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Sialic acid (SA) is present at the terminal ends of carbohydrate chains in glycoproteins and glycolipids and is involved in various biological phenomena. The biological function of the disialyl-T (SAα2-3Galβ1-3(SAα2-6)GalNAcα1-O-Ser/Thr) structure is largely unknown. To elucidate the role of disialyl-T structure and determine the key enzyme from the N-acetylgalactosaminide α2,6-sialyltransferase (St6galnac) family involved in its in vivo synthesis, we generated St6galnac3- and St6galnac4-deficient mice. Both single-knockout mice developed normally without any prominent phenotypic abnormalities. However, the St6galnac3::St6galnact4 double knockout (DKO) mice showed spontaneous hemorrhage of the lymph nodes (LN). To identify the cause of bleeding in the LN, we examined podoplanin, which modifies the disialyl-T structures. The protein expression of podoplanin in the LN of DKO mice was similar to that in wild-type mice. However, the reactivity of MALII lectin, which recognizes disialyl-T, in podoplanin immunoprecipitated from DKO LN was completely abolished. Moreover, the expression of vascular endothelial cadherin was reduced on the cell surface of high endothelial venule (HEV) in the LN, suggesting that hemorrhage was caused by the structural disruption of HEV. These results suggest that podoplanin possesses disialyl-T structure in mice LN and that both St6galnac3 and St6galnac4 are required for disialyl-T synthesis., (© 2023. The Author(s).)

Chronic BK polyomavirus (BKPyV) infection is recognized as a potential oncogenic factor of urothelial carcinoma (UC) in renal transplant recipients. Recent studies have reported a positive correlation among BKPyV integration, persistent overexpression of viral large T antigen (TAg), and malignancy, yet little is known about the specific integration mechanisms and the impacts of viral integration. Here, we performed whole-genome sequencing (WGS) and viral capture-based sequencing on high-grade immunohistochemically TAg-positive UCs in two renal transplant recipients. A total of 181 integration sites, including the three found by WGS, were identified by viral capture-based sequencing, indicating its enhanced sensitivity and ability in identifying low-read integration sites in subpopulations of the tumor cells. The microhomologies between human and BKPyV genomes were significantly enriched in the flanking regions of 84.5% the integration sites, with a median length of 7 bp. Notably, 75 human genes formed fusion sequences due to viral insertional integration. Among them, the expression of 15 genes were statistically associated with UC based on GEO2R expression analysis. Our results indicated a multisite and multifragment linear integration pattern and a potential microhomology or nonhomologous end joining integration mechanism at the single-nucleotide level. We put forward a potential selection mechanism driven by immunity and centered on viral integration in the carcinogenesis of BKPyV.

Whereas large T antigen (LT) of simian virus 40 (SV40) promotes oncogenesis by inactivating the tumor suppressor proteins p53 and pRb, SV40 small T antigen (ST) has been thought to be dispensable for this process. However, here we show that LT promotes both oncogenic growth and senescence in human cells expressing oncogenic Ras and that this latter effect is antagonized by ST. Inactivation of p53 by LT alone promoted the senescence-associated secretory phenotype (SASP), whereas the additional expression of ST attenuated this phenotype, allowing cells to avoid oncogene-induced senescence (OIS) and thereby promoting efficient oncogenesis. ST interacts with and inhibits the function of heterochromatin protein 1-binding protein 3 (HP1BP3), a positive regulator of global microRNA biogenesis, and it thereby triggers aberrant upregulation of B-cell translocation gene 2 (BTG2), which is essential for prevention of SASP and OIS by ST. Collectively, our results indicate that the HP1BP3-BTG2 axis constitutes a fail-safe system to prevent oncogenesis by means of OIS induction, and that this system is hijacked by ST.

In the normal mammary gland, the basal epithelium is known to be bipotent and can generate either basal or luminal cells, whereas the luminal epithelium has not been demonstrated to contribute to the basal compartment in an intact and normally developed mammary gland. It is not clear whether cellular heterogeneity within a breast tumor results from transformation of bipotent basal cells or from transformation and subsequent basal conversion of the more differentiated luminal cells. Here we used a retroviral vector to express an oncogene specifically in a small number of the mammary luminal epithelial cells and tested their potential to produce basal cells during tumorigenesis. This in-vivo lineage-tracing work demonstrates that luminal cells are capable of producing basal cells on activation of either polyoma middle T antigen or ErbB2 signaling. These findings reveal the plasticity of the luminal compartment during tumorigenesis and provide an explanation for cellular heterogeneity within a cancer.

Polyoma small T antigen (PyST), an early gene product of the polyoma virus, has been shown to cause cell death in a number of mammalian cells in a protein phosphatase 2A (PP2A)-dependent manner. In the current study, using a cell line featuring regulated expression of PyST, we found that PyST arrests cells in mitosis. Live-cell and immunofluorescence studies showed that the majority of the PyST expressing cells were arrested in prometaphase with almost no cells progressing beyond metaphase. These cells exhibited defects in chromosomal congression, sister chromatid cohesion and spindle positioning, thereby resulting in the activation of the spindle assembly checkpoint. Prolonged mitotic arrest then led to cell death via mitotic catastrophe. Cell cycle inhibitors that block cells in G1/S prevented PyST-induced death. PyST-induced cell death that occurs during M is not dependent on p53 status. These data suggested, and our results confirmed, that PP2A inhibition could be used to preferentially kill cancer cells with p53 mutations that proliferate normally in the presence of cell cycle inhibitors.

Replicative DNA helicases generally unwind DNA as a single hexamer that encircles and translocates along one strand of the duplex while excluding the complementary strand (known as steric exclusion). By contrast, large T antigen, the replicative DNA helicase of the simian virus 40 (SV40), is reported to function as a pair of stacked hexamers that pumps double-stranded DNA through its central channel while laterally extruding single-stranded DNA. Here we use single-molecule and ensemble assays to show that large T antigen assembled on the SV40 origin unwinds DNA efficiently as a single hexamer that translocates on single-stranded DNA in the 3'-to-5' direction. Unexpectedly, large T antigen unwinds DNA past a DNA-protein crosslink on the translocation strand, suggesting that the large T antigen ring can open to bypass bulky adducts. Together, our data underscore the profound conservation among replicative helicase mechanisms, and reveal a new level of plasticity in the interactions of replicative helicases with DNA damage.

The introduction of the Simian virus 40 (SV40) early region, the telomerase catalytic subunit (hTERT) and an oncogenic allele of H-Ras directly transforms primary human cells. SV40 small T antigen (ST), which forms a complex with protein phosphatase 2A (PP2A) and inhibits PP2A activity, is believed to have a critical role in the malignant transformation of human cells. Recent evidence has shown that aberrant microRNA (miRNA) expression patterns are correlated with cancer development. Here, we identified miR-27a as a differentially expressed miRNA in SV40 ST-expressing cells. miR-27a is upregulated in SV40 ST-transformed human bronchial epithelial cells (HBERST). Suppression of miR-27a expression in HBERST cells or lung cancer cell lines (NCI-H226 and SK-MES-1) that exhibited high levels of miR-27a expression lead to cell growth arrested in the G(0)-G(1) phase. In addition, suppression of miR-27a in HBERST cells attenuated the capacity of such cells to grow in an anchorage-independent manner. We also found that suppression of the PP2A B56γ expression resulted in upregulation of miR-27a similar to that achieved by the introduction of ST, indicating that dysregulation of miR-27a expression in ST-expressing cells was mediated by the ST-PP2A interaction. Moreover, we discovered that Fbxw7 gene encoding F-box/WD repeat-containing protein 7 was a potential miR-27a target validated by dual-luciferase reporter system analysis. The inverse correlation between miR-27a expression levels and Fbxw7 protein expression was further confirmed in both cell models and human tumor samples. Fbxw7 regulates cell-cycle progression through the ubiquitin-dependent proteolysis of a set of substrates, including c-Myc, c-Jun, cyclin E1 and Notch 1. Thus, promotion of cell growth arising from the suppression of Fbxw7 by miR-27a overexpression might be responsible for the viral oncoprotein ST-induced malignant transformation. These observations demonstrate that miR-27a functions as an oncogene in human tumorigenesis.

Serous borderline ovarian tumors (SBOTs) are slow-growing, non-invasive ovarian epithelial neoplasms. SBOTs are considered to be distinct entities that give rise to invasive low-grade serous carcinomas (LGCs), which have a relatively poor prognosis and are unrelated to high-grade serous carcinomas (HGCs). The mechanisms underlying the progression of non-invasive SBOTs to invasive epithelial ovarian carcinomas are not understood. We recently established short-term cultures of SBOT cells from tumor biopsies and showed that inactivation of p53, retinoblastoma (Rb) and/or PP2A by the simian virus 40 (SV40) large (LT) and small T antigens extends the life span of the cells and endows them with the ability to invade Matrigel-coated transwells. In this study, we show that concurrent inhibition of p53 and Rb by the SV40 LT produces cells (referred to as SBOT4-LT) with increased life span and cell invasion. To distinguish the roles of p53 and Rb in the progression from SBOTs to invasive ovarian carcinomas, we performed small interfering RNA-mediated knockdown of endogenous p53 in a spontaneously immortalized SBOT cell line, SBOT3.1, which increased cell invasion. This increased invasive activity was associated with the transcriptional downregulation of E-cadherin, correlated with an increase in PIK3CA levels and the increased activation of Akt. Conversely, in invasive LGC-derived MPSC1 cells, enhancing the levels of p53 decreased cell invasion and diminished the phosphatidylinositol 3-kinase (PI3K)/Akt-mediated downregulation of E-cadherin. Inhibition of Rb also enhanced invasiveness, but did not affect the levels of PIK3CA and E-cadherin in SBOT3.1 cells, suggesting that it functions by a different pathway. To our knowledge, this study is the first to show that p53 has an important role in the progression from SBOTs to invasive carcinomas. In addition, our findings suggest that downregulation of E-cadherin by the PI3K/Akt pathway contributes to this progression.

Wnt signaling follows canonical and non-canonical pathways to regulate a variety of processes during cellular homeostasis and development. The large T-antigen (T-Ag) of the human neurotropic JC virus, has been shown to modulate the Wnt-signaling pathway via interaction with beta-catenin, one of the most important components of the canonical Wnt pathway. Here, we have identified an alternative non-canonical pathway that allows T-Ag to recruit Rac1 for stabilizing beta-catenin by inhibiting its ubiquitin-dependent proteasomal degradation. We demonstrate that inhibition of Rac1 by its dominant negative mutant, RacN17, abrogates T-Ag-mediated stabilization of beta-catenin yet exhibits no impact on the transcriptional activity of beta-catenin. Results from immunocytochemistry revealed that together with T-Ag, a pool of beta-catenin appears at the cell surface, particularly at the membrane ruffles where active Rac1 is positioned. Interestingly, cooperativity between T-Ag and beta-catenin leads to activation of Rac1, which in turn, stimulates its association with beta-catenin. These observations unravel the interplay between beta-catenin and Rac1 that is initiated by T-Ag and results in stabilization of beta-catenin and its presence in cell membrane ruffles.

Hepatoblasts are bipotent progenitors of both hepatocytes and cholangiocytes. The lack of stable in vitro culture systems for such cells makes it necessary to generate liver progenitor cell lines by means of immortalization. In this study, we describe the long-term behaviour of a clone of simian foetal hepatic progenitor cells immortalized by Simian virus 40 (SV40) large T-antigen (T-Ag) flanked by loxP sites. Immortalization was associated with the re-expression of telomerase activity, which decreased at late passages (population doubling 120) after more than a year in culture. This decrease was concomitant to telomere shortening and karyotypic instability. However, the chromosomes carrying the p53 gene remained intact and long-term immortalized progenitor cells maintained contact inhibition and proliferative properties. They also displayed the features of a normal bipotent phenotype. We constructed a retroviral vector expressing an inducible Cre recombinase and transferred it into the immortalized progenitors. Activation of the Cre recombinase by 4-hydroxy-tamoxifen induced SV40 T-Ag excision, leading to the death of cells expressing Cre recombinase. Immortalized progenitors at late passages stopped growing and eventually disappeared after transplantation into the livers of immunocompromised mice. These cells provide a novel model to study hepatic differentiation and carcinogenesis.

The human polyomavirus, JC virus, has recently been associated with several human CNS tumors, including medulloblastomas and a broad range of glial-origin tumors. This ubiquitous virus is the causative agent of the rare demyelinating disease, progressive multifocal leukoencephalopathy in immunocompromised individuals. Expression of the viral protein, T-antigen, which possesses the ability to transform cells of neural origin, has been detected in human CNS tumors. In an effort to further understand the transforming potential of JCV T-antigen, transgenic mice expressing JCV T-antigen under the control of the Mad-4 promoter were generated. As described previously, approximately 50% of the animals developed pituitary tumors by 1 year of age. However, a small subset of the animals developed solid masses arising from the soft tissues surrounding the salivary gland, the sciatic nerve, and along the extremities that histologically resemble malignant peripheral nerve sheath tumors, rare neoplasms that occur in individuals with neurofibromatosis type 1 (NF1). JCV T-antigen was detected in tumor tissue by immunohistochemistry and immunoprecipitation/Western blotting, but not in normal tissues and was colocalized with NF2, the putative tumor suppressor protein associated with neurofibromatosis type 2, in the nucleus of some cells. In addition, T-antigen was co-precipitated with NF2, but not with NF1 protein, although NF1 was detectable in tumor tissue. Furthermore, precipitated immunocomplexes contained T-antigen, NF2, and p53, suggesting that these three proteins may form a ternary complex. The importance of these findings on mechanisms of T-antigen-mediated tumorigenesis and the pathogenesis of neurofibromatosis are discussed. Oncogene (2004) 23, 5459-5467. doi:10.1038/sj.onc.1207728 Published online 10 May 2004

Expression of the JCV early protein T-antigen in transgenic mice leads to the development of cerebellar primitive neuroectodermal tumors (PNETs). In light of earlier reports on the association of JCV with PNETs in humans and the involvement of the Wnt signaling pathway in the development of cerebellar tumors, we investigated the interplay between T-antigen and beta-catenin, the key protein of the Wnt pathway. Our results demonstrate the physical interaction of T-antigen with beta-catenin through the central domain of T-antigen spanning residues 82-628, and the C-terminus of beta-catenin located between amino acids 695 and 781. The association of T-antigen with beta-catenin elevates the level of beta-catenin in the cells due to increased in the stability of the protein. In the presence of T-antigen, beta-catenin was found in the nuclei of cells, suggesting that the interaction of beta-catenin with T-antigen facilitates its nuclear import. In cells expressing mutant T-antigen with no nuclear localization domain, beta-catenin was found in the cytoplasm. Coexpression of T-antigen with beta-catenin increased the transcription of the c-myc promoter, a known downstream target of beta-catenin, and artificial promoter whose activity is beta-catenin dependent. These observations ascribe a new oncogenic pathway for T-antigen, and offer an alternative mechanism for the deregulation of the Wnt pathway through stabilization of beta-catenin upon its association with the viral oncoprotein.

Maspin is a unique serpin involved in the suppression of tumor growth and metastasis. To investigate whether increased levels of maspin protect against tumor progression in vivo, we established a transgenic model in which maspin is targeted to mammary epithelial cells by the Whey Acidic Protein (WAP) promoter for overexpression. We crossed these WAP-maspin transgenic mice with the WAP-TAg mouse model of tumor progression. Maspin overexpression increased the rate of apoptosis of both preneoplastic and carcinomatous mammary epithelial cells. Maspin reduced tumor growth through a combination of reduced angiogenesis and increased apoptosis. The number of pulmonary metastases was reduced in the presence of maspin overexpression. These data demonstrate that targeted overexpression of maspin can inhibit tumor progression in vivo, likely through a combination of increased apoptosis, decreased angiogenesis, and inhibition of tumor cell migration.

The BRCT region, the carboxyl-terminus of BRCA1 (the breast cancer susceptibility gene 1 product), is ubiquitous in several proteins that participate in cell cycle checkpoints and DNA repair. We have previously shown that the BRCT regions of TopBP1 (DNA topoisomerase II binding protein 1) and BRCA1 bound DNA breaks. A BRCT-related region, BRCT-W1, in the retinoblastoma susceptibility gene product (Rb) also could bind DNA fragments, independently of DNA sequences. Five BRCT-W regions were found in the Rb family. All BRCT-Ws of Rb bound DNA fragments. Electron microscopy and treatment with an exonuclease showed that BRCT-Ws bound double-strand DNA breaks. Since some BRCTs are exceptional common relating elements in tumor suppression, our findings reveal novel aspects of the tumor suppression mechanism.

The simian double strand DNA tumor virus SV40 encodes the 90-kDa multi-functional protein, large T antigen (LT). LT functions by binding to DNA, as well as to many cellular target proteins such as p53 and retinoblastoma protein (pRB). We report here the identification of a cellular heat shock protein, HSP90, as a previously undescribed LT-associated protein. Immunoprecipitates by anti-HSP90 antibodies from LT-expressing cell lysates contained LT protein, as revealed by Western blotting. Conversely, anti-LT antibody co-immunoprecipitated HSP90. Co-immunoprecipitation of HSP90 and LT was observed even after complete immuno-depletion of p53, indicating that the association of LT with HSP90 is p53-independent. LT-HSP90 complexes can be reconstituted from purified HSP90 and unfolded-LT in vitro in an ATP-independent manner but not from HSP90 and native LT, suggesting that non-mature conformation of LT is required for the efficient association with HSP90. Moreover, geldanamycin, an anti-tumor drug that specifically binds and inhibits HSP90, reduced the intracellular concentration of LT by destabilizing newly synthesized LT. The above results suggest that HSP90 associates with immature forms of LT both in vivo and in vitro, and thus might assist LT in the formation of a functional, mature structure.

The retinoblastoma tumor suppressor RB and its related proteins, p107 and p130, are targets of several viral oncoproteins, including SV40 large T-antigen (T-Ag) and adenovirus E1A. T-Ag and E1A each contains an LXCXE-peptide motif through which binding to the conserved 'A/B pocket' present in RB, p107 and p130 is achieved. The LXCXE-binding activity of RB, we have previously shown, is inhibited by phosphorylation at Thr 821 and 826 in the C-terminal region of RB. Thr 821 and its surrounding sequence is unique to RB and not found in p107 or p130. Interestingly, hyperphosphorylation of p107 does not disrupt its ability to bind T-Ag or to inhibit the transactivating function of E1A. Insertion of a fourteen amino acid sequence of RB containing Thr 821 into p107 prevents binding of T-Ag and E1A to phosphorylated p107. These results show that the RB sequence surrounding Thr 821 plays a critical role in the regulation of the 'A/B pocket' function. In addition, these data indicate that the protein binding functions of RB and p107 are not equivalently regulated by phosphorylation.

SV40 large T antigen (T) inactivates the tumor suppressor proteins p53 and pRb, and can induce cells to enter DNA replication at inappropriate times. We show here that T also compromises three cell cycle checkpoints that regulate the entry into and exit from mitosis. Human diploid fibroblasts infected with a retrovirus expressing T displayed an attenuated radiation-induced mitotic delay, were more susceptible to chemical-induced uncoupling of mitosis from the completion of DNA replication, and were more likely to exit mitosis and rereplicate their DNA when mitotic spindle assembly was inhibited. Consistent with altered mitotic checkpoint control, cells expressing T displayed elevated protein levels and/or associated activities of the mitotic regulatory proteins cyclin A, cyclin B, Cdc25C and p34(cdc2). These changes in mitotic control were evident within 5-10 population doublings after retroviral infection, indicating a direct effect of T expression. Cells acutely infected with the T-expressing retrovirus suffered numerical and structural chromosome aberrations, including increases in aneuploidy, dicentric chromosomes, chromatid exchanges and chromosome breaks and gaps. These findings indicate that T rapidly disrupts mitotic checkpoints that help maintain genomic stability, and suggest mechanisms by which T induces chromosome aberrations and promotes the immortalization and neoplastic transformation of human cells.

Hepatocyte Growth Factor (HGF) receptor, encoded by the protooncogene c-met, is overexpressed in many human tumours, including those of thyroid epithelium. The absence in most cases of any primary structural abnormality of the met gene suggests that overexpression is secondary to mutation of other gene(s). To test this hypothesis we investigated the effect on met expression of two activated oncogenes known to play a major role in thyroid oncogenesis, ras and ret. To minimize the possibility of unknown co-operating events, we introduced these genes directly into normal human thyrocytes in primary culture using amphotropic retroviral vectors and assessed met expression as early as possible in the resulting epithelial colonies. Double immunofluorescence revealed expression of met protein, strictly localized to cells expressing the mutant ras and ret vectors, expression in background normal cells being barely detectable. In contrast, colonies induced to proliferate at a comparable rate by a vector expressing SV40 T showed no increase in met expression. To permit quantitation by Western blotting we also extended these findings to a thyroid cell line (R18) containing a zinc-inducible mutant ras gene. Induction of the oncogene led to a fourfold increase in met protein expression. We conclude that overexpression of met is induced by activation of the ras or ret signalling pathway and not simply by deregulation of the cell cycle per se. The data suggest that the proliferative advantage conferred by these oncogenes may be in part due to the resulting sensitization of tumour epithelium to paracrine HGF secreted by stromal cells.

Although established cell lines can be transformed with oncogenic ras, primary epithelial cells cannot, but require the coexpression of an immortalizing oncogene, such as the E1A region of adenovirus. We have previously shown that immortalization of primary epithelial cells by E1A 12S requires the expression of five regions encoded by both the first and second exons of the gene. However, only three of these regions, located in the first exon, are required for cotransformation of primary cells with an activated ras oncogene. Thus, the expression of oncogenic ras is able to abrogate the need for the E1A function(s) encoded by the second exon that are required for immortalization. This suggested the possibility that the functions encoded by the second exon of E1A may involve or interact with the normal ras signal transduction pathway. The results described herein demonstrate that immortalization-competent 12S gene products induce the expression of a novel 110 kD protein, p110, that forms a stable complex with rasGAP. Failure to induce the p110-rasGAP complex results in the concomitant loss of ability of 12S to immortalize primary epithelial cells. The appearance of this complex parallels the expression of the 12S protein and is sensitive to the levels of E1A 12S. p110 induction is independent of the ability of 12S to activate the cell cycle and of the presence of adenovirus E1B and is not observed in the presence of the large T antigen of SV40. Thus, it is not a general response to proliferation or tumorigenic transformation, but rather seems to be specific to the immortalization function(s) of E1A 12S.

The effects of heat treatment on key cell cycle regulatory molecules is currently unknown. Using primary mouse kidney cell cultures induced to re-enter S-phase by transforming infection with SV40, the effects of thermal treatment on the phosphorylation state of the Retinoblastoma gene product (pRb) and on its ability to bind T-antigen were examined. Time course analysis showed no detectable inhibition of cellular protein and T-antigen synthesis up to 180 min treatment at 42.5 degrees C, while a gradual disappearance of the phosphorylated forms of pRb was observed after 90 min. Dephosphorylation of pRb was shown to correlate with inhibition of SV40-induced DNA synthesis. Also, large T-antigen/pRb protein complex was affected since a gradual dissociation of the hypophosphorylated pRb from large T-antigen was observed concomitant to the inhibition of DNA synthesis. The effects of heat treatment were found to be reversible after shifting the cultures to 37 degrees C. Cells were shown to resume DNA synthesis concomitant to the reappearance of the phosphorylated forms of pRb and rebinding of the hypophosphorylated form to large T-antigen within 6-8 h after recovery at 37 degrees C. In addition, no evidence for a bona fide hsp71/large T-antigen protein complex was seen under the experimental conditions used. The data strongly suggest that inhibition of DNA synthesis by heat treatment might involve either an increase in phosphatase activity or the inhibition of a pRb kinase activity.

Simian virus 40 (SV40) large tumor antigen (T) is an oncoprotein whose biological and biochemical functions appear to be modulated by phosphorylation. Recently, SV40 DNA replication in vitro has been shown to be activated by dephosphorylation involving the activity of a serine/threonine phosphoprotein phosphatase belonging to the type 2A class (PP2A) [Virshup, D.M., Kauffman, M.G. & Kelly, T.J. (1989) EMBO J., 8, 3891-3898]. To address the question of how specificity of PP2A activity towards T is regulated, an in vitro assay to study the process of T dephosphorylation was developed. Unlabeled extracts from cells enriched for various stages of the cell cycle were incubated with 32P-labeled, immunocomplexed T. Extracts from a population of cells enriched for S phase demonstrated a selective ability to dephosphorylate this labeled protein when compared with extracts prepared from G1- and M-phase cells. The time course of release from growth arrest demonstrated that this T-specific phosphatase activity occurred at the onset of host-cell DNA synthesis. In contrast, when using 32P-labeled phosphorylase a as the substrate, phosphatase activity appeared to be present throughout the cell cycle. The data presented here are consistent with the notion that PP2A activity towards T is regulated in a cell cycle-dependent manner.

The adenovirus early region 1A (E1A) proteins associate with several cellular proteins in adenovirus infected or transformed cells. Recently, two of the cellular proteins that bind to E1A encoded proteins have been identified. p105 has been shown to be the product of the retinoblastoma tumor suppressing gene. p60 has been shown to be a human cyclin A. Previously studies have shown that E1A protein sequences encoded by conserved domains 1 and 2 are required for interactions with the retinoblastoma protein (pRB). We have demonstrated here that amino acids 30 to 60 and 121 to 127 within the E1A proteins are required for interaction with p60/cyclin A. These are the same sites within conserved domains 1 and 2 that are required for E1A protein association with pRB. However, the association of p60/cyclin A does not appear to require pRB. We also demonstrate that another cellular protein, 130K, interacts with E1A at essentially the same sites. It is interesting that mutations in these regions destroy the ability of E1A to function as an oncogene, thereby raising the possibility that interaction with several different cellular proteins may be needed for transformation by E1A.

Using analytical flow cytometry we have monitored changes in the conformation of p53 through the cell cycle under different conditions of cell growth. Conformational variants of murine p53 are characterized by reactivity with monoclonal antibodies PAb246, PAb248 and PAb421. In proliferating cells, p53 conformation was independent of the cell cycle. Addition of fresh medium, however, resulted in loss of p53 reactivity with PAb246 (p53-246(0]. Mutant p53, which lacks suppressor function, is also p53-246(0). Thus in SV3T3 cells p53-246(0) may reflect a change in p53 tertiary structure that is induced by growth stimulation and is compatible with the normal cell growth response. We argue that p53-246(0) and p53-246+ may exert positive and negative constraints in cell growth control. By stabilising p53-246(0) activating mutants of p53 would favour cell proliferation with dominant effect.

Expression of the middle-T antigen of polyomavirus is sufficient to induce transformation of fibroblasts in culture and tumour formation in whole animals. Middle-T can form a complex with the cellular src gene product (p60c-src) and can be phosphorylated by p60c-src in vitro. Studies using middle-T mutants have suggested that the association of middle-T with p60c-src may be necessary but not sufficient for transformation. Therefore, we addressed the possibility that middle-T could interact with other tyrosine protein kinases structurally related to p60c-src. Using antibody raised against a fusion protein between beta-galactosidase and amino-terminal sequences of p90gag-yes from Y73 virus (anti-yes antibody), we have found that middle-T can associate with and be phosphorylated by the c-yes proto-oncogene product, a protein of relative molecular mass (Mr) 62,000 (62K). This raises the possibility that the middle-T-p62c-yes complex contributes to transformation by polyomavirus.

Simian virus 40 (SV40) large T antigen, a multifunctional protein necessary for lytic growth and cell transformation, is located mainly in the nucleus and in small amounts on the cell surface (surface T). Surface T may have a passive role in SV40 tumour rejection by cytotoxic T cells as a component of SV40-TSTA (tumour-specific transplantation antigen). The unusual induction of this immune response by immunizing mice with soluble T antigen led us to investigate the in vitro binding of T antigen to the surface of living cells in more detail. Our results show that native surface T and a minor subset of large T antigen having a high cell surface binding affinity in vitro, behave like integral membrane proteins. Several viral proteins including SV40 T antigen and cellular proteins seem to be linked to fatty acids (acylation). To analyse whether this mechanism is involved in the stable attachment of in vitro-bound T antigen to the plasma membrane of living target cells, we determined the degree of labelling of this molecule by using target cells prelabelled with 3H-fatty acid. Here we report that T antigen extracted from unlabelled SV40-transformed cells (SV80) becomes 3H-labelled after in vitro binding to the cell surface of 3H-palmitate-prelabelled HeLa cells. These results suggest that T antigen attached externally to living cells, may be anchored by tightly linked lipids.

Polyoma middle-T antigen is required for viral transformation of cultured cells and for tumorigenesis in animals. Like many other transforming gene products, middle-T is bound to the membrane and has an associated tyrosine kinase activity in vitro. This activity seems to result from the interaction of middle-T with pp60c-src, the cellular homologue of the transforming gene product of the Rous sarcoma virus, pp60v-src (refs 3-5). Both pp60v-src (ref. 6) and another retrovirus transforming gene product, pp68v-ros (ref. 7) were shown recently to have an associated phosphatidylinositol (PI) kinase activity in vitro and to increase PI turnover in vivo. These results suggest that viral transformation may be directly connected to a complex network of second messengers generated from PI turnover. Here, we assayed for PI kinase activity in immunoprecipitates made with middle-T- or pp60c-src-specific antisera of cells infected with polyoma virus. A PI kinase activity was detected in those immunoprecipitates which contained middle-T. Studies of mutants of middle-T defective in transformation indicate a close correlation between PI kinase activity and transformation.

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