Overexpression of epidermal growth factor receptor in urothelium elicits urothelial hyperplasia and promotes bladder tumor growth

Although urothelium is constantly bathed in high concentrations of epidermal growth factor (EGF) and most urothelial carcinomas overexpress EGF receptor (EGFr), relatively little is known about the role of EGFr signaling pathway in urothelial growth and transformation. In the present study, we used the uroplakin II gene promoter to drive the urothelial overexpression of EGFr in transgenic mice. Three transgenic lines were established, all expressing a higher level of the EGFr mRNA and protein in the urothelium than the nontransgenic controls. The overexpressed EGFr was functionally active because it was autophosphorylated, and its downstream mitogen-activated protein kinases were highly activated. Phenotypically, the urinary bladders of all transgenic lines developed simple urothelial hyperplasia that was strongly positive for proliferative cell nuclear antigen and weakly positive for bromodeoxyuridine incorporation. When coexpressed with the activated Ha-ras oncogene in double transgenic mice, EGFr had no apparent tumor-enhancing effects over the urothelial hyperplastic phenotype induced by Ha-ras oncogene. However, when coexpressed with the SV40 large T antigen, EGFr accelerated tumor growth and converted the carcinoma in situ of the SV40T mice into high-grade bladder carcinomas, without triggering tumor invasion. Our studies indicate that urothelial overexpression of EGFr can induce urothelial proliferation but not frank carcinoma formation. Our results also suggest that, whereas EGFr and Ha-ras, both of which act in the same signal transduction cascade, stimulated urothelial hyperplasia, they were not synergistic in urothelial tumorigenesis, and EGFr overexpression can cooperate with p53 and pRB dysfunction (as occurring in SV40T transgenic mice) to promote bladder tumor growth.     

Efficient replication of plasmids containing the SV40 origin in N-myc overexpressing human neuroblastoma cells.

Using test plasmids containing the SV40 origin, we found a wide spectrum of permissiveness to their replication in different human cell lines. N-myc overexpressing neuroblastoma cells were highly permissive. LA-N-1 neuroblastoma cells were the most permissive of all the cell lines that we tested including the homologous CV-1 or COS-1 monkey kidney cells. Other human cell lines expressing various amounts of c-myc, and the 293 cell line expressing adenovirus E1A and E1B exhibited intermediate levels of permissiveness. T24 and EJ bladder carcinoma cells, which do not express the myc genes, were nonpermissive. Transient expression of c-myc or N-myc from plasmid vectors resulted in a modest stimulation of replication. Replication of test plasmids containing different configurations of the SV40 origin region was activated by the myc proteins. The high efficiency of replication in LA-N-1 cells is due to a combination of reasons including the overproduction of N-myc, high efficiency of expression of the SV40 replication initiator protein large T antigen from a cotransfected expression plasmid (containing the T antigen gene under the RSV LTR control), and other unknown host cell replication stimulatory factors. Replication of test plasmids was not detected in N-myc or c-myc overexpressing cells when the T antigen expression plasmid was not provided, showing that the myc proteins cannot substitute for T antigen in SV40 DNA replication.     

Influence of a hamster tumor suppressor gene on transformation by viral and cellular oncogenes

To determine if the tumor suppressor gene active in BHK hamstercells acts to maintain the normal phenotype by influencing oncogenetransformation, careful, quantitative transfections with a varietyof oncogenes were performed on four closely related BHK subclones.Two of the clones had an active suppressor gene (sup+ clones)and two of them had lost the suppressor (sup- clones) yet remainedanchorage dependent. Both sup+ and sup- clones could be transformedto anchorage independence by ras, src, mos, neu, polyoma mTand SV40 suggesting that neither the presence nor the absenceof the suppressor gene in BHK limits the transforming abilityof these common oncogenes. All lines were resistant to transformationby N-myc, E1A and c-sis, oncogenes that may perform redundantfunctions in the immortal, fast growing BHK cell. SV40 smallt antigen which has previously been considered unable to transformcultured cells by itself, was nevertheless able to transformsup+ BHK lines to anchorage independence in the absence of theviral large T antigen. Clones of sup- cells expressing highlevels of small t antigen protein could be isolated, but theyremained anchorage dependent and in tumorigenicity assays retainedthe long latent period characteristic of normal BHK cells. Suchlines should enable the identification of cellular targets vitalto the transforming function of SV40 small t.     

Alterations of the thymic selection process in transgenic mice expressing SV40 large T antigen

We generated SV40 T antigen transgenic mice (lines SVT125, SVT127, and SVT248) which developed unique thymic carcinomas originating from thymic cortical epithelial cells. In these mice we observed alterations in the thymic selection process not reported before in SV40 T antigen transgenic mice. Along with tumor cell growth, thymocytes increased in number and the proportion of CD4 or CD8 single positive cells rose to 10 times the normal level. Expression of SV40 T antigen was detectable by Northern analysis in thymic stromal cells but not in thymocytes. Thymic stromal cell lines, derived from the thymic tumor, produced high levels of cytokines which caused morphological transformation and growth stimulation in hematopoietic stem cells, including fetal liver cells and bone marrow cells. These observations suggest that the unusual multiplication of thymocytes and the alterations in thymic selection are the result of the activity of thymic stromal cells transformed by SV40 T antigen. The cell lines derived from the tumor can thus be used to study cytokines involved in thymic differentiation of T cells. © 1996 Wiley-Liss, Inc.     

Enhanced expression of vascular endothelial growth factor (VEGF) plays a critical role in the tumor progression potential induced by simian virus 40 large T antigen

Vascular endothelial growth factor (VEGF), an important angiogenic factor, regulates cell proliferation, differentiation, and apoptosis through activation of its tyrosine-kinase receptors, such as Flt-1 and Flk-1/Kdr. Human malignant mesothelioma cells (HMC), which have wild-type p53, express VEGF and exhibit cell growth increased by VEGF. Here, we demonstrate that early transforming proteins of simian virus (SV) 40, large tumor antigen (Tag) and small tumor antigen (tag), which have been associated with mesotheliomas, enhanced HMC proliferation by inducing VEGF expression. SV40-Tag expression potently increased VEGF protein and mRNA levels in several HMC lines. This effect was suppressed by the protein synthesis inhibitor, cycloheximide. Inactivation of the VEGF signal transduction pathway by expression of soluble form of Flt-1 inhibited Flk-1/Kdr activation and HMC proliferation induced by SV40 early genes. Experiments with SV40 mutants revealed that SV40-Tag, but not -tag, is involved in the VEGF promoter activation. However, concomitant expression of SV40-tag enhanced Tag function. In addition, SV40-Tag expression sustained VEGF induction in colon carcinoma cell line (CCL)-233, which have wild-type p53, but not in CCL-238, which lack functional p53. These data indicate that VEGF regulation by SV40 transforming proteins can represent a key event in SV40 signaling relevant for tumor progression.     

Surface proteins of simian-virus-40-transformed cells

Mammalian cells transformed in tissue culture by SV40 were shown to contain, in addition to the SV40-coded 94,000 d large T antigen and the 20, 000 d small t antigen, a ～ 56,000 d cellular protein, which specifically precipitates with sera of animals bearing SV40-induced tumor (s) (tumor or T serum). We investigated the presence of these three proteins at the surface of logarithmically growing SV40-transformed cloned mouse cells, after metabolic labelling with [³⁵S]-methionine for 3 h. The 56,000 d protein was found to be susceptible to digestion by trypsin under conditions which did not disrupt the cells, while no small t antigen was found to be digested. Both the 56,000 d cellular protein and the SV40 large T antigen were susceptible to lactoperoxi-dase-catalyzed iodination from the outside of intact cells. Trypsin treatment removed both the iodinated 56,000 d protein and the iodinated SV40 large T antigen. These experiments indicated that (a certain amount of) the 56,000 d protein and a relatively small amount of the large T antigen (which is present mainly in the nucleus) are present on the cell surface. The results confirm and extend independent experiments using subcellular frac-tionation techniques (Luborsky and Chandrasekaran, 1980; Soule and Butel, 1979). After heat treatment (at 50°C for 30 min) of the whole-cell extract the 56,000 d cellular protein was precipitated by the tumor serum in the absence of precipitation of SV40 large T antigen. This result showed that the 56,000 d protein is more (thermostable (in the whole-cell extract) than the SV40 large T antigen, and also indicated that the tumor serum employed had antibodies against the 56,000 d cellular antigen. The heat-treated whole-cell extract of SV40-transformed mouse cells was able to immunize and fully protect mice against a lethal tumorigenic dose of SV40-transformed cells. These results suggest the need for further experiments to characterize the chemical and immunologic properties of the 56,000 d protein.     

Tumor suppressor protein WT1 inhibits autonomous DNA replication directly, as well as indirectly by causing loss of replicated DNA as a consequence of cell death induced by the protein.

The effects of the Wilms' tumor suppressor protein WT1 on autonomous DNA replication under stable transfection conditions were investigated. COS ts2 monkey kidney cells, which express the simian virus 40 (SV40) replication initiator protein large tumor antigen (TAg) as a temperature-sensitive protein, were stably transfected with SV40 origin-containing plasmids expressing WT1 from the Zn2+ and Cd2+-inducible metallothionein promoter. Stable transformant clones of cells containing the integrated plasmids were isolated at the non-permissive temperature, expanded, and shifted to the permissive temperature to allow autonomous replication of the plasmid and overexpression of WT1. Expression of WT1 triggered apoptosis of the cells. Analysis of the kinetics of occurrence of cell death and accumulation of the replicated plasmid indicated that WT1 inhibited replication directly, and also indirectly by causing loss of replicated plasmid as a consequence of WT1-induced cell death.     

Thyroid hormone receptor β suppresses SV40-mediated tumorigenesis via novel nongenomic actions

Accumulated evidence suggests that thyroid hormone receptor β (TRβ) could function as a tumor suppressor, but the detailed mechanisms by which TRβ inhibits tumorigenesis are not fully understood. The present studies explored the mechanisms by which TRβ acted to inhibit thyroid tumor development mediated by simian virus-40 (SV40). In mouse xenograft models, SV40 large T antigen (SV40Tag)-immortalized human thyroid epithelial (HTori) cells rapidly induced tumors, but the tumor development was totally blocked by TRβ stably expressed in HTori cells. Previous studies showed that the SV40Tag oncoprotein binds to and inactivates tumor suppressors p53 and retinoblastoma protein (Rb), thereby inducing tumorigenesis. Here we showed that one of the mechanisms by which TRβ suppressed tumor development was by competing with p53 and Rb for binding to SV40Tag. The interaction of TRβ with SV40Tag led to reactivation of Rb to inhibit cell cycle progression. TRβ- SV40Tag interaction also resulted in reactivating p53 to increase the expression of Pten, thus attenuating PI3K-AKT signaling to decrease cell proliferation and to induce apoptosis. The present study uncovered a novel action of TRβ as a tumor suppressor initiated via interfering with the recruitment of Rb and p53 by SV40Tag oncoprotein through protein-protein interaction, thereby acting to block tumor development.     

Establishment of human fibroma cell lines from a MEN1 patient by introduction of either hTERT or SV40 early region

Establishment of tumor cell lines as model systems for studying tumor biology or as a part of immunotherapeutic anti-cancer strategies is of high importance, whereby the highest possible preservation of the original tumor cell phenotype is a prerequisite for these aims. Since overexpression of the catalytic subunit of human telomerase (hTERT) is known to minimally alter the cellular phenotype, we focused on the establishment of cell lines derived from human fibroma from a MEN1 patient by ectopic expression of hTERT. Additionally, a cell line was generated by introduction of the early region of SV40 (SV40 ER). Both approaches resulted in continuous cell lines, and neither T1-LOHG (hTERT) nor SV1-LOHG (SV40 ER) showed a transformed phenotype. While SV40 ER-transfected cells underwent dramatic changes in morphology and growth characteristics, hTERT-expressing cells indeed retained a phenotype highly similar to the parental cells. Nevertheless, hTERT overexpression resulted in increased growth rates after about 70 population doublings (PD) and alterations of mRNA levels of genes associated with tumor pathogenesis. Thus, our data suggest that ectopic hTERT expression leads to immortalization of LOHG-F, sustaining many characteristics of the non-transfected counterparts, but continuous growth in vitro is associated with changes of the cellular phenotype.     

Human kin17 protein directly interacts with the simian virus 40 large T antigen and inhibits DNA replication

Kin17 is an evolutionarily conserved DNA-binding protein, which forms intranuclear foci in proliferating cells. Recent data have suggested that human kin17 protein is associated with cell proliferation and unrepaired DNA lesions. Herein, we show that human fibroblasts (MRC5-V2 and CHSV4) immortalized with SV40 overexpress endogenous kin17 protein, as compared with normal diploid human fibroblasts. We observed that certain carcinoma cell lines also up-regulated kin17 protein, suggesting that increased kin17 protein levels may be a consequence of the immortalized phenotype. We report here that the endogenous kin17 protein is located in nucleoplasmic foci and colocalizes with SV40 large T antigen. Purification of human kin17 protein allowed analysis of the physical interaction with T antigen by several in vitro and in vivo assays. Large T antigen and human kin17 protein are part of the same high molecular weight multiprotein complex in human cells. Furthermore, human kin17 protein interacts with T antigen bound to the SV40 DNA origin of replication. Strikingly, the overexpression of human kin17 protein in vivo and the introduction of increased amounts of human kin17 protein in an in vitro assay reduced T-antigen-dependent DNA replication, suggesting that kin17 protein may be involved in the DNA replication process in human cells.     

Manganese superoxide dismutase overexpression inhibits the growth of androgen-independent prostate cancer cells

This study investigates the role of the antioxidant enzyme manganese superoxide dismutase (MnSOD) in androgen-independent human prostate cancer (PC-3) cells' growth rate in vitro and in vivo. MnSOD levels were found to be lower in parental PC-3 cells compared to nonmalignant, immortalized human prostate epithelial cells (P69SV40T). To unravel the role of MnSOD in the prostate cancer phenotype, PC-3 cells were stably transfected with MnSOD cDNA plasmid. The MnSOD protein and activity levels in clones overexpressing MnSOD were increased seven- to eightfold. These cell lines showed elongated cell doubling time, reduced anchorage-independent growth in soft agar compared to parental PC-3 (Wt) cells, and reduced growth rate of PC-3 tumor xenografts in athymic nude mice. Flow cytometric studies showed an increase in membrane potential in the MnSOD-overexpressing clone (Mn32) compared to Wt and Neo cells. Also, production of extracellular H(2)O(2) was increased in the MnSOD-overexpressing clones. As determined by DNA cell cycle analysis, the proportion of cells in G(1) phase was enhanced by MnSOD overexpression. Therefore, MnSOD not only regulates cell survival but also affects PC-3 cell proliferation by retarding G(1) to S transition. Our results are consistent with MnSOD being a tumor suppressor gene in human prostate cancer.     

Single-steep transformation of human breast epithelial cells by SV40 large T oncogene.

Normal human mammary epithelial cell (HMEC) cultures originating from 2 mammoplasty reduction surgical samples were transfected with replication-defective SV 40 DNA. Two independent cell lines designated as S2T2 and S1T3, selected for their increased proliferation potential and lifespan, were propagated for greater than 22 months in culture. They maintained a near-diploid karyotype with few chromosomal markers such as trisomy 1q (S1T3) and trisomy 8q (S2T2), which are most common in breast cancer in vivo. Immortalized S1T3 cells were not tumorigenic, whereas S2T2 cells produced slowly growing tumors in nude mice. One tumor was propagated in vitro and the transformed NS2T2 cell line subsequently raised 100% large tumors in the nude mouse. Rearrangement of the SV40 genome was observed in NS2T2 cells, which was not associated with increased expression of large T antigen. S1T3, S2T2 and transformed NS2T2 cell lines expressed cytokeratins CK18, CK19, the mammary-specific antigen DF3, and functional EGF receptors. Single-step immortalization and malignant transformation of human breast epithelial cells can thus occur upon transfection with SV40 large T oncogene. The chromosomal abnormalities observed in these cell lines suggest that they could offer a model for the study of breast-tumor progression in vitro.     

Enhanced kinase-activity in sv40-transformed cells may be compensated by enhanced phosphatase-activity in revertants as reflected by phosphorylation of p53

Transformation of rat cells with SV40 large T antigen results in activation of protein kinases and hyperphosphorylation of the tumor suppressor protein p53. In searching for cellular targets involved in SV40-mediated transformation, flat revertants of SV40-transformed rat cells had been isolated carrying a presumptive defect in a cellular gene (Bauer et al: J Virol 61: 1821, 1987). In this investigation, we asked whether the phosphorylation state of p53 might be affected in the revertants. Two-dimensional phosphopeptide analyses revealed indeed a characteristic reduction of phosphorylation of p53 compared to the parental transformed cells. However, when we employed the phosphatase inhibitor okadaic acid in vivo hyperphosphorylation of p53 resumed indicating that the kinases involved in phosphorylation of p53 were fully active but counterbalanced by enhanced phosphatase activity. Indeed, the phosphate turnover of p53 in vivo and phosphatase activity towards p53 in vitro was higher in the revertants than in the parental transformants. These findings demonstrate that the transformation state of a cell correlates with the phosphorylation state of p53 which in turn can be regulated in different ways, enhanced kinase activity in transformed cells may be counteracted by enhanced phosphatase activity in revertant cells.     

Evidence against a role for SV40 in human mesothelioma

SV40 has been implicated in the etiology of 40% to 60% of human mesotheliomas. These studies could have important medical implications concerning possible sources of human infection and potential therapies if human tumors are induced by this agent. We did PCR-based analysis to detect SV40 large T antigen DNA in human mesotheliomas. None of 69 tumors in which a single copy gene was readily amplified contained detectable SV40 large T antigen sequences. Under these conditions, it was possible to detect one copy of integrated SV40 DNA per cell in a mixture containing a 5,000-fold excess of normal cells using formalin-fixed preparations. Kidney, a known reservoir of SV40 in monkeys, from some of these individuals were also negative for SV40 large T antigen sequences. A subset of mesotheliomas was analyzed for SV40 large T antigen expression by immunostaining with a highly specific SV40 antibody. These tumors as well as several human mesothelioma cell lines previously reported to contain SV40 large T antigen were negative for detection of the virally encoded oncoprotein. Moreover, mesothelioma cell lines with wild-type p53 showed normal p53 function in response to genotoxic stress, findings inconsistent with p53 inactivation by the putative presence of SV40 large T antigen. Taken together, these findings strongly argue against a role of SV40 by any known transformation mechanism in the etiology of the majority of human malignant mesotheliomas.     

Properties of transformed hamster cells containing SV40 tumor antigen in the cytoplasm

The properties of hamster cells containing SV40 tumor (T) antigen in the cytoplasm, rather than the nucleus, were determined. Eight cell lines were established from eight tumors induced by hamster embryo fibroblasts transformed in vitro by PARA (2cT)-adenovirus 7. Six cell lines contained only cytoplasmic SV40 T-positive cells while two were a mixture of nuclear T-positive and cytoplasmic T-positive cells. All the cell lines contained SV40 S antigen, all caused the production of SV40 T antibody in vivo, and four elicited the production of adenovirus T antibody. The cell lines seem to have acquired an infinite life span in vitro. The localization of T antigen apparently can be a stable phenomenon because five of the cell lines have retained T antigen exclusively in the cytoplasm for over 40 passages in tissue culture. The cytoplasmic T antigen could be detected by complement fixation in addition to immunofluorescence. Cytoplasmic T-positive cells were readily transplantable in vivo and contained SV40 TSTA demonstrable by both immunogenicity and immunosensitivity procedures. Two of the three cytoplasmic variants of PARA appear to be weakly oncogenic in newborn hamsters.     

SV40-induced transformation of cells of the lizard Gekko gecko

Attempts to infect four cell lines of reptilian origin, GL1 (Gekko gecko), IgH-2 (Iguana iguana) and VH2 and VSW (both Vipera russelli), with SV40 led to successful infection of the lizard cell line GL1 only. SV40 infection, indicated by acquisition of persisting T antigen in 100% of the cell population, was obtained in cells inoculated and maintained at 23, 30 or 35°C. (The optimal temperature for GL1 cell growth is 30°C). In early passages, infected GL1 cells exhibited a more orderly growth pattern and a more uniform cell morphology than did control cells. However, at about the 50th passage level after infection, a more exuberant growth pattern developed, characterized by cell saturation densities exceeding those of control cell cultures approximately threefold. Infectious virus in low titer was recovered from cells and media of infected cell lines tested during the first 21 passages post infection. At subsequent passage levels, virus could be rescued from cells maintained at each of the three different incubation temperatures by co-cultivation with AGMK cells, but not directly from the cultures. The presence of SV40 tumor-specific transplantation antigen was demonstrated by experiments in which hamsters inoculated with infected GL1 cells received marked protection against SV40 tumor induction. Further evidence of the altered state of the SV40-infected cells was provided by demonstration of increased resistance to inhibition of cell growth by dextran sulfate, and an enhanced ability of the cells to multiply at supraoptimal temperatures. The evidence indicates that cells of a poikilothermic vertebrate have been transformed in vitro by the mammalian papovavirus SV40. This is the first demonstration of which we are aware of viral transformation of poikilothermic cells in vitro.     

The SV40 S antigen; A carcinoembryonic-type antigen of the hamster?

In addition to hamster tumor cells induced or transformed by SV40, hamster anti-SV40 S (Surface) antiserum also reacted with non-SV40-exposed cell lines spontaneously induced (BHK 21) or transformed by heterologous oncogenic DNA and RNA viruses in the indirect membrane fluorescent antibody test. The antiserum titered equally with both BHK and SV40-transformed cells and the reaction could be absorbed with either of these cell lines, or with hamster embryo. The antiserum also reacted with early hamster embryo cells, although various organ cells from late fetuses, newborns and adults were negative. Mouse cell lines spontaneously induced (BALB/c-3T3) or transformed by SV40 and adenovirus 12 were also non-reactive. The results suggest that the antigen in question is a hamster somatic antigen present during embryonic life and derepressed in lines of actively growing cells.     

Transformation of late passage insulin-like growth factor-I receptor null mouse embryo fibroblasts by SV40 T antigen

There is evidence that the insulin-like growth factor-I (IGF-I) receptor is required for transformation by a variety of viral and cellular oncogenes in a mouse embryo fibroblast model. To further investigate the IGF-I receptor signaling pathways that are required for the permissive effect of the receptor on transformation by SV40 T antigen, we established three independent fibroblast cell lines each from wild-type and IGF-I receptor null embryos (R-). We transfected the wild-type and R- cell lines with an SV40 T antigen plasmid and selected three clones from each cell line that expressed T antigen. As in previous reports, none of the cloned R- cell lines expressing T antigen were transformed as measured by the ability to form large colonies in soft agar. However, with further passage, all three T antigen-expressing clones from one of the R- cell lines (R(-)3) formed large colonies in soft agar and the transformation of these T antigen-expressing clones was confirmed by tumorigenesis experiments in immunodeficient mice. DNA microarray analysis comparing gene expression between early passage and late passage R(-)3/T antigen clones showed, among other changes, an increase in the expression of ErbB-3 mRNA in the late passage clones. Also, the expression of ErbB-3 protein was dramatically increased in the late passage R(-)3/T antigen clones. We conclude that late passage IGF-I receptor null mouse embryo fibroblasts can be transformed by SV40 T antigen, and that ErbB-3 may play a role in permitting transformation by T antigen.     

Altered phosphorylation at specific sites confers a mutant phenotype to SV40 wild-type large T antigen in a flat revertant of SV40-transformed cells

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