Laboratory of Molecular Oncology Dept. of Experimental and Clinical Medicine "G. Salvatore" University of Catanzaro Magna Graecia Biosciences Building, Level 7 University Campus 'Salvatore Venuta' - Germaneto 88100 Catanzaro - Italy
Education and training 1987 Degree in Biological Sciences University of Naples, Naples, Italy. 1988-1990 Postdoctoral fellow, Institute of Genetics and Byophisics (IGB), National Research Council, Naples. 1990-94: Residency in Genetics, at the University "La Sapienza", Rome, Italy. 1985: Visiting fellow, National Institutes of Health (Bethesda, MD, USA) 2003: Visiting fellow, Kimmel Cancer Center (Philadelphia, PA, USA) 2004: Visiting fellow, Kimmel Cancer Center (Philadelphia, PA, USA) 2006: Visiting fellow, CNIO (Madrid, Spain).
Positions held and academic responsibilities 1992-2001: Research Assistant, National Cancer Institute “G Pascale”, Naples, Italy. 2001-2004: Research Assistant, Institute of Experimental Oncology and Endocrinology (IEOS), National Research Council, Naples, Italy. Since 2004: Full Professor of General Pathology, University “Magna Graecia”, Catanzaro, Italy. Since 2007: Dean of Research, University “Magna Graecia”, Catanzaro, Italy.
Faculty of the following PhD programmes and Schools of Specialization • School of Clinical Pathology at University Magna Graecia, Catanzaro, Italy. • School of Clinical Biochemistry at University Magna Graecia, Catanzaro, Italy. • School of Internal Medicine at University Magna Graecia, Catanzaro, Italy. • School of Neurology at University Magna Graecia, Catanzaro, Italy. • School of Pediatry at University Magna Graecia, Catanzaro ,Italy. • School of Radiology at University Magna Graecia, Catanzaro, Italy.
• PhD Programme in Molecular Oncology, Immunology and Experimental Therapeutic Approaches, University Magna Graecia, Catanzaro, Italy. • PhD Programme in Experimental Oncology and Endocrinology, University Federico II, Naples, Italy.
Scientific interests 1985-90: Cloning of the human gene encoding Glucose-6-phosphate-dehydrogenase (G6PD) and characterization of the pathological variants from the area of South Italy. 1990-96: Cloning and characterization of the endothelial growth factors PlGF; definition of the molecular mechanisms that regulate neo-angiogenesis in human cancer. 1996-05: Molecular mechanisms that induce cell cycle disregulation in human cancer; In this field Giuseppe Viglietto’s group has contributed to develop the concept that tumour suppressor KIP1 (a powerful inhibitor of cell cycle progression) is inactivated in cancer cells through sequestration in the cytoplasmic compartment. Since 2005: Role of the PI3K/AKT pathway in the development of human cancer; in this field G Viglietto has identified a novel mutation (E17K) in the gene encoding the ser/thr kinase AKT1 in a subset of lung carcinoma.
Scientific Societies Since 1996: Italian Society for Cancerology (SIC) Since 2002: European Thyroid Association (ETA) Since 2007: American Association for Cancer Research (AACR).
Cited in the list of the 1255 TOP ITALIAN SCIENTISTS of the VIA-Academy (H-index > 30)
Abstract: Malignant pleural effusions (MPEs) could represent an excellent source to culture a wide variety of cancer cells from different donors. In this study, we set up culture conditions for cancer cells deriving from MPEs of several patients affected by the most frequent form of lung cancer, namely the subset of non small cell lung cancers (NSCLC) classified as Lung Adenocarcinomas (AdenoCa) which account for approximately 40% of lung cancer cases. AdenoCa malignant pleural effusions gave rise to in vitro cultures both in adherent and/or in spheroid conditions in almost all cases analyzed. We characterized in greater detail two samples which showed the most efficient propagation in vitro. In these samples we also compared gene profiles of spheroid vs adherent cultures and identified a set of differentially expressed genes. Finally we achieved efficient tumor engraftment in recipient NOD/SCID mice, also upon inoculation of small number of cells, thus suggesting indirectly the presence of tumor initiating cells.
Abstract: HIPK2 is a serine/threonine kinase that acts as a coregulator of an increasing number of factors involved in cell survival and proliferation during development and in response to different types of stress. Here we report on a novel target of HIPK2, the cyclin-dependent kinase inhibitor p27(kip1). HIPK2 phosphorylates p27(kip1) in vitro and in vivo at serine 10, an event that accounts for 80% of the total p27(kip1) phosphorylation and plays a crucial role in the stability of the protein. Indeed, HIPK2 depletion by transient or stable RNA interference in tumor cells of different origin was consistently associated with strong reduction of p27(kip1) phosphorylation at serine 10 and of p27(kip1) stability. An initial evaluation of the functional relevance of this HIPK2-mediated regulation of p27(kip1) revealed a contribution to cell motility, rather than to cell proliferation, but only in cells that do not express wild-type p53.
Abstract: Human DNA mismatch repair (MMR) is involved in the removal of DNA base mismatches that arise either during DNA replication or are caused by DNA damage. In this study, we show that the activation of the MMR component hMLH1 in response to doxorubicin (DOX) treatment requires the presence of BRCA1 and that this phenomenon is mediated by an ATM/ATR dependent phosphorylation of the hMLH1 Ser-406 residue. BRCA1 is an oncosuppressor protein with a central role in the DNA damage response and it is a critical component of the ATM/ATR mediated checkpoint signaling. Starting from a previous finding in which we demonstrated that hMLH1 is able to bind to BRCA1, in this study we asked whether BRCA1 might be the bridge for ATM/ATR dependent phosphorylation of the hMLH1 molecular partner. We found that: (i) the negative modulation of BRCA1 expression is able to produce a remarkable reversal of hMLH1 stabilization, (ii) BRCA1 is required for post-translational modification produced by DOX treatment on hMLH1 which is, in turn, attributed to the ATM/ATR activity, (iii) the serine 406 phosphorylatable residue is critical for hMLH1 activation by ATM/ATR via BRCA1. Taken together, our data lend support to the hypothesis suggesting an important role of this oncosuppressor as a scaffold or bridging protein in DNA-damage response signaling via downstream phosphorylation of the ATM/ATR substrate hMLH1.
Abstract: Periostin (POSTN), an osteoblast-specific secreted protein known to be associated with cell adhesion activity for bone formation and development by the epithelial cell-derived tumors, leads to a significant enhancement in angiogenesis and tumorigenesis. At present, little is known about the mechanisms underlying its transcriptional control either in physiological or neoplastic conditions. In this study we demonstrate that the ability of the human POSTN promoter to drive transcription mostly depends on the activity of YingYang-1 (YY1) zinc finger transcription factor. YY1, whose regulatory role in biology includes, besides transcriptional control, also chromatin remodeling, DNA damage repair and tumorigenesis, acts as a strong negative modulator of the POSTN expression. We retain that the identification of the functional role of YY1 in the transcriptional control of the human POSTN gene adds new insights in the studies focused on gene expression in normal and transformed cells.
Abstract: The family of serine/threonine kinases B/Akt (hereafter Akt) represents a central node in signalling pathways downstream of growth factors, cytokines, and other cellular stimuli. In mammalian cells the Akt family comprises three highly homologous members -known as Akt1/PKBalpha, Akt2/PKBbeta, and Akt3/PKBgamma- that regulate several processes including cell proliferation and survival, growth and response to nutrient availability, migration, tissue invasion and angiogenesis. Aberrant activation of Akt is involved in a variety of human cancers including those arising in the thyroid gland. Here, we review the contribution of Akt-dependent pathway in the proliferation of normal thyrocytes, the different pathogenic mechanisms underlying aberrant Akt signalling in thyroid malignancies as well as the relative roles of Akt substrates that most likely contribute to the onset and/or progression of thyroid cancer. Finally, we discuss the current therapeutic strategies targeting the components of the PI3K/Akt pathway in the context of thyroid malignancy.
Abstract: Here we show that a subpopulation of MCF-7 breast cancer cells which stains pale to Toluidine Blue (Light Cells- LCs), is endowed with features of CSCs. LCs give rise to self-renewing mammospheres and express typical CSC markers; moreover this subpopulation is chemoresistant and highly tumorigenic in vivo. LCs can be identified in several other breast cancer cell lines, irrespectively of their histological origin (luminal vs. mesenchymal vs. basal) and represent an heterogeneous cell population composed mainly of CSC-like and early progenitor cells. By a limited in vitro drug screening assay, we identify compounds which can specifically interfere with the viability of LCs from multiple breast cancer cell lines. Analysis of the Sphere-Forming Efficiency (SFE) and of the distribution of ALDH(bright) cells within the treated cell lines suggest that one of the identified compounds acts in vitro by modulating the CSC phenotype. Interestingly, a subset of the identified compounds is known to affect directly or indirectly the NFkappaB pathway which is emerging as an important modulator of CSC proliferation and chemoresistance.
Abstract: Genomic copy number (CN) information is useful to study genetic traits of many diseases. Using array comparative genomic hybridization (aCGH), researchers are able to measure the copy number of thousands of DNA loci at the same time. Therefore, a current challenge in bioinformatics is the development of efficient algorithms to detect the map of aberrant chromosomal regions.
Abstract: Loss of the PTEN tumor suppressor gene occurs frequently in non-small-cell lung carcinoma (NSCLC), although neither genetic alterations nor epigenetic silencing are significant predictors of PTEN protein levels. Since recent reports implicated neural precursor cell expressed, developmentally down-regulated 4-1 (NEDD4-1) as the E3 ubiquitin ligase that regulates PTEN stability, we investigated the role of NEDD4-1 in the regulation of PTEN expression in cases of NSCLC. Our findings indicate that NEDD4-1 plays a critical role in the development of NSCLC and provides novel insight on the mechanisms that contribute to inactivate PTEN in lung cancer. Immunohistochemical analysis on tissue microarrays containing 103 NSCLC resections revealed NEDD4-1 overexpression in 80% of tumors, which correlated with the loss of PTEN protein (n=98; P<0.001). Accordingly, adoptive NEDD4-1 expression in NSCLC cells decreased PTEN protein stability, whereas knock-down of NEDD4-1 expression decreased PTEN ubiquitylation and increased PTEN protein levels. In 25% of cases, NEDD4-1 overexpression was due to gene amplification at 15q21. In addition, manipulation of NEDD4-1 expression in different lung cell systems demonstrated that suppression of NEDD4-1 expression significantly reduced proliferation of NSCLC cells in vitro and tumor growth in vivo, whereas NEDD4-1 overexpression facilitated anchorage-dependent and independent growth in vitro of nontransformed lung epithelial cells that lack pRB and TP53 (BEAS-2B). NEDD4-1 overexpression also augmented the tumorigenicity of lung cancer cells that have an intact PTEN gene (NCI-H460 cells).
Abstract: The aim of this study is to assess if common genetic variants located in the CDKN1B locus, coding for the cell cycle inhibitor p27(Kip1), are involved in thyroid cancer susceptibility. Based on the literature and functional predictions, we selected three polymorphisms within the CDKN1B gene (rs2066827 (T326G, V109G), rs34330 (-79C>T) and rs36228499 (-838C>A)) to perform the first case-control study in thyroid cancer involving this locus. We had 649 Spanish patients with sporadic thyroid cancer and 385 healthy representative controls available. Luciferase reporter gene assays, real-time quantitative reverse transcription-PCR and immunoblot experiments were carried out to demonstrate the putative effect of the associated variant. The polymorphism rs34330 (-79C>T) was identified as a risk factor for developing the follicular variant of papillary thyroid carcinoma (FVPTC), fitting a recessive model (odds ratio=2.12; 95% confidence interval=1.09-4.15; P value=0.023). The risk allele (T) of this single nucleotide polymorphism led to a lower transcription rate in cells transfected with a luciferase reporter driven by the polymorphic p27(Kip1) promoter (P value <0.001). This effect was observed in -79TT genotype control carriers, who showed a tendency towards lower CDKN1B mRNA levels in lymphocytes, as well as at the protein level. This is the first study that identifies CDKN1B as a low-penetrance gene in thyroid cancer, and specifically in FVPTC subtype. We propose a reduced CDKN1B gene transcription depending on the genotype of the -79C>T (rs34330) variant as a novel mechanism underlying p27(Kip1) downregulation.
Abstract: Impairment of the p27(kip1) function, caused by a drastic reduction of its expression or cytoplasmic mislocalization, has been frequently observed in thyroid carcinomas. To understand the role of p27(kip1) impairment in thyroid carcinogenesis, we investigated the consequences of the loss of p27(kip1) expression in the context of a mouse modeling of papillary thyroid cancer, expressing the TRK-T1 oncogene under the transcriptional control of thyroglobulin promoter. We found that double mutant mice homozygous for a p27(kip1) null allele (TRK-T1/p27(-/-)) display a higher incidence of papillary thyroid carcinomas, with a shorter latency period and increased proliferation index, compared with p27(kip1) wild-type compounds (TRK-T1/p27(+/+)). Consistently, double mutant mice heterozygous for a p27(kip1) null allele (TRK-T1/p27(+/-)) show an incidence of thyroid carcinomas that is intermediate between TRK-T1/p27(-/-) and TRK-T1/p27(+/+) mice. Therefore, our findings suggest a dose-dependent role of p27(kip1) function in papillary thyroid cancer development.
Abstract: OBJECTIVE: Emerging evidence suggests that specific sub-populations of cancer cells with stem cell characteristics within the bulk of tumours are implicated in the pathogenesis of heterogeneous malignant tumours. The cells that drive tumour growth have been denoted cancer-initiating cells or cancer stem cells (hereafter CSCs). CSCs have been isolated initially from leukaemias and subsequently from several solid tumours including brain, breast, prostate, colon and lung cancer. This study aimed at isolating and characterising the population of tumour-initiating cells in non-small-cell lung cancer (NSCLC). METHODS: Specimens of NSCLC obtained from 89 patients undergoing tumour resection at the Cancer National Institute of Naples were analysed. Three methods to isolate the tumour-initiating cells were used: (1) flow cytometry analysis for identification of positive cells for surface markers such as CD24, CD29, CD31, CD34, CD44, CD133 and CD326; (2) Hoechst 33342 dye exclusion test for the identification of a side-population characteristic for the presence of stem cells; (3) non-adherent culture condition able to form spheres with stem cell-like characteristics. Definition of the tumourigenic potential of the cells through soft agar assay and injection into NOD/SCID mice were used to functionally define (in vitro and in vivo) putative CSCs isolated from NSCLC samples. RESULTS: Upon flow cytometry analysis of NSCLC samples, CD133-positive cells were found in 72% of 89 fresh specimens analysed and, on average, represented 6% of the total cells. Moreover, the number of CD133-positive cells increased markedly when the cells, isolated from NSCLC specimens, were grown as spheres in non-adherent culture conditions. Cells from NSCLC, grown as spheres, when assayed in soft agar, give rise to a 3.8-fold larger number of colonies in culture and are more tumourigenic in non-obese diabetic (NOD)/severe combined immunodeficiency (SCID) mice compared with the corresponding adherent cells. CONCLUSIONS: We have isolated and characterised a population of CD133-positive cells from NSCLC that is able to give rise to spheres and can act as tumour-initiating cells.
Abstract: Regulation of receptor-type phosphatases can involve the formation of higher-order structures, but the exact role played in this process by protein domains is not well understood. In this study we show the formation of different higher-order structures of the receptor-type phosphatase PTPRJ, detected in HEK293A cells transfected with different PTPRJ expression constructs. In the plasma membrane PTPRJ forms dimers detectable by treatment with the cross-linking reagent BS(3) (bis[sulfosuccinimidyl]suberate). However, other PTPRJ complexes, dependent on the formation of disulfide bonds, are detected by treatment with the oxidant agent H(2)O(2) or by a mutation Asp872Cys, located in the eighth fibronectin type III domain of PTPRJ. A deletion in the eighth fibronectin domain of PTPRJ impairs its dimerization in the plasma membrane and increases the formation of PTPRJ complexes dependent on disulfide bonds that remain trapped in the cytoplasm. The deletion mutant maintains the catalytic activity but is unable to carry out inhibition of proliferation on HeLa cells, achieved by the wild type form, since it does not reach the plasma membrane. Therefore, the intact structure of the eighth fibronectin domain of PTPRJ is critical for its localization in plasma membrane and biological function.
Abstract: Somatic mutation (E17K) that constitutively activates the protein kinase AKT1 has been found in human cancer patients. We determined the role of the E17K mutation of AKT1 in lung cancer, through sequencing of AKT1 exon 4 in 105 resected, clinically annotated non-small cell lung cancer specimens. We detected a missense mutations G-->A transition at nucleotide 49 (that results in the E17K substitution) in two squamous cell carcinoma (2/36) but not in adenocarcinoma (0/53). The activity of the endogenous kinase carrying the E17K mutation immunoprecipitated by tumour tissue was significantly higher compared with the wild-type kinase immunoprecipitated by the adjacent normal tissue as determined both by in vitro kinase assay using a consensus peptide as substrate and by in vivo analysis of the phosphorylation status of AKT1 itself (pT308, pS473) or of known downstream substrates such as GSK3 (pS9/S22) and p27 (T198). Immunostaining or immunoblot analysis on membrane-enriched extracts indicated that the enhanced membrane localization exhibited by the endogenous E17K-AKT1 may account for the observed increased activity of mutant E17K kinase in comparison with the wild-type AKT1 from adjacent normal tissue. In conclusion, this is the first report of AKT1 mutation in lung cancer. Our data provide evidence that, although AKT1 mutations are apparently rare in lung cancer (1.9%), the oncogenic properties of E17K-AKT1 may contribute to the development of a fraction of lung carcinoma with squamous histotype (5.5%).
Abstract: Loss of expression of the cyclin-dependent kinase inhibitor p27 through enhanced protein degradation frequently occurs in human cancer. Degradation of p27 requires ubiquitination by the S-phase kinase-associated protein 2 (Skp2), a member of the F-box family of Skp1-Cullin-F-box protein ubiquitin ligases. In the present study, we have investigated the role of Skp2 in human thyroid tumours. Immunohistochemistry analysis showed that Skp2 was overexpressed significantly in thyroid carcinomas (26 out of 51) compared with goitres (0 out of 12, P<0.001) or adenomas (1 out of 10, P<0.05), and that high Skp2 expression was detected more often in anaplastic thyroid (ATC; 83%, n=12) than follicular thyroid (FTC; 40%, n=20) or papillary thyroid (PTC; 42%, n=19) carcinomas (P<0.05). Thyroid cancer cell lines and tissues with high levels of Skp2 protein presented high p27 degradation activity and there was an inverse correlation between Skp2 and p27 expression in thyroid cancer tissues (n=68; P<0.05). In most cases, the observed overexpression of Skp2 protein was paralleled by an increase in the levels of Skp2 mRNA, and we observed Skp2 gene amplification at 5p13 in 2 out of 6 cell lines and in 9 out of 23 primary tumours (six out of eight ATCs, two out of nine PTCs and one out of six FTCs) using Q-PCR and/or fluorescence in situ hybridization analysis. Finally, in vitro experiments demonstrated that suppression of Skp2 expression drastically reduced proliferation of thyroid cancer cells and, conversely, forced expression of Skp2 circumvented serum dependency and contact inhibition in Skp2-negative cells by promoting p27 degradation. These findings indicate that Skp2 plays an important role for the development of thyroid cancer.
Abstract: In the present study, we report that the RAS/BRAF/MAP kinase cascade plays a crucial role in the regulation of the Skp2/p27 pathway in thyroid cancer cells and that this is critical for cell proliferation. In vitro studies with cellular models of human thyroid carcinoma cells demonstrated that the adoptive expression of oncogenic RET/PTC1, Ha-RASV12 or BRAFV600E enhances Skp2 and reduces p27 protein expression in a MAP kinase-dependent manner; that RAS/BRAF/MAP kinase-dependent control of p27 expression in thyroid cancer cells occurs by regulating the stability of Skp2 and p27 protein; and that antisense oligonucleotides to p27 suppress growth arrest induced by MEK inhibitors. Finally, analysis of human thyroid carcinomas indicated that MAP kinase-positive thyroid tumors-as detected by immunostaining for p-ERK - presented high p27 degradative activity and low levels of p27 protein (n = 30; p < 0.05). In summary, our results indicate that constitutive signalling of the MAP kinase cascade contributes to the development of thyroid cancer promoted by activated RAS and BRAF oncogenes and that this occurs, at least in part, by compromising the inhibitory function of p27.
Abstract: BACKGROUND: The down regulation of protein p27(kip1) (p27) in most cases of thyroid cancer has relevant diagnostic and prognostic implications. However, the oxyphilic (Hurthle cell) variant of follicular thyroid carcinoma expresses more p27 than benign oxyphilic lesions do. AIM: To evaluate the mechanism underlying this difference in expression of p27. METHODS: Because high levels of cyclin D3 lead to p27 accumulation in cell lines and clinical samples of thyroid cancer, the immunocytochemical pattern of cyclin D3 in oxyphilic (n = 47) and non-oxyphilic (n = 70) thyroid neoplasms was investigated. RESULTS: In the whole study sample, there was a significant correlation between p27 and cyclin D3 expression (Spearman's r: 0.64; p<0.001). The expression of cyclin D3 and p27 was significantly higher in the oxyphilic variant of follicular carcinomas than in non-oxyphilic carcinomas (p<0.001). In the former, cyclin D3 overexpression and p27 accumulation were observed in a median of 75% and 55% of cells, respectively. In co-immunoprecipitation experiments, the level of p27-bound cyclin D3 was much higher in oxyphilic neoplasias than in normal thyroids and other thyroid tumours. CONCLUSION: These results show that increased p27 expression in the oxyphilic (Hurthle cell) variant of follicular thyroid carcinoma results from cyclin D3 overexpression.
Abstract: We investigated the role of the MEK/MAPK pathway in the sensitivity/resistance of breast carcinoma cells to the EGFR tyrosine kinase inhibitor gefitinib (IRESSA). We assessed the effects of gefitinib on the growth of three breast cancer cell lines that showed high (SK-Br-3; IC50 4 microM), intermediate (MDA-MB-361; IC50 5.3 microM), and low (MDA-MB-468; IC50 6.8 microM) sensitivity to the drug. Although treatment with gefitinib inhibited EGFR activation in the three cell lines in a similar fashion, significant reduction of both p42/p44-MAPK and AKT phosphorylation was observed in SK-Br-3 and MDA-MB-361, but not in MDA-MB-468 cells. The growth of MDA-MB-468 cells was significantly inhibited by treatment with either the PI3K-inhibitor LY294002 or the MEK-inhibitor PD98059. In agreement with these findings, treatment of MDA-MB-468 cells with a combination of PD98059 and gefitinib produced a synergistic anti-tumor effect, whereas this combination was only additive in SK-Br-3 and MDA-MB-361 cells. The combination of gefitinib and PD98059 also produced a significant increase in the levels of apoptosis in MDA-MB-468 cells as compared with treatment with a single agent. This phenomenon was associated with a profound decrease in MAPK activation, reduction of BAD (ser112) phosphorylation and a paradoxical increase in the levels of AKT activation. Finally, overexpression of a constitutively activated form of p42-MAPK in MCF-10A non-transformed human mammary epithelial cells resulted in a two- to three-fold increase in the IC50 to gefitinib. Taken together, these data strongly support the role of the MEK/MAPK pathway in the resistance to gefitinib, and provide the rationale for novel therapeutic approaches based on combinations of signal transduction inhibitors.
Abstract: Thyroid cancer is frequently associated with the oncogenic conversion of the RET receptor tyrosine kinase. RET gene rearrangements, which lead to the generation of chimeric RET/papillary thyroid carcinoma (PTC) oncogenes, occur in PTC, whereas RET point mutations occur in familial multiple endocrine neoplasia type 2 (MEN2) and sporadic medullary thyroid carcinomas (MTC). We showed previously that the expression of the receptor-type protein tyrosine phosphatase J (PTPRJ) is suppressed in neoplastically transformed follicular thyroid cells. We now report that PTPRJ coimmunoprecipitates with wild-type RET and with the MEN2A-associated RET(C634R) oncoprotein but not with the RET/PTC1 and RET-MEN2B isoforms. Using mutated forms of PTPRJ and RET-MEN2A, we show that the integrity of the respective catalytic domains is required for the PTPRJ/RET-MEN2A interaction. PTPRJ expression induces dephosphorylation of the RET(C634R) and, probably via an indirect mechanism, RET/PTC1 oncoproteins on two key RET autophosphorylation sites (Tyr1062 and Tyr905). This results in a significant decrease of RET-induced Shc and extracellular signal-regulated kinase 1/2 phosphorylation levels. In line with this finding, adoptive PTPRJ expression reduced the oncogenic activity of RET(C634R) in an in vitro focus formation assay of NIH3T3 cells. As expected from the coimmunoprecipitation results, the RET(M918T) oncoprotein, which is associated to MEN2B and sporadic MTC, was resistant to the dephosphorylating activity of PTPRJ. Taken together, these findings identify RET as a novel substrate of PTPRJ and suggest that PTPRJ expression levels may affect tumor phenotype associated with RET/PTC1 and RET(C634R) mutants. On the other hand, resistance to PTPRJ may be part of the mechanism of RET oncogenic conversion secondary to the M918T mutation.
Abstract: The HMGA1 protein is a major factor in chromatin architecture and gene control. It plays a critical role in neoplastic transformation. In fact, blockage of HMGA1 synthesis prevents rat thyroid cell transformation by murine transforming retroviruses, and an adenovirus carrying the HMGA1 gene in the antisense orientation induces apoptotic cell death in anaplastic human thyroid carcinoma cell lines, but not in normal thyroid cells. Moreover, both in vitro and in vivo studies have established the oncogenic role of the HMGA1 gene. In this study, to define HMGA1 function in vivo, we examined the consequences of disrupting the Hmga1 gene in mice. Both heterozygous and homozygous mice for the Hmga1-null allele show cardiac hypertrophy due to the direct role of HMGA1 on cardiomyocytic cell growth regulation. These mice also developed hematologic malignancies, including B cell lymphoma and myeloid granuloerythroblastic leukemia. The B cell expansion and the increased expression of the RAG1/2 endonuclease, observed in HMGA1-knockout spleen tissues, might be responsible for the high rate of abnormal IgH rearrangements observed in these neoplasias. Therefore, the data reported here indicate the critical role of HMGA1 in heart development and growth, and reveal an unsuspected antioncogenic potential for this gene in hematologic malignancies.
Abstract: The expression of the receptor protein tyrosine phosphatase r-PTPeta is drastically reduced in rat and human malignant thyroid cells, whereas its restoration reverts the neoplastic phenotype of retrovirally transformed rat thyroid cells. Moreover, reduced levels and loss of heterozygosity of DEP-1, the human homolog of r-PTPeta, have been found in many human neoplasias. Here, we report that the r-PTPeta protein binds to c-Src in living cells and dephosphorylates the c-Src inhibitory tyrosine phosphorylation site (Tyr 529), thereby increasing c-Src tyrosine kinase activity in malignant rat thyroid cells stably transfected with r-PTPeta. Tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin was enhanced in r-PTPeta-expressing cells. This was associated with increased adhesion of malignant r-PTPeta-transfected thyroid cells vs both untransfected cells and cells stably transfected with an inactive r-PTPeta mutant. Treatment of rat thyroid cells with the c-Src inhibitor PP2 decreased cell adhesion to a higher extent in r-PTPeta-transfected cells than in mock-transfected or stably transfected cells with the inactive r-PTPeta mutant, indicating that r-PTPeta regulates cell-substratum adhesion by activating c-Src. Interestingly, the extent of both c-Src dephosphorylation at Tyr 529, FAK and paxillin phosphorylation, and the increased cell adhesion were associated with the degree of r-PTPeta expression.
Abstract: Functional inactivation of the tumor suppressor p27(kip1) in human cancer occurs either through loss of expression or through phosphorylation-dependent cytoplasmic sequestration. Here we demonstrate that dysregulation of the PI3K/AKT pathway is important in thyroid carcinogenesis and that p27(kip1) is a key target of the growth-regulatory activity exerted by this pathway in thyroid cancer cells. Using specific PI3K inhibitors (LY294002, wortmannin, and PTEN) and a dominant active AKT construct (myrAKT), we demonstrated that the PI3K/AKT pathway controlled thyroid cell proliferation by regulating the expression and subcellular localization of p27. Results obtained with phospho-specific antibodies and with transfection of nonphosphorylable p27(kip1) mutant constructs demonstrated that PI3K/AKT-dependent regulation of p27(kip1) mislocalization in thyroid cancer cells occurred via phosphorylation of p27(kip1) at T157 and T198 (but not at S10 or T187). Finally, we evaluated whether these results were applicable to human tumors. Analysis of 100 thyroid carcinomas indicated that p27(kip1) phosphorylation at T157/T198 and cytoplasmic mislocalization were preferentially associated with activation of the PI3K/AKT pathway. Thus the PI3/AKT pathway and its effector p27(kip1) play major roles in thyroid carcinogenesis.
Abstract: Overexpression of HMGA1 proteins is a constant feature of human carcinomas. Moreover, rearrangements of this gene have been detected in several human benign tumors of mesenchymal origin. To define the role of these proteins in cell transformation in vivo, we have generated transgenic mice overexpressing ubiquitously the HMGA1 gene. These mice developed mixed growth hormone/prolactin cell pituitary adenomas and natural killer (NK)-T/NK cell lymphomas. The HMGA1-induced expression of IL-2 and IL-15 proteins and their receptors may account for the onset of these lymphomas. At odds with mice overexpressing a wild-type or a truncated HMGA2 protein, adrenal medullar hyperplasia and pancreatic islet cell hyperplasia frequently occurred and no increase in body size and weight was observed in HMGA1 mice. Taken together, these data indicate an oncogenic role of the HMGA1 gene also in vivo.
Abstract: Downstream regulatory element antagonistic modulator (DREAM) was originally identified in neuroendocrine cells as a calcium-binding protein that specifically binds to downstream regulatory elements (DRE) on DNA, and represses transcription of its target genes. To explore the possibility that DREAM may regulate the endocrine activity of the thyroid gland, we analyzed its mRNA expression in undifferentiated and differentiated thyroid cells. We demonstrated that DREAM is expressed in the normal thyroid tissue as well as in differentiated thyroid cells in culture while it is absent in FRT poorly differentiated cells. In the present work, we also show that DREAM specifically binds to DRE sites identified in the 5' untranslated region (UTR) of the thyroid-specific transcription factors Pax8 and TTF-2/FoxE1 in a calcium-dependent manner. By gel retardation assays we demonstrated that thapsigargin treatment increases the binding of DREAM to the DRE sequences present in Pax8 and TTF-2/Foxe1 5' UTRs, and this correlates with a significant reduction of the expression of these genes. Interestingly, in poorly differentiated thyroid cells overexpression of exogenous DREAM strongly inhibits Pax8 expression. Moreover, we provide evidence that a mutated form of DREAM unable to bind Ca(2+) interferes with thyroid cell proliferation. Therefore, we propose that in thyroid cells DREAM is a mediator of the calcium-signaling pathway and it is involved in the regulation of thyroid cell function.
Abstract: The hybridisation of an Affymetrix HG_U95Av2 oligonucleotide array with RNAs extracted from six human thyroid carcinoma cell lines and a normal human thyroid primary cell culture led us to the identification of the UbcH10 gene that was upregulated by 150-fold in all of the carcinoma cell lines in comparison to the primary culture cells of human normal thyroid origin. Immunohistochemical studies performed on paraffin-embedded tissue sections showed abundant UbcH10 levels in thyroid anaplastic carcinoma samples, whereas no detectable UbcH10 expression was observed in normal thyroid tissues, in adenomas and goiters. Papillary and follicular carcinomas were only weakly positive. These results were further confirmed by RT-PCR and Western blot analyses. The block of UbcH10 protein synthesis induced by RNA interference significantly reduced the growth rate of thyroid carcinoma cell lines. Taken together, these results would indicate that UbcH10 overexpression is involved in thyroid cell proliferation, and may represent a marker of thyroid anaplastic carcinomas.
Abstract: In the present study, we have characterized several human thyroid cancer cell lines of different histotypes for their responsiveness to contact inhibition. We found that cells derived from differentiated carcinoma (TPC-1, WRO) arrest in G(1) phase at confluence, whereas cells derived from anaplastic carcinoma (ARO, FRO and FB1) continue to grow after reaching confluence. Furthermore, we provide experimental evidence that the axis, E-cadherin/beta-catenin/p27(Kip1), represents an integral part of the regulatory mechanism that controls proliferation at a high cell density, whose disruption may play a key role in determining the clinical behaviour of thyroid cancer. This conclusion derives from the finding that: (i) the expression of p27(Kip1) is enhanced at high cell density only in cells responsive to contact inhibition (TPC-1, WRO), but not in contact-inhibition resistant cells (ARO, FRO or FB1 cells); (ii) the increase in p27(Kip1) also resulted in increased levels of p27(Kip1) bound to cyclin E-Cdk2 complex, a reduction in cyclin E-Cdk2 activity and dephosphorylation of the retinoblastoma protein; (iii) antisense inhibition of p27(Kip1) upregulation at high cell density in confluent-sensitive cells completely prevents the confluence-induced growth arrest; (iv) proper expression and/or membrane localization of E-cadherin is observed only in cells responsive to contact inhibition (TPC-1, NPA, WRO) but not in unresponsive cells (ARO, FRO or FB1); (v) disruption of E-cadherin-mediated cell-cell contacts at high cell density induced by an anti-E-cadherin neutralizing antibody, inhibits the induction of p27(kip1) and restores proliferation in contact-inhibited cells; (vi) re-expression of E-cadherin into cells unresponsive to contact inhibition (ARO, FB1) induces a p27(kip1) expression and growth arrest. In summary, our data indicate that the altered response to contact inhibition exhibited by thyroid anaplastic cancer cells is due to the failure to upregulate p27(Kip1) in response to cell-cell interactions.
Abstract: PTEN/MMAC1/TEP1: (hereafter PTEN) is a tumor suppressor gene (located at 10q23) that is frequently mutated or deleted in sporadic human tumors. PTEN encodes a multifunctional phosphatase, which negatively regulates cell growth, migration and survival via the phosphatidylinositol 3'-kinase/AKT signalling pathway. Accordingly, Pten+/- mice develop various types of tumors including teratocarcinomas and teratomas. We have investigated PTEN expression in 60 bioptic specimens of germ cell tumors (32 seminomas, 22 embryonal carcinomas and six teratomas) and 22 intratubular germ cell neoplasias (ITGCN) adjacent to the tumors for PTEN protein and mRNA expression. In total, 10 testicular biopsies were used as controls. In the testis, PTEN was abundantly expressed in germ cells whereas it was virtually absent from 56% of seminomas as well as from 86% of embryonal carcinomas and virtually all teratomas. On the contrary, ITGCN intensely expressed PTEN, indicating that loss of PTEN expression is not an early event in testicular tumor development. The loss of PTEN expression occurs mainly at the RNA level as determined by in situ hybridization of cellular mRNA (17/22) but also it may involve some kind of post-transcriptional mechanisms in the remaining 25% of cases. Analysis of microsatellites D10S551, D10S541 and D10S1765 in GCTs (n=22) showed LOH at the PTEN locus at 10q23 in at least 36% of GCTs (three embryonal carcinoma, three seminoma, two teratoma); one seminoma and one embryonal (9%) carcinoma presented an inactivating mutation in the PTEN gene (2/22). Finally, we demonstrated that the phosphatidylinositol 3'-kinase/AKT pathway, which is regulated by the PTEN phosphatase, is crucial in regulating the proliferation of the NT2/D1 embryonal carcinoma cells, and that the cyclin-dependent kinase inhibitor p27(kip1) is a key downstream target of this pathway.
Abstract: In this issue of the JCI, Ciampi et al. report the identification of a novel oncogene in patients affected by radiation-associated thyroid papillary carcinomas. This oncogene derives from a paracentric inversion of the long arm of chromosome 7, which results in an in-frame fusion of the N-terminus of the A-kinase anchor protein 9 (AKAP9) gene with the C-terminal catalytic domain (exons 9-18) of the serine-threonine kinase BRAF. The resulting AKAP9-BRAF fusion protein shows constitutive kinase activity, and it is able to transmit mitogenic signals to the MAPK pathways and to promote malignant transformation of NIH3T3 cells.
Abstract: Glioblastoma is one of the most aggressive tumors in mankind with 50% of patients dying within the 1st year of diagnosis, and being refractory to conventional therapies. The aim of our work has been to analyse the expression of the HMGA1 proteins in human astrocytomas and glioblastomas in order to verify whether the detection of these proteins might be of some help in the diagnosis of these neoplasias. Here we report the analysis of 27 cases, including 12 astrocytomas and 15 glioblastomas, for HMGA1 expression. All the neoplastic samples showed positive staining even though the number of positive cells and the staining intensity was higher in glioblastomas compared to astrocytomas. Conversely, HMGA1 proteins were not detected in normal brain. Accordingly, expression of the hmga1 gene, analysed by RT-PCR, was higher in glioblastomas than in astrocytomas.
Abstract: Pancreatic carcinoma is one of the most aggressive tumors, and, being refractory to conventional therapies, is an excellent target for new therapeutic approaches. Based on our previous finding of high HMGA1 expression in pancreatic cancer cells compared to normal pancreatic tissue, we evaluated whether suppression of HMGA1 protein expression could be a treatment option for patients affected by pancreatic cancer. Here we report that HMGA1 proteins are overexpressed in pancreatic carcinoma cell lines, and their downregulation through an adenovirus carrying the HMGA1 gene in an antisense orientation (Ad Yas-GFP) results in the death of three human pancreatic carcinoma cell lines (PANC1, Hs766T and PSN1). Pretreatment of PANC1 and PSN1 cells with Ad Yas-GFP suppressed and reduced, respectively, their ability to form xenograft tumors in nude mice. To further verify the role of HMGA1 in pancreatic tumorigenesis, we used a HMGA1 antisense phosphorothioate oligodeoxynucleotide (ODN); its addition induced a decrease in HMGA1 protein levels and a significant reduction of the proliferation rate of PANC1-, Hs766T- and PSN1-treated cells. Therefore, suppression of HMGA1 protein synthesis by an HMGA1 antisense approach seems to be a feasible treatment strategy in pancreatic carcinomas.
Abstract: The localization of the cyclin-dependent kinase inhibitor p27(kip1) is dependent on the phosphorylation of one of three key amino acid residues: S10, T157 and T198. However, it was unclear whether endogenous p27(kip1) is phosphorylated at T198 in the living cell. In the present work we describe the generation and characterization of a polyclonal antibody able to recognize recombinant, transfected as well as endogenous T198-phosphorylated p27(kip1). Using this antibody, we demonstrate that: (1) endogenous p27(kip1) is phosphorylated at T198 in 4 breast cancer cells lines (MCF7, MDA-MB231, MDA- MB436 and MDA-MB468); (2) T198 phosphorylation is increased in breast cancer cells compared with normal mammary epithelial cells (HMEC); (3) T198-phosphorylated p27(kip1) is exclusively cytoplasmic; (4) T198 phosphorylation is dependent on the activity of the PI3K-PKB/Akt pathway, being it drastically reduced by the pharmacological PI3K inhibitor LY294002 or stimulated by the constitutive activation of PKB/Akt. Finally, in primary human breast carcinomas, cytoplasmic accumulation of T198-phosphorylated p27(kip1) parallels Akt activation. We conclude that in breast cancer cells p27(kip1) is phosphorylated at T198 in a PI3K/Akt dependent manner and that this phosphorylation may contribute to p27(kip1) cytoplasmic mislocalization observed in breast cancer.
Abstract: We show that treatment of a panel of thyroid carcinoma cell lines naturally harboring the RET/PTC1 oncogene, with the RET kinase inhibitors PP1 and ZD6474, results in reversible G(1) arrest. This is accompanied by interruption of Shc and mitogen-activated protein kinase (MAPK) phosphorylation, reduced levels of G(1) cyclins, and increased levels of the cyclin-dependent kinase inhibitor p27Kip1 because of a reduced protein turnover. MAP/extracellular signal-regulated kinase 1/2 inhibition by U0126 caused G(1) cyclins down-regulation and p27Kip1 up-regulation as well. Forced expression of RET/PTC in normal thyroid follicular cells caused a MAPK- and proteasome-dependent down-regulation of p27Kip1. Reduction of p27Kip1 protein levels by antisense oligonucleotides abrogated the G(1) arrest induced by RET/PTC blockade. Therefore, in thyroid cancer, RET/PTC-mediated MAPK activation contributes to p27Kip1 deregulation. This pathway is implicated in cell cycle progression and in response to small molecule kinase inhibitors.
Abstract: The high mobility group A (HMGA) proteins are non-histone chromosomal proteins implicated in the organization of chromatin structure and in the assembly of protein complexes on the promoters of several inducible genes. Rearrangements of HMGA1 and HMGA2 genes, consequent to chromosomal translocation, have been frequently detected in human benign tumours of mesenchymal origin including lipomas. We have demonstrated previously that 3T3-L1 adipocytic differentiation is associated with increased HMGA1 protein levels, and that the block of HMGA1 synthesis dramatically increases the growth rate of 3T3-L1 cells and suppresses adipocytic differentiation. Here we have examined the role of a truncated HMGA1 gene in adipocytic cell growth. We have found that expression of the truncated Hmga1 gene (Hmga1/T) dramatically increases 3T3-L1 cell growth without blocking adipocytic differentiation. The Hmga1/T 3T3-L1 cells had higher E2F activity than the wild-type cells, and a deregulated cell cycle. In fact, the Hmga1/T cells had a reduced G0/G1 fraction, and a greater number of cells in S-phase. However, consistent with the benign nature of tumours associated with HMGA1 rearrangements, the Hmga1/T 3T3-L1 cells did not acquire the malignant phenotype. These results suggest a critical role played by HMGA1 rearrangements in the generation of human lipomas.
Abstract: High mobility group A 1 (HMGA1) proteins are chromatinic factors, which are absent or expressed at very low levels in normal adult tissues, while they are over-expressed in several human malignant tumors. In this study, HMGA1 protein expression was investigated by immunohistochemistry in a series of 44 epithelial ovarian specimens, which included four normal ovarian tissues, 29 primary invasive carcinomas, one metastatic ovarian tumor and 10 low malignant potential (LMP) tumors. HMGA1 staining was not detected in normal ovarian surface epithelium, which is the area from which ovarian adenocarcinoma frequently arises. HMGA1 proteins were expressed at low levels in some LMP tumors, whereas they were present in abundance in most of the primary ovarian adenocarcinomas. RT-PCR and western blot analysis correlated with immunohistochemical data. We demonstrated that the suppression of HMGA1 protein synthesis by an adenovirus carrying the HMGA1 gene in antisense orientation (Ad-Yas-GFP) inhibited the growth of two human ovarian carcinoma cell lines (OVCAR-5 and OVCAR-8). These results confirm HMGA1 over-expression as a general feature of human malignant neoplasias, including ovarian cancer and suggest that suppression of HMGA1 protein synthesis by an antisense adenoviral vector may represent a new and promising gene therapy for the treatment of ovarian cancer.
Abstract: We report that cyclin D3 is rate limiting for G1 progression in thyroid follicular cells and that its constitutive upregulation by chronic stimulation of the TSH/cAMP pathway plays a role in human and experimental hyperproliferative diseases of the thyroid gland. These conclusions are supported by in vitro and in vivo studies. In rat thyrocytes (PC Cl 3 cells), cyclin D3 expression is enhanced in response to activation of the TSH/cAMP pathway. Interference with the expression of G1 cyclins (in particular cyclin D3) by the antisense methodology strongly reduced TSH-dependent proliferation of PC Cl 3 cells, indicating that proper progression through G1 requires cyclin D3. Accordingly, PC Cl 3 cells engineered to overexpress cyclin D3 (PC-D3 cells) show enhanced growth rate and elude hormone-dependence and contact inhibition. Using an animal experimental model of thyroid stimulation, we demonstrate that cyclin D3 is a key mediator of TSH-dependent proliferation of thyroid follicular cells also in vivo. Cyclin D3 protein levels were higher in the thyrocytes from glands of propylthiouracil-treated rats compared with control animals. The increase in cyclin D3 expression occurred after the propylthiouracil-induced increase in TSH levels and preceded the burst of cell proliferation. Finally, we found that cyclin D3 protein is expressed in a fraction of human goiters but it is strongly overexpressed in most follicular adenomas.
Abstract: We demonstrated previously that rat tyrosine phosphatase r-PTPeta expression was suppressed in rat and human thyroid neoplastic cells, and that restoration of r-PTPeta expression reverted the malignant phenotype. To investigate the potential of this gene for cancer therapy, we generated an adenovirus carrying the r-PTPeta cDNA (Ad-r-PTPeta). This virus infected human thyroid carcinoma cells and overexpressed the r-PTPeta protein. Overexpression of r-PTPeta significantly inhibited the growth of four thyroid carcinoma cell lines. Cell growth inhibition was associated with down-regulation of extracellular signal-regulated kinase 1/2 activity, with increased levels of the cell-cycle inhibitor p27(kip1) protein and with dephosphorylation of PLCgamma1, a substrate of DEP-1, the human homologue of r-PTPeta. Finally, the growth of xenograft tumors induced in athymic mice by anaplastic thyroid carcinoma ARO cells transduced with the Ad-r-PTPeta virus was drastically reduced. These data suggest that gene therapy based on restoration of PTPeta function has potential in the treatment of human thyroid malignant neoplasias.
Abstract: Atypical lipomatous tumors (ALTs)/well-differentiated liposarcomas represent a distinctive subset of mesenchymal neoplasms featuring mature adipocytic proliferation. These tumors are characterized cytogenetically by the presence of supernumerary ring and/or long marker chromosomes that contain several copies of the chromosomal region 12q13-15, in which the HMGA2 gene is located. Deregulation of the HMGA2 gene is a common molecular alteration implicated in the development of a variety of benign tumors, such as lipomas, uterine leiomyomas, and pulmonary chondroid hamartomas. In this study, we observed HMGA2 overexpression in 7 of 12 ALT primary cell cultures examined. Subsequently, we generated an adenovirus containing the HMGA2 gene in the antisense orientation (Ad-A2as) to study the effect of HMGA2 protein suppression in ALT cells. The infection of six ALT cells, three of which were positive for HMGA2 expression, resulted in growth inhibition coupled with a significant increase in apoptosis. In addition, the growth of the ALT cells negative for HMGA2 expression was not affected by the infection with either the Ad-A2as or the control virus. On the basis of these findings, the targeting of the HMGA2 protein expression may represent a promising approach for treating the well-differentiated liposarcomas resistant to conventional therapies.
Abstract: We have analyzed 18 families with high incidence of breast cancer or breast and ovarian cancer for the presence of BRCA1 mutations. We identified 4 mutations in the BRCA1 gene in 4 unrelated probands who belong to families with at least 1 case of breast and 1 case of ovarian cancer. Two of the mutations reported in this study are novel (GAA(1172)-->TAA in family Naples 14, GAA(1765)-->TAA in family Naples 20) whereas the others are already present in the Breast Cancer Information Core Electronic Database (http://nchgr.nih.gov/ Intramural research/Lab transfer/Bic/) (5382insC in family Naples 18 and 2080delA in family Naples 19). Conversely, no mutation in the BRCA1 gene was detected in 14 families characterized by 2 or more cases of breast cancer only, even if bilateral and with early-onset. These results indicate that germline mutations in the BRCA1 gene highly predispose for a cancer syndrome that involves the presence of both breast and ovarian cancer.
Abstract: The cyclin-dependent kinase inhibitor p27(kip1) is a putative tumor suppressor for human cancer. The mechanism underlying p27(kip1) deregulation in human cancer is, however, poorly understood. We demonstrate that the serine/threonine kinase Akt regulates cell proliferation in breast cancer cells by preventing p27(kip1)-mediated growth arrest. Threonine 157 (T157), which maps within the nuclear localization signal of p27(kip1), is a predicted Akt-phosphorylation site. Akt-induced T157 phosphorylation causes retention of p27(kip1) in the cytoplasm, precluding p27(kip1)-induced G1 arrest. Conversely, the p27(kip1)-T157A mutant accumulates in cell nuclei and Akt does not affect p27(kip1)-T157A-mediated cell cycle arrest. Lastly, T157-phosphorylated p27(kip1) accumulates in the cytoplasm of primary human breast cancer cells coincident with Akt activation. Thus, cytoplasmic relocalization of p27(kip1), secondary to Akt-mediated phosphorylation, is a novel mechanism whereby the growth inhibitory properties of p27(kip1) are functionally inactivated and the proliferation of breast cancer cells is sustained.
Abstract: There is considerable evidence that the inactivation of the cyclin-dependent kinase inhibitor p27(kip1) is a fundamental step for the development of human malignancies. In particular, reduced expression of p27(kip1), due to increased protein degradation, correlates with poor prognosis of patients affected by various types of cancer. The purpose of this mini-review is to present an overview of the current understanding of the alteration of p27(kip1) function in human cancer and to describe the different mechanisms that contributes to it. Particular emphasis is placed on the novel finding of p27(kip1) mislocalization in tumor cells and on the biochemical pathways responsible for p27(kip1) cytosolic accumulation. Finally, we review the possible clinical implications of these observations with respect to prognosis and novel anticancer therapies.
Abstract: We used subtractive library screening to identify the changes that occur in gene expression during thyroid cell neoplastic transformation. Complementary DNA from normal thyroid cells (HTC 2) was subtracted from a complementary DNA library constructed from a human thyroid papillary carcinoma cell line. The library was screened for genes upregulated in human thyroid papillary carcinoma cell line cells, and several cDNA clones were isolated. One of these clones has a sirtuin core and high homology with the human silent information regulator protein family. This clone, designated "SIR-T8", was overexpressed in human thyroid carcinoma cell lines and tissues, but not in adenomas. The human SIR-T8 protein has a molecular weight of 39 kDa and is primarily located in the cytoplasm under the nuclear membrane. The SIR-T8 gene is located on chromosome 17q25-1.
Abstract: Growth factors of the glial cell line-derived neurotrophic factor (GDNF) family control the differentiation of neuronal cells of the central and peripheral nervous systems. Intracellular signalling of these growth factors is, at least in part, mediated by activation of the RET receptor tyrosine kinase. Here, we demonstrate that GDNF triggering inhibits the proliferation of the embryonal carcinoma cell line NT2/D1. This anti-proliferative effect is accompanied by down-regulation of the SSEA-3 antigen, a marker typical of undifferentiated NT2/D1 cells. We show that these effects are mediated by activation of RET signalling. The block of RET by a kinase-deficient dominant negative mutant impairs GDNF-dependent growth inhibition, whereas the adoptive expression of a constitutively active RET, the RET-MEN2A oncogene, promotes effects similar to those exerted by GDNF. We show that RET signalling increases the expression of the cyclin-dependent kinase inhibitor p27(kip1) in NT2/D1 cells. Both DNA synthesis inhibition and SSEA-3 down-regulation are prevented if p27(kip1) expression is blocked by an antisense construct, which demonstrates that RET-triggered effects are mediated by p27(kip1).
Abstract: The tyrosine phosphatase r-PTPeta is able to suppress the malignant phenotype of rat thyroid tumorigenic cell lines. To identify r-PTPeta interacting proteins, a yeast two-hybrid screening was performed and an insert corresponding to the full-length syntenin cDNA was isolated. It encodes a protein containing two PDZ domains that mediates the binding of syntenin to proteins such as syndecan, proTGF-alpha, beta-ephrins and neurofascin. We show that r-PTPeta is able to interact with syntenin also in mammalian cells, and although syntenin is a tyrosine-phosphorylated protein it is not a substrate of r-PTPeta. The integrity of both PDZ domains of syntenin and the carboxy-terminal region of r-PTPeta are required for the interaction between syntenin and r-PTPeta.
Abstract: The human teratocarcinoma-derived growth factor (TDGF)-1 gene encodes a 188-amino acid protein, cripto-1. The TDGF-1 gene is overexpressed in the majority of human primary colorectal carcinomas and hepatic metastases, in breast carcinomas and in testicular nonseminoma germ cell embryonal carcinomas. In the human embryonal carcinoma cell line NTERA-2 clone D1, a 2-kb TDGF-1 mRNA transcript is expressed. The present study shows that a 1.7-kb mRNA transcript lacking the first two exons of the TDGF-1 gene is expressed in the human colon carcinoma cell line GEO. This shorter mRNA is the only TDGF-1 transcript that is present in the majority of primary human colorectal carcinomas and hepatic metastases and in adult human tissues such as the pancreas, heart, stomach, mammary gland, skeletal muscle, liver and placenta. In contrast, in the kidney, brain, testis, ovary and spleen, the longer 2-kb TDGF-1 mRNA transcript is expressed. The putative shorter protein starts at a CUG codon 129 nucleotides downstream of the starting AUG codon of the longer protein. These data indicate the potential for differential transcriptional regulation of the TDGF-1 gene in different normal and tumor tissues.
Abstract: The RING-finger protein RNF4 modulates both steroid-receptor-dependent and basal transcription and interacts with a variety of nuclear proteins involved in cell growth control. RNF4 is expressed at very high levels in testis and at much lower levels in several other tissues. We show that in germ cells RNF4 expression is strongly modulated during progression of spermatogonia to spermatids, with a peak in spermatocytes. Analysis of human testicular germ cell tumors shows that RNF4 is not expressed in all tumors analyzed including seminomas, the highly malignant embryonal carcinomas, yolk sac, and mixed germ cell tumors. We also show that the ectopically expressed RNF4 gene inhibits cell proliferation of both somatic and germ cell tumor-derived cells. Mutation of critical cysteine residues in the RING finger domain abolished the RNF4 growth inhibition activity. Our results suggest that the lack of RNF4 expression may play a role in the progression of testicular tumors.
Abstract: The high mobility group (HMG) proteins (HMGA1a, HMGA1b, and HMGA2) bind to DNA and interact with various transcriptional factors. Therefore, they play an important role in chromatin organization. HMGA protein expression is low in normal adult tissues, but abundant during embryonic development and in several experimental and human tumors. Blockage of HMGA expression inhibits the transformation of rat thyroid PC Cl 3 cells treated with oncogene-carrying retroviruses, thus implicating HMGA in rat thyroid transformation. To better understand the role of HMGA and to establish whether its up-regulated expression is sufficient to induce the transformed phenotype, we generated PC Cl 3 cells that overexpress the protein. We demonstrate that HMGA1b protein overexpression does not transform normal rat thyroid PC Cl 3 cells, but it deregulates their cell cycle: cells enter S-phase earlier and the G(2)-M transition is delayed. HMGA1-overexpressing cells undergo apoptosis through a pathway involving caspase-3 activation, probably consequent to the conflict between mitogenic pressure and the inability to proceed through the cell cycle. Using various HMGA1b gene mutations, we found that the third AT-hook domain and the acetylation site K60 are the protein regions required for induction of apoptosis in PC Cl 3 cells. In conclusion, although HMGA1 protein overexpression is associated with the malignant phenotype of rat and human thyroid cells, it does not transform normal thyroid cells in culture but leads them to programmed cell death.
Abstract: The proteins of the Ets family are transcription factors involved in signal transduction, cell cycle progression, and differentiation. In this study, we report that thyroid cell neoplastic transformation is associated with a dramatic increase in ETS transcriptional activity, which is dependent on the accumulation of Ets-1, Ets-2, and other Ets-related proteins. Inhibition of ETS transactivation activity by the Ets-dominant negative construct (Ets-Z) induced programmed cell death in human thyroid carcinoma cell lines but not in normal thyroid cells. Apoptotic cell death induced by Ets-Z was dependent on the reduction of c-MYC protein levels, because it was prevented by overexpression of c-myc. Taken together, these data indicate that the induction of Ets-1 and Ets-2 transcription factors plays a pivotal role in thyroid cell neoplastic transformation.
Abstract: HMGI(Y) proteins are overexpressed in experimental and human malignancies, including colon, prostate and thyroid carcinomas. To determine at which step of the carcinogenic process HMGI(Y) induction occurs, we analysed the expression of the HMGI(Y) proteins in hyperplastic, preneoplastic and neoplastic tissues of colorectal origin by immunohistochemistry. All the colorectal carcinomas were HMGI(Y)-positive, whereas no expression was detected in normal colon mucosa tissue. HMGI(Y) expression in adenomas was closely correlated with the degree of cellular atypia. Only 2 of the 18 non-neoplastic polyps tested were HMGI(Y)-positive. These data indicate that HMGI(Y) protein induction is associated with the early stages of neoplastic transformation of colon cells and only rarely with colon cell hyperproliferation.
Abstract: The high-mobility group I (HMGI) nonhistone chromosomal proteins HMGI(Y) and HMGI-C have been implicated in defining chromatin structure and in regulating the transcription of several genes. These proteins have been implicated in adipocyte homeostasis: a severe deficiency of fat tissue is found in mice with targeted disruption of the HMGI-C locus, and lipomagenesis in humans is frequently associated with somatic mutations of HMGI genes. The aim of this study was to examine the role of HMGI(Y) proteins in adipocytic cell growth and differentiation. First, we found that differentiation of the preadipocytic 3T3-L1 cell line caused early induction of HMGI(Y) gene expression. Suppression of HMGI(Y) expression by antisense technology dramatically increased the growth rate and impaired adipocytic differentiation in these cells. The process of adipogenic differentiation involves the interplay of several transcription factors, among which is the CCAAT/enhancer-binding protein (C/EBP) family of proteins. These factors are required for the transcriptional activation of adipocyte-specific genes. We also tested the hypothesis that HMGI(Y) might participate in transcriptional control of adipocyte-specific promoters. We found that HMGI(Y) proteins bind C/EBPbeta in vivo and in vitro. Furthermore, we show that HMGI(Y) strongly potentiates the capacity of C/EBPbeta to transactivate the leptin promoter, an adipose-specific promoter. Taken together, these results indicate that the HMGI(Y) proteins play a critical role in adipocytic cell growth and differentiation.
Abstract: The HMGI proteins (HMGI, HMGY and HMGI-C) have an important role in the chromatin organization and interact with different transcriptional factors. The HMGI genes are expressed at very low levels in normal adult tissues, whereas they are very abundant during embryonic development and in several experimental and human tumours. In order to isolate proteins interacting with the HMGI(Y) proteins, a yeast two-hybrid screening was performed using the HMGI(Y) protein as bait. This analysis led to the isolation of homeodomain-interacting protein kinase-2 (HIPK2), a serine/threonine nuclear kinase. HIPK2 co-immunoprecipitates with the HMGI(Y) protein in 293T cells. The interaction between HIPK2 and HMGI(Y) occurs through the PEST domain of HIPK2 and it is direct because in vitro translated HIPK2 binds HMGI(Y). We also show that HIPK2 is able to phosphorylate the HMGI(Y) protein by an in vitro kinase assay. In order to understand a possible role of HIPK2 gene in cell growth we performed a colony assay which showed an impressive HIPK2 inhibitory effect on normal thyroid cells. Flow cytometric analysis would indicate the block of cell growth at the G2/M phase of the cell cycle. Since normal thyroid cells do not express detectable HMGI(Y) protein levels, we assume that the HIPK2 inhibitory effect is independent from the interaction with the HMGI(Y) protein.
Abstract: Retinoic acid (RA) treatment of embryonal carcinoma cell line NTERA-2 clone D1 (NT2/D1) induces growth arrest and terminal differentiation along the neuronal pathway. In the present study, we provide a functional link between RA and p27 function in the control of neuronal differentiation in NT2/D1 cells. We report that RA enhances p27 expression, which results in increased association with cyclin E/cyclin-dependent kinase 2 complexes and suppression of their activity; however, antisense clones, which have greatly reduced RA-dependent p27 inducibility (NT2-p27AS), continue to synthesize DNA and are unable to differentiate properly in response to RA as determined by lack of neurite outgrowth and by the failure to modify surface antigens. As to the mechanism involved in RA-dependent p27 upregulation, our data support the concept that RA reduces p27 protein degradation through the ubiquitin/proteasome-dependent pathway. Taken together, these findings demonstrate that in embryonal carcinoma cells, p27 expression is required for growth arrest and proper neuronal differentiation.
Abstract: The r-PTPeta gene encodes a rat receptor-type protein tyrosine phosphatase whose expression is negatively regulated by neoplastic cell transformation. Here we first demonstrate a dramatic reduction in DEP-1/HPTPeta (the human homolog of r-PTPeta) expression in a panel of human thyroid carcinomas. Subsequently, we show that the reexpression of the r-PTPeta gene in highly malignant rat thyroid cells transformed by retroviruses carrying the v-mos and v-ras-Ki oncogenes suppresses their malignant phenotype. Cell cycle analysis demonstrated that r-PTPeta caused G(1) growth arrest and increased the cyclin-dependent kinase inhibitor p27(Kip1) protein level by reducing the proteasome-dependent degradation rate. We propose that the r-PTPeta tumor suppressor activity is mediated by p27(Kip1) protein stabilization, because suppression of p27(Kip1) protein synthesis using p27-specific antisense oligonucleotides blocked the growth-inhibitory effect induced by r-PTPeta. Furthermore, we provide evidence that in v-mos- or v-ras-Ki-transformed thyroid cells, the p27(Kip1) protein level was regulated by the mitogen-activated protein (MAP) kinase pathway and that r-PTPeta regulated p27(Kip1) stability by preventing v-mos- or v-ras-Ki-induced MAP kinase activation.
Abstract: Rat thyroid differentiated cells (PC Cl 3) are an excellent model system with which to study the interaction between differentiation and cell transformation. We previously demonstrated that PC Cl 3 cells expressing the adenovirus E1A gene no longer depend on thyrotropin for growth and do not express thyroid differentiation markers. Here we show that an E1A mutant unable to bind the RB protein failed to transform the PC Cl 3 cells. Conversely, mutations in the E1A p300 interacting region did not affect its transforming ability. The pivotal role of RB family proteins in the thyroid cell transformation is supported by the thyrotropin independence induced by the E7 gene of human papilloma virus type 16, but not by a mutated form in the RB-binding region.
Abstract: The Akt/protein kinase B serine/threonine kinase is a downstream effector of phosphoinositide 3-kinase (PI3K). Akt is an important component of mitogenic and antiapoptotic signaling pathways and is implicated in neoplastic transformation. Thyroid cells in culture retain a differentiated phenotype consisting of epithelial cell morphology and the expression of several tissue-specific genes. The survival and proliferation of these cells depend on thyrotropin and a mixture of five additional hormones that includes insulin. The regulation of proliferation and the expression of the thyroid differentiation program are intimately connected processes. As a result, oncogenes that induce hormone-independent proliferation invariably impair the expression of the thyroid-specific differentiation markers. Given that thyrotropin and insulin stimulate Akt activation in thyroid cells, we set out to determine the effects of Akt on thyroid cell proliferation, survival, and differentiation. To this end, we expressed constitutively active myristylated Akt (myrAkt) in PC Cl 3 thyroid cells. The myrAkt-expressing cells continued to proliferate, even in the absence of hormones, and they were resistant to programmed cell death induced by starvation. These effects were paralleled by the induction of the G1 cyclins D3 and E and by the inhibition of induction of the proapoptotic Fas, Fas ligand, and BAD genes in starved cells. However, in marked contrast with several other oncogenes, myrAkt did not interfere with the expression of thyroid differentiation functions. These results unveil the existence of an Akt-triggered thyroid cell pathway that modulates proliferation and survival without affecting the expression of the thyroid cell differentiated phenotype.
Abstract: The dual-specificity phosphatase PTEN/MMAC1/TEP1 has recently been identified as the tumor suppressor gene most frequently mutated and/or deleted in human tumors. Germline mutations of PTEN give rise to Cowden Disease (CD), an autosomal dominantly-inherited cancer syndrome which predisposes to increased risk of developing breast and thyroid tumors. However, PTEN mutations have rarely been detected in sporadic thyroid carcinomas. In this study, we confirm that PTEN mutations in sporadic thyroid cancer are infrequent as we found one point mutation and one heterozygous deletion of PTEN gene in 26 tumors and eight cell lines screened. However, we report that PTEN expression is reduced both at the mRNA and at the protein level - in five out of eight tumor-derived cell lines and in 24 out of 61 primary tumors. In most cases, decreased PTEN expression is correlated with increased phosphorylation of the PTEN-regulated protein kinase Akt/PKB. Moreover, we demonstrate that PTEN may act as a suppressor of thyroid cancerogenesis as the constitutive re-expression of PTEN into two different thyroid tumor cell lines markedly inhibits cell growth. PTEN-dependent inhibition of BrdU incorporation is accompanied by enhanced expression of the cyclin-dependent kinase inhibitor p27kip1 and can be overcome by simultaneous co-transfection of an excess p27kip1 antisense plasmid. Accordingly, in a subset of thyroid primary carcinomas and tumor-derived cell lines, a striking correlation between PTEN expression and the level of p27kip1 protein was observed. In conclusion, our findings demonstrate that inactivation of PTEN may play a role in the development of sporadic thyroid carcinomas and that one key target of PTEN suppressor activity is represented by the cyclin-dependent kinase inhibitor p27kip1.
Abstract: Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) are related growth factors which exert trophic effects on several neuronal populations and developing kidney. GDNF-family ligands interact with membrane receptors designated GFRalphas which, in turn, mediate stimulation of the Ret receptor tyrosine kinase. Here we show that Ret, GFRalpha-1 (the GDNF receptor), and GFRalpha-2 (the NTN receptor) are expressed by testicular germ cells, while GDNF and NTN are expressed by Sertoli cells. Both GDNF and NTN stimulate DNA synthesis in spermatogonia. Furthermore, Ret, ligands and co-receptors are expressed in germ cell tumors. Thus, GDNF-family ligands may act as paracrine factors in spermatogenesis and this circuit may be active in germ cell tumors.
Abstract: The beta-thymosins comprise a family of structurally related, highly conserved acidic polypeptides, originally isolated from calf thymus. Recently, we have demonstrated the overexpression of thymosin beta-10 (TB10) in rat thyroid transformed cell lines and in human thyroid carcinoma tissues and cell lines. To verify whether TB10 overexpression is a general event in the process of carcinogenesis, we have analyzed TB10 mRNA levels in human colon carcinomas, germ cell tumors of different histological types, breast carcinomas, ovarian carcinomas, uterine carcinomas, colon and esophageal carcinoma cell lines. Overexpression of the TB10 gene was detected in all of the neoplastic tissues and cell lines compared to the respective normal tissues. Moreover, the mouse model of skin carcinogenesis induced by the combined action of chemical carcinogens and phorbol esters was used to identify the stage of TB10 gene induction. The expression was almost undetectable in normal keratinocytes, its induction occurred even at the papilloma stage, however a further increased expression was observed in the carcinoma derived cell lines. Finally, immunohistochemical analysis of some breast, colon and ovary carcinoma samples by using specific anti-TB10 antibodies revealed the presence of the TB10 protein in all of the neoplastic tissues, but not in the respective normal tissues. Therefore the TB10 detection may be considered a potential tool for the diagnosis of several human neoplasias.
Abstract: Hexamethylen-bisacetamide (HMBA) represents the prototype of a group of hybrid polar compounds, which induce differentiation in a variety of transformed cells including human embryonal carcinoma cells. Therefore, HMBA has been used in the differentiation therapy of cancer for patients with both hematological and solid malignancies. Upon HMBA treatment, the embryonal carcinoma cell line NTERA-2 clone D1 (NT2/D1) accumulates in G1 and undergoes terminal differentiation. Here we demonstrate that growth arrest and differentiation of NT2/D1 cells induced by HMBA involve increased expression of the cyclin-dependent kinase inhibitor p27, enhanced association of p27 with cyclin E/CDK2 complexes and suppression of kinase activity associated to cyclin E/CDK2 (but not to cyclin D3/CDK4). When HMBA differentiation was induced in the presence of p27 antisense oligonucleotides, NT2/D1 cells failed to arrest growth properly and, in parallel with the reduction of the anti-apoptotic Bcl-2 gene expression, cells underwent massive programmed cell death. Conversely, constitutive expression of p27 into NT2/D1 cells induced a marked reduction in the growth potential of these cells and partially reproduced HMBA-induced modification of surface antigen expression (down-regulation of SSEA-3 expression and up-regulation of VINIS-53 expression). Expression of p21 induced growth arrest but not differentiation. Likewise, inhibition of CDK2 by transfection of a dominant negative CDK2 in NT2/D1 cells or treatment with the kinase inhibitor olomucine induced growth arrest but not differentiation. Therefore, we propose that p27 represents a crucial molecule in HMBA signaling that cannot be replaced by p21. Furthermore, the results obtained with CDK2 inhibitors demonstrate that the block of CDK2 activity is sufficient for growth arrest but not for cell differentiation and suggest that, at least in these cells, growth arrest and differentiation are regulated by two overlapping but different pathways.
Abstract: A sharp increase in the incidence of pediatric thyroid papillary cancer was documented after the Chernobyl power plant explosion. An increased prevalence of rearrangements of the RET protooncogene (RET/PTC rearrangements) has been reported in Belarussian post-Chernobyl papillary carcinomas arising between 1990 and 1995. We analyzed 67 post-Chernobyl pediatric papillary carcinomas arising in 1995-1997 for RET/PTC activation: 28 were from Ukraine and 39 were from Belarus. The study, conducted by a combined immunohistochemistry and RT-PCR approach, demonstrated a high frequency (60.7% of the Ukrainian and 51.3% of the Belarussian cases) of RET/PTC activation. A strong correlation was observed between the solid-follicular subtype of papillary carcinoma and the RET/PTC3 isoform: 19 of the 24 RET/PTC-positive solid-follicular carcinomas harbored a RET/PTC3 rearrangement, whereas only 5 had a RET/PTC1 rearrangement. Taken together these results support the concept that RET/PTC activation plays a central role in the pathogenesis of thyroid papillary carcinomas in both Ukraine and Belarus after the Chernobyl accident.
Abstract: The majority of thyroid carcinomas maintain the expression of the cell growth suppressor p27, an inhibitor of cyclin-dependent kinase-2 (Cdk2). However, we find that 80% of p27-expressing tumors show an uncommon cytoplasmic localization of p27 protein, associated with high Cdk2 activity. To reproduce such a situation, a mutant p27 devoid of its COOH-terminal nuclear-localization signal was generated (p27-NLS). p27-NLS accumulates in the cytoplasm and fails to induce growth arrest in 2 different cell lines, indicating that cytoplasm-residing p27 is inactive as a growth inhibitor, presumably because it does not interact with nuclear Cdk2. Overexpression of cyclin D3 may account in part for p27 cytoplasmic localization. In thyroid tumors and cell lines, cyclin D3 expression was associated with cytoplasmic localization of p27. Moreover, expression of cyclin D3 in thyroid carcinoma cells induced cytoplasmic retention of cotransfected p27 and rescued p27-imposed growth arrest. Endogenous p27 also localized prevalently to the cytoplasm in normal thyrocytes engineered to stably overexpress cyclin D3 (PC-D3 cells). In these cells, cyclin D3 induced the formation of cytoplasmic p27-cyclin D3-Cdk complexes, which titrated p27 away from intranuclear complexes that contain cyclins A-E and Cdk2. Our results demonstrate a novel mechanism that may contribute to overcoming the p27 inhibitory threshold in transformed thyroid cells.
Abstract: The human teratocarcinoma derived growth factor 1 (TDGF1) gene maps on chromosome (Chr) 3p21.3. One pseudogene (TDGF3) maps on Chr Xq21-->q22. We now report the nucleotide sequence and chromosome location of three additional TDGF pseudogenes. The three new sequences (TDGF2, TDGF4 and TDGF5) are truncated at the 5' end and have accumulated several point mutations, deletions and insertions. TDGF2, TDGF4 and TDGF6 map on Chrs 2q37, 6p25 and 3q22, respectively. Finally, Southern blot analysis of DNA from normal individuals shows a highly variable restriction pattern of the TDGF sequences.
Abstract: OBJECTIVE: To investigate the expression of thymosin beta10 - a small conserved acidic protein involved in the inhibition of actin polymerization - in human and experimental thyroid goiters as well as the regulation exerted by TSH on thymosin beta10 expression in thyroid follicular cells both in vivo and in vitro. DESIGN: To this aim, we have used 5 bioptic specimens from patients affected by thyroid goiter, a well known experimental model of thyroid goitrogenesis (rat fed with the drug propylthiouracil) and a cultured rat thyroid cell line (PC Cl 3 cells) as a model system. RESULTS: We report that the mRNA expression of thymosin beta10 is markedly enhanced in human goiters compared with normal thyroid. In vivo results showed that the steady-state level of thymosin beta10 mRNA is up-regulated in the thyroid gland of propylthiouracil-fed rats in parallel with follicular cell proliferation: iodide administration to goitrous rats, which induced a marked involution of thyroid hyperplasia, reduced the mRNA level of thymosin beta10. Finally, in vitro studies showed that in cultured rat thyrocytes, the expression of thymosin beta10 mRNA is induced in a time- and dose-dependent manner by the activation of pathways which are mitogenic for thyroid cells (i.e. the protein kinase (PK) A and PKC pathways). CONCLUSION: Taken together, the findings reported here demonstrate that thymosin beta10 expression is regulated by extracellular signals that stimulate growth of thyroid cells both in vitro and in vivo, and suggest a role for this protein in thyroid diseases characterized by proliferation of follicular cells.
Abstract: Vascular endothelial growth factor A (VEGF) is a potent mitogen for endothelial cells in vitro and promotes neo-angiogenesis in vivo. VEGF overexpression occurs in most human malignancies including thyroid carcinomas in which elevated VEGF expression is associated with a high tumorigenic potential. To investigate the role of VEGF in angiogenesis associated with development of thyroid carcinomas, we constitutively expressed VEGF121 into a poorly tumorigenic cell line (NPA) expressing minimal levels of endogenous VEGF. Here we report that VEGF overexpressing NPA cells showed the same growth potential as untransfected NPA in vitro but formed well-vascularized tumors when injected subcutaneously into nude mice with markedly reduced latency compared to parental cells. A complementary approach was to suppress VEGF expression in a highly tumorigenic anaplastic cell line (ARO) by the transfection of an antisense construct. Antisense-transfected ARO cells expressed reduced constitutive levels of VEGF, showed the same growth potential as untransfected ARO cells in vitro and formed small tumors characterized by minimal vascularization, extensive necrosis and longer latency compared to parental or vector-transfected ARO cells in vivo. Finally, we investigated the expression of both VEGF tyrosine kinase receptors (Flt-1 and Flk-1/KDR) in tumor specimens by RT - PCR. Expression of (Flt-1 and Flk-1/KDR) was low in tissue specimens derived from NPA tumors, but was found enhanced in NPA VEGF tumors; conversely, the expression of VEGF receptors was high in tissue specimens derived from ARO tumors but was decreased in tumors derived from VEGF depleted ARO cells. These results clearly demonstrate that VEGF indirectly promotes the growth of thyroid tumors by stimulating angiogenesis.
Abstract: We have recently reported the isolation of a rat cDNA encoding a receptor-type tyrosine phosphatase, which appears to be a marker of thyroid differentiation. To elucidate the molecular mechanisms underlying r-PTPeta expression in normal thyroid cells both in vitro and in vivo, we investigated the regulation of r-PTPeta expression in cultured thyrocytes (the rat cell line PC Cl 3) and in an animal model of TSH-dependent thyroid goitrogenesis. In vitro studies showed that mRNA expression of r-PTPeta in thyroid cells is induced in a time- and dose-dependent manner by the activation of growth- and differentiation-linked PKA pathways (TSH and forskolin), whereas it is down-regulated by the activation of the proliferative dedifferentiating PKC-dependent transduction pathway (TPA). However, the regulation of r-PTPeta expression by TSH and TPA, respectively, is observed only in normal thyroid cells, but is lost in transformed thyroid cells. In vivo studies with thiouracil-fed rats demonstrated that increased serum levels of TSH up-regulated r-PTPeta mRNA expression in parallel with the stimulation of thyroid growth and function. The reduction of blood TSH levels due to iodide refeeding to goitrous rats determined a marked down-regulation of r-PTPeta expression, in parallel with involution of thyroid hyperplasia. Taken together these results demonstrate that the phosphatase r-PTPeta is regulated by the two main thyroid regulatory pathways and suggest that it may play an important role in the growth and differentiation of thyroid cells.
Abstract: Cowden disease (CD) is an autosomal dominant multiple hamartoma syndrome with an elevated risk of thyroid and breast cancers. The CD susceptibility gene has recently been identified as the PTEN/MMAC1/TEP1 gene localized at 10q23 and coding for a dual specificity protein phosphatase. We report the mutational analysis of the PTEN gene in one Italian CD kindred. By using the single strand conformation polymorphism technique and subsequent direct DNA sequencing of the polymerase chain reaction product, we identified a novel mutation in the exon 5 of the PTEN gene. A heterozygous germline TGT-TAT transition was detected at the nucleotide 407; this causes the amino acid substitution cys136-tyr136 and the generation of a new NSI I restriction site. This mutation was not detected in the unaffected member of the family thereby indicating that it is causally linked to the disease. We ruled out that this mutation is a polymorphic variant because it was not detected in over 100 chromosomes analyzed. Using reverse trancriptase-polymerase chain reaction, we detected the expression of the mutant allele in lymphocytes and pathological tissues from an affected member of the family.
Abstract: We have recently reported that neoplastic transformation of two rat thyroid epithelial cell lines by retroviruses carrying the v-mos and v-ras Ki oncogenes is associated with a drastic increase of AP-1 activity. The most important effects were represented by the dramatic junB and fra-1 gene induction, which was abolished by the block of the transformation-induced HMGI-C protein synthesis. Here, we have further characterized the transformation-dependent AP-1 activity, by analysing the expression of different jun- and fos-related components, in rat thyroid cell lines transformed by several oncogenes, in human thyroid carcinoma cell lines, and in naturally occurring human thyroid tumours. A significant increase of Fra-1 and JunB protein levels was detected in all oncogene transformed rat thyroid cell lines. Fra-1 gene induction was demonstrated to occur also in human thyroid carcinoma cell lines and tissues. Conversely, c-Jun and JunD proteins, rather than JunB, accumulated in human thyroid carcinoma cell lines. An induction of AP-1 target genes was also detected both in rat and human thyroid transformed cell lines. Therefore, in vivo and in vitro thyroid cell transformation is associated with important compositional changes in the AP-1 complex and an increased transcriptional activity.
Abstract: A subtractive thyroid cDNA library was constructed from two human thyroid carcinoma cell lines originating from an anaplastic carcinoma and a papillary thyroid carcinoma. The library was used to identify genes correlated with the progression to a highly malignant phenotype. The thymosin beta-10 gene was isolated and found to be expressed at much higher levels in the anaplastic cell line than in the papillary cells. The thymosin beta-10 gene was overexpressed in five carcinoma cell lines compared with normal thyroid tissue and normal thyroid primary culture cells. The highest expression occurred in the most malignant cell lines. Thymosin beta-10 gene expression was also increased in surgically removed human thyroid carcinomas and was highest in the anaplastic carcinomas. Thymosin beta-10 gene expression was correlated with the degree of the malignant phenotype also in rat thyroid cells transfected with cellular and viral oncogenes of different tumorigenicity. These results show that thymosin beta-10 overexpression is a general event of thyroid cell neoplastic transformation and suggest that the gene is involved in the progression of thyroid carcinogenesis. Finally, the thymosin beta-10 gene was located on chromosome 2q37 by fluorescence in situ hybridization analysis.
Abstract: Overexpression of the high mobility group I (HMGI) proteins is often associated with the malignant phenotype. Moreover, many benign human tumors, mainly of mesenchymal origin, are characterized by rearrangements of the HMGI-C gene. In most cases, HMGI-C alterations involve breaks within the third intron of the gene resulting in aberrant transcripts carrying exons from 1-3, which encode the three DNA binding domains, fused to ectopic sequences. Here, we show that the expression of a truncated form of HMGI-C protein carrying only the three DNA-binding domains, or of a fusion protein carrying the three DNA-binding domains of HMGI-C and the LIM domains of the lipoma preferred partner gene (LPP) protein, causes malignant transformation of NIH3T3 cells. The unrearranged wild-type HMGI-C cDNA did not exert any transforming activity. These findings indicate that rearranged forms of HMGI-C play a role in cell transformation.
Abstract: The teratocarcinoma-derived growth factor-1 (TDGF-1) gene codes for a 188-aminoacid glycoprotein that shares structural homology with the epidermal growth factor (EGF) family of growth factors. TDGF-1 is highly expressed in the undifferentiated embryonal carcinoma stem cell line NTERA2 clone D1 (NT2/D1) and its expression is downregulated in response to differentiating agents such as retinoic acid (RA) and hexamethylen-bisacetamide (HMBA). To assess the role of TDGF-1 in the onset and/or progression of human germ cell tumors, we analysed TDGF-1 expression by Northern blot and immunostaining in a panel of 59 human germ cell tumors of different histological origins. We show that TDGF-1 expression is markedly elevated in a subset of human testicular germ cell tumors as compared to normal testes. TDGF-1 overexpression occurs in about 100% of tumors with non-seminomatous phenotype, such as embryonal carcinomas and malignant undifferentiated teratocarcinomas. To address the questions of how TDGF-1 (previously called CRIPTO) may affect the growth and/or the differentiation of embryonal carcinoma cells, we have characterized the effects of exogenous recombinant TDGF-1 protein on the proliferation rate and differentiation 'potential of NT2/D1. Exogenous TDGF-1 protein stimulated DNA synthesis and cell proliferation in both undifferentiated and differentiated NT2/D1 cells. However, TDGF-1 protein treatment was unable to block differentiation induced by both RA and HMBA. These results suggest that TDGF-1 growth factor may represent an autocrine growth factor that may be involved in the process of development of testicular neoplasms.
Abstract: Placenta growth factor (PlGF) and vascular endothelial growth factor (VEGF) represent two closely related angiogenic growth factors active as homodimers or heterodimers. Since goiters of the thyroid gland are extremely hypervascular, we investigated the expression of PlGF, VEGF and their receptors, Flt-1 and Flk-1/KDR, in a small panel of human goiters from patients with Graves's disease, in an animal model of thyroid goitrogenesis and in in vitro cultured thyroid cells. Here we report that the mRNA expression of PlGF, VEGF and their receptors is markedly enhanced in biopsies of goiters resected from Graves's patients. In vivo studies demonstrated that in the thyroid gland of thiouracil-fed rats, increased mRNA and protein expression of PIGF, VEGF, Flt-1 and Flk-1/KDR occurred subsequent to the rise in the serum thyroid stimulating hormone (TSH) levels and in parallel with thyroid capillary proliferation. In vitro studies confirmed the existence of such TSH-dependent paracrine communication between thyroid epithelial cells and endothelium since the conditioned medium collected from TSH-stimulated thyrocytes acquired mitogenic activity for human umbilical vein endothelial (HUVE) cells. Altogether, these data suggest that PlGF and VEGF, released by thyrocytes in response to the chronic activation of the TSH receptor pathway, may act through a paracrine mechanism on thyroid endothelium.
Abstract: The expression of the high mobility group I (HMGI)-C chromatin component was shown previously to be essential for the establishment of the neoplastic phenotype in retrovirally transformed thyroid cell lines. To identify possible targets of the HMGI-C gene product, we have analyzed the AP-1 complex in normal, fully transformed and antisense HMGI-C-expressing rat thyroid cells. We show that neoplastic transformation is associated with a drastic increase in AP-1 activity, which reflects multiple compositional changes. The strongest effect is represented by the dramatic junB and fra-1 gene induction, which is prevented in cell lines expressing the antisense HMGI-C. These results indicate that the HMGI-C gene product is essential for the junB and fra-1 transcriptional induction associated with neoplastic transformation. The inhibition of Fra-1 protein synthesis by stable transfection with a fra-1 antisense RNA vector significantly reduces the malignant phenotype of the transformed thyroid cells, indicating a pivotal role for the fra-1 gene product in the process of cellular transformation.
Abstract: We have isolated a rat thyroid cDNA encoding a novel rat receptor-type tyrosine phosphatase protein. This gene, on the basis of its homology to another tyrosine phosphatase, the recently isolated human DEP-1/HPTPeta, has been named r-PTPeta. In rat thyroid cells the r-PTPeta gene acts as a differentiation marker. Indeed, the block of thyroid cell differentiation induced by viral and cellular oncogenes is associated with the inhibition or marked reduction of the expression of this gene, and its expression is positively regulated by thyrotropin, the physiological stimulator of thyroid cell growth.
Abstract: The placental-derived growth factor (PIGF) is a dimeric glycoprotein showing a high degree of sequence similarity to the vascular endothelial growth factor. Alternative splicing of the PIGF primary transcript gives rise to two forms, named PIGF-1 and PIGF-2, which differ only in the insertion of a highly basic 21-amino acid stretch at the carboxyl end. The presence of the PIGF mRNA in thyroid, placenta, lung, and goiter has indicated the tissues where this factor functions. However, the role of PIGF in vascular development has not yet been clearly established. In the present study, we described the purification of PIGF-1 from overexpressing eukaryotic cells and then measured the angiogenic activity of the purified PIGF-1 in vivo in the rabbit cornea and the chick chorioallantoic membrane assays. In both in vivo assays, PIGF-1 induced a strong neovascularization process that was blocked by affinity-purified anti-PIGF-1 antibody. In the avascular cornea, PIGF-1 induced angiogenesis in a dose-dependent manner and seemed to be at least as effective (if not more effective) than vascular endothelial growth factor and basic fibroblast growth factor under the same conditions and at the same concentration. PIGF-1 was shown to induce cell growth and migration of endothelial cells from bovine coronary postcapillary venules and from human umbilical veins. In these two in vitro assays, PIGF-1 seemed to have a comparable effect to that of vascular endothelial growth factor and basic fibroblast growth factor on the cultured microvascular endothelium (eg, capillary venule endothelial cells). In summary, this is the first study to demonstrate that PIGF-1 can induce angiogenesis in vivo and stimulate the migration and proliferation of endothelial cells in vitro.
Abstract: Placenta growth factor (PlGF) and vascular endothelial growth factor (VEGF) are angiogenic factors containing the 8-cysteine motif of platelet-derived growth factor (PDGF). Both PlGF and VEGF are mitogens for endothelial cells in vitro and promote neoangiogenesis in vivo. In addition, PlGF strongly potentiates the proliferative and the permeabilization effects exerted by VEGF on the vascular endothelium. We have now isolated the cDNA coding for mouse Plgf by screening a mouse heart cDNA library with the human PlGF sequence as probe. The human PlGF protein has two forms, PlGF-1 and PlGF-2, that arise from alternative splicing of a single gene mapping on Chromosome (Chr) 14; the isolated mouse Plgf cDNA encodes the longer of these two forms (PlGF-2). We show that the mouse Plgf-2 mRNA is the only transcript present in the normal tissues analyzed. Mouse Plgf-2 is a 158-amino-acid-long protein that shows 78% similarity (65% identity) to the human PlGF-2. Computer analysis reveals a putative signal peptide and three probable N-glycosylation sites, two of which are also conserved in human PlGF. The mouse Plgf gene was isolated and characterized; the gene is encoded by 7 exons spanning a 13-kb DNA interval. Finally, we have mapped the mouse Plgf gene to Chr 12, one cM from D12Mit5, and the human PlGF gene to 14q24, using both FISH and genetic crosses.
Abstract: A correlation has previously been demonstrated between the presence of the three HMGI proteins (HMGI, HMGY, and HMGI-C) and the expression of a highly malignant phenotype in epithelial and fibroblastic rat thyroid cells; this being subsequently extended to experimental thyroid, lung, prostate, mammary, and skin carcinomas. Recently, we have demonstrated that expression of HMGI and HMGY proteins, coded for by the HMGI(Y) gene, is associated with the malignant phenotype of human thyroid neoplasias. Here, we show that HMGI(Y) gene expression is present both at the RNA and protein level in human colorectal carcinoma cell lines and tissues examined in this study. Conversely, no HMGI(Y) proteins were detected in normal intestinal mucosa. Therefore, these results suggest an involvement of HMGI and HMGY proteins overexpression in colorectal tumorigenesis.
Abstract: OBJECTIVE: p53 is a well-known nuclear phosphoprotein encoded by a suppressor gene know to be mutated in various kinds of human tumours. A relationship between p53 gene mutation and tumour progression seems to be a common feature of several neoplasias. DESIGN: In order to investigate the role of p53 mutations in human thyroid tumours, DNA samples derived from fifty-six neoplastic tissues, ranging from benign adenomas to undifferentiated carcinomas, were examined for the presence of p53 gene mutations. METHODS: The analysis has been conducted using polymerase chain reaction (PCR) amplification of the exons 5-9 of the p53 gene followed by single strand conformation polymorphism (SSCP) and sequence analyses. RESULTS: One anaplastic carcinoma and one papillary carcinoma showed p53 gene mutations in exons 5 and 8, respectively. A cell line established from the papillary carcinoma showed the same mutation present in the original tumour. Both p53 mutations were heterozygous. The p53 positive samples were analysed for other genetic alterations frequently detected in human thyroid carcinomas (mutations of the RET, TRK, and ras oncogenes): both p53-mutated samples proved to be mutated at level of codon 13 of the c-Ki-ras gene. CONCLUSIONS: Our data confirm that p53 gene alterations are rare in well-differentiated thyroid tumours, that they are an important requirement for the establishment in culture of human thyroid carcinoma cell lines, and that they can be associated with other genetic alterations, namely ras mutations, in the malignant progression of thyroid tumours.
Abstract: Neoangiogenesis is a prerequisite for tumor growth and metastasis. In germ cell cancer patients with the disease limited to the testicle (stage A), tumor-associated neovascularization is predictive of metastatic disease (stage B). To investigate the molecular mechanisms underlying neovascularization in human germ cell tumors (GCTs), we analysed the expression of two angiogenic growth factors, vascular endothelial growth factor (VEGF) and placenta growth factor (P1GF), and of their receptors (FLT-1) and Flk-1/KDR) in a panel of testicular tumors. In this study we show a marked increase in VEGF expression in 36/44 (81.8%) primary testicular-derived GCTs, as compared to normal testis, that significantly correlates with a high density of intratumor microvessels (r = 0.72461, P < 0.001; n = 24). As determined by RT - PCR and/or Western blot, the predominant VEGF isoforms expressed in GCTs are the VEGF121 and VEGF165, which are more efficiently secreted by the cells, and thus more active in eliciting angiogenesis. Conversely, in the case of PIGF, only a weak correlation with the vascular density of tumors is observed (r = 0.26599, P < 0.05; n = 24). Northern blot analysis also revealed significant up-regulation of VEGF/ PIGF receptors in highly vascularized germ cell tumors, compared to normal testes. These findings suggest that VEGF may act in a paracrine manner to induce neovascularization, oedema extravasation and cyst formation in human germ cell tumors. The correlation between VEGF expression and the vascular density of tumors, suggest that the evaluation of VEGF expression may be of help in predicting patients at risk for metastatic diseases. Finally, we demonstrate that VEGF up-regulation may occur at the RNA level since no gene amplification is observed; conversely, in in vitro models such as the embryonal stem cell line NTERA-2 and the choricarcinoma JEG-3 cell line, VEGF (but not PIGF) mRNA expression is regulated by hypoxic stress.
Abstract: Mutations in any of the genes encoding the alpha, beta or gamma-sarcoglycan components of dystrophin-associated glycoproteins result in both sporadic and familial cases of either limb-girdle muscular dystrophy or severe childhood autosomal recessive muscular dystrophy. The collective name 'sarcoglycanopathies' has been proposed for these forms. We report the identification of a fourth member of the human sarcoglycan family. We named this novel cDNA delta-sarcoglycan. Its mRNA expression is abundant in striated and smooth muscles, with a main 8 kb transcript, encoding a predicted basic transmembrane glycoprotein of 290 amino acids. Antibodies specifically raised against this protein recognized a single band at 35 kDa on western blots of human and mouse muscle. Immunohistochemical staining revealed a unique sarcolemmal localization. FISH, radiation hybrid and YAC mapping concordantly linked the delta-sarcoglycan gene to 5q33, close to D5S487 and D5S1439. The gene spans at least 100 kb and is composed of eight exons. The identification of a novel sarcoglycan component modifies the current model of the dystrophin-glycoprotein complex.
Abstract: p53 is the gene most frequently found mutated in human neoplasias. In the majority of tumors, p53 mutations contribute to the progression towards stages of increasing malignancy with the appearance of an undifferentiated phenotype. Also in thyroid cancerogenesis, p53 mutations correlate with the loss of the differentiated phenotype. The results presented here, suggest a direct involvement of p53 in the molecular mechanisms regulating cellular differentiation in thyroid since a mutated p53 gene markedly affects the growth potential and differentiated functions of the rat thyroid cell line PC Cl 3. Blockage in the expression of the PAX-8 transcription factor seems to be a key event in the loss of thyroid differentiated functions induced by the mutated p53 gene. Thyroid cells carrying a mutated p53 gene did not form colonies in soft agar or tumors in athymic mice, suggesting that a mutation of the p53 gene is not sufficient for the induction of the malignant phenotype and probably a cooperation with other oncogenes is necessary to accomplish full malignancy. No effect on either growth or differentiation of thyroid cells was exerted either by overexpression of the wild-type p53 gene, or by the vector alone.
Abstract: Vascular endothelial growth factor (VEGF) is a potent mitogen for endothelial cells in vitro, promotes neoangiogenesis in vivo and increases the permeability of the vascular endothelium. VEGF overexpression occurs in several cultured tumor cell lines and in certain human malignancies. Placenta growth factor (PlGF) is a recently identified growth factor for endothelial cells (EC); PlGF strongly potentiates both the proliferative and the permeabilization effects exerted by VEGF on the vascular endothelium. To uncover the molecular mechanisms underlying neoangiogenesis in human thyroid tumors, we have analysed VEGF and PlGF expression in a panel of thyroid carcinoma cell lines with different tumorigenic potential as well as in human primary thyroid tumors. We show that a high tumorigenic potential is associated with an elevated VEGF expression in human thyroid tumor cell lines. Furthermore, VEGF overexpression occurs in 5/5 highly malignant anaplastic carcinomas. Papillary and follicular carcinomas express intermediate levels of VEGF mRNA. In contrast, PlGF expression is severely down regulated in the majority of thyroid tumor cell lines and in tumors. Furthermore, we show that both the VEGF receptors, FLT-1 and flk/KDR, are expressed in endothelial cells that line tumor-embedded microvascular vessels, suggesting that VEGF but not PlGF, contributes to thyroid tumor development.
Abstract: RET/PTC oncogene activation occurs in about 20% of human thyroid papillary carcinomas. However, it is not known yet whether it is an early or late event in the process of thyroid carcinogenesis. Here we demonstrate, by using a combined immunohistochemical and reverse transcriptase-polymerase chain reaction based approach, that RET/PTC activation is present in 11 out of 26 occult thyroid papillary carcinomas analysed. Therefore, we conclude that it represents an early event in the process of thyroid cell transformation.
Abstract: We have previously reported on the identification of a cDNA (placenta growth factor, PlGF) coding for a novel angiogenic factor expressed in placental tissue that is similar to vascular permeability factor/vascular endothelial growth factor (VPF/VEGF). Biochemical and functional characterization of PlGF derived from transfected COS-1 cells revealed that it is a glycosylated dimeric secreted protein able to stimulate endothelial cell growth in vitro. Here, we report the isolation and characterization of the PlGF gene located on chromosome 14. At least two different mRNAs are produced from this single-copy gene in different cell lines and tissues. Sequence comparison of the polypeptides encoded by the two different isolated cDNAs indicates that they are identical except for the insertion of a highly basic 21 amino acid stretch at the carboxyl end of the protein. RNA expression analysis of several tissues, tumors and cell lines indicates differential distribution of the two PlGF mRNAs. Finally, preliminary results indicate that the PIGF gene has been conserved in evolution, since the human PlGF cDNA hybridizes to sequences present in the genomic DNA of Drosophila, Xenopus, chicken and mouse.
Abstract: A human cDNA coding for a protein related to the vascular permeability factor (VPF) was isolated from a term placenta cDNA library; we therefore named its product placenta growth factor (PlGF). PlGF is a 149-amino-acid-long protein and is highly homologous (53% identity) to the platelet-derived growth factor-like region of human VPF. Computer analyses reveal a putative signal peptide and two probable N-glycosylation sites in the PlGF protein, one of which is also conserved in human VPF. By using N-glycosidase F, tunicamycin, and specific antibodies produced in both chicken and rabbit, we demonstrate that PlGF, derived from transfected COS-1 cells, is actually N-glycosylated and secreted into the medium. In addition, PlGF, like VPF, proves to be a dimeric protein. Finally, a conditioned medium from COS-1 cells containing PlGF is capable of stimulating specifically the growth of CPA, a line of endothelial cells, in vitro.
Abstract: By biochemical characterization of glucose-6-phosphate dehydrogenase (G6PD) from the red cells of seventeen subjects of the population of Matera (Southern Italy) we have identified six genetically determined common variants. Among these, G6PD Metaponto and G6PD A(-) Matera had been already fully characterized. We have now found that A(-) Matera is genetically heterogeneous since one of two subjects examined had the two mutations at codons 68 and 126 characteristic of a typical A(-) variant, while the other subject had only the codon 126 mutation. G6PD Pisticci and G6PD Tursi are two new variants whose molecular lesion is not yet known. G6PD Cagliari-like has biochemical characteristics reminiscent of G6PD Cagliari, isolated in Sardinia, and was found to have the same nucleotide substitution as G6PD Mediterranean. G6PD Montalbano is a new variant, with nearly normal properties, due to a G----A transition which causes an Arg----His amino acid replacement at position 285.
Abstract: We report the isolation and analysis of human genomic DNA clones spanning about 100 kb of the X chromosome and comprising the entire gene coding for the enzyme glucose 6-phosphate dehydrogenase (G6PD). The G6PD gene is 18 kb long and consists of 13 exons: the protein-coding region is divided into 12 segments ranging in size from 12 to 236 bp; an intron is present in the 5' untranslated region. Mature G6PD mRNA has a single polyadenylation site in HeLa cells. The major 5' end of mature G6PD mRNA in several cell lines is located 177 bp upstream of the translation initiating codon; longer mRNA molecules extending further in the 5' direction could be identified by S1 mapping and by comparing genomic and cDNA sequences. The DNA sequence around the major mRNA start is very GC rich; as to putative transcription regulatory sequences, a non-canonical TATA box and 9 CCGCCC elements are present, but no CAAT element could be identified. The genomic DNA we have isolated includes another ubiquitously transcribed region, provisionally named the GdX gene. Although the function of GdX is as yet unknown, we have established that this gene is located about 40 kb downstream of G6PD and is transcribed in the same direction. A comparative analysis of the promoter region of G6PD and 10 other housekeeping enzyme genes has confirmed the presence of a number of common features. In particular, in the eight cases in which a 'TATA' box is present, a conserved sequence of 25 bp is seen immediately downstream.
Abstract: Glucose-6-phosphate dehydrogenase (G6PD) is an ubiquitous enzyme which by determining the NADPH level has a crucial role in NADPH-mediated reductive processes in all cells (1). The structural gene for G6PD, Gd, is X-linked in mammals and on the basis of its expression in many tissues, it can be regarded as a typical "housekeeping" gene (2). Over 300 variants of the protein are known, many of which have deficient enzyme activity. Nearly 100 of these variants are polymorphic in various populations (3). The mammalian enzyme is a homodimer or a homotetramer with a subunit molecular weight of approximately 56000 daltons (4). Here we report the isolation of cDNA clones from HeLa cells, SV40-transformed human fibroblasts, human placenta and human teratocarcinoma cell lines. These clones have enabled us to sequence the entire coding region of Gd. Thus, the entire amino acid sequence of human G6PD is provided for the first time. This work is the first step for structural analysis of G6PD variants and for an understanding of the biological features of this enzyme at the molecular level.
