Abstract:

Abstract: Previous work had suggested that recombinant CCN3 was partially inhibiting cell proliferation. Here we show that native CCN3 protein secreted into the conditioned medium of glioma transfected cells indeed induces a reduction in cell proliferation. Large amounts of CCN3 are shown to accumulate both cytoplasmically and extracellularly as cells reach high density, therefore highlighting new aspects on how cell growth may be regulated by CCN proteins. Evidence is presented establishing that the amount of CCN3 secreted into cell culture medium is regulated by post-translational proteolysis. As a consequence, the production of CCN3 varies throughout the cell cycle and CCN3 accumulates at the G2/M transition of the cycle. We also show that CCN3-induced inhibition of cell growth can be partially reversed by specific antibodies raised against a C-terminal peptide of CCN3. The use of several clones expressing various portions of CCN3 established that the CT module of CCN3 is sufficient to induce cell growth inhibition. J. Cell. Biochem. 101: 1475-1491, 2007. (c) 2007 Wiley-Liss, Inc.

Abstract: We elucidate the role of CCN3/NOV, a member of the CCN family proteins, in osteoblast differentiation using MC3T3-E1 osteoblastic cells. Transduction with CCN3 adenovirus (AdCCN3) alone induced no apparent changes in the expression of osteoblast-related markers, whereas cotransduction with BMP-2 adenovirus (AdBMP-2) and AdCCN3 significantly inhibited the AdBMP-2-induced mRNA expression of Runx2, osterix, ALP, and osteocalcin. Immunoprecipitation-western analysis revealed that CCN3 associated with BMP-2. Compared to transduction with AdBMP-2 alone, cotransduction with AdBMP-2 and AdCCN3 attenuated the expression of phosphorylated Smad1/5/8 and the mRNA for Id1, Id2, and Id3. Transduction with AdCCN3 stimulated the expression of cleaved Notch1, the mRNA expression of Hes1 and Hey1/Hesr1, and the promoter activities of Hes1 and Hey1. The inhibitory effects of CCN3 on the expression of BMP-2-induced osteoblast-related markers were nullified in Hey1-deficient osteoblastic cells. These results indicate that CCN3 exerts inhibitory effects on BMP-2-induced osteoblast differentiation by its involvement of the BMP and Notch signaling pathways.

Abstract: PURPOSE: We encountered a case of lipofibromatosis in a 21-month-old male patient and examined its histopathologic properties. METHODS: We examined morphological aspects of the tumor and immunohistochemical patterns. RESULTS: Tumor proliferation was infiltrative, which did not show apparent encapsulation. Positive immunoreactivity was found for CD-34, CD-99, Ki-67, and connective tissue growth factor/CCN2 in the fibrous region, S-100 in the adipose region, and Notch1 stain was observed in the eccrine sweat gland cells juxtaposed to the tumor adipose tissue, but no reactivity for Bcl-2, alphaSMA, Notch 2-4, CCN1, and CCN3. CONCLUSIONS: The clinical process and immunohistochemical pattern of this case was consistent with the criteria of reported lipofibromatosis. Specific expression of CCN2 might be significant for the development of the tumor.

Abstract: CCN3 is a founding member of the CCN (Cyr61, Ctgf, Nov) family of cell growth and differentiation regulators. These secreted proteins are key regulators in embryonic development, and are associated with severe pathologies including fibrotic diseases and cancers. CCN3 was discovered as a MAV integration site in an avian nephroblastoma. Previous work established that the amino-truncated protein expressed in this tumor was inducing morphological transformation of chicken embryo fibroblasts, whereas the full-length secreted CCN3 protein was inhibiting cell growth. Amino-truncated variants were identified in cancer cell lines. Since the lack of signal peptide was expected to alter the fate of the truncated proteins, we hypothesized that modifications of CCN3 subcellular addressing could be responsible for the oncogenic activities of CCN3. The CCN proteins are composed of four structural modules (IGFBP, TSP1, VWC, and CT). We report that amino-truncated variants of CCN3 are addressed to the nucleus and that the carboxyterminal (CT) module of CCN3 is responsible for the nuclear addressing. Furthermore, our data identify nuclear CCN3 variants as potential transcriptional regulators. In this context, the CT module confers on nuclear CCN3 proteins a negative regulatory effect on transcription. We propose that the nuclear localization of amino-truncated CCN3 proteins be correlated to oncogenicity.

Abstract: The principal aim of this historical review- the first in a new series- is to present the basic concepts that led to the discovery of NOV and to show how our ideas evolved regarding the role and functions of this new class of proteins. It should prove particularly useful to the new comers and to students who are engaged in this exciting field. It is also a good opportunity to acknowledge the input of those who participated in the development of this scientific endeavour.

Abstract: AIMS: Myeloblastosis Associated Virus type 1 (N) [MAV 1(N)] induces specifically nephroblastomas in 8-10 weeks when injected to newborn chicken. The MAV-induced nephroblastomas constitute a unique animal model of the pediatric Wilms' tumor. We have made use of three independent nephroblastomas that represent increasing tumor grades, to identify the host DNA regions in which MAV proviral sequences were integrated. METHODS: Cellular sequences localized next to MAV-integration sites in the tumor DNAs were used to screen a Bacterial Artificial Chromosomes (BACs) library and isolate BACs containing about 150 kilobases of normal DNA corresponding to MAV integration regions (MIRs). These BACs were mapped on the chicken chromosomes by Fluorescent In Situ Hybridization (FISH) and used for molecular studies. RESULTS: The different MAV integration sites that were conserved after tumor cell selection identify genes involved in the control of cell signaling and proliferation. Syntenic fragments in human DNA contain genes whose products have been involved in normal and pathological kidney development, and several oncogenes responsible for tumorigenesis in human. CONCLUSION: The identification of putative target genes for MAV provides important clues for the understanding of the MAV pathogenic potential. These studies identified ADAMTS1 as a gene upregulated in MAV-induced nephroblastoma and established that ccn3/nov is not a preferential site of integration for MAV as previously thought. The present results support our hypothesis that the highly efficient and specific MAV-induced tumorigenesis results from the alteration of multiple target genes in differentiating blastemal cells, some of which are required for the progression to highly aggressive stages. This study reinforces our previous conclusions that the MAV-induced nephroblastoma constitutes an excellent model in which to characterize new potential oncogenes and tumor suppressors involved in the establishment and maintenance of tumors.

Abstract: Chronic myeloid leukemia (CML) is characterized by the presence of the constitutively active BCR-ABL protein tyrosine kinase. Using a multipotent hemopoietic cell line, FDCP-Mix, expressing BCR-ABL tyrosine kinase, we investigated the initial effects of this kinase in primitive hematopoietic stem cells. We identified down-regulation of a novel gene, CCN3, as a direct consequence of BCR-ABL kinase activity. CCN3 has been reported to function as a tumor suppressor gene in solid tumors. Northern and Western blotting plus immunocytochemical analysis confirmed CCN3 expression is decreased and is tyrosine-phosphorylated in BCR-ABL kinase active FDCP-Mix cells. Decreased cellular CCN3 correlated with increased CCN3 secretion in BCR-ABL kinase active cells. In vitro treatment of human CML cell lines with imatinib or siRNA directed against BCR-ABL significantly reduced BCR-ABL while increasing CCN3 expression. Cells from patients responding to imatinib showed a similar decrease in BCR-ABL and increase in CCN3. CML CD34+ cells treated with imatinib in vitro demonstrated increased CCN3 protein. Transfecting CCN3 into BCR-ABL+ cells inhibited proliferation and decreased clonogenic potential. CCN3 plays an important role in internal and external cell-signaling pathways. Thus, BCR-ABL can regulate protein levels by governing secretion, a novel mechanism for this tyrosine kinase.

Abstract: IGF binding proteins (IGFBPs) are a family of structurally homologous proteins that bind IGFs with high affinities and can modulate IGF activity. The IGF binding site has been shown to comprise residues in both the aminoterminal and carboxyterminal domains. In recent years several proteins including members of the CCN (connective tissue growth factor, Cyr61, and nephroblastoma overexpressed) family were recognized as having structural homology in their aminoterminal domains to the IGFBPs. Despite their low or undetectable IGF binding ability, a proposal was made to rename them as IGFBP-related proteins. To test whether the aminoterminal domain of a CCN protein can fulfill the high-affinity IGF binding function of an IGFBP, we created a chimera in which the aminoterminal domain of IGFBP-3 was substituted with the aminoterminal domain of CCN3 (previously known as Nov). The CCN3-IGFBP-3 chimera bound IGFs and inhibited IGF activity very weakly, similar to CCN3 itself. Although structurally similar, the aminoterminal domain of CCN3 is unable to replace the aminoterminal domain of IGFBP-3 in forming a high-affinity IGF-binding site. These results argue against a direct role of CCN3 in the regulation of IGF bioavailability and indicate that the nomenclature of IGFBP-related proteins (which implies functional relationship to the classical IGFBPs) is inappropriate for CCN proteins.

Abstract: A new family of cell growth and differentiation regulators has emerged in the past decade. These signaling proteins grouped under the CCN acronym, participate to fundamental biological functions, during normal development and in adulthood, from birth to death. Disregulation of their expression has been associated to tumorigenesis. Even though part of their physiological properties may be related to their capacity to bind several integrins, the CCN protein also interact with several other receptors and ligands that play critical roles in the regulation of cell signaling and communication. The multimodular structure of the CCN proteins provides the ground for the myriad of roles in which they participate, but it remains a challenge to those who wish to decipher the structure-function relationship that govern their multifunctional properties. The recent discovery of CCN variants whose expression is associated to the development of cancer raises fascinating questions regarding their role in the establishment and maintenance of the tumor state. Identifying the pathways in which the CCN proteins act and establishing the role of these proteins in intercellular communication will constitute new promising avenues among the trends for 21st century in cancer research.

Abstract: The identification of potential partners for CCN3(NOV) sheds new light on the biological activity of this signaling protein. In particular, the physical interaction of CCN3 with the IL33 cytokine combined with previous data indicating that CCN3 expression was regulated by TNFalpha and IL1 cytokines, point to CCN3 as a potent player in a variety of inflammatory responses, including neurodegenerative disease, and arthritis. Nuclear proteins that are involved in the regulation of RNA processing and chromatin remodeling were also found to interact with CCN3. These observations reinforce the concept that routing of CCN3 to the cell nucleus where it acts as a transcription regulator, might constitute a key element in the balance between the anti- and pro-proliferative activities of CCN3 proteins.

Abstract: Melanocytes reside within the basal layer of the human epidermis, where they attach to the basement membrane and replicate at a rate proportionate to that of keratinocytes, maintaining a lifelong stable ratio. In this study, we report that coculturing melanocytes with keratinocytes up-regulated CCN3, a matricellular protein that we subsequently found to be critical for the spatial localization of melanocytes to the basement membrane. CCN3 knockdown cells were dissociated either upward to the suprabasal layers of the epidermis or downward into the dermis. The overexpression of CCN3 increased adhesion to collagen type IV, the major component of the basement membrane. As the receptor responsible for CCN3-mediated melanocyte localization, we identified discoidin domain receptor 1 (DDR1), a receptor tyrosine kinase that acts as a collagen IV adhesion receptor. DDR1 knockdown decreased melanocyte adhesion to collagen IV and shifted melanocyte localization in a manner similar to CCN3 knockdown. These results demonstrate an intricate and necessary communication between keratinocytes and melanocytes in maintaining normal epidermal homeostasis.

Abstract: A cell line of murine osteogenic progenitor cells, Kusa, was established from femoral bone marrow stromal cells with other types of mesenchymal progenitor cells. We characterized two sublines of Kusa (Kusa-A1 and Kusa-O) from several aspects, including the use of an expression profiling system, a cDNA microarray. The original Kusa subline (Kusa-A1) had high alkaline phosphatase activity and high accumulation of calcium deposits in a condition inducing mineralization, with ascorbic acid and beta-glycerophosphate. Kusa-O, a low osteogenic subline of Kusa, had high alkaline phosphatase activity but slow accumulation of calcium deposits even in the inducing condition. These two Kusa sublines differed in the expression of the osteogenic marker genes, osteocalcin and osteopontin, during mineralization. A type of cDNA microarray revealed marked downregulation of gene expression in the inducing condition in both Kusa-A1 and Kusa-O. Another type of high-throughput microarray was performed to examine the difference in gene expression patterns between Kusa-A1 and Kusa-O. By this analysis, periostin, which would be involved in a stage of osteogenesis, was low in Kusa-A1. On the contrary, Myocyte enhancer factor 2C (MEF2C), a myogenic transcriptional factor, was high in Kusa-A1, although no expression of any other myogenic genes was shown.

Abstract: The CCN genes encode secreted proteins, associated to the extracellular matrix. They are involved in diverse biological processes such as regulation of cell- adhesion, migration, proliferation, differentiation and survival. They play important roles in pregnancy, development, angiogenesis, wound repair and inflammation. Several lines of evidence support a role for CCN genes in fibrotic disorders and tumorigenesis. We will focus our attention in this review on two CCN proteins: CCN1 and CCN3, that appear to exert distinct and opposite effects. Recent data suggest that CCN1 acts as a tumor-promoting factor and a key regulator in cancer progression, while CCN3 exhibits suppressive capabilities. The possible opposite functions of CCN1 and CCN3 in tumorigenesis, and the relevance of the distinct expression profiles of these two genes observed in many cancers are discussed below.

Abstract:

Abstract: Altered expression of CCN3 has been observed in a variety of musculoskeletal tumours, including Ewing's sarcoma (ES). Despite its widespread distribution, very little is known about its biological functions and molecular mechanisms of action. We transfected CCN3 gene into a CCN3-negative ES cell line and analysed the in vitro and in vivo behaviours of stably transfected clones. Forced expression of CCN3 significantly reduced cell proliferation in vitro, growth in anchorage-independent conditions, and tumorigenicity in nude mice. Despite the antiproliferative effect, CCN3-transfected ES cells displayed increased migration and invasion of Matrigel. The decreased expression of alpha2beta1 integrin receptor and the increased amount of cell surface-associated matrix metalloproteinase (MMP)-9 following the expression of CCN3 may be the basis for the increased migratory abilities of transfected cells. Cells lacking alpha2beta1 are less facilitated to have stable anchorage since the predominant collagen extracted from ES tissue is indeed type I collagen, and proMMP-9 was recently found to provide a cellular switch between stationary and migratory ES cell phase. Our findings are in line with those recently obtained in glioblastoma. However, the underlying molecular mechanisms appear to be different, further highlighting the importance of the cellular context in the regulation of function of CCN proteins.

Abstract: Long-term survivors of glioblastoma multiforme, the most common form of primary intracranial malignancy in adults, are extremely rare. Experimental animal models that more closely resemble human disease are essential for the identification of effective novel therapies. We report here an extensive analysis of the 4C8 glioma model to assess its suitability for evaluating novel type 1 herpes simplex virus (HSV-1) therapies of malignant glioma. We first determined that expression of major histocompatibility complex I and II and of alphavbeta3 in the 4C8 model was comparable to that seen in human glioma cells. Next, using a panel of Delta(gamma1)34.5 HSVs, we demonstrated that, in vitro, 4C8 cells were as sensitive as human glioma cells to both infection and lysis and that the 4C8 cells supported the production of foreign gene products. Replication competence of HSV was demonstrated in vitro. Finally, 4C8 intracranial gliomas were established in immunologically competent syngeneic B6D2F1 mice, treated by intratumoral injection of selected engineered HSVs, including the interleukin-12-expressing virus, M002. Survival data from these studies demonstrated that 4C8 cells in vivo are sensitive to both direct oncolysis and HSV-mediated interleukin-12 expression. Fluorescence-activated cell sorting analyses of immune-related infiltrating cells supported the concept that survival was prolonged in part because of antitumor actions of these cells. We conclude that the 4C8/B6D2F1 syngeneic glioma model is suitable for preclinical evaluation of HSV-based therapies and that M002 is a superior virus for the treatment of murine glioma in this model.

Abstract: BACKGROUND/AIMS: Connective tissue growth factor (CCN2) is expressed during activation of hepatic stellate cells (HSC) and promotes HSC proliferation, adhesion, and collagen production. The aim of the study was to investigate CCN2 signaling pathways in HSC. METHODS: Primary HSC were obtained by enzymatic perfusion of rat liver. DNA synthesis was evaluated by [(3)H]thymidine incorporation. Phosphorylation of Elk-1, extracellular signal-regulated kinase (ERK1/2) and focal adhesion kinase (FAK) was evaluated by Western blot. Transcriptional factor binding activity was determined by gel mobility shift assay while c-fos promoter and CCN2 promoter activity was evaluated using luciferase reporters. c-fos mRNA expression was evaluated by Northern blot. RESULTS: CCN2 stimulated DNA synthesis and phosphorylation of FAK, Elk-1 and ERK1/2, the latter of which was blocked by heparin. The serum response element binding activity and luciferase reporter activity of the c-fos promoter, together with expression of c-fos, were enhanced by CCN2. CCN2-induced c-fos gene activation, expression and cell proliferation were blocked by inhibiting ERK1/2 with PD98059. CCN2 promoter activity was enhanced by TGF-beta1 or PDGF via a Smad7-dependent pathway. CONCLUSIONS: CCN2-stimulated HSC DNA synthesis is associated with transient induction of c-fos gene activation and expression as well as activation of the ERK1/2 signal pathway.

Abstract: CONTEXT: Although little is known as yet about the processes that coordinate cell-signalling pathways, matrix proteins are probably major players in this type of global control. The CCN (cyr61, ctgf, nov) proteins are an important family of matricellular regulatory factors involved in internal and external cell signalling. This family participates in angiogenesis, chondrogenesis, and osteogenesis, and they are probably involved in the control of cell proliferation and differentiation. STARTING POINT: Runping Gao and David Brigstock (Hepatol Res 2003; 27: 214-20) recently showed that CCN2 (CTGF, connective tissue growth factor) is a cell-adhesion factor for hepatic stellate cells. On exposure to transforming growth factor beta, hepatic stellate cells produce distinct CCN2 isoforms. Gao and Brigstock assign to CCN2 module 3 the capacity to mediate binding to low-density-lipoprotein receptor-related protein (LRP), which was previously reported to interact with CCN2 and to be involved in various types of signalling. They also establish that CCN2 binding to LRP is heparin dependent and that module 4 of CCN2 promotes LRP-independent adhesion of hepatic stellate cells. The differential binding of CCN2 isoforms to LRP highlights the importance of functional interactions between individual modules, and reinforces the concept that different module combinations might confer agonistic or antagonistic activities. WHERE NEXT? It is essential to understand how the distinct configuration of the various CCN isoform affects their biological activities and bioavailability, and to explore the mechanisms and the regulatory processes involved in the production of truncated CCN isoforms. A better understanding of the structural basis for their multifunctionality is a prerequisite to wider use of CCN proteins in molecular medicine.

Abstract: The gap junction protein connexin43 (Cx43) is thought to be involved in growth control in several tissues. Using the doxycycline inducible tet-on system, we generated human malignant trophoblast Jeg3 cells transfected with either Cx40, Cx43, or C-terminal truncated Cx43 (trCx43). Cx43, but not Cx40 or trCx43, displayed a reduced cell growth of Jeg3 cells in vitro and tumor growth in nude mice, suggesting a role of the C terminus of Cx43 in growth regulation. Using gene array analysis, the growth regulator NOV (CCN3), a member of the CCN gene family, was found to be up-regulated only in the Cx43-transfected cells. Validation by reverse transcriptase-PCR confirmed an up-regulation of the NOV transcript exclusively upon Cx43 induction. In contrast to Cx40 or trCx43, induction of Cx43 led to a switch in localization of NOV from the nucleus to the cell membrane, where it is colocalized with Cx43. Coimmunoprecipitation showed a binding of NOV to the C terminus of Cx43 in vitro as well as in transfected cells. Jeg3 cells transfected only with NOV revealed that NOV itself acts as a growth regulator. We suggest that Cx43 is able to regulate cell growth via an up-regulation of NOV transcription, a change in localization of the NOV protein and a binding of NOV to the C terminus of Cx43.

Abstract: Many tumor cells exhibit aberrant gap junctional intercellular communication, which can be restored by transfection with connexin genes. We have previously discovered that overexpression of connexin43 (Cx43) in C6 glioma cells not only reduces proliferation but also leads to production of soluble growth-inhibitory factors. We identified that several members of the CCN (Cyr61/connective tissue growth factor/nephroblastoma-overexpressed) family are up-regulated following Cx43 expression, including CCN3 (NOV). We now report evidence for an association between CCN3 and Cx43. Western blot analysis demonstrated that the 48-kDa full-length CCN3 protein was present in the lysate and conditioned medium of growth-suppressed C6-Cx43 cells, as well as primary astrocytes, but not in C6 parental and human glioma cells. Immunocytochemical examination of CCN3 revealed diffuse localization in parental C6 cells, whereas transfection of C6 cells with Cx43 (C6-Cx43) or with a modified Cx43 tagged to green fluorescent protein on its C terminus (Cx43-GFP) resulted in punctate staining, suggesting that CCN3 co-localizes with Cx43 in plaques at the plasma membrane. In cells expressing a C-terminal truncation of Cx43 (Cx43Delta244-382), this co-localization was lost. Glutathione S-transferase pull-down assay and co-immunoprecipitation demonstrated that CCN3 was able to physically interact with Cx43. In contrast, CCN3 was not found to associate with Cx43Delta244-382. Similar experiments revealed that CCN3 did not co-localize or associate with other connexins, including Cx40 or Cx32. Taken together, these data support an interaction of CCN3 with the C terminus of Cx43, which could play an important role in mediating growth control induced by specific gap junction proteins.

Abstract: AIM: To study the cellular distribution of CCN3(NOV) and to determine if the carboxyterminus of CCN3 is hidden or masked due to high affinity interactions with other partners. CCN3 was detected using affinity purified antibodies (anti-K19M-AF) as well as a Protein A purified anti-K19M antibodies (anti-K19M IgG) against a C-terminal 19-aminoacid peptide (K19M) of human CCN3 protein. The antibodies were applied in indirect immunofluorescence tests and immunoenzyme assays on glial tumor cell line, G59, and its CCN3-transfected variant G59/540 and the adrenocortical cell line, NCI-H295R. RESULTS: Anti-K19M-AF antibodies reacted against K19M peptide in ELISA and recognized two bands of 51 kDa and 30 kDa in H295R (adrenocortical carcinoma) cell culture supernatants by immunoblotting. H295R culture supernatants which contained CCN3 as shown by immunoblotting did not react with anti-CCN3 antibodies in liquid phase. Anti-CCN3 antibodies stained the surface membranes of non-permeabilized H295R and cytoplasm in permeabilized H295R cells. Similarly, anti-CCN3 stained surface membranes of G59/540, but did not react with G59 cells. Prominent cytoplasmic staining was observed in G59/540, as well as the cell footprints of G59/540 and H295R were strongly labeled. CONCLUSIONS: The K19M-AF antibody directed against the C-terminal 19-aminoacid peptide of CCN3 recognized the secreted protein under denaturing conditions. However, the C-terminal motif of secreted CCN3 was not accessible to K19M-AF in liquid phase. These anti-CCN3 antibodies stained CCN3 protein which was localized to cytoplasmic stores, cell membranes and extracellular matrix. This would suggest that cytoplasmic and cell membrane bound CCN3 has an exposed C-terminus while secreted CCN3 has a sequestered C-terminus which could be due to interaction with other proteins or itself (dimerization). Thus the K19M-AF antibodies revealed at least two conformational states of the native CCN3 protein.

Abstract: In this article we briefly review the reasons and advantages that underly our publisher's decision to introduce article-processing charges (APC) for manuscripts submitted to Cell Communication and Signaling. The charge is an attempt to develop a new business model for distributing biomedical information and has been accepted in a number of other journals. APCs will enable BioMed Central to continue to provide their excellent service and will help to establish our journal.

Abstract: The molecularly cloned myeloblastosis-associated virus type-1(N) (MAV-1(N)) strain induces specifically nephroblastomas in chicken. MAV-induced nephroblastoma constitutes a unique animal model of the human Wilms' tumor. We have previously shown that the MAV-1(N) long terminal repeats (LTR) were necessary and sufficient for nephroblastoma induction. Since major determinants for oncogenesis have been mapped in the U3 region of several other retroviruses, we have analyzed the tumorigenic potential of five recombinant viruses partially deleted in their U3 region. The results obtained indicated that deletions of the LTRs resulted in a modification of the pathogenic spectrum of MAV-1(N) and a decreased efficiency for nephroblastoma induction.

Abstract: The CCN (CYR61 [Cystein-rich61]/CTGF [connective tissue growth factor]/NOV [Nephroblastoma overexpressed]) proteins constitute a family of regulatory factors involved in many aspects of cell proliferation and differentiation. An increasing body of evidence indicates that abnormal expression of the CCN proteins is associated to tumourgenesis. The multimodular architecture of the CCN proteins, and the production of truncated isoforms in tumours, raise interesting questions regarding the participation of each individual module to the various biological properties of these proteins. In this article, we review the current data regarding the involvement of CCN proteins in tumourigenesis. We also attempt to provide structural basis for the stimulatory and inhibitory functions of the full length and truncated CCN proteins that are expressed in various tumour tissues.

Abstract: A proposal is put forth to unify the nomenclature of the CCN family of secreted, cysteine rich regulatory proteins. In the order of their description in the literature, CCN1 (CYR61), CCN2 (CTGF), CCN3 (NOV), CCN4 (WISP-1), CCN5 (WISP-2), and CCN6 (WISP-3) constitute a family of matricellular proteins that regulate cell adhesion, migration, proliferation, survival, and differentiation, at least in part through integrin mediated mechanisms. This proposal is endorsed by the International CCN Society and will serve to eliminate confusion from the multiple names that have been given to these molecules.

Abstract: For the second time, researchers from leading laboratories in the CCN field gathered in Saint-Malo, France, to participate in the Second International Workshop on the CCN family of genes. In addition to the regular research communications, meeting highlights included the inauguration of the first CCN newsletter (http://ccnnewsletter.free.fr) and the recognition of the International CCN Society (http://www.ccnsociety.jussieu.fr) as an important medium for the exchange of scientific knowledge and resources in the CCN field. Once more, the high quality of scientific communications and individual interactions set the stage for an extremely fruitful meeting.

Abstract: The CCN genes encode secreted signaling proteins that participate in fundamental processes including cell adhesion, proliferation and differentiation, embryogenesis, tissue remodeling and patterning. Abnormal expression of CCN proteins is associated with several pathological conditions, including vascular diseases, fibrosis and cancer. Understanding the structural and functional basis for the variety of biological properties attributed to CCN proteins is an important challenge. It will help the understanding of their roles in the control of cellular proliferation, differentiation and death, thereby allowing their use for early diagnosis and therapy. In an attempt to evaluate the relevance of CCN3 as a useful tool in modern biomedical technologies, the biological properties of the CCN proteins and the data that established their potential usefulness will briefly be reviewed.

Abstract: The CCN family of genes consists presently of six members in human (CCN1-6) also known as Cyr61 (Cystein rich 61), CTGF (Connective Tissue Growth Factor), NOV (Nephroblastoma Overexpressed gene), WISP-1, 2 and 3 (Wnt-1 Induced Secreted Proteins). Results obtained over the past decade have indicated that CCN proteins are matricellular proteins, which are involved in the regulation of various cellular functions, such as proliferation, differentiation, survival, adhesion and migration. The CCN proteins have recently emerged as regulatory factors involved in both internal and external cell signaling. CCN3 was reported to physically interact with fibulin-1C, integrins, Notch and S100A4. Considering that, the conformation and biological activity of these proteins are dependent upon calcium binding, we hypothesized that CCN3 might be involved in signaling pathways mediated by calcium ions.In this article, we review the data showing that CCN3 regulates the levels of intracellular calcium and discuss potential models that may account for the biological effects of CCN3.

Abstract: Growth plate chondrocytes undergo a coordinated differentiation process resulting in terminal differentiation and new bone formation. Enchondromas are pre-malignant, benign cartilaginous lesions that arise from growth plate chondrocytes that fail to undergo terminal differentiation. NOV (nephroblastoma overexpressed) is a member of the CCN family of proteins, which share a common multi-modular organization. While the role of NOV in chondrocyte development and cartilage neoplasia is not known, other CCN family members play a role in chondrocyte differentiation, or are differentially regulated in cartilage neoplasia. In embryonic murine growth plates, NOV was expressed in pre-hypertrophic and early hypertrophic chondrocytes. PTHrP treatment (which inhibits terminal differentiation) decreased NOV expression in murine femurs maintained in organ culture, and decreased the activity of a NOV reporter construct in vitro. Expression of the CCN family members NOV, CTGF, CYR61, and WISP-1 was examined in 15 chondrosarcomas of various grades and in three enchondromas. Expression of all of the family members was lower in the higher-grade tumours. As identification of the grade of cartilage neoplasia can sometimes be difficult using histology alone, the level of expression of CCN family members could be a useful adjunct in the determination of tumour grade.

Abstract: All forms of communication between human beings have long been recognized as a requirement for reciprocal understanding, transfer of knowledge, and productive development of societies. This also applies to living cells who are organized in <<microsocietiest; that constantly adjust to their environment through a complex network of signaling pathways. The chemical communication which occurs at various levels results in an integrated exchange of information that is essential for coordinated responses.We wish to present a few features of Cell Communication and Signaling: an open access, peer-reviewed journal devoted to the publication of manuscripts covering all aspects of cell communication, with a particular focus on molecular processes that govern intercellular signaling and events that sustain cellular communication, both in normal and pathological conditions.The launching of Cell Communication and Signaling provides us the opportunity to present a brief overview of basic processes underlying cell communication and the signaling processes that take place within and between cells to permit an efficient communication.

Abstract: We demonstrate a novel interaction of the nephroblastoma overexpressed gene (NOV), a member of the CCN gene family, with the Notch signaling pathway. NOV associates with the epidermal growth factor-like repeats of Notch1 by the CT (C-terminal cysteine knot) domain. The promoters of HES1 and HES5, which are the downstream transducers of Notch signaling, were activated by NOV. Expressions of NOV and Notch1 were concomitant in the presomitic mesoderm and later in the myocytes and chondrocytes, suggesting their synergistic effects in mesenchymal cell differentiation. In C2/4 myogenic cells, elevated expression of NOV led to down-regulation of MyoD and myogenin, resulting in inhibition of myotube formation. These results indicate that NOV-Notch1 association exerts a positive effect on Notch signaling and consequently suppresses myogenesis.

Abstract: The CCN3(NOV) protein belongs to the CCN [cysteine-rich CYR61, connective tissue growth factor (CTGF), nephroblastoma overexpressed gene (Nov)] family of growth regulators, sharing a strikingly conserved multimodular organization but exhibiting distinctive functional features. Although previous studies have revealed an expression of CCN3 protein in several normal tissues, including kidney, nervous system, lung, muscle, and cartilage, less is known about its expression in tumors. In this study, we analyzed the expression of CCN3 in musculoskeletal tumors, using a panel of human cell lines and tissue samples. An association between CCN3 expression and tumor differentiation was observed in rhabdomyosarcoma and cartilage tumors, whereas, in Ewing's sarcoma, the expression of this protein seemed to be associated with a higher risk to develop metastases. CCN3 expression was found in 15 of 45 Ewing's sarcoma tissue samples. In particular, we did not observe any expression of CCN3 in the 15 primary tumors that did not develop metastases. In contrast, 15 of the 30 primary tumors that developed lung and/or bone metachronous metastases showed a high expression of the protein (P < 0.001, Fisher's test). Our studies indicate that CCN3 is generally expressed in the cells of the musculoskeletal system. This protein may play a role both in normal and pathological conditions. However, the regulation of CCN3 expression varies in the different neoplasms and depends on the type of cells. Thus, as reported for other CCN genes, the biological properties and regulation of expression of CCN3 are dependent on the cellular context and the nature of the cells in which it is produced. Further studies will help to clarify the biological role of this protein in musculoskeletal neoplasms.

Abstract: AIMS: Lymphoblastoid cell lines derived from Marek's disease virus (MDV) induced tumours have served as models of MDV latency and transformation. They are stable and can be cultured with no detectable MDV genomic alterations upon repeated passaging. An MDV transformed lymphoblastoid T cell line (T9 cell line) has been reported to contain a disrupted MDV BamHI-H fragment and a Rous associated virus insertional activation of the c-myb protooncogene. In an attempt to define the respective participation of c-myb and MDV in the transformed phenotype of T9 cells, an analysis of MDV oncogenic sequences (BamHI-H, BamHI-A, and EcoQ fragments) was performed in these cells. METHODS: Using two different passages of the T9 cell line (late and early passages), the organisation of the MDV oncogenic regions and their expression in these cells were analysed. In vivo assessment of the oncogenicity of the virus contained within these cells was assessed by injecting them into 1 day old chickens. RESULTS: In T9 cells maintained in culture for up to six months (late T9), the MDV ICP4 gene was disrupted, whereas the meq gene was actively transcribed. The alterations of the MDV genome in these cells correlated with the inability of the virus to induce the classic signs of Marek's disease in 1 day old chickens. However, early T9 cells submitted to a limited number of passages induced classic MDV pathogenicity, as efficiently as the MDV control cell line (T5), and did not show gross structural changes in the oncogenic MDV sequences. CONCLUSIONS: Although the expression pattern of the MDV oncogenes in early T9 cells was identical to the one reported for other MDV transformed cells, longterm culture of an MDV transformed cell line containing a RAV insertional activation of the c-myb protooncogene led to the disruption of the MDV BamHI-H and BamHI-A oncogenic regions. In the late T9 cells MEQ was the only detected MDV oncoprotein. These results suggest that in the late T9 cells the truncated MYB protein compensates for the loss of MDV oncoproteins and reinforce the possibility that MEQ and MYB cooperate in the maintenance of the transformed state and the tumorigenic potential of these cells.

Abstract: AIMS: In animals and humans increased expression of CCN3 (NOV) is detected in tissues where calcium is a key regulator, such as the adrenal gland, central nervous system, bone and cartilage, heart muscle, and kidney. Because the multimodular structure of the CCN proteins strongly suggests that these cell growth regulators are metalloproteins, this study investigated the possible role of CCN3 in ion flux and transport during development, control of cell proliferation, differentiation, and pathobiology. METHODS: The isolation of CCN3 partners was performed by means of the two hybrid system. Yeasts were cotransfected with an HL60 cDNA library fused to the transactivation domain of the GAL4 transcription factor, and with a plasmid expressing CCN3 fused to the DNA binding domain of GAL4. Screening of the recombinant clones selected on the basis of leucine, histidine, and tryptophan prototrophy was performed with a beta-galactosidase assay. After the interaction between CCN3 and its putative partners was checked with a GST (glutathione S-transferase) pull down assay, the positive clones were identified by cloning. To establish whether the CCN3 protein affected calcium ion flux, a dynamic imaging microscopy system was used, which allowed the fluorometric measurement of the intracellular calcium concentration. The proteins used in the assays were GST fused with either CCN3 or CCN2 (CTGF) and GST alone as a control. RESULTS: The two hybrid system identified the S100A4 (mts1) calcium binding protein as a partner of CCN3 and the use of the GST fusion proteins showed that the addition of CCN3 and CCN2 to G59 glioblastoma and SK-N-SH neuroblastoma cells caused a pronounced but transient increase of intracellular calcium, originating from both the entry of extracellular calcium and the mobilisation of intracellular stores. CONCLUSIONS: The interaction of CCN3 with S100A4 may account, in part, for the association of CCN3 with carcinogenesis and its pattern of expression in normal conditions. The increased intracellular calcium concentrations induced by CCN3 and CCN2 both involve different processes, among which voltage independent calcium channels might be of considerable importance in regulating the calcium flux associated with cell growth control, motility, and spreading. These observations assign for the first time a biological function to the CCN3 protein and point out a broader role for the CCN proteins in calcium ion signalling.

Abstract: AIMS: To establish whether the MYB protein expressed in HL-60 variant cells, which are cells resistant to 12-O-tetradecanoylphorbol-13-acetate (TPA) induced differentiation, is able to bind MYB recognition elements (MREs) involved in the transcriptional regulation of myb target genes. In addition, to determine whether alterations in the binding of the MYB protein to MREs affects HL-60 cell proliferation and differentiation. METHODS: Nuclear extracts of HL-60 variant cells exhibiting different degrees of resistance to TPA induced monocytic differentiation were used in electrophoretic mobility shift experiments (EMSAs), bandshift experiments performed with labelled oliogonucleotides containing the MYB consensus binding sequences. RESULTS: The MYB protein contained in nuclear extracts from HL-60 variant cells did not bind efficiently to the MYB recognition elements identified in the mim-1 and PR264 promoters. Molecular cloning of the myb gene and analysis of the MYB protein expressed in the HL-60 variant cells established that the lack of binding did not result from a structural alteration of MYB in these cells. The lack of MRE binding did not abrogate the ability of variant HL-60s to proliferate and to undergo differentiation. Furthermore, the expression of the PR264/SC35 splicing factor was not affected as a result of the altered MYB DNA binding activity. CONCLUSIONS: Because the MYB protein expressed in HL-60 variant cells did not appear to be structurally different from the MYB protein expressed in parental HL-60 cells, it is possible that the HL-60 variant cells contain a MYB binding inhibitory factor (MBIF) that interferes with MYB binding on MREs. The increased proliferation rate of HL-60 variant cells and their reduced serum requirement argues against the need for direct MYB binding in the regulation of cell growth.

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Abstract: AIMS: To establish the expression pattern of ccn3 (nov) in the central nervous system (CNS) of adult rats and to determine whether spatiotemporal variations in the expression of ccn3 (nov) are related to specific developmental stages and/or specific CNS functions. METHODS:The sites of ccn3 (nov) expression have been identified by in situ hybridisation using didoxigenin labelled cRNA and by the reverse transcription-polymerase chain reaction (RT-PCR). The rat CCN3 (NOV) protein was characterised by western blotting performed on brain extracts. The localisation of the CCN3 (NOV) protein in the brain was established by immunocytochemistry. RESULTS: Increased expression of ccn3 (nov) was detected in the developing brain of rats after birth, as shown by RT-PCR and immunocytochemistry analysis performed on a series of samples taken between day 5 (P5) and day 300 (P300), with a pronounced peak between P15 and P150, suggesting that CCN3 (NOV) might play a role in the maintenance or establishment of specific brain functions. The relatively high amounts of an N-terminal truncated CCN3 (NOV) related protein detected both in the brain tissues and cerebrospinal fluid suggested that post translational processing of CCN3 (NOV) might be particularly prevalent in the brain. Such processing might be of biological importance in the light of the previously reported growth stimulatory effects of N-terminal truncated CCN3 (NOV) isoforms. CONCLUSIONS: The postnatal differential expression of ccn3 (nov) in the brain of developing rats suggests that CCN3 (NOV) might be involved in the acquisition of specific functions. The rat species provides an as yet unequalled system for these studies. Because the CCN3 (NOV) protein is detected in restricted areas of the brain, it will be interesting to establish whether variations of ccn3 (nov) expression are associated with normal cognitive processes and whether ccn3 (nov) expression is affected by aging. In addition, because CCN3 (NOV) is found in the spinal cord and along the axonal processes, it will be of interest to determine the expression of the normal and truncated isoforms of CCN3 (NOV) in various pathological conditions, such as neurodegenerative diseases.

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Abstract: The CCN family of genes presently consists of six distinct members encoding proteins that participate in fundamental biological processes such as cell proliferation, attachment, migration, differentiation, wound healing, angiogenesis, and several pathologies including fibrosis and tumorigenesis. Whereas CYR61 and CTGF were reported to act as positive regulators of cell growth, NOV (nephroblastoma overexpressed) provided the first example of a CCN protein with negative regulatory properties and the first example of aberrant expression being associated with tumour development. The subsequent discovery of the ELM1, rCOP1, and WISP proteins has broadened the variety of functions attributed to the CCN proteins and has extended previous observations to other biological systems. This review discusses fundamental questions regarding the regulation of CCN gene expression in normal and pathological conditions, and the structural basis for their specific biological activity. After discussing the role of nov and other CCN proteins in the development of a variety of different tissues such as kidney, nervous system, muscle, cartilage, and bone, the altered expression of the CCN proteins in various pathologies is discussed, with an emphasis on the altered expression of nov in many different tumour types such as Wilms's tumour, renal cell carcinomas, prostate carcinomas, osteosarcomas, chondrosarcomas, adrenocortical carcinomas, and neuroblastomas. The possible use of nov as a tool for molecular medicine is also discussed. The variety of biological functions attributed to the CCN proteins has led to the proposal of a model in which physical interactions between the amino and carboxy portions of the CCN proteins modulate their biological activity and ensure a proper balance of positive and negative signals through interactions with other partners. In this model, disruption of the secondary structure of the CCN proteins induced by deletions of either terminus is expected to confer on the truncated polypeptide constitutive positive or negative activities.

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Abstract: NOV, located on human chromosome 8q24.1, was originally cloned following discovery of its avian homolog as a consequence of over-expression in virally induced nephroblastoma. The gene product is a secreted, modular, protein and a member of the CCN gene family. Evidence to date indicates that the expression of the wild type protein is associated with cellular quiescence in normal embryonic fibroblasts yet produces growth stimulatory effects on established murine NIH 3T3 cells. Here we report the expression of NOV in the first trimester of human embryogenesis, between 5 and 10 weeks. In situ hybridisation and immunohistochemistry reveal widespread expression in derivatives of all three germ layers. The most abundant sites of expression are in the motor neurons and floor plate of the spinal cord, adrenal cortex, fusing skeletal, and smooth muscle, the urogenital system and the developing heart. Additionally, expression is seen in the cranial ganglia, differentiating chondrocytes, gonads, and lung. The sites of expression suggest strongly that autocrine or paracrine expression of NOV is associated with the process of cell differentiation.

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Abstract: AIMS: To investigate the expression of the human ccn3 (hccn3; nov) proto-oncogene, a member of the CCN family of proteins, in prostate epithelial cells and prostate tissue samples. METHODS: Normal adult prostate luminal epithelial cells immortalised by SV40 large T (PNT1A and PNT1B), metastatic tumours (LNCaP, DU-145, and PC-3), and prostate tissue samples from patients with benign prostatic hyperplasia (BPH) and prostatic adenocarcinoma were used. hccn3 (nov) mRNA was measured by the reverse transcription polymerase chain reaction (RT-PCR) and hCCN3 (NOV) protein expression was determined by immunochemistry. RESULTS: hccn3 (nov) RNA values were higher in PC-3 cells than in the other prostate cell lines. The immortalised normal cell lines either did not express hccn3 (nov) RNA (PNT1B) or expressed very low amounts (PNT1A). BPH samples expressed variable amounts of hccn3 (nov) RNA. With the use of immunocytochemistry, all cell lines except PNT1A and PNT1B were shown to contain hCCN3 (NOV) protein. hCCN3 (NOV) was localised mainly in the epithelial compartment of BPH and adenocarcinoma samples, and there was evidence of luminal secretion. CONCLUSION: The overexpression of hccn3 (nov) RNA in cancer cell lines compared with other cell lines and its epithelial localisation in human prostate samples are consistent with a role for hCCN3 (NOV) in prostatic tumorigenesis.

Abstract: In this study we present biochemical and immunocytochemical results on NOVH protein secretion and localization in NCI-H295R cells, as well as results on the ultrastructural characteristics of NCI-H295R cells. NCI-H295R cells were characterized by small quantities of rough and smooth endoplasmic reticulum, many free ribosomes, large nuclei with prominent nucleoli, numerous elongated mitochondria, a few Golgi complexes, and a small number of lipid droplets. Large numbers of coated pits and coated vesicles were present, but no secretory granules or exocytotic profiles were seen. Best ultrastructural preservation of NCI-H295R cells was achieved when fixation was done directly on the culture dishes and the cells were detached by scraping. Our biochemical results showed that NCI-H295R cells secreted large amounts of NOVH protein. The immunocytochemical localization of NOVH protein showed that the protein was localized in the cytoplasm, the plasma membrane and the nuclear envelope. This localization pattern, along with the ultrastructural and biochemical findings raise interesting questions on the function(s) and the mode of secretion of NOVH protein.

Abstract: A series of 13 sporadic renal cell carcinomas was analyzed for the specific chromosome rearrangements after serial xenografting into immunodeficient mice. Seven tumors displayed genetic traits of the conventional subtype and 5 showed genetic features of the papillary subtype. In all the xenografted conventional tumors, we observed loss of 3p, as well as loss of the 9p21 region and of the long arm of chromosome 14, both considered as markers of a poor prognosis. In the xenografted papillary tumors, a duplication of chromosome arm 8q was observed concomitant with the duplication of the 7q31 region. The association of the 7q31 and 8q22 approximately qter duplicated regions was also observed for one conventional tumor. The latency of tumor take was found to be reduced and the median time to passage statistically shorter for all tumors which presented the associated duplication of the 7q31 and 8q22 approximately qter regions. The proto-oncogene NOV (nephroblastoma overexpressed gene) maps to 8q24.1 and is overexpressed in some Wilms tumors. It could be an interesting candidate gene, since its level of expression and release in the culture medium was found to be increased in all of the fast growing tumors analyzed.

Abstract: AIMS: To establish whether the ectopic expression of CCN3 (NOV) in glioma cells can interfere with their tumorigenic potential and assess its potential value in molecular medicine. METHODS: Glioma cell lines were used to assess whether differences in the degree of intracellular communication induced by the expression of the gap junction protein connexin 43 (Cx43) is related to the differential expression of CCN3 (NOV). The antiproliferative activity of rat CCN3 (rCCN3; NOV) in glioma cells, has been assessed both in vitro and in vivo with glioma cell lines expressing different amounts of CCN3 (NOV). RESULTS: Upon ectopic expression of Cx43, the growth of C6 glioma cells is decreased. An increase of CCN3 (NOV) expression matches the reduced tumorigenic potential of these transfected cells. The localisation of CCN3 (NOV) is affected by the increased expression of Cx43 in the Cx-13 transfected cells, in which it is detected at areas of cell-cell contact. In a xenograft model, CCN3 (NOV) transfected glioma cells were found to induce tumours to a lesser degree than their parental counterparts, which do not express detectable amounts of CCN3 (NOV). CONCLUSIONS: Previous observations had suggested an inverse relation between CCN3 (NOV) expression in glioma cells and their tumorigenicity. These results establish a direct association between the establishment of functional gap junctional intercellular communication and the expression of rCCN3 (NOV). In addition to a negative effect on murine and human cell growth, CCN3 (NOV) has antiproliferative activity on tumour cells in vivo. Thus, the antiproliferative activity of the CCN3 (NOV) protein might involve reorganisation of cellular contacts that play a crucial role in tumorigenesis. The antiproliferative activity of CCN3 (NOV) established in this work sets the stage for the potential use of CCN proteins in molecular oncology.

Abstract: The nephroblastoma overexpressed gene, NOV, is a member of the CCN gene family. We investigated the NOV gene expression pattern in the chicken during early stage embryogenesis. Several embryonic structures showed a distinct expression pattern. The initial expression was detected in Hensen's node (Hamburger and Hamilton stage (HH) 5). The expression was noted in the presumptive notochord and floor plate forming cells. The expression on the left side was more elongated posteriorly, a type of left-right asymmetry. Chicken NOV gene expression in the forming notochord and floor plate was observed until HH 18. The expression was also detected in the ventral area of the mesencephalon and isthmus at HH 14-16.

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Abstract: During the establishment of learning and memory of adult rats with active avoidance reaction expression of nov gene, nov mRNA positive neurons and NOV protein immunoreactive neurons were found in hippocampus, cingulate cortex, globus pallidus, caudate putamen and hypothalamus. The strongest positive reaction of NOV protein was observed in high ability group of learning and memory (HALM). Basic expression was found in pseudoconditioning (PC) group. The expression of NOV protein was higher in low ability group of learning and memory (LALM) than in PC group. No significant difference was detected in nov mRNA positive reaction between the three groups. The results indicate that nov gene may play an important role in learning and memory of adult rats. This regulation occurs at the level of NOV protein translation.

Abstract: The NOVH protein belongs to the emerging CCN [Connective tissue growth factor (CTGF), Cyr61/Cef10, nephroblastoma overexpressed gene] family of growth regulators sharing a strikingly conserved multimodular organization but exhibiting distinctive functional features. Two members of the family (CYR61 and CTGF) are positive regulators of cell proliferation, whereas NOVH and two other members (ELM1 and RCOP-1) exhibit features of negative regulators of growth. The multimodular structure of these proteins suggests that their biological role(s) may depend on interactions with several factors as well as proteins constitutive of the extracellular matrix. To gain insight into the functionality of these domains, we have used a two-hybrid system to identify proteins interacting with NOVH. We report here that the C-terminal domain confers on the full-length NOVH protein the capacity to bind fibulin 1C, a protein of the extracellular matrix that interacts with several other regulators of cell adhesion. Furthermore, we show that a natural N-truncated isoform of NOVH produced by cells expressing the full-length NOVH protein also binds fibulin 1C with a high affinity, and we hypothesize that the production of truncated isoforms of NOVH (and probably of other CCN proteins) may be a critical aspect in the modulation of their biological activity. These results set the stage for a study of NOVH-fibulin 1C interactions and their potential significance in cell-adhesion signaling in normal and pathological conditions.

Abstract: AIMS: To identify the NOV protein detected by immunofluorescence in the nucleus of human cancer cell lines to establish whether targeting to the nucleus reflects dual paracrine and intracrine biological functions of NOV, as has been reported previously for several signalling peptides and proteins. METHODS: Nuclear and cytoplasmic fractions were prepared from 143 and HeLa cells in which nuclear NOV protein was detected. Western blotting analysis of NOV proteins in both types of fractions was performed using two NOV specific antibodies. Confocal microscopy was used to visualise the nuclear NOV protein in HeLa and 143 cells. A yeast two hybrid screening system was used to isolate cDNAs encoding proteins able to interact with the human NOV protein. RESULTS: A 31/32 kDa doublet of NOV protein was identified in the nuclear fraction of 143 and HeLa cells. Because the antibodies were directed against the C-terminus of NOV, the 31/32 kDa NOV isoform is probably truncated at the N-terminus and might correspond to the secreted 32 kDa NOV isoform detected in cell culture medium. Confocal microscopy indicated that in addition to the cytoplasmic NOV protein already identified, a nuclear NOV protein was present in both the nucleoplasm and nucleoli of Hela and 143 cells. Screening of cDNA libraries prepared from HeLa cells, Epstein-Barr virus transformed lymphocytes, and normal human brain showed that the NOV protein interacts with the rpb7 subunit of RNA polymerase in a yeast two hybrid system. CONCLUSIONS: The NOV protein detected in the nucleus of 143 and HeLa cells is probably an N-terminus truncated isoform of the secreted 48 kDa NOV protein. A growing body of evidence suggests that novH expression is closely associated with differentiation in normal human tissues and that the nov gene encodes a signalling protein that belongs to an emerging family of cell growth regulators. The nuclear localisation of a NOV isoform potentially provides an additional degree of signalling specificity. The interaction of the NOV protein and the rpb7 subunit of RNA polymerase II in the two hybrid system suggests that NOV might be involved in regulating gene expression at the transcriptional level. As has already been suggested for several other nuclearly located cytokines, the NOV protein does not contain a typical nuclear localisation signal. Therefore, it is possible that it combines with either a receptor or a chaperone during its translocation. Disruption of the balance between the secreted and nuclear NOV isoforms might affect the putative autocrine and paracrine functions of NOV and might be of considerable importance in the development of cancers in which the expression of novH has been shown to be impaired.

Abstract: We previously established that the expression of the human nov gene (novH) was altered in Wilms' tumors and that levels of novH and WT1 mRNA were inversely correlated in individual Wilms' tumors. Insofar as novH has been shown to be a target for WT1 regulation, novH might play an important role during normal nephrogenesis and in the development of Wilms' tumors. We now show that during normal nephrogenesis novH protein is tightly associated with differentiation of glomerular podocytes. NovH expression is not restricted to renal differentiation but is also detected in endothelium and neural tissue of the kidney. Our results establish that alteration of novH expression in sporadic and heritable Wilms' tumors is associated with dysregulated expression of both novH mRNA and protein. In general, the highest novH expression was noted in the Wilms' tumor, genitourinary anomalies, aniridia, and mental retardation (WAGR)-associated Wilms' tumors. Expression in the Denys-Drash syndrome (DDS)-associated Wilms' tumors fell within the variable spectrum observed in sporadic Wilms' tumor cases. As in developing kidney podocytes, novH protein was also prominent in the abnormal hypoplastic podocytes from DDS cases and in kidney podocytes adjoining Wilms' tumors. In Wilms' tumors exhibiting heterotypic differentiation, novH protein was expressed at high levels in tumor-derived striated muscle and at lower levels in tumor-derived cartilage. These observations taken together indicate that novH may represent both a marker of podocytic differentiation in kidney and a marker of heterotypic mesenchymal differentiation in Wilms' tumors. In addition, absence or very low levels of WT1 are correlated with higher novH expression, and its variable expression in cases with mutant WT1 (sporadic and DDS) suggests that the potential activation and repression transcriptional functions possessed by WT1 are likely dependent on the specific mutation incurred.

Abstract: The expression pattern of the human nephroblastoma overexpressed (novH) gene in the fetal human central nervous system was examined by in situ hybridization using digoxigenin-labeled novH-specific riboprobes. In the spinal cord, the nov-expressing neurons were first detected both in the ventral region at 16 weeks of gestation (G16W) and in the dorsal region at G38W. In the medulla, nov-expressing neurons were detected in the principal nucleus of the inferior olive, the hypoglossal nucleus and the dorsal motor nucleus of vagus at G16W. Nov-positive neurons were detected at G28W in the nucleus of the spinal tract of the trigeminal and cuneate nucleus, and at G38W in the abducens nucleus of pons, the red nucleus and the substantia nigra of the midbrain, the ventral posterolateral and the mediodorsal thalamic nucleus. A strong labeling was also detected in the striatum of the cerebrum and the cerebral cortex of the parietal lobe. These data established that novH is mainly expressed in somato-motor neurons in the lower central nervous system at early developmental stages and in the higher central nervous system at later stages, suggesting that nov may play an important role in neuronal differentiation.

Abstract: The highly conserved SR family contains a growing number of phosphoproteins acting as both essential and alternative splicing factors. In this study, we have cloned human genomic and cDNA sequences encoding a novel SR protein designated SRp46. Nucleotide sequence analyses have revealed that the SRp46 gene corresponds to an expressed PR264/SC35 retropseudogene. As a result of mutations and amplifications, the SRp46 protein significantly differs from the PR264/SC35 factor, mainly at the level of its RS domain. Northern and Western blot analyses have established that SRp46 sequences are expressed at different levels in several human cell lines and normal tissues, as well as in simian cells. In contrast, sequences homologous to SRp46 are not present in mice. In vitro splicing studies indicate that the human SRp46 recombinant protein functions as an essential splicing factor in complementing a HeLa cell S100 extract deficient in SR proteins. In addition, complementation analyses performed with beta-globin or adenovirus E1A transcripts and different splicing-deficient extracts have revealed that SRp46 does not display the same activity as PR264/SC35. These results demonstrate, for the first time, that an SR splicing factor, which represents a novel member of the SR family, is encoded by a functional retropseudogene.

Abstract: Although several cytogenetic alterations have been associated with development of Wilms' tumor, a multigenic neoplasia, molecular mechanisms of its induction, development and maintenance remain to be elucided. In order to characterize these different steps we have developed a unique animal model of Wilms' tumor constituted by the MAV-1(N) induced avian nephroblastoma. This animal model led to the discovery in our laboratory of a new gene now (nephroblastoma overexpressed gene) which is overexpressed in all avian nephroblastoma. Expression of the human nov gene (novH), which is down-regulated by WT1, is also deregulated in Wilms' tumors. Nov characteristics suggest that it would play a role in the control of cellular proliferation and differentiation. Our observations also indicate that nov could be involved in the development of Wilms' tumors, and represent a marker of their differentiation state.

Abstract: The PR264/SC35 splicing factor belongs to the family of SR proteins which function as essential and alternative splicing factors. Here, we report that the human PR264/SC35 locus is bidirectionally transcribed. Double in situ hybridization experiments have allowed simultaneous detection of sense and antisense RNA in human CCRF-CEM cells, suggesting that expression of the corresponding genes is not mutually exclusive. We have characterized three main classes of ET RNAs encoded by the opposite strand of the PR264/SC35 gene and containing PR264/SC35-overlapping sequences, PR264/SC35-non overlapping sequences or a combination of both. We show that their expression results from the use of alternative promoters, exons and polyadenylation signals. PR264/SC35-non overlapping ET mRNA species potentially encode two protein isoforms (449 and 397 amino acids) and are expressed from the PR264/SC35 promoting region. Northern blots and RNase protection analyses indicate that ET polyadenylated RNAs are differentially expressed in several human cell lines. Similar studies performed in the mouse have revealed that the bidirectional transcription of the PR264/SC35 locus is a conserved mechanism and that the open reading frame identified in a subset of human ET mRNAs is highly conserved (93% homology). Northern blot analyses performed with several murine tissues confirmed the differential expression of the ET gene and revealed that it is predominantly expressed in the testis.

Abstract: AIMS: To characterise the human cyr61 gene (cyr61H) and determine its chromosomal locality. To compare expression of cyr61H in human tumour cell lines with that of two other structurally related genes, novH (nephroblastoma overexpressed gene) and CTGF (connective tissue growth factor), that are likely to play a role in the control of cell proliferation and differentiation. METHODS: To isolate the human cyr61 gene, placental genomic and HeLa cDNA libraries were screened with murine cyr61 cDNA. The nucleotide sequence of the complete cyr61H cDNA was established. Both Southern blotting of a panel of somatic cell hybrids and in situ hybridisation on chromosomes were performed to map the cyr61H gene. Expression of cyr61H, novH, CTGF, and novH was analysed by northern blotting in both human neuroblastomas and glioblastoma cell lines. RESULTS: Genomic and cDNA clones encompassing the cyr61H gene were isolated and characterised. Comparison of mouse and human cyr61 sequences indicated that their genomic organisation is highly conserved. Alignment of coding sequences highlighted the conservation of cyr61 regions that might be critical for its biological function. The data showed that the cyr61H gene is assigned to chromosome 1p22.3 and that different levels of cyr61H, CTGF, and novH mRNA have been detected in several human tumour cell lines derived from the nervous system. CONCLUSIONS: The human cyr61 gene belongs to an emerging family of genes including CTGF/fisp12 and nov. The murine cyr61 encodes an extracellular cysteine rich protein that exhibits chemotactic activity, promotes attachment and spreading of cells, and potentiates the mitogenic effect of growth factors. Assignment of the cyr61H gene to chromosome 1p22.3 will allow studies to determine whether human pathologies derived from the nervous system or from other tissues are associated with chromosomal abnormalities involving this region. Although the coding regions of cyr61H, CTGF, and novH are highly homologous, a growing body of evidence suggests that expression of these genes is regulated differentially, and that a balance between expression of these genes might represent a key element in determining the stage of differentiation and/or the malignant potential of tumour cells.

Abstract: The use of chimeric viruses allowed us to establish that myeloblastosis-associated virus long terminal repeat sequences are necessary and sufficient for induction of nephroblastoma in chickens and that the blastemal hyperplasia induced by env SU is not a prerequisite for tumor development but rather constitutes a predisposing stage.

Abstract: We have sought to identify genes whose expression is altered as a consequence of transformation by p60v-src. Using the mRNA differential display method, we have identified the nov proto-oncogene as one gene that is down regulated in chicken embryo fibroblasts (CEFs) transformed by p60v-src. nov transcripts were also found to be present at only very low levels in proliferating CEFs in comparison with quiescent CEFs. Serum stimulation of quiescent CEFs also resulted in a decline in the steady-state level of nov transcripts. Taken together, these findings suggest that the nov gene is expressed only in quiescent fibroblasts and that its down regulation may contribute to cellular transformation by the v-src oncogene. Down regulation of the nov gene appears to occur at both the transcriptional and posttranscriptional levels. Results obtained from experiments with a protein kinase inhibitor suggest that protein kinase C may be a key downstream effector in mediating the down regulation of nov transcripts in response to activation of p60src or serum stimulation. In addition, we found that transcription of an unknown gene is required for the decline in the steady-state level of nov transcripts in response to serum stimulation.

Abstract: Aims-(1) To investigate the expression in human derived glioblastoma cell lines of two structurally related genes, novH (nephroblastoma overexpressed gene) and CTGF (connective tissue growth factor), which encode putative insulin-like growth factor binding proteins of a novel type. (2) To investigate whether the same transcription factors regulate CTGF and novH expression.Methods-Expression of novH and CTGF was analysed in 24 glioblastoma derived cell lines by northern blotting. The CTGF promoter region was characterised by nucleotide sequencing, RNase protection experiments, by transient transfections, and CAT assays.Results-CTGF and novH mRNA levels differed in the glioma cell lines studied. NovH and CTGF genes were not co-expressed in all cell lines. The CTGF promoter region was highly conserved compared with the corresponding region in the mouse (FISP12) and exhibited in vitro transcriptional activity.Conclusions-Although the coding regions of novH and CTGF are highly homologous, their promoter regions are substantially different, suggesting that these two genes may be regulated by different mechanisms. Considering that novH and CTGF are likely to be, respectively, negative and positive regulators of growth and that some glioma cell lines expressing novH are not tumorigenic, expression of these two genes might represent a key element in determining the stage of differentiation or the malignant potential, or both, of some tumour cell lines.

Abstract: The nov gene encodes a putative Insulin-like-Growth Factor-Binding-Protein (IGFBP) of a novel type which is structurally related to a family of growth-factors likely to play a role in the control of cell proliferation. In the kidney, nov is expressed essentially at the embryonic stage and alterations of nov expression, relative to the normal kidney, have been detected in both avian nephroblastomas and human Wilms' tumors. The levels of human nov (novH) and WT1 mRNA in individual Wilms' tumors have been shown to be inversely correlated, suggesting that the expression of novH could be under the negative control of WT1. We have now established the nucleotide sequence of the 5' flanking region and identified two transcription start sites by RNase protection assays and primer extension. We report that in transient cotransfection experiments the transcription activity of novH promoter constructs was repressed by two isoforms of WT1 proteins (WT1 and WT1+KTS). Repression of the novH promoter required both intact zinc finger regions and the NH2 transcription repression domain of WT1. Inasmuch as the minimal region of novH promoter required to mediate WT1 repression in vivo failed to bine recombinant WT1 protein in in vitro footprinting assays this repression may be mediated by either (i) low affinity sites cooperative interactions or (ii) indirectly via protein-protein interactions with another factor(s). Furthermore, constitutive expression of wild type WT1 into 293 cells resulted in a decrease of endogenous NOVH protein levels, suggesting that novH may be a physiological target for WT1. The downregulation of novH expression by WT1 might represent a key element in normal and tumoral nephrogenesis.

Abstract: The nov gene encodes a cysteine-rich protein that is overexpressed in avian nephroblastomas. It is a member of the CCN family of proteins, all of which are involved in cell growth. Genomic and cDNA clones encompassing the mouse nov gene have been isolated and characterized. The mouse nov gene is highly conserved with the human and chick nov genes at the level of nucleotide sequence and genomic organization. The exon structure reflects the modular organization of the NOV protein in a number of structural domains. These are highly conserved with other members of the CCN family, as is the distribution of 38 of its 40 cysteine residues. The nov gene maps to chromosome 15, between D15 Mit 153 and D15 Mit 183, in a region of conserved synteny with human chromosome 8.

Abstract: Marek's disease virus (MDV) is an avian herpesvirus that causes, in chickens, a lymphoproliferative disease characterized by malignant transformation of T lymphocytes. The rapid onset of polyclonal tumors indicates the existence of MDV-encoded oncogenic products. However, the molecular basis of MDV-induced lymphoproliferative disease and latency remains largely unclear. Several lines of evidence suggest that MDV and Rous-associated virus (RAV) might cooperate in the development of B-cell lymphomas induced by RAV. Our present results indicate for the first time that MDV and RAV might also act synergistically in the development of T-cell lymphomas. We report an example of an MDV-transformed T-lymphoblastoid cell line (T9) expressing high levels of a truncated C-MYB protein as a result of RAV integration within one c-myb allele. The chimeric RAV-c-myb mRNA species initiated in the 5' long terminal repeat of RAV are deprived of sequences corresponding to c-myb exons 1 to 3. The attenuation of MDV oncogenicity has been strongly related to structural changes in the MDV BamHI-D and BamHI-H DNA fragments. We have established that both DNA restriction fragments are rearranged in the T9 MDV-transformed cells. Our results suggest that retroviral insertional activation of the c-myb proto-oncogene is a critical factor involved in the maintenance of the transformed phenotype and the tumorigenic potential of this T-lymphoma cell line.

Abstract: Myeloblastosis-associated viruses (MAV) are replication competent avian retroviruses responsible for the induction of lymphoid leukosis, osteopetrosis, and nephroblastoma. Although both the route of infection and the strain of host used has been reported to be a critical factor in determining the outcome of viral infection, genetically distinct strains of MAV that exhibit a multiple pathogenic potential have been molcularly cloned. Osteopetrosis is a proliferative disease of the bones and nephroblastoma is a kidney cancer. Both diseases occur in chickens a few weeks after MAV injection. In both cases, the nature of the target cells and mechanisms of transformation induced by MAV remain to be established. Molecular cloning and sequencing of three MAV proviral genomes inducing both osteopetrosis and nephroblastoma or only nephroblastoma have allowed the identification of viral determinants essential for osteopetrosis induction. For the last decade we have focused our attention on the MAV-induced nephroblastoma because it is a unique animal model of the human Wilms' tumor. Studies that we have conducted to understand the molecular basis of MAV tumorigenic potential have led to the identification of viral sequences required for tumor induction and to the discovery of a new cellular gene (nov) likely to play a critical role in avian and human nephroblastoma development.

Abstract: We have cloned and sequenced the nov gene (novH) which is the homolog of the chicken nov proto-oncogene overexpressed in avian nephroblastomas. The novH gene is highly conserved and encodes a putative IGF-binding protein similar to that of chicken. We report that relative to autologous normal kidney expression of novH is elevated in Wilms tumors containing predominantly stromal elements and is inversely correlated in these tumors to the expression of WT1. Our results suggest that the regulation of IGFII expression by WT1 and increase of novH in Wilms tumors might be interrelated and represent a key element in tumor development in human.

Abstract: The avian nephroblastoma induced by myeloblastosis-associated virus constitutes a unique animal model in which to study genes involved in the initiation and progression of the human Wilms' tumor. Along this line, we have identified a new cellular gene (nov, for nephroblastoma overexpressed) whose expression is regulated in vitro by the WT1 proteins and is stimulated in all avian nephroblastomas. The nov gene is also conserved in human and its expression is altered in Wilms' tumors. In both species, the nov gene encodes a potential insulin growth factor (IGF)-binding protein associated tot he extracellular matrix and likely to be involved in cell growth regulation. The recent data that are discussed in this review strongly suggest that the nov protein might play a critical role in normal and pathological nephrogenesis.

Abstract: We have recently established that several mRNAs encode the PR264/SC35 splicing factor and that their expression is related to that of c-Myb in human hematopoietic cells. We now report that the various PR264 mRNAs whose expression is transactivated by c-Myb proteins encode an identical PR264/SC35 polypeptide. These mRNAs differ only in their 3' untranslated sequences and exhibit significantly different half-lives. We also show that there is a direct correlation between the length of the 3' untranslated region and the stability of the mRNA species. Given that 5' and 3' splice site selection mediated by the PR264/SC35 splicing factor is concentration-dependent, we postulate that alternative splicing of the 3' untranslated sequences might represent another key element in regulating the levels of PR264/SC35 expression during hematopoietic differentiation.

Abstract: To identify the nucleotide sequences responsible for the tumorigenic specificity of myeloblastosis-associated virus (MAV) we have established the complete nucleotide sequences of three infectious clones inducing either both osteopetrosis and nephroblastoma [MAV2(O)/2 and MAV2(O)p9] or only nephroblastoma [MAV1(N)], and compared their biological properties in the same chicken host strain. The MAV2(O)p9 originally described as a type 2 strain was found to carry a hybrid env gene containing sequences of both the types 1 and 2, and it induced milder and less rapid osteopetrosis than the original MAV2(O) clone when injected into Brown Leghorn chickens. These results, together with sequence comparisons between the MAV strains examined, suggest that subtle changes in the primary structure of the TM env protein's extracellular domain are likely to affect the tumorigenic potential of MAV.

Abstract: We have shown previously that c-myb expressed in the yeast S. cerevisiae mediated efficient transcriptional activation of reporter genes designed with specific Myb Recognition Elements (MRE's), confirming that this proto-oncogene is able to function as a regulator of transcription in that heterologous context. Here we show that in yeast, as in higher eucaryotic cells, the central domain of c-Myb displays transactivating capacity. In yeast, however, the carboxy-terminal region, defined as a negative regulatory domain in higher cells, activates transcription as well and appears to be a more potent transactivating domain than the central domain itself. Within this region two domains, namely C1 and C2, have been defined that contribute about equally to the activity of the carboxy-terminal region. C1 spans the sequences missing in AMV v-myb while C2, which contains the leucine-zipper motif is specifically absent in the E26 v-myb in addition to C1. The c-Myb DNA-binding domain itself has no effect on the level of transcription in yeast. We also show that AMV v-Myb stimulates gene expression in yeast with about half the efficiency of full length c-Myb. The fact that the carboxy-terminal region either stimulates or inhibits transactivation properties of c-Myb, depending on the cellular context, stresses the participation of putative c-Myb partner proteins in Myb regulated processes and reopens the question of whether the oncogenic activation of c-myb is indeed due to the increased transactivation capacity of its onco derivatives.

Abstract: HL-60 cells isolated for resistance to vincristine are multidrug resistant and defective in the cellular accumulation of drug. Further studies demonstrate that these cells are also highly defective in 12-O-tetradecanoylphorbol-13-acetate (TPA) induced differentiation to macrophages. Analysis of this system demonstrates that certain protooncogenes which may contribute to differentiation are expressed at similar levels in sensitive and resistant cells. Thus, treatment of cells with TPA results in a reduction in the levels of c-myb and c-myc mRNA, while the expression of c-fos, c-jun, and junB is greatly enhanced. Immunoprecipitation experiments also demonstrate a TPA induced increase in the c-jun protein in both sensitive and resistant cells. Gel mobility shift assays show that TPA induces AP-1 formation in sensitive cells, whereas in parallel experiments with the HL-60/Vinc isolate, AP-1 is essentially absent. It has been found, however, that in resistant cells which have reverted to drug sensitivity, the levels of TPA inducible AP-1 is essentially identical to that of sensitive cells. Revertant and sensitive cells differentiate at similar levels in the presence of TPA. These studies therefore demonstrate that HL-60/Vinc cells are defective in the TPA induction of a functional AP-1 complex and that this may account for the inability of these cells to differentiate to macrophages. The molecular basis of the finding that AP-1 is not formed in resistant cells remains to be determined.

Abstract: Histological and anatomopathological studies performed on 152 independent myeloblastosis-associated virus type 1 (MAV1)-induced nephroblastomas allowed us to precisely define the chronology of tumor development in chickens. Three tumors representing increasing developmental stages were used to construct genomic libraries and to study both the state of proviral genomes and the sites of MAV1 integration in genomic DNA. We established that increasing levels of proviral rearrangement, eventually leading to the elimination of infectious MAV genomes, were associated with tumor progression and that 22 individual tumors, representative of different developmental stages, did not contain any common MAV1 integration site. Cloning of cellular fragments flanking the MAV1-related proviruses in tumor DNA showed that each one of eight nephroblastomas tested expressed a high level of an as yet unidentified cellular gene (nov) whose transcription is normally arrested in adult kidney cells. Cloning of the normal nov gene established that in one tumor, fused long terminal repeat-truncated nov mRNA species were expressed, indicating that at least in that case, the high level of nov expression was under the control of the MAV long terminal repeat promoter. The normal nov gene encodes a putative 32-kDa secreted polypeptide, which is a member of a new family of proteins likely to be involved in cell growth regulation. We also showed that the expression of an amino-terminal-truncated nov product in chicken embryo fibroblasts was sufficient to induce their transformation.

Abstract: We have previously reported that truncated forms of the v-myb oncogene of avian myeloblastosis virus (AMV) are expressed in transformed chicken embryo fibroblasts (CEF). In this paper, we show that deletion mutants encoding v-myb products altered in either the DNA-binding or the negative regulatory domains are able to induce CEF transformation. In addition, we report that recombinant plasmids expressing gag-myb fusion proteins are maintained as extrachromosomal forms in transfected cells. This observation provides an important clue for a possible role of myb in the DNA replication processes.

Abstract: We have previously reported that expression of the c-myb gene in normal avian thymic cells proceeds through the intermolecular recombination of ET (thymus-specific) and c-myb coding sequences, thereby generating a novel type of c-myb product. Antisense transcripts expressed from the ET locus encode the extremely well-conserved splicing factor PR264/SC35. We now show that the human PR264 promoter sequences contain several myb-recognition elements that efficiently interact in vitro with the c-myb DNA-binding domain. Moreover, expression from the PR264 promoter is transactivated, both in vitro and in cultured cells, by different c-myb products. Thus, the PR264 gene is most likely a physiological target for the c-myb family of transcription factors.

Abstract: We previously established that the expression of a thymic c-myb mRNA species requires the intermolecular recombination of coding sequences expressed from transcriptional units localized on different chromosomes, in both chicken and human. We now report that a putative splicing factor (PR264), extremely well conserved in chicken and human, is encoded by the opposite strand of the c-myb trans-spliced exon. The PR264 polypeptide, which contains a typical ribonucleoprotein 80 and an arginine/serine-rich domain, is highly homologous to the Drosophila splicing regulators tra, tra-2, and su(wa) and to the human alternative splicing factor ASF/SF2. Furthermore, we show that PR264-specific mRNAs are expressed in normal hematopoietic cells of chicken and human origin and that the relative proportion of the PR264 transcripts is developmentally regulated in chicken.

Abstract: The human locus (novH) corresponding to the nov protooncogene overexpressed in avian nephroblastoma has been identified and mapped on chromosome 8q24.1. Another locus sharing homology with novH and corresponding to the connective tissue growth factor (CTGF) gene has also been mapped on chromosome 6q23.1. The chromosomal assignment of nov and CTGF proximal to c-myc and c-myb respectively is of interest because chromosomal abnormalities involving these regions have been associated with different human tumors including Wilms'.

Abstract: The ras oncogenes alone fully transform established (immortalized) rodent fibroblasts in a few days, but generally transform early-passage fibroblasts only partially, unless their action is complemented by that of a nuclear, immortalizing, oncogene. Here we show that transfection of second-passage Syrian hamster embryo fibroblasts (HEFs) by the EJ-H-ras oncogene coupled to the neo gene, followed by selection with G418, gives rise to apparently normal, or only slightly transformed, clonal colonies, only a few of which become established. The study of two established clonal lines showed that they acquired only after some weeks, and stepwise, the main characteristics of full neoplastic transformation, i.e. anchorage independence, reduced requirement for serum growth factors and tumorigenicity. Later both clonal lines became increasingly tumorigenic and completely independent of exogenous growth and attachment factors, without increase in the expression of the H-ras oncogene. Transfection of one of the clones, early after its isolation, with a truncated derivative of the nuclear v-myb oncogene devoid of its transcriptional negative regulatory domain and able to partially transform chicken embryo fibroblasts [(myb(KXANM)] gave rise to more transformed cells, expressing both EJ-H-ras and myb(KXANM), which became tumorigenic earlier than the controls and remained more tumorigenic later on. With more efficient transfection techniques, numerous foci of fully transformed cells were subsequently obtained, in a few days, in cultures transfected sequentially with EJ-H-ras(neo) and myb(KXANM) and in cultures co-transfected with the two oncogenes. Highly tumorigenic, serum-independent and immortalized clones expressing both oncogenes were obtained from these cultures. Hence, the truncated myb(KXANM) oncogene accelerate the stepwise transformation of unestablished HEFs by the EJ-HH-ras oncogene and, together with this oncogene, fully transforms these same cells in a single step. The two oncogenes acting in cooperation also induce cell immortalization, but myb(KXANM), by itself, is not an immortalizing oncogene. No cooperation was observed between EJ-H-ras(neo) and the unaltered v-myb oncogene.

Abstract: Recently, we reported evidence suggesting that expression of c-myb thymic mRNA species involves the intermolecular recombination of coding sequences (ET and c-myb) localized on two different chromosomes, both in chicken and human. Our present studies demonstrate that the ET locus encodes, in the antisense orientation, a novel member of the RNA binding protein family in these two species.

Abstract: We have shown in a previous study that the expression of an as yet unidentified, embryonic gene (nov) encoding a potential IGF binding protein was upregulated in all of eight virally-induced avian nephroblastomas tested. We now report that homologous sequences are conserved in human DNA and are expressed in normal human bone marrow, thymic cells and in one nephroblastoma.

Abstract: We have previously reported evidence suggesting that intermolecular recombination events are involved in the tissue specific expression of the c-myb proto-oncogene in chicken. We show in this paper that recombined c-myb mRNA species are also expressed in human thymic cells, therefore indicating that intermolecular recombination of coding sequences occurs in higher eucaryotes.

Abstract: We assayed in the yeast S. cerevisiae the transcriptional transactivation activity of the c-myb products encoded by a normal thymus cDNA and of an aminoterminally truncated version of it (minus 58 amino acids) corresponding to the cDNAs isolated from lymphoma and leukemia cells from different origins. Both proto-oncogene products were expressed under the control of the galactose inducible GAL10 promoter. The reporter system used to monitor the transactivation potential of the myb products consisted of a CYCl-lacZ gene fusion in which the UASCYC signals were replaced by one or multiple copies of the myb recognition element (mRE). As shown by Northern blot analyses and by primer extension experiments both c-myb products increase the level of beta-galactosidase transcription. Interestingly, the c-myb product corresponding to lymphoma cDNAs stimulates transcription four to five times more efficiently than does the normal thymic c-myb product.

Abstract: We have characterized a novel chicken c-myb exon whose sequences are specifically expressed in thymic cells. In situ hybridization experiments indicate that this thymus-specific coding exon is localized on a small chromosome, distinct from the large acrocentric chromosome 3 on which we recently mapped the bulk of 15 exons, common to the c-myb mRNA species expressed in hematopoietic cells of both B and T lineages. These observations indicate that intermolecular recombination is required for the tissue-specific expression of the c-myb proto-oncogene. We also show that these thymus-specific sequences are conserved in human DNA and lie on chromosome 17q25, whereas the human c-myb locus is localized on chromosome 6q22-23. Sequencing data obtained from genomic DNA and PCR analyses performed with c-myb mRNA species expressed in chicken thymic cells strongly suggest that a repeated decameric sequence plays a key role in the recombination process.

Abstract: Human Xeroderma pigmentosum "normal" fibroblasts AS16 (XP4 VI) were transformed after transfection with a recombinant v-myb clone. In this clone (pKXA 3457) derived from avian myeloblastosis virus (AMV), the expression of the oncogene sequences is driven by the AMV U-5 LTR promoter. The transformed cells (ASKXA), which have integrated a rearranged v-myb oncogene, grow in agar, are not tumorigenic in nude mice, and express a 45-kDa v-myb protein. The HMW DNA of these cells transform chicken embryo fibroblasts. The c-Ha-ras oncogene is overexpressed in the ASKXA cells but not in the parental "normal" AS16 cells and a revertant clone (ASKXA Cl 1.1 G). Our results lead to the conclusion that the XP fibroblasts are phenotypically transformed by the presence of the transfected v-myb oncogene, which is able to induce an overexpression of the c-Ha-ras gene.

Abstract: The physical separation of c-myb coding exons on two different chromosomes, both in chicken and human, indicated that intermolecular recombination events might occur in higher eucaryotes. We present evidence in this paper suggesting that the expression of "antisens" mRNA species could be involved in the regulation of the intermolecular recombination process.

Abstract: In the course of our studies concerning the tissue-specific expression of the c-myb proto-oncogene, we have established the nucleotide sequence of the chicken c-myb 3'-proximal coding exons. In situ hybridization performed with different genomic DNA probes corresponding to nearly all the c-myb gene allowed us to localize the corresponding locus on the large acrocentric chromosome 3 in chicken. Our sequencing data also indicate that the 3'-proximal noncoding sequences represented in c-myb mRNA species are derived from non-contiguous exons.

Abstract: Myeloblastosis-associated virus type 1 (MAV1) derived from a molecular clone of infectious proviral DNA (B. Perbal, J. S. Lipsick, J. Svoboda, R. F. Silva, and M. A. Baluda, J. Virol. 56:240-244, 1985) was shown to specifically induce nephroblastoma in chickens and therefore belongs to the MAV-N class. We show that nephroblastomas are polyclonal tumors containing rearranged proviral genomes. Rearrangements occur preferentially in the gag-pol region of the MAV1 proviral genome, and similar rearrangements can be detected in well-developed independent tumors.

Abstract: A 70 kDa protein of Halobacterium halobium cross-reacts with an antiserum directed against the v-myc gene product of the avian myelocytomatosis virus (MC29). This cross-reaction is in agreement with hybridization studies which indicate that H. halobium possesses DNA and RNA sequences homologous to the v-myc gene.

Abstract: The organization of 5'-proximal c-myb exons in chicken DNA has been established by restriction enzyme mapping and nucleotide sequencing. Hybridization studies performed with cDNA probes revealed that yolk sac and thymic c-myb RNAs differ in their 5'-termini. A comparison of the genomic c-myb sequence with that of cDNAs isolated from normal thymic and lymphoma avian cells suggests that different promoter regions are used to initiate c-myb transcription in hematopoietic cells of different origins.

Abstract:

Abstract: Transformed cells have been isolated after transfection of chicken embryo fibroblasts (CEF) with the DNA of a recombinant clone (KXA 3457) in which the v-myb sequences are flanked by the two AMV-LTRs. Abnormal myb-specific RNA species and myb-related polypeptides were found to be expressed in these cells, suggesting that transformation of CEF by v-myb might require alterations of the oncogene product.

Abstract: The potential regulatory signals contained in the v-myb oncogene of avian myeloblastosis virus have been inserted upstream to the herpes simplex type 1 thymidine kinase gene in order to test their promoter activity. The isolation of TK+ transformants after transfection of clone 1D(TK-) mouse cells with the resulting recombinant DNAs indicated that the expression of the TK gene was made possible by the myb-derived sequences. Analysis of the TK specific RNA expressed in different TK+ transformants revealed that the regulatory signals contained in v-myb correspond to a weak functional promoter.

Abstract: Transfection of brown leghorn chicken embryo fibroblasts by DNA containing v-myb sequences cloned either in a complete AMV proviral DNA or in a retroviral derived vector has led to the isolation of two kinds of transformed cells. A characterization of the proviral sequences retained and expressed in these transformed cells revealed that they contained either new or altered v-myb-related RNA species. The experiments presented in this paper also show that both types of transformants expressed truncated myb-related polypeptides, suggesting that alterations of the v-myb product may result in a new target specificity, leading to the transformation of chicken embryo fibroblasts.

Abstract: The presence of DNA sequences homologous to the v-myb oncogene in the genome of both halophilic and methanogenic archaebacteria was revealed after hybridization of restriction fragments with cloned probes. No myb-related sequences were detected in the DNA from S. acidocaldarius.

Abstract: Brown Leghorn chicken embryo fibroblasts were transfected with a mixture of avian myeloblastosis virus (AMV) and myeloblastosis-associated virus type 1 (MAV1) proviral DNA purified from lambda-Charon 4A recombinant clones. A transformed cell line (T1AM) able to grow without anchorage in semisolid medium was obtained. The presence of both proviral AMV and MAV sequences was detected in T1AM DNA by hybridization with v-myb- and MAV1-specific probes. Altered AMV and MAV1 proviral genomes were found in T1AM genome. Characterization of the RNA species expressed in transformed cells showed that in addition to a 2.5-kilobase (kb) putative subgenomic v-myb-specific RNA, three other myb-containing RNAs (9.4, 8.4, and 7.0 kb) were present in T1AM cells. No AMV genomic RNA was detected. Also, a new 5.0-kb MAV1-specific RNA species was expressed in transformed cells in addition to MAV1 genomic RNA species (7.8 kb). No infectious AMV virions are released by T1AM cells. Chicken embryo fibroblasts infected by T1AM-released virions contained and expressed all MAV1 sequences detected in T1AM transformed cells but did not express any transformation parameter. These results indicated that the presence of AMV proviral sequences in T1AM cells is responsible for their transformed phenotype.

Abstract: A biologically active myeloblastosis-associated virus (MAV) provirus was cloned from a bacteriophage recombinant library constructed from leukemic chicken myeloblast DNA. The restriction endonuclease map of this clone was consistent with that of a type 1 MAV (MAV-1). Interference assays of virus recovered from cultured chicken embryo fibroblasts after DNA transfection established that the provirus was infectious and confirmed that it belonged to avian retrovirus subgroup A (type 1). Antipeptide antibodies raised against the env-encoded carboxyl terminus of p48myb, the transforming protein of avian myeloblastosis virus, specifically immunoprecipitated the gp37env from quail cells transfected with MAV-1 proviral DNA but not from cells infected with MAV-2. This suggests that MAV-1 rather than MAV-2 is the progenitor helper virus from which avian myeloblastosis virus arose by the transduction of cellular proto-oncogene sequences.

Abstract: Expression of endogenous retrovirus genes and two different cellular oncogenes (c-onc genes) was examined at the transcriptional level in a variety of normal and lymphoma/leukemia tissues of the domestic cat. The two oncogenes, c-myb(related to avian myeloblastosis virus) and c-myc(related to avian myelocytomatosis virus) were selected for their association with the induction of hematopoietic malignancies, when present in the transforming retroviruses. Tissue-specific expression of endogenous feline leukemia virus (FeLV)-related genes was detected in cellular subpopulations of the cat placenta by in situ method of hybridization. Gel blotting analysis of placental poly(A)-selected RNA revealed that the FeLV-related RNA species were primarily subgenomic, representing the env gene region of the endogenous provirus elements. Like the endogenous retrovirus genes, c-myb and c-myc loci of the cat genomic DNA were also transcribed at differential levels in normal tissues of the cat. Dot-blot hybridization analysis showed that the expression of these two oncogenes was linked to growth and development as it varied with the gestational age of the fetus and from fetal to adult tissues. Among the major hematopoietic organs, spleen and bone marrow contained both c-myb and c-myc transcripts, while thymus preferentially expressed the c-myb gene. In contrast to the low level of c-myc expression in fetal thymus tissues, enhanced c-myc expression was detected in all five thymomas tested and also in several other neoplasms including two granulocytic leukemias. The feline c-myb gene was not very active in granulocytic leukemias or in three of the five thymomas. RNA gel blotting analysis of poly(A)-selected RNA of a thymoma and its lymph node metastasis showed identical pattern of c-myc transcripts.

Abstract:

Abstract: Several lambda proto-amv recombinants isolated from a lambda Charon 4A library of leukemic chicken DNA were analyzed by using various restriction endonucleases and hybridization with specific probes representing different regions of the transforming gene of avian myeloblastosis virus. The position of 30 sites for 11 different restriction endonucleases was established in the proto-amv region of chicken DNA. Identical restriction endonuclease maps were obtained for the normal and leukemic DNAs in the proto-amv domain, which covers 8 to 9 kilobases of DNA. The cellular genetic elements homologous to the cellular sequence (amv) inserted into the avian myeloblastosis virus genome are contained within six major proto-amv segments which are interrupted by at least five large DNA regions lacking homology with amv.

Abstract: Avian myeloblastosis virus (AMV) is a replication-defective acute leukemia virus, requiring a helper virus to provide the viral proteins essential for synthesis of new infectious virus. The genome of the AMV has undergone a sequence substitution in which a portion of the region normally coding for the "env" protein has been replaced by chicken cellular sequences. These latter sequences are essential for the transforming activity of the virus. We have determined the complete nucleotide sequence of this region. Examination of the AMV oncogenic sequence revealed an open reading frame starting with the initiation codon ATG within the acquired cellular sequences and terminating with the triplet TAG at a point 33 nucleotides into helper viral sequences to the right of helper-viral-cellular junction. The stretch of 795 nucleotides would code for a protein of 265 amino acids with a molecular weight of 30,000 daltons. The eleven amino acids at the carboxy terminus of such a protein would be derived from the env gene of helper virus.

Abstract: Identification of several additional restriction endonuclease sites within the cellular substitution (amv) inserted into the avian myeloblastosis virus proviral genome has permitted us to isolate different regions of the amv sequence. These subsets of the avian myeloblastosis virus transforming gene have been cloned in the plasmid pBR322 and used as hybridization probes to investigate the topology of homologous (proto-amv) normal chicken DNA sequences. The results showed that the cellular proto-amv sequences in C/O chicken DNA are interrupted by at least one intervening sequence. A partial arrangement of the proto-amv sequences is presented.

Abstract: Replication-defective acute leukemia viruses E26 and myeloblastosis virus (AMV) cause distinct leukemias although they belong to the same subgroup of oncogenic avian tumor viruses based on shared transformation-specific (onc) RNA sequences. E26 causes predominantly erythroblastosis in chicken and in quail, whereas AMV induces a myeloid leukemia. However, upon cultivation in vitro for >1 month, a majority of surviving hemopoietic cells of E26-infected animals bear myeloid markers similar to those of AMV-transformed cells. We have analyzed the genetic structure and gene products of E26 virus for a comparison with those of AMV. An E26/helper virus complex was found to contain two RNA species: a 5.7-kilobase (kb) RNA that hybridizes with cloned AMV-specific proviral DNA and hence is probably the E26 genome; and an 8.5-kb RNA that is unrelated to AMV and represents helper virus RNA. Thus, E26 RNA is smaller than 7.5-kb AMV RNA. Hybridization of size-selected poly(A)-terminating E26 RNA fragments with AMV-specific DNA indicated that the shared specific sequences are located in the 5' half of the E26 genome as opposed to a 3' location in AMV RNA. In nonproducer cells transformed in vitro by E26, a gag-related nonstructural 135,000-dalton protein (p135) was found. No gag(Pr76) or gag-pol (Pr180) precursors of essential virion proteins, which are present in AMV nonproducer cells, were observed. p135 was also found in cultured E26 virus producing cells of several leukemic chickens, and its intracellular concentration relative to that of the essential virion proteins encoded by the helper virus correlates with the ratio of E26 to helper RNA in virions released by these cells. p135 is phosphorylated but not glycosylated; antigenically it is not related to the pol or env gene products. It appears to be coded for by a partial gag gene and by E26-specific RNA sequences, presumably including those shared with AMV. Hence, AMV and E26 appear to use different strategies for the expression of related onc sequences: AMV is thought to encode a transforming protein via a subgenomic mRNA, whereas E26 codes for a gag-related polyprotein via genomic RNA. It is speculated that differences in the oncogenic properties of E26 and AMV are due to differences in their genetic structures and gene products.

Abstract: Avian myeloblastosis virus is defective in reproductive capacity, requiring a helper virus to provide the viral proteins essential for synthesis of new infectious virus. This virus arose by recombination of the nondefective helper virus and host cellular sequences present within the normal avian genome. These latter sequences are essential for leukemogenic activity. The complete nucleotide sequence of this region is reported. Within the acquired cellular sequences there is an open reading frame of 795 nucleotides starting with the initiation codon ATG (adenine, thymine, guanine) and terminating with the triplet TAG. This open reading frame could code for the putative transforming protein of 265 amino acids with a molecular weight of approximately 30,000.

Abstract: The avian myeloblastosis virus provirus inserted in a lambda bacteriophage, recombinant clone 11A1-1 (Souza et al., Proc. Natl. Acad. Sci. U.S.A. 77:3004-3008, 1980), was transfected into chicken embryo fibroblasts which had been preinfected with either Rous-associated virus type 61 or the transformation-defective avian sarcoma virus tdB77. Within 4 to 5 h after transfection, the cells were injected into 16-day-old chicken embryos or 1-day-old chicks. Acute myeloblastic leukemia developed after a long latent period. Filtered (0.22-micrometer pores) supernatant of transformed buffy-coat cell cultures from one leukemic chicken of the lambda 11A1-1 (tdB77) group rapidly transformed yolk sac cells in vitro. Results from an infectivity interference assay and analysis of proviral DNA fragments generated with restriction endonucleases were consistent with the presence in leukemic cells of defective avian myeloblastosis virus and tdB77 as the helper virus.

Abstract: Hybridization probes consisting of cloned DNA recombinants which represent different regions of the leukemogenic sequence (amv) from avian myeloblastosis virus were used to carry out a more detailed restriction endonuclease analysis of the homologous sequences (proto-amv) present in normal and leukemic chicken DNA. The results show that four large introns interrupt the normal cellular proto-amv sequences and that there is no major rearrangement of these sequences in leukemic myeloblasts.

Abstract: 1. Two strains of independently purified Herpes simplex virus type 1 were irreversibly inactivated after incubation with the pregnene derivative methyl-p-toluene sulphonate of diethylamino-3-beta-ethoxy-20-hydroxy-pregn-5-ene (TX 3047) in vitro. 2. RNA and DNA synthesis (both cellular and viral) were studied in the presence of TX 3047. Viral and cellular DNA synthesis but not viral RNA synthesis was reduced. 3. Formation of infectious particles was not affected by TX 3047 if the parental virus was absorbed to the cell before it was incubated with TX 3047. This suggests that TX 3047 acts at the level of the membrane.

Abstract: Several transformed cell lines established from Fisher rat cells (FR 3T3) infected with wild-type polyoma virus or simian virus 40 or early temperature-sensitive mutants (polyoma tsa and simian virus 40 tsA30) were studied for their transformation phenotypes. The distinct patterns which were obtained for polyoma and simian virus 40 transformants led to the conclusion that these two viruses express different transforming abilities in rat cells. The results obtained with temperature-sensitive mutant-derived transformants indicate that all of the transformation characteristics studied so far may be under the control of a viral function in polyoma tsa-transformed cells.

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Abstract: The expression of two transformation parameters, namely, ability to grow in agar and plasminogen activator production, was studied in several rat fibroblasts transformed by either wild-type or thermo-sensitive (tsa and ts25) polyoma viruses. The production of plasminogen activator was found to be dependent upon the growth state of the infected cells during a period of several days after infection. The analysis of the transformed phenotype of 25 tsa transformants and of 19 ts25 transformants independently isolated under various growth conditions led to the conclusion that there is no correlation between the regulation processes involved in plasminogen activator production and ability to grow without anchorage. The results obtained also suggested that the production of plasminogen activator is under the control of a functional large T antigen.

Abstract: Amino acid-requiring mutants capable of producing derepressed levels of aspartate transcarbamylase (carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) were obtained and used for the incorporation in this enzyme of eight different amino acid analogs. These amino acid replacements enabled the biosynthesis of a series of modified aspartate transcarbamylases altered in their catalytic or regulatory properties. The enzyme in which phenylalanine was rereplaced by 2-fluorophenylalanine was purified to homogeneity and appeared to have the same specific activity as normal asparate transcarbamylase but lacking both homotropic and heterotropic interactions.

Abstract: Fisher rat fibroblasts (FR 3T3), transformed with the tsA30 mutant of simian virus 40 and selected by colony formation in soft agar, maintained the transformed phenotype at high temperature, whereas most transformants isolated from foci were found to undergo a phenotypic reversion toward the normal state in their saturation density, ability to grow in soft agar, and rate of 2-deoxyglucose transport. The temperature-independent phenotype observed in agar-selected transformants was not due to a reversion of the viral mutation. These results, similar to those previously obtained with polyoma virus tsa mutants, further suggest that two distinct mechanisms may operate in both cases for maintaining the transformed phenotype. Immunofluorescence studies suggested a different regulation of T antigen synthesis in these two classes of transformants.

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Abstract: LMTK-cells infected with u.v.-irradiated herpes simplex virus type 1 have been selected for the presence of the enzyme thymidine kinase. These cells have an altered morphology compared to the control cells and contain herpes-specific antigens in their cytoplasm. The thymidine kinase activity present in these cells has been shown, on the basis of a number of biochemical properties, to be identical to the herpes virus specified deoxypyrimidine kinase found during lytic infection of this virus. In addition it has been possible to detect herpes simplex-specific RNA sequences in the transformed cells and this occurs in both the polyadenylated and non-polyadenylated cytoplasmic and nuclear fractions.

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