F111 (D. Pintel et al., J. Virol. 38, 518-528, 1981). Characterization of the selected cell lines demonstrated that cells of the N-type transformant [FR(tsA58)A] exhibited properties similar to those of the corresponding SV40 wild-type transformant [FR(wt648)] at the permissive growth temperature (32 degrees ), but reverted to a phenotype indistinguishable from the parental F111 cells at the nonpermissive growth temperature (39 degrees). At both growth temperatures, cells of the A-type transformant [FR(tsA58)57] were very similar to FR(wt648) cells in all properties analyzed. Both mutant-transformed cell lines expressed authentic tsA58 mutant large T antigens at comparable steady-state levels. Analysis of the subnuclear distribution of large T antigens in wild-type and in mutant-transformed cells kept at permissive or at nonpermissive growth temperature, respectively, revealed an important biological difference between the mutant T antigens in N- and A-type transformants: Whereas the subnuclear distribution of wild-type large T in FR(wt648) cells remained unchanged at both growth temperatures, mutant large T in FR(tsA58)A cells (N-type transformant) already 1 day after the shift to the nonpermissive growth temperature no longer stably associated with nuclear substructures, notably the cellular chromatin. In contrast, mutant large T in FR(tsA58)57 cells (A-type transformant) retained this ability. The ability (or inability) of the mutant T antigens to associate with the cellular chromatin in vivo was paralleled by different DNA binding properties of the mutant large T antigens in vitro. Large T in FR(tsA58)A cells no longer bound to the SV40 ORI in vitro after the shift to the nonpermissive growth temperature, whereas large T in FR(tsA58)57 cells at the elevated growth temperature had preserved this activity to a degree similar to its ability to associate with the cellular chromatin. We suggest that in the system of matched pairs of N- and A-type transformants analyzed in this study, expression of the transformed phenotype in FR(tsA58)57 (A-type) cells at the nonpermissive growth temperature is due to the preservation of a biologically active conformation of the mutant large T, allowing it to maintain its interaction with specific targets at the cellular chromatin.

Abstract: We analyzed the subcellular distribution of nuclear transport-defective simian virus 40 Lys-128-mutant (cT-3 [R. E. Lanford and J. S. Butel, Cell 37:801-813, 1984] and d10 [D. Kalderon, W. D. Richardson, A. F. Markham, and A. E. Smith, Nature (London) 311:33-38, 1984]) large T antigens in various Lys-128-mutant-transformed rodent cells and in Lys-128-mutant d10-infected TC7 cells. Small but significant amounts of the mutant large T antigens were found in association with nuclear substructures, both in mutant-transformed and in mutant-infected cells. Experiments with TC7 cells made incompetent for cell division by 60Co irradiation supported the assumption that Lys-128-mutant large T antigen did not associate with nuclear components during mitosis but most likely was transported into the nucleus because the Lys-128 mutation was leaky for nuclear transport. Low-level simian virus 40 DNA replication and production of infectious mutant virus progeny in TC7 cells indicated that the association of Lys-128-mutant large T antigen with nuclear substructures is functional.

Abstract: To address the question whether phosphorylation of p53 might be functionally involved in its metabolic stabilisation and in cellular transformation processes, we have analysed the phosphorylation of the cellular protein p53 in normal and transformed cells of Balb/c mouse origin. Two-dimensional tryptic peptide maps of metabolically unstable p53 from normal Balb/c 3T3 cells and of metabolically stable p53 from 3T3 cells transformed by Simian virus 40 (SV3T3 cells) revealed no qualitative differences between their phosphorylation sites. Except for the unique lack of one or possibly two sites, the phosphopeptide map of p53 from cells transformed by the chemical carcinogen methylcholanthrene (MethA cells), expressing two different, metabolically stable mutant forms of p53, was identical to that of wild-type p53 from normal and SV40-transformed 3T3 cells. These results suggest that no direct relationship exists between phosphorylation of p53, its metabolic stabilisation, and cellular transformation processes. We have included in our analyses p53 from primary Balb/c mouse embryo fibroblast (MEF) cells and the immortalised MEFP27 cell line, established from primary cells after 27 passages. The phosphorylation sites of p53 from these cells were found to be identical to that of p53 from 3T3 cells. In addition, primary and established (immortalised) Balb/c mouse cells revealed no major differences in abundance of p53 mRNA and p53 protein, as well as stability of p53. These results indicate that immortalisation of normal cells involves neither gross alterations of p53 expression nor changes in specific phosphorylation of p53.

Abstract: In addition to controlling the transition of resting normal cells from the G0-state of the cell cycle into S-phase, expression of the cellular protein p53 also seems to be necessary for the proliferation of cycling normal cells in an as yet undefined manner. To further elaborate the role of p53 in growing cells, we analysed p53 expression and its regulation in cells going into, and after release from, growth arrest at the restriction point (R-point) in the G1-phase of the cell cycle, induced by isoleucine depletion. Since growth arrest at the R-point is subject to internal control mechanisms of the cell cycle, this approach allowed us to include in our analyses normal Balb/c 3T3 fibroblasts, as well as cells of the chemically induced Balb/c fibrosarcoma cell line Meth A, expressing mutated p53. Isoleucine depletion induced a viable growth arrest at the R-point in cells of both cell lines, marked by a synchronous shut-down of DNA synthesis when the cells went into growth arrest, and a synchronous resumption of DNA synthesis after a lag period of about 2-4 h when the cells were released from growth arrest, as well as a shift to a G1 DNA content at the R-point. p53 expression in both cell lines showed a phenotypically similar regulation, as its synthesis was specifically reduced at the R-point. At the molecular level, however, p53 expression in growth arrested 3T3 cells was controlled at the transcriptional/post-transcriptional level, whereas control in growth arrested Meth A cells seemed to be at the level of mRNA translation. After release from growth arrest, p53 synthesis in both types of cells was rapidly restored, preceding resumption of total protein synthesis, and exhibiting a p53-specific profile.

Abstract: To further characterize the role of p53 in growing normal Balb/c 3T3 fibroblasts, as well as of p53 in cells of the methylcholanthrene induced fibrosarcoma cell line Meth A, we analysed the effect of inhibition of p53 synthesis by microinjection of p53-specific monoclonal antibody PAb 122 into the nuclei of these cells after release from growth arrest induced by isoleucine starvation (see preceding paper [Steinmeyer et al., this issue] ). We show that microinjection of PAb 122, but not of control immunoglobulins, into the nuclei of both types of cells effectively blocked their re-entry into the S-phase of the cell cycle. Since isoleucine depletion of these cells was shown to lead to a growth arrest at the restriction point (R-point) in the G1-phase of the cell cycle, our results (i) define more precisely the role of p53 in growing cells as a protein controlling transition of the cells through this restriction point, and (ii) demonstrate that mutated p53 in Meth A cells still is functional with regard to cell cycle control at this restriction point. We suggest that p53 acts as a 'gate-keeping' protein at restriction points in the cell cycle, exerting a positive effect on the transition of cells through the cell cycle.

Abstract: The plasma membrane associated subclass of simian virus 40 large T antigen is specifically acylated with palmitic acid in vivo. To further analyze possible biological functions of fatty acid acylation, we developed a target-bound cell free in vitro acylation assay, in which immunopurified large T, bound to protein A-sepharose, was incubated with [3H]fatty acid. In this assay, large T was efficiently labeled with [3H]palmitic acid, but not with [3H]myristic acid. Thus the specificity of the in vivo labeling was preserved in vitro, too. The specific acylation of large T in vitro seemed to occur by an autocatalytic reaction, since it was found to be independent of added acyltransferases and exogenous energy. The energy for this reaction must be provided by the large T molecule itself, probably by an energy-rich internal ester bond. Our results provide evidence for a novel mechanism for the covalent attachment of fatty acids to proteins, which might also operate in vivo.

Abstract:

Abstract: Simian virus 40 large T antigen (large T) in the early and the late phases of infection differs significantly in its sequence-specific DNA-binding and ATPase activities, indicating that different large-T populations participate in virus-specific events at various stages of the infectious cycle. To further characterize these large-T populations, we have analyzed nuclear subclasses of large T, isolated from their in vivo location, for their biochemical activities. We show that chromatin- and nuclear matrix-associated large-T molecules exhibit different simian virus 40 control region (ORI) DNA-binding and ATPase activities. The association of large T with a certain nuclear substructure, therefore, subcompartmentalizes large-T molecules exerting different biochemical activities. Nuclear subcompartmentalization thus may provide a higher-order level for the regulation of biochemical activities of large T in vivo.

Abstract: We have established a sensitive assay for the quantitative determination of large T antigen determinants on the surface of living simian virus 40 (SV40)-transformed cells (mKSA). Cells in suspension culture were incubated with monoclonal antibodies specific for large T antigen (KT3, directed against the carboxyterminus of large T antigen, and PAb 108, directed against an aminoterminal determinant on large T antigen). After incubation with secondary antibody (rabbit anti-mouse IgG), followed by incubation with 3H-protein A, the cells were sequentially extracted first with the nonionic detergent NP-40, followed by ultrasonication and extraction with the zwitterionic detergent Empigen BB. NP-40 solubilized large T antigen associated with NP-40-soluble constituents of the plasma membrane, whereas Empigen BB solubilized the plasma membrane lamina-associated subclass of large T antigen (U. Klockmann and W. Deppert, 1983, EMBO J., 7, 1151-1157). The amount of cell surface-bound 3H-protein A in the NP-40 and Empigen BB extracts was determined by liquid scintillation counting. In agreement with earlier reports, cell surface large T antigen was mainly found in association with the plasma membrane lamina (PML). Since the specific activity of 3H-protein A was known, it was possible to calculate the number of surface-bound 3H-protein A molecules, and thus to estimate the average number of surface-exposed amino- and carboxyterminal determinants of large T antigen per cell. KT3 recognized about 450-900 carboxyterminal determinants, while PAb 108 bound to about 1200-2400 aminoterminal determinants on the surface of a single mKSA cell. The cellular protein p53 also was detected on the surface of mKSA cells and was found to be present in amounts comparable to cell surface large T antigen.

Abstract: The bulk of simian virus 40 (SV40) large T antigen in SV40-infected and -transformed cells localizes within the cell nucleus, while a minor fraction specifically associates with the plasma membrane (PM) and is exposed on the cell surface. PM-associated large T seems to span the lipid bilayer but, on the other hand, does not display typical features of a transmembrane protein. To further characterize the postulated transmembrane orientation of large T, we asked whether all large T molecules associated with the plasma membrane indeed are exposed on the cell surface. We compared the amount of cell surface-exposed large T, determined on living cells by a sensitive 3H-protein A-binding assay and by external immunoprecipitation, with that of total PM-associated large T extracted from isolated PM. We demonstrate that in mKSA cells (SV40-transformed BALB/c mouse fibroblasts), total PM-associated large T accounted for a substantial portion (ca. 2%) of total cellular large T. However, only 0.1 to 0.2% of it could be detected on the cell surface. Thus, only a minor fraction of PM-associated large T (less than 10%) is exposed on the surface of these cells. Interior PM-associated large T is stably associated with the plasma membrane, while the small fraction of surface-exposed large T is rapidly released from the cell surface.

Abstract: We analysed large T antigen expression and metabolic stabilisation of the cellular protein p53 in cells of a matched pair of SV40 tsA mutant (tsA58) N-type or A-type transformants, respectively. At the permissive growth temperature (32 degrees C), cells of both transformants, like SV40 wild-type transformed cells, were phenotypically transformed and expressed large T antigen, as well as metabolically stable p53 (both complexed and free p53). At the nonpermissive growth temperature (39 degrees C), cells of the N-type transformant reverted to a normal phenotype, whereas cells of the A-type transformant still displayed a transformed phenotype. Under these growth conditions, the mutant large T antigens in both cell types were no longer able to complex p53 (both in vivo and in vitro), but the metabolic stabilities of the free p53 in these cells correlated with their phenotypes: p53 in cells of the N-type transformant was rapidly degraded, whereas it was metabolically stable in cells of the A-type transformant. This difference in p53 stability correlated with an in vivo functional difference between the mutant large T antigens at the nonpermissive growth temperature: large T antigen in cells of the N-type transformant no longer stably associated with the cellular chromatin and the nuclear matrix, but accumulated in the nucleoplasm. In contrast, large T antigen in cells of the A-type transformant at least partially had retained this ability. Maintenance of SV40 cell transformation thus seems to require both a functional large T antigen and a metabolically stabilised p53.

Abstract: We have characterized the interactions of simian virus 40 (SV40) large tumor antigen (large T) with the control region of the SV40 genome, the SV40 ORI, by analyzing the specific binding of large T antigen to SV40 wild-type origin DNA and to isolated binding sites I and II, respectively. DNA binding affinities of large T antigen were determined under standardized conditions and DNA excess, using a target-bound DNA binding assay (M. Hinzpeter, E. Fanning, and W. Deppert, 1986, Virology 148, 159-167). Our results show that large T antigen exhibits similar affinities for isolated binding sites I and II and for combined sites I and II on wild-type ORI DNA. When the fraction of large T antigen molecules (calculated per large T antigen monomers) able to bind specifically to these sites was determined (DNA binding activity of large T antigen) we found that only 2% of large T antigen molecules present in extracts of lytically infected cells were able to bind to isolated site II, whereas about 50% bound to isolated site I. However, only about 10% of large T antigen molecules bound to the complete wild-type ORI, containing combined binding sites I and II. Thus, a much larger proportion of large T antigen molecules is capable of binding specifically to site I as is suggested by analysis of large T antigen binding to combined sites I and II on the SV40 wild-type ORI. These findings indicate that the interaction of large T antigen with the SV40 wild-type ORI is restricted on one hand by the ability of large T antigen to bind to site II, and on the other hand by the spatial arrangement of binding sites I and II on the SV40 wild-type ORI.

Abstract: We have analyzed by immunofluorescence microscopy and by biochemical cell fractionation the subcellular distribution of the adenovirus type 2 72K DNA binding protein (DBP) during the course of infection in HeLa cells. Early in infection, the 72K DBP was strictly localized in the cell nucleus. However, as infection progressed, the 72K DBP was additionally found in other subcellular fractions, notably in association with the cytoskeletal framework of the plasma membrane, the plasma membrane lamina. Pulse-chase experiments demonstrated that this association was specific. Control experiments excluded the possibility of an artificial redistribution of the 72K DBP during cell fractionation. Our data, therefore, demonstrate that a significant portion of the 72K DBP during late times of infection associates specifically with the cytoskeletal framework of plasma membranes of infected cells.

Abstract: We analysed the in vitro binding of p53 from normal (3T3) and from chemically transformed (Meth A) Balb/c mouse cells to double-stranded (ds-) DNA and to single-stranded (ss-) DNA by DNA-cellulose chromatography. We confirm previous findings that p53 in cellular extracts exhibits ds-DNA-binding activity (Lane and Gannon, 1983). In addition, we demonstrate that such p53 also binds to ss-DNA. Analyses with immunopurified p53 protein provide evidence that this DNA-binding activity is intrinsic to p53. DNA binding of p53 could not be inhibited by a monoclonal antibody specific for the C-terminal region. An N-terminal deletion mutant of p53 (Rovinski et al., 1987) exhibited similar DNA-binding properties as wild-type p53, indicating that the N-terminus also is dispensable for DNA binding. We further show a close correlation between the DNA-binding activity of p53 from 3T3 cells and its association with nuclear substructures.

Abstract: We analysed biological and biochemical parameters for the association of the simian virus 40 (SV40) large tumor antigen (large T) with the cellular chromatin and the nuclear matrix in SV40-transformed cells. Nuclear subclasses of large T were isolated by in situ cell fractionation (Staufenbiel & Deppert, 1983) and first analysed for possible biological functions in the maintenance of cellular transformation. Like large T in SV40 wild-type transformants, large T in SV40 tsA mutant (tsA58)-transformed cells, expressing a temperature-dependent phenotype, was present in all nuclear subfractions (nucleoplasm, chromatin and nuclear matrix), when cells were kept at the growth temperature permissive for the expression of the transformed phenotype (32 degrees C). When tsA mutant-transformed cells were shifted to the non-permissive growth temperature (39 degrees C), they reverted to the normal phenotype. Concomitantly, large T lost its ability to associate with the cellular chromatin and the nuclear matrix, indicating that an association of large T with these subcellular structures may be important for the maintenance of cellular transformation. We next analysed the DNA-binding properties (sequence-specific binding to the SV40 origin of replication, ORI) of the nuclear subclasses of SV40 wild-type and of SV40 mutant large T defective in SV40 ORI binding in order to determine the influence of sequence-specific DNA binding on the association of large T with the chromatin and the nuclear matrix. Our detailed analyses show distinct differences in the ability of the various nuclear subclasses of large T to bind to the SV40 ORI, but suggest that the association of large T with the chromatin and the nuclear matrix is mediated by protein-protein interactions rather than by sequence-specific DNA binding.

Abstract: We analyzed the subnuclear distribution of the simian virus 40 (SV40) large tumor (large T) antigen during the course of viral infection. Three distinct nuclear subclasses were detected in SV40 lytically infected TC7 cells (large T antigen in the nucleoplasm, at the cellular chromatin, and at the nuclear matrix). During the course of infection the relative subnuclear distribution of large T antigen changed significantly at about the switch from the early to late phase of infection: at early times postinfection, large T antigen was present mainly in the nucleoplasm and at the cellular chromatin, and nuclear-matrix-associated large T antigen was barely detectable. Concomitant with the onset of viral DNA replication, the amount of nuclear-matrix-associated large T antigen increased drastically. During the further course of infection large T antigen accumulated at the cellular chromatin and nuclear matrix, paralleling the increase in viral DNA synthesis. The biological significance of this correlation was corroborated by analysis of cells infected with the SV40 mutant tsA58 at permissive (32 degrees C) and restrictive (39 degrees C) temperatures. tsA58 large T antigen failed to initiate viral DNA replication in infected cells kept at the restrictive temperature and also failed to associate with the cellular chromatin and nuclear matrix. By blocking viral DNA synthesis with aphidicolin, an inhibitor of DNA polymerase alpha, we were able to show that the accumulation of large T antigen at these structures does not result from the binding of large T antigen to viral chromatin but reflects an association with cellular components of the chromatin and nuclear matrix of infected cells.

Abstract: We analyzed the relation of metabolic stabilization of the p53 protein during cellular transformation by simian virus 40 (SV40) to (i) expression of the transformed phenotype and (ii) expression of the large tumor antigen (large T). Analysis of SV40-tsA28-mutant-transformed rat cells (tsA28.3 cells) showed that both p53 complexed to large T and free p53 (W. Deppert and M. Haug, Mol. Cell. Biol. 6:2233-2240, 1986) were metabolically stable when the cells were cultured at 32 degrees C and expressed large T and the transformed phenotype. At the nonpermissive temperature (39 degrees C), large-T expression is shut off in these cells and they revert to the normal phenotype. In such cells, p53 was metabolically unstable, like p53 in untransformed cells. To determine whether metabolic stabilization of p53 is directly controlled by large T, we next analyzed the metabolic stability of complexed and free p53 in SV40 abortively infected normal BALB/c mouse 3T3 cells. We found that neither p53 in complex with large T nor free p53 was metabolically stable. However, both forms of p53 were stabilized in SV40-transformed cells which had been developed in parallel from SV40 abortively infected cultures. Our results indicate that neither formation of a complex of p53 with large T nor large-T expression as such is sufficient for a significant metabolic stabilization of p53. Therefore, we suggest that metabolic stabilization of p53 during cellular transformation with SV40 is mediated by a cellular process and probably is the consequence of the large-T-induced transformed phenotype.

Abstract: We isolated 16 new monoclonal antibodies that recognize large T antigen of simian virus 40 and mapped the epitopes to three distinct regions of the large T antigen. Also, 3 of the 16 recognized the large T antigen of the human papovavirus BKV.

Abstract: We have developed a new sensitive target-bound DNA binding assay (TB assay) for SV40 large T antigen (large T). The major advantage of this assay is that in contrast to commonly used DNA binding assays, DNA binding is not performed in large T extracts, but instead is performed with immunopurified target-bound large T. Thereby interference of cellular components present in large T extracts is avoided. Thus the TB assay allows DNA binding analysis of large T from different sources (extracts, cell lines) under standardized conditions. Large T is first immunopurified with an anti-T monoclonal antibody not interfering with DNA binding and protein A-Sepharose. Then SV40 DNA is added to the large T immune complex. For analysis of bound DNA and large T, we developed a two-step elution procedure by which bound DNA and large T in the immune complex can be analyzed separately and which allows the determination of the actual amounts of bound DNA and large T. Binding data obtained with the TB assay allowed us to determine an equilibrium dissociation constant (Kd). As a further application of this assay, we analyzed the ORI binding of SVR9D mutant large T which has been reported to exhibit no ORI binding activity. We found that a small percentage of SVR9D large T binds specifically to the SV40 ORI.

Abstract: Large T antigen (large T) extracted from SV40-infected or transformed cells exhibits an in vitro protein kinase activity, whose origin and biological significance up to now had been obscure. We have addressed the questions of whether this activity is intrinsic to large T or arises by association with a cellular kinase, and, furthermore, whether this activity might play a biological role in vivo. Instead of analyzing large T from whole-cell lysates, where non-specific association of a cellular kinase(s) with large T might easily occur, we analyzed individual cellular subclasses of large T, isolated from their in vivo locations. In contrast to large T isolated from whole-cell lysates which was always kinase positive, none of the cellular subclasses of large T prepared by in situ fractionation of SV40-transformed mKSA cells exhibited detectable in vitro kinase activity. We could demonstrate that our fractionation conditions neither inactivated the large T-associated kinase activity nor dissociated it from large T when they were applied to kinase-positive large T isolated from whole-cell lysates. We conclude that large T does not contain an intrinsic kinase activity. This conclusion was further supported by our finding that it was possible to remove the large T-associated kinase activity from kinase-positive large T preparations and to reconstitute it by incubating the kinase-negative large T with cell lysates from various cell lines. Therefore, the simplest way of interpreting our results is that the in vitro kinase activity measured with large T preparations from whole-cell lysates is the result of an in vitro association of a cellular kinase(s) with large T during certain conditions of cell lysis.

Abstract: To determine functional subcellular loci of p53, a cellular protein associated with cellular transformation, we analyzed the nucleoplasmic, chromatin, and nuclear matrix fractions from normal mouse 3T3 cells, from methylcholanthren-transformed mouse (MethA) cells, and from various simian virus 40 (SV40)-transformed cells for the presence of p53. In 3T3 and MethA cells, p53 was present in all nuclear subfractions, suggesting an association of p53 with different structural components of the nucleus. In 3T3 cells, p53 was rapidly turned over, whereas in MethA cells, p53 was metabolically stable. In SV40-transformed cells, p53 complexed to large tumor antigen (large T) was found in the nucleoplasmic and nuclear matrix fractions, as described previously (M. Staufenbiel and W. Deppert, Cell 33:173-181, 1983). In addition, however, metabolically stable p53 not complexed to large T (free p53) was also found in the chromatin and nuclear matrix fractions of these cells. This free p53 did not arise by dissociation of large T-p53 complexes, suggesting that stabilization of p53 in SV40-transformed cells can also occur by means other than formation of a complex with large T.

Abstract: Infection of cells with adenovirus lead to a characteristic reorganization of all cytoskeleton systems, starting with alterations at the microtubuli of the cells. During this progress, the flat, extended, and polar morphology of the cytoskeleton became nonpolar and rounder. These rearrangements were initiated before the appearance of adenovirus structural proteins hexon and fiber, as well as before the shutoff of host protein synthesis. We conclude that these alterations reflect a specific reorganization rather than an unorganized breakdown of the cell during adenovirus infection.

Abstract: Cells of Dictyostelium discoideum were incubated with [H]palmitic acid during development, and recovery of the fatty acid label in soluble and membrane-associated proteins was investigated. One of the major labeled proteins was found exclusively in the soluble fraction. This protein, with an apparent mol. wt. of 44 kd, was identified as actin based on its labeling with a monoclonal anti-actin antibody, its coincidence with the major [S]methionine-labeled protein after two-dimensional electrophoresis and its binding to a DNase I affinity column. The H-label was resistant to chloroform-methanol extraction and boiling in SDS-containing buffer. After partial purification by preparative SDS-polyacrylamide gel electrophoresis, the 44-kd protein was treated with KOH, the fatty acids released were derivatized to methyl esters and palmitic acid methylester was identified by gas-liquid chromatography.

Abstract: Human adenoviruses fail to multiply effectively in monkey cells. The block to the replication of these viruses can be overcome by coinfection with simian virus 40 (SV40) or when part of the SV40 genome is integrated into and expressed as part of the adenovirus type 2 (Ad2) genome, as occurs in several Ad2+SV40 hybrid viruses, such as Ad2+ND1, Ad2+ND2, and Ad2+ND4. The SV40 helper-defective Ad2+SV40 hybrid viruses Ad2+ND5 and Ad2+ND4del were analyzed to determine why they are unable to grow efficiently in monkey cells even though they contain the appropriate SV40 genetic information. Characterization of the Ad2+ND5-SV40-specific 42,000-molecular-weight (42K) protein revealed that this protein is closely related, but not identical, to the SV40-specific 42K protein of the SV40 helper-competent Ad2+ND2 hybrid virus. Although the minor differences between these proteins may be sufficient to account for the poor growth of Ad2+ND5 in monkey cells, the most striking difference between helper-competent Ad2+ND2 and helper-defective Ad2+ND5 is in the production of the SV40-specific protein after infection of monkey cells. Whereas synthesis of the SV40-specific proteins of Ad2+ND2 is very similar in human and in monkey cells, production of the 42K protein of Ad2+ND5 is dramatically reduced in monkey cells compared with human cells. Similarly, the synthesis of the SV40-specific proteins of Ad2+ND4del is markedly reduced in monkey cells. Thus, it is likely that both Ad2+ND5 and Ad2+ND4del are helper defective because of a block in the production of their SV40-specific proteins rather than because their SV40-specific proteins are nonfunctional. This block, like the block to adenovirus fiber synthesis, is overcome by coinfection with SV40, with helper-competent hybrid viruses, or with host range mutants of adenoviruses. This suggests that the synthesis of fiber and the synthesis of SV40-specific proteins are similarly regulated in Ad2+SV40 hybrid viruses.

Abstract: In mKSA cells (a simian virus 40-transformed BALB/c mouse tumor cell line), plasma membrane-associated large T antigen (large T) is found in two subfractions of the plasma membrane; a minor amount of large T is recovered from the Nonidet P-40 (NP-40)-soluble plasma membrane fraction, whereas the majority is tightly bound to a substructure of the plasma membrane, the plasma membrane lamina (PML). Only PML-associated large T is fatty acid acylated (U. Klockmann and W. Deppert, EMBO J. 2:1151-1157, 1983). We have analyzed whether these two forms of plasma membrane-associated large T might differ in features like cell surface expression or metabolic stability. In addition, we have asked whether one of the two large Ts might represent the hypothetic, large T-related protein T* (D. F. Mark and P. Berg, Cold Spring Harbor Symp. Quant. Biol. 44:55-62, 1979). We show that in mKSA cells grown in suspension culture, large T associated with the PML is also exposed on the cell surface. This form of large T, therefore, exhibits properties of a transmembrane protein. Large T in the NP-40-soluble plasma membrane fraction could not be labeled with radioiodine on the cell surface and, for this reason, does not seem to be oriented towards the cell surface. In contrast, when mKSA cells were grown on substratum (culture dish), we found that in these cells both NP-40-soluble large T as well as large T anchored in the PML could be cell surface iodinated. We also have analyzed the plasma membrane association of surface T antigen in mKSA cells grown in a mouse as ascites tumor. In tumor cells, only PML-bound large T is cell surface associated. We conclude that differences in extractibility of cell surface-associated large T most likely depend on cell shape and are not an artifact of cell culture. Both NP-40-soluble and PML-bound large Ts are associated with the plasma membrane in a metabolically stable fashion. Neither of the two large Ts represents T*.

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Abstract: To sort out possible influences of protein sequences and fatty acid acylation on the plasma membrane association of simian virus 40 large T-antigen, we have analyzed the membrane interactions of carboxy-terminal fragments of large T-antigen, encoded by the adenovirus type 2 (Ad2+)-simian virus 40 hybrid viruses Ad2+ND1 and Ad2+ND2. The 28,000 (28K)-molecular-weight protein of Ad2+ND1 as well as the 42K and 56K proteins of Ad2+ND2 associate preferentially with membranous structures and were found in association with the membrane system of the endoplasmic reticulum and with plasma membranes. Neither the endoplasmic reticulum membrane- nor the plasma membrane-associated 28K protein of Ad2+ND1 is fatty acid acylated. We, therefore, conclude that fatty acid acylation is not necessary for membrane association of this protein and suggest that an amino acid sequence in this protein is responsible for its membrane interaction. In contrast, the 42K and 56K proteins of Ad2+ND2 in plasma membrane fractions contain fatty acid. However, the interaction of these proteins with the plasma membrane differs from that of the 28K protein of Ad2+ND1: whereas the 28K protein of Ad2+ND1 interacts stably with Nonidet P-40-soluble constituents of the plasma membrane, the 42K and 56K proteins of Ad2+ND2 are tightly bound to the Nonidet P-40-insoluble plasma membrane lamina. Thus, an amino acid sequence in the amino-terminal region of the 28K protein confers membrane affinity to these proteins, whereas a region between the amino-terminal end of the 42K protein of Ad2+ND2 and the amino-terminal end of the 28K protein of Ad2+ND1 contains a reactive site for fatty acid acylation. This posttranslational modification correlates with the stable association of the 42K and 56K proteins with the plasma membrane lamina. We suggest that the same sequences also mediate the proper plasma membrane association of large T-antigen in simian virus 40-transformed cells.

Abstract: Analyses of the different structural systems of the nucleus and the proteins associated with them pose many problems. Because these systems are largely overlapping, in situ localization studies that preserve the in vivo location of proteins and cellular structures often are not satisfactory. In contrast, biochemical cell fractionation may provide artifactual results due to cross-contamination of extracts and structures. To overcome these problems, we have developed a method that combines biochemical cell fractionation and in situ localization and leads to the preparation of a residual cellular skeleton (nuclear matrix and cytoskeletal elements) from cultured cells. This method's main feature is that cell fractionation is performed in situ. Therefore, structures not solubilized in a particular extraction step remain attached to the substrate and retain their morphology. Before and after each extraction step they can be analyzed for the presence and location of the protein under study by using immunological or cytochemical techniques. Thereby the in vivo origin of a protein solubilized in a particular extraction step is determined. The solubilized protein then may be further characterized biochemically. In addition, to allow analyses of proteins associated with the residual cellular skeleton, we have developed conditions for its solubilization that do not interfere with enzymatic and immunological studies.

Abstract: Analysis of cellular extracts of HeLa cells infected with adenovirus type 2 (Ad2) by immunoprecipitation with antiserum against the late nonstructural 100,000-dalton (100K) protein revealed the presence of a specific complex between the 100K protein and newly synthesized hexon molecules. Serological analysis of the hexon molecule in the 100K/hexon complex with antibodies specific for hexon monomers or trimers showed that only monomeric hexon molecules were associated with the 100K protein. By immunofluorescence microscopy this monomeric hexon was primarily found in the cytoplasm, whereas the trimeric form was mainly confined to the nucleus of infected cells. We conclude that in the cytoplasm of Ad2-infected cells newly synthesized, monomeric hexon molecules can interact with the 100K protein. This suggests that the 100K protein may play some role either in trimerization of newly synthesized, monomeric hexon molecules and/or in its transport from the cytoplasm into the nucleus.

Abstract: SV40 transformed mouse cells (mKSA) were labeled in parallel with either [35S]methionine or [3H]palmitate and subfractionated. Nuclear extracts and solubilized plasma membranes were analyzed for the presence of either 35S- or 3H-labeled SV40 large tumor antigen by immunoprecipitation and SDS polyacrylamide gel electrophoresis. The majority of the [35S]methionine labeled large T was recovered from the nuclear fraction, only minor amounts were detected in plasma membranes. In contrast, large T labeled specifically with [3H]palmitate was found only in the plasma membrane fraction. Our results demonstrate a specific acylation of large T associated with plasma membranes, suggesting that the membrane location of this predominantly nuclear protein is specific.

Abstract: We have analyzed the plasma membrane association of the SV40 large tumor antigen (large T) in SV40-transformed BALB/c mouse tumor cells (mKSA). Isolated plasma membranes were subfractionated: treatment with the non-ionic detergent Nonidet P40 (NP40) resulted in a NP40-resistant plasma membrane lamina, which could be further extracted with the zwitterionic detergent Empigen BB. Analysis of the different plasma membrane fractions revealed that only about one third of large T associated with isolated plasma membranes could be solubilized with NP40. The residual plasma membrane-associated large T was tightly bound to the NP40-resistant lamina of the plasma membrane from which it was released by treatment with the zwitterionic detergent Empigen BB. Further evidence for a specific interaction of a distinct subclass of large T with the plasma membrane was provided by showing that only T associated with the NP40-resistant lamina of the plasma membrane contained covalently bound fatty acid. Neither nuclear large T nor large T in the NP40-soluble plasma membrane fraction could be labeled with [3H]palmitic acid. Our results indicate that an acylated subclass of large T interacts specifically with a structure of the plasma membrane, suggesting that it might be involved in a membrane-dependent biological function.

Abstract: HeLa cells infected with the adenovirus 2-simian virus 40 (Ad2+SV40) hybrid virus Ad2+ND2 were labeled with either [35S]methionine or [3H]palmitate and fractionated into cytoplasmic, nuclear, and plasma membrane fractions. Analysis of these fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the SV40-specific proteins in the plasma membrane fraction were specificially acylated.

Abstract: The major polypeptides of nuclear matrix preparations from vertebrate tissues are rather similar. However, nuclear matrix fractions isolated from cultured vertebrate cells of different origin show variations in their major polypeptides. We demonstrate that cytoplasmic intermediate filaments copurify with nuclear matrices from these cells. Because of their abundance, their subunit proteins form major bands on SDS-polyacrylamide gels. The tissue specificity of the intermediate filament proteins then gives rise to the variations observed. To explain the differences in major proteins of nuclear matrices isolated from vertebrate cells grown in tissue and in culture we have analyzed the distribution of intermediate filaments during isolation of nuclei from liver tissue. We show that during homogenization of liver intermediate filaments are torn off and can be separated from the nuclei. Nuclear matrix preparations from these nuclei, therefore, do not contain intermediate filaments and true nuclear matrix proteins (e.g. lamins) are the major protein species. Our results suggest that the major nuclear matrix polypeptides (lamins) are similar in all vertebrate cells, since lamin like proteins were identified in cultured cells, too. Using antisera we demonstrate an immunological difference between lamins A/C and lamin B.

Abstract: To define the interaction of SV40 large T with different structural systems in the nuclei of SV40-transformed cells (BALB/c mKSA), we have employed an in situ cell fractionation procedure leading to the preparation of the nuclear matrix, and giving rise to defined nuclear extracts comprising soluble nuclear proteins (nucleoplasm) and the solubilized chromatin. Large T could be detected in the nucleoplasmic fraction and in the chromatin fraction, as well as in tight association with the nuclear matrix. From the nuclear matrix, large T could be solubilized by treatment with a zwitterionic detergent. Different solubility properties, differences in the amount of the cellular phosphoprotein p53 coprecipitating with large T, and different stabilities in its association with the nuclear structural systems indicate that distinct subclasses of large T were isolated from their in vivo location in SV40-transformed cells.

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Abstract: A recombinant plasmid based on pBR322 has been constructed which carries the replicator proximal early region of SV40 DNA, including the viral origin of replication (ORI). It lacks a major part of the tumour antigen 3'-coding region, the large T-antigen termination codon and the polyadenylation site. The recombinant plasmid was transferred together with the herpes simplex virus thymidine kinase gene, as a selectable marker into mouse LTK- cells. Integration and expression of the cloned SV40 gene fragment in TK+ transformants could be demonstrated by DNA restriction and blot hybridization and by immunofluorescence techniques.

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Abstract: The recombinant plasmid p102 based on pBR322 carrying approximately equal to 50% of the replicator proximal early region of simian virus 40 (SV40) DNA, including the viral origin of replication, has been constructed. It lacks a major part of the large tumor (T) antigen 3'-coding region, the T-antigen termination codon, and the polyadenylylation site. The plasmid was transferred together with the herpes simplex virus thymidine kinase (TK) gene as a selectable marker to mouse LTK- cells. TK+ cell clones were isolated and their high molecular weight DNAs were shown by DNA blotting and hybridization experiments to contain the SV40 DNA fragment from the recombinant. In some of these clones, heterogeneous expression of the SV40 DNA fragment could be detected by immunofluorescence while, in control experiments in which a plasmid containing the complete SV40 early DNA region was used, this extensive heterogeneity of T-antigen expression was not observed. RNA . DNA hybridization experiments showed that the SV40-specific RNA of those clones is polyadenylylated. The molecular weight of the T-antigen-related protein coded by p102 corresponded well to the expected coding capacity of the SV40 DNA fragment. Small tumor antigen was not expressed.

Abstract: The antigenic binding sites of two monoclonal antibodies are located in the COOH-terminal region (clone 412) and probably in an internal region (clone 7) of simian virus 40 large T antigen. A third monoclonal antibody (clone 122), which has been shown to bind nonviral T antigen, does not react with HeLa cells infected with nondefective adenovirus type 2 (Ad2)-simian virus 40 hybrid viruses Ad2+ND1, Ad2+ND2, or Ad2+ND4.

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Abstract: We analyzed the subcellular locations of the late adenovirus type 2 nonstructural 100,000-dalton (100K) and 33K proteins in adenovirus type 2-infected HeLa cells both by biochemical cell fractionation and by immunofluorescence microscopy, using specific antisera against purified sodium dodecyl sulfate-denatured 100K and 33K polypeptides. Both methods showed that the 100K protein was present in the cytoplasm as well as in the nuclei of infected cells and that it accumulated in the nuclei during the course of infection. Phosphorylated 100K protein also was found both in the cytoplasm and in nuclei. However, the nuclear 100K protein pool was phosphorylated to a higher degree than the cytoplasmic pool. In all experiments the 33K protein, which also is a phosphoprotein, was present exclusively in the nuclei of infected cells. The 100K and 33K proteins were associated with different nuclear substructures; this was demonstrated serologically by an analysis of infected cells in which double color immunofluorescence microscopy was used. In these experiments antibodies against the 100K protein decorated different nuclear structures than antibodies against the 33K protein.

Abstract: Simian virus 40 (SV40)-transformed cells express the SV40-specific tumour transplantation antigen (TSTA) on the cell surface and the SV40-coded tumour antigen in their nuclei. TSTA is defined by SV40-specific transplantation immunity, whereas T-antigen (T-Ag) can be detected serologically by indirect immunofluorescence. Both antigens, however, are derived from the A gene of SV40. We therefore analysed SV40-transformed cells for the presence of serologically detectable T-Ag-related molecules. Such antigens could not be detected on the surface of living SV40-transformed cells in monolayers. However, after a short formaldehyde fixation it was possible to stain the cell surfaces of SV40-transformed cells with sera from rabbits immunized with purified SDS-denatured T-Ag, but not with sera from hamsters bearing SV40-induced tumours. T-Ag-related antigens could be detected with both types of antisera by applying a more sensitive 125I-protein A assay. The T-Ag specificity of the binding of hamster SV40 tumour sera was demonstrated be a 125I-IgG-blocking assay in which preincubation of formaldehyde-fixed SV40-transformed cells with rabbiet anti-SDS-T-Ag serum inhibited the binding of hamster SV40 tumour serum by about 70%. The localization of T-Ag-related antigens on the outside of plasma membranes of formaldehyde-fixed cells was shown by an anti-SDS-T-Ag serum-specific binding of fluorescein isothiocyanate-labelled Staphylococcus aureus to the cell surface. Out results are consistent with the hypothesis that SV40 T-Ag-related antigens are involved in the formation of TSTA.

Abstract: For an immunological analysis of the late adenovirus type 2 nonstructural 100,000-dalton (100K) and 33K proteins, we prepared antisera against sodium dodecyl sulfate-denatured, gel-purified 100K and 33K proteins. These antisera were tested for potential cross-reactivity, since according to a previous report (Axelrod, Virology 87:366--383, 1978) these two proteins exhibit extensive amino acid homologies. However, immunoprecipitations of 100K and 33K proteins, as well as a sensitive immune replica technique, did not reveal any immunological relationship between these proteins. Therefore, using fingerprint peptide analysis, we investigated the structural relationship between 100K and 33K proteins labeled with a 14C-amino acid mixture or with [14C]proline after digestion with trypsin. We detected only minor, if any, amino acid homologies, indicating that the 100K and 33K proteins are not structurally related.

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Abstract: Simian virus 40 (SV40)-transformed monolayer cells were analyzed in situ by indirect immunofluorescence microscopy for the postulated cell surface location of SV40 T-antigen-related molecules. With antisera prepared against purified, sodium dodecyl sulfate-denatured SV40 T-antigen, positive surface staining was obtained when the cells had been treated with formaldehyde before immunofluorescence analysis. In contrast, living SV40-transformed cells analyzed in monolayer were surface fluorescence negative. The fixation procedure developed in this study combined with a double staining immunofluorescence technique allowed the simultaneous analysis of the same cells for the expression of both SV40 T-antigen-related surface antigen and nuclear T-antigen. The localization of SV40 T-antigen-related surface antigen on the outer surface of the plasma membrane of formaldehyde-fixed SV40-transformed cells was demonstrated directly by the protein A-mediated binding of Staphylococcus aureus bacteria on formaldehyde-fixed SV40-transformed cells precoated with antiserum against sodium dodecyl sulfate-denatured T-antigen. Both cell surface staining and S. aureus binding were found to be highly specific for SV40 T-antigen-related binding sites. These results indicate that T-antigen-related molecules in a cryptic form are located on the surface of SV40-transformed monolayer cells and can be detected in situ after modification of the cell surface architecture.

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Abstract: Simian virus 40 (SV40)-transformed cells and cells infected by the nondefective adenovirus 2(Ad2)-SV40 hybrid viruses Ad2+ND1 and Ad2+ND2 were analyzed for SV40 T- and U-antigens, respectively, using individual hamster SV40 tumor sera or serum for which U-antibodies were removd by absorption. These studies showed that (i) T- and U-antigens can be defined by separate classes of antigenic determinants and (ii) the U-antigenic determinants in SV40-transformed cells and in hybrid virus-infected cells are similar. The apparent discrepancy in the subcellular location of U-antigen in SV40-transformed cells (nuclear location) and in hybrid virus-infected cells (perinuclear location) as determined by immunofluorescence staining of methanol/acetone-fixed cells could be resolved by treating hybrid virus-infected cells with a hypotonic KCl solution before fixation. Upon this treatment hybrid virus-infected cells also showed nuclear U-antigen staining. The possibility of an association of T- and U-antigens with different nuclear subfractions in SV40-transformed cells was investigated. Detergent-cleaned nuclei of SV40-transformed cells were fractionated into nuclear matrices and a DNase-treated, high-salt nuclear extract. Analysis of the nuclear matrices by immunofluorescence microscopy with T+U+ and T+U- hamster SV40 tumor serum revealed that U-antigen remained associated with the nuclear matrices, whereas T-antigen could not be detected in this nuclear subfraction. T-antigen, however, could be immunoprecipitated from nuclear extracts of the SV40-transformed cells.

Abstract: HeLa cells infected with the nondefective adenovirus type 2-simian virus 40 hybrid viruses Ad2+ND1 or Ad2+ND2 were analyzed for cell surface location of the SV40-specific hybrid virus proteins by indirect immunofluorescence microscopy. Two different batches of sera from SV40 tumor-bearing hamsters, serum from SV40 tumor-bearing mice, or two different antisera prepared against purified sodium dodecyl sulfate-denatured SV40 T-antigen, respectively, were used. All sera were shown to exhibit comparable T- and U-antibody titers and to specifically immunoprecipitate the SV40-specific proteins from cell extracts of Ad2+ND2-infected cells. Whereas analysis of living, hybrid virus-infected HeLa cells did not yield conclusive results, analysis of Formalin-fixed cells resulted in positive cell surface fluorescence with both Ad2+ND1- and Ad2+ND2-infected HeLa cells when antisera prepared against sodium dodecyl sulfate-denatured SV40 T-antigen were used as first antibody. In contrast, sera from SV40 tumor-bearing animals were not or only very weakly able to stain the surfaces of these cells. The fact that the tumor sera had comparable or even higher T- and U-antibody titers than the antisera against sodium dodecyl sulfate-denatured T-antigen but were not able to recognize SV40-specific proteins on the cell surface suggests that SV40 tumor-specific transplantation antigen may be an antigenic entity different from T- or U-antigen.

Abstract: The presence of glycosyltransferases on surfaces of mammalian cells has been reported by many investigators and a biological role for these enzymes in cell adhesion and cell recognition has been postulated. Critical analysis, however, showed 2 major complications regarding the assay for cell surface glycosyltransferases: 1) hydrolysis of the nucleotide sugar by cell surface enzymes and subsequent intracellular use of the free sugar and 2) loss of cell integrity if trypsinized or EDTA-treated cells were used in suspension assays. We have assayed intact, viable cells in monolayer for cell surface glycosyltransferases using conditions under which intracellular utilization of free sugars generated by hydrolysis of the nucleotide sugar was prevented. Our data demonstrate that the presence of galactosyltransferases on the surface of a variety of cells, including established (normal and virally transformed) as well as nonestablished cells, is unlikely. No evidence for the existence of cell surface fucosyl- and sialytransferases could be obtained, but our data do not exclude the possibility that low levels of these enzymes are present.

Abstract: The distribution of simian virus 40 (SV40)-specific proteins in nuclear subfractions of pulse-chase-labeled HeLa cells infected with nondefective adenovirus type 2 (Ad2)-SV40 hybrid viruses was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The SV40-specific proteins of Ad2+ND1, Ad2+ND2, and Ad2+ND5 specifically associate with the nuclear matrix and are virtually absent from the high-salt nuclear extract. In Ad2+ND4-infected HeLa cells, the SV40-specific proteins with molecular weights of 64,000 (64K) and lower also specifically associate with the nuclear matrix. The SV40-specific 72K, 74K, and 95K proteins were found both in the nuclear matrix and in the high-salt nuclear extract. Analyses of the nuclear matrices isolated from hybrid virus-infected cells by immunofluorescence microscopy showed that SV40 U-antigen-positive sera from SV40 tumor-bearing hamsters react with SV40-specific proteins integrated into nuclear matrices of HeLa cells infected by Ad2+ND1, Ad2+ND2, and Ad2+ND4, but not with nuclear matrices of HeLa cells infected by Ad2+ND5. This suggests that SV40-specific proteins of Ad2+ND1, Ad2+ND2, and Ad2+ND4 integrated into the nuclear matrix carry SV40 U-antigen determinants. The apparent discrepancy in the subcellular localization of SV40-specific proteins in hybrid virus-infected cells when analyzed by biochemical cell fractionation procedures and when analyzed by immunofluorescence staining is discussed.

Abstract: HeLa cells infected with adenovirus type 2 (Ad2)-simian virus 40 (SV40) hybrid viruses produce several SV40-specific proteins. These include the previously reported 28,000-dalton protein of Ad2+ND1, and 42,000- and 56,000-dalton proteins of Ad2+ND2, the 56,000-dalton protein of Ad2+ND4, and the 42,000-dalton protein of Ad2+ND5. In this report, we extend the list of SV40-specific proteins induced by Ad2+ND4 to include proteins of apparent molecular weights of 28,000 42,000, 60,000, 64,000, 72,000, 74,000, and a doublet of 95,000. Cell fractionation studies demonstrate that the SV40-specific proteins are detectable in the nuclear, cytoplasmic, and plasma membrane fractions. By pulse-chase and cell fractionation experiments, three classes of SV40-specific proteins can be distinguished with regard to metabolic stability: (i) unstable in the cytoplasmic but stable in the nuclear and plasma membrane fractions; (ii) stable in the nuclear, cytoplasmic, and plasma membrane fractions; and (iii) unstable in all subcellular fractions. Immunoprecipitation of infected cell extracts demonstrates that most of the above proteins share antigenic determinants with proteins expressed in hamsters bearing SV40-induced tumors. Only the 42,000-dalton protein of Ad2+ND5 is not immunoprecipitable.

Abstract: Low passage BHK 21/13 cells contain two cell surface enzymes, a nucleotide pyrophosphatase and a monophosphoester hydrolase, which together hydrolyze exogenous UDP-galactose to free galactose. During serial passage, BHK cells successively lose both enzymes. Concomitant with the loss of these enzymatic activities, changes in cell morphology, as well as in the serum requirement for the initiation of DNA synthesis, were observed. Clonal sublines of BHK cells were isolated, which differed qualitatively in their ability to hydrolyze UDP-galactose. Clonal BHK sublines, which exhibited both enzymatic activities on their cell surface, resembled low passage BHK cells in morphology and serum requirement for the initiation of DNA synthesis. Sublines not containing these enzymes resembled BHK cells of high passage cultures. The ability of intact BHK cells to hydrolyze exogenous nucleotide sugars may serve as an indicator for the progression of BHK cells from a normal to a more transformed state.

Abstract: HeLa cells infected with the nondefective adenovirus 2-SV40 hybrid virus (Ad2+ND2) have previously been shown to synthesize two SV40-specific proteins with molecular weights of 56,000 (56 K protein) and 42,000 (42 K protein). The present study demonstrates that these proteins are immunoprecipitable with serum from hamsters bearing SV40 tumors. By peptide analysis, it is shown that the 56 K and 42 K proteins share common amino acid sequences. Both proteins are metabolically stable in nuclei and plasma membranes but unstable in the cytoplasm, as shown by pulse-chase experiments and cell fractionation studies.

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Abstract: Intact BHK (baby hamster kidney) cells catalyze the hydrolysis of UDP-galactose to free galactose. The generation of galactose from UDP-galactose and its intracellular utilization impede the detection of possible galactosyl transferases on the cell surface of intact cells. Several independent procedures have been used to distinguish between intracellular and cell surface glycosyl transferases. With these procedures, no evidence was obtained for the presence of detectable amounts of galactosyl transferase activity on the surface of BHK cells. The data suggest that galactosyl transferases do not play a general role in the phenomena of cell adhesion and contact inhibition.

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