Fluctuation of simian virus 40 (SV40) super T-antigen expression in tumors induced by SV40-transformed mouse mammary epithelial cells.

Higher-molecular-weight forms of the simian virus 40 (SV40) large tumor antigen (T-Ag), designated super T-Ag, are commonly found in SV40-transformed rodent cells. We examined the potential role of super T-Ag in neoplastic progression by using a series of clonal SV40-transformed mouse mammary epithelial cell lines. We confirmed an association between the presence of super T-Ag and cellular anchorage-independent growth in methylcellulose. However, tumorigenicity in nude mice did not correlate with the expression of super T-Ag. In the tumors that developed in nude mice, super T-Ag expression fluctuated almost randomly. Cell surface iodination showed that super T-Ag molecules were transported to the epithelial cell surface. The biological functions of super T-Ag remain obscure, but it is clear that it is not important for tumorigenicity by SV40-transformed mouse mammary epithelial cells. Super T-Ag may be most important as a marker of genomic rearrangements by the resident viral genes in transformed cells.     

Tumorigenicity of revertants from an SV40-transformed line

A syndrome of in vitro properties correlates with the tumorigenicity of SV40-transformed rodent cells. These properties are plasminogen activator production, loss of large actin cables, and anchorage-independent growth. An established rat fibroblast line, its SV40 transformant, several T-antigen negative revertants, and a spontaneous retransformant isolated form one of the revertants were analyzed in vivo for their tumorigenicity and in vitro for the syndrome. The two transformed lines were highly tumorigenic, and had clearly abnormal in vitro properties. The parental rat line was weakly tumorigenic in nude mice and demonstrated a slightly transformed response in the in vitro assays. The revertants were completely nontumorigenic. Expression of the in vitro syndrome was not uniform for all revertants; however, most cell lines maintained the correlation of the syndrome and tumorigenicity.     

"Transformation" of human endothelial cells by SV40 virions

Human endothelial cells derived from the umbilical vein were transformed with SV40 virions. A cell line subcultured for over 60 serial passages was characterized in comparison with its untransformed counterpart which was culturable for less than five passages. The SV40-transformed human endothelial cells, designated SV-HUVEC, were positive not only for tumor (T) antigen specific to the SV40-transformed cell, but also for two markers of endothelial cells, Factor VIII-related antigen and a receptor for Ulex europaeus agglutinin I. By transformation the growth potential of the human endothelial cells was increased and their serum requirement was decreased. The SV40-transformed endothelial cells were, however, unable to form colonies in soft agar or to form tumors in athymic nude mice, although a small nodule was produced at the site of inoculation. Subcultivation of these cells up to the 62nd passage eventually resulted in crisis and loss of further cell division. Thus, the human endothelial cells were transformed by SV40 while retaining certain normal functions but without showing tumorigenicity.     

Viral gene inhibition of class I major histocompatibility antigen expression: not a general mechanism governing the tumorigenicity of adenovirus type 2-, adenovirus type 12-, and simian virus 40-transformed Syrian hamster cells

The association between the level of class I major histocompatibility (MHC) antigen expression and the tumorigenic phenotype was determined for cells from a series of 15 lines of adenovirus type 2 (Ad2)-, Ad12-, and simian virus 40 (SV40)-transformed hamster cells and 16 lines of cells established from hamster tumors induced by SV40 mutants. These cells range from nontumorigenic to highly tumorigenic in both syngeneic and allogeneic adult hamsters. The Ad2-transformed cells--cells that were nontumorigenic in syngeneic adult hamsters--expressed either high levels or low levels of class I MHC antigens. The SV40-transformed cells--cells transformed in vitro that produced tumors with equal efficiency in both syngeneic and allogeneic adult hamsters--or cells derived from SV40-induced tumors expressed very high levels of class I MHC antigens. The Ad12-transformed cells uniformly expressed low levels of class I MHC antigens; these cells produced tumors 200- to 1,000-fold less efficiently in allogeneic adult hamsters than in syngeneic adult hamsters and produced tumors with about the same efficiency in immunoimmature newborns and immunocompetent syngeneic adult hamsters. We conclude that the expression of either high levels or low levels of class I MHC antigens is, at most, a minor factor in the differences observed among these adenovirus- and SV40-transformed cells in their tumor-inducing capacity in naive, immunocompetent hamsters.     

Immunologic selection of simian virus 40 (SV40) T-antigen-negative tumor cells which arise by excision of early SV40 DNA.

A clonal line of highly oncogenic spontaneously transformed mouse cells (104C) was transformed in tissue culture by simian virus 40 (SV40) and subsequently recloned (106CSC). This 106CSC cell line expressed T antigen and transplantation antigen but was about 100 times less tumorigenic than the 104C parent. When 10(5) 106CSC cells were injected into immunocompetent syngeneic mice, tumors were produced. From such tumors, cell lines were established in culture, all of which were consistently negative for T antigen. We found previously by solution DNA hybridization methods that the tumor cells were depleted in the early region of SV40 DNA which codes for the T antigen. We postulated that this loss occurs through a DNA rearrangement of unknown mechanism in one or a few 106CSC cells and that the tumors are then produced from such a cell or cells, whereas all the T-antigen-positive 106CSC cells are rejected by immunologic means. In this investigation we showed by the DNA transfer method using appropriately selected SV40 DNA probes that indeed the tumor cell clone (130CSCT) we selected to investigate came from one 106CSC cell in which the T-antigen-coding SV40 DNA sequences (but not all the early SV40 DNA sequences) were lost by an excision and recombination mechanism. We also showed that the 130CSCT cells, which are highly tumorigenic, could again be transformed by SV40 and that the resulting T-antigen-positive cloned derivative cells became much less tumorigenic (approximately 10(5)-fold), apparently again because of immunologic recognition and rejection. Indeed, when 10(7) T-antigen-positive cloned cells were injected, all the T-antigen-positive cells were rejected and the tumor was produced again from one or more T-antigen-negative cells. Thus, a one-step in vivo transplantation experiment allowed a selection (for tumorigenicity and against the SV40 T antigen) of a mutant mammalian cell with a DNA deletion at a definable site.     

Properties of T antigen-positive phenotypic revertants isolated from SV40-transformed rat and mouse cells

Four T antigen-positive phenotypic revertants were isolated by negative selection with BUdR from SV40-transformed rat and mouse cells which contain six and two viral genome equivalents per cell, respectively. Karyological analysis indicated that one rat and one mouse revertant had a hyperploid number of chromosomes, while the remaining two rat revertants had a subtetraploid number similar to those of the transformed parent cells. The hyperploid revertants were unable to grow in soft agar medium and were nontumorigenic in nude mice. One of the subtetraploid revertants formed large colonies at a very low frequency and induced tumors after a prolonged incubation period. These results indicate that there is a good correlation between the capacity of cells to grow without anchorage and the capacity to form tumors in nude mice and suggest that the revertant phenotype is stable in the presence of T antigen when the number of chromosomes is greatly increased as compared with that of the transformed parent cells.     

Characterization of a 54K dalton cellular SV40 tumor antigen present in SV40-transformed cells and uninfected embryonal carcinoma cells.

SV40 infection or transformation of murine cells stimulated the production of a 54K dalton protein that was specifically immunoprecipitated, along with SV40 large T and small t antigens, with sera from mice or hamsters bearing SV40-induced tumors. The same SV40 anti-T sera immunoprecipitated a 54K dalton protein from two different, uninfected murine embryonal carcinoma cell lines. These 54K proteins from SV40-transformed mouse cells and the uninfected embryonal carcinomas cells had identical partial peptide maps which were completely different from the partial peptide map of SV40 large T antigen. An Ad2+ND4-transformed hamster cell line also expressed a 54K protein that was specifically immunoprecipitated by SV40 T sera. The partial peptide maps of the mouse and hamster 54K protein were different, showing the host cell species specificity of these proteins. The 54K hamster protein was also unrelated to the Ad2+ND4 SV40 T antigen. Analogous proteins immunoprecipitated by SV40 T sera, ranging in molecular weight from 44K to 60K, were detected in human and monkey SV40-infected or -transformed cells. A wide variety of sera from hamsters and mice bearing SV40-induced tumors immunoprecipitated the 54K protein of SV40-transformed cells and murine embryonal carcinoma cells. Antibody produced by somatic cell hybrids between a B cell and a myeloma cell (hybridoma) against SV40 large T antigen also immunoprecipitated the 54K protein in virus-infected and -transformed cells, but did not do so in the embryonal carcinoma cell lines. We conclude that SV40 infection or transformation of mouse cells stimulates the synthesis or enhances the stability of a 54K protein. This protein appears to be associated with SV40 T antigen in SV40-infected and -transformed cells, and is co-immunoprecipitated by hybridomas sera to SV40 large T antigen. The 54K protein either shares antigenic determinants with SV40 T antigen or is itself immunogenic when in association with SV40 large T antigen. The protein varies with host cell species, and analogous proteins were observed in hamster, monkey and human cells. The role of this protein in transformation is unclear at present.     

Isolation of novel cDNA transformation markers from SV40-transformed human keratinocytes

Differential screening of a cDNA library was used to isolate probes for mRNAs that are induced in simian virus 40 (SV40)-transformed human keratinocytes. Several of these cDNAs hybrid select mRNAs which encode transformation-induced proteins found in the cytoskeletal component of SV40-transformed keratinocytes. One of these cDNAs was used to study the phenotype of normal and transformed cell lines derived from various tissues. We found that mRNA encoding the novel transformation-induced proteins is expressed in two squamous carcinoma cell lines derived from the oral epithelium, four SV40-transformed keratinocyte cell lines, and two SV40-transformed fibroblasts. Normal or transformed lymphoid cells or cell lines derived from carcinoma of the cervix do not express mRNAs which hybridize to these probes. The results from this study suggest that these probes may be used to detect markers of transformation in certain cell types.     

Differentiation of human epidermal cells transformed by SV40

Human epidermal cells were transformed with DNA from wild-type SV40 virus or with DNA from a temperature-sensitive A mutant (tsA209). The SV40-transformed cells differed from nontransformed cells in their morphologic appearance, growth properties, and expression of certain characteristics associated with differentiation. The transformed cells were more variable in size and shape than their nontransformed counterparts and were less stratified and less keratinized. While the growth properties of the cells were similar under optimal growth conditions, the transformed cells could be propagated under stringent growth conditions that did not support the growth of nontransformed human epidermal cells. The transformants still required a 3T3 feeder layer for growth, remained anchorage dependent as assayed in soft agar, and were not tumorigenic in athymic nude mice. The expression of certain differentiated functions of the human epidermal cell, the presence of keratins and cross-linked envelopes, was decreased in the transformed cells, and these functions could be restored at the nonpermissive temperature in the tsA209 transformed cells.     

Evidence for the involvement of cytolytic macrophages in rejection of SV40-induced tumors

The potential role of cytolytic macrophages in in vivo resistance to tumors induced by simian virus 40 (SV40) was evaluated in two experimental systems. First, a cell line produced by sequential in vivo passage of SV40-transformed fibroblasts through syngeneic C3H/HeJ mice was found to develop both increased neoplastic character and resistance to macrophage-mediated lysis, suggesting in vivo selection pressure against the macrophage-sensitive phenotype. In the second approach, SV40-transformed cells from C3H.OL mice, a strain that fails to produce SV40-specific cytolytic T lymphocytes (CTL), were cloned, and the cloned cells were tested for susceptibility to macrophage cytolysis in vitro. Two clones SV-COL-E8 and SV-COL-F5, which represent the extremes of macrophage susceptibility and resistance, respectively, were tested for progressive growth in syngeneic C3H.OL recipients. Progression in vivo was found to correlate with resistance to macrophage cytolysis in vitro. Other in vitro measures of the neoplastic phenotype, cell division rate and anchorage-independent growth, did not predict the relative abilities of clones E8 and F5 to form tumors. Likewise, the cells were indistinguishable in their sensitivity to cytolysis by allogeneic CTL and by natural killer cells. Finally, the presence of activated macrophages in the peritoneum of mice rejecting a challenge of syngeneic SV40-transformed cells was confirmed in both CTL responder and nonresponder strains. These studies suggest that cytolytic macrophages are indeed generated during rejection of SV40-induced mouse tumors and that, in the absence of an effective anti-SV40 CTL response, resistance of the transformed cell to macrophage-mediated cytolysis can be a determining factor in in vivo tumor growth.     

Rat embryo fibroblasts transformed by c-Jun display highly metastatic and angiogenic activities in vivo and deregulate gene expression of both angiogenic and antiangiogenic factors.

The comparative tumorigenicity in rats and nude mice of cell lines derived from FR3T3 and transformed by either c-jun, ras, SV40 lt, or bovine papilloma virus type 1 (BPV1) oncogenes was investigated. c-Jun-transformed cells were as tumorigenic and metastatic as Ras-transformed cells. Latencies were short, and numerous pulmonary metastases were observed in all injected animals. In contrast, tumors induced by s.c. injection of SV40-transformed cells developed slower, and none of the animals who received injections i.v. presented with metastases. BPV1-transformed cells had an intermediate tumorigenic and metastatic activity. Microvessels present in the different tumors were revealed by immunostaining with Griffonia (Bandeiraea) Simplicifolia lectin 1. Tumors obtained with c-Jun-transformed cells exhibited more neovascularization than those induced by the other oncogenes. By comparison to FR3T3 cells or SV40- or BPV1-transformed cells, c-Jun-transformed fibroblasts repress the antiangiogenic thrombospondin-1 and SPARC genes, whereas we found that they express higher levels of gene expression of the angiogenic vascular endothelial growth factor. Finally, as compared with cells before passage in animals, thrombospondin-1, SPARC, and VEGF gene expression was also deregulated in cell lines isolated from primary tumors induced by BPV1-transformants. Our results indicate that the high transforming potential of c-Jun, evidenced as soon as transformation is established in vitro, correlates with deregulation of gene expression of both angiogenic and antiangiogenic factors leading to rapid neovascularization of tumors.     

Karyotype evolution of the human HBL-100 cell line and mapping of the integration site of SV40 DNA

Cytogenetic analysis of the human HBL-100 cell line, that we have previously shown to harbour SV40 genetic information (Caron de Fromentel et al., 1985), reveals numerous chromosomal rearrangements as soon as the 30th in vitro passage. The karyotype is relatively stable during in vitro maintenance and even at late passages (approximately 70) when the cells have acquired the capacity to form tumors in nude mice. In all the somatic cell hybrids obtained after fusion of mouse 3T3-4E cells with HBL-100 cells, several human chromosomes are maintained and a derivative from chromosome 15-der(15)- is the most frequently observed. The der(15) marker is present in the HBL-100 cell line at every passage studied as well as in different cell lines derived from tumors induced by HBL-100 cells. The various hybrids, originally isolated for a transformed phenotype on the basis of their ability to grow in soft-agar, were all found to express the SV40 T-antigen. In situ hybridization of an SV40 DNA probe to chromosome spreads obtained from one of these hybrids shows that the integration site of the viral genome is located on the der(15) marker chromosome, at band 15q24. The possible cooperation of SV40 T-antigen with some other oncogene(s), required by human HBL-100 cells in order to express a malignant phenotype, is discussed.     

Transforming viruses directly reduce the cellular growth requirement for a platelet derived growth factor

Early passage mouse embryo fibroblasts, mouse 3T3 cell lines, and early passage diploid human fibroblasts grew to higher cell densities in tissue culture medium supplemented with serum than in medium supplemented with defibrinogenated platelet-poor plasma (PPP). Unlike the mouse cells, the human fibroblasts displayed this differential growth response only in the presence of hypophysiologic concentrations of calcium. The addition of heat-treated extracts of human platelets to PPP-supplemented medium stimulated the replication of both the normal mouse cells and early passage human embryo fibroblasts. Human or mouse fibroblasts transformed by either retroviruses or by SV40, including SV40 infected “serum revertants” and “flat transformants,” grew to equal cell densities in medium supplemented with either serum or PPP. Infection of Balb/c-3T3 cells with SV40 rapidly induced them to grow in PPP-supplemented medium demonstrating that the ability of SV40-transformed cell lines to proliferate in PPP-supplemented medium does not arise from the cell culture selection procedures usually employed to obtain stable virus-transformed cell lines. 3T3 cells infected but not transformed by retroviruses do not replicate in PPP-supplemented medium demonstrating that reduction of the growth requirement for the platelet growth factor(s) by retroviruses is a transformation-specific response. Cell cultures that did not proliferate well in PPP-supplemented medium did not form tumors when inoculated into athymic nude mice. Many, although not all, of the lines which grew well in PPP medium were tumorigenic in nude mice. Together, these findings indicate that: (1) normal fibroblast-like cells display a growth requirement for factor(s) present in serum but not found in PPP; (2) this serum specific growth factor is derived from platelets; (3) a primary response to viral transforming genes is a reduction in the growth requirement for these platelet-derived factors; and (4) cells that have a reduced requirement for the platelet-derived growth factor are often tumorigenic.     

Reactivity to SV40 T antigen in athymic (nude), anti-thymocyte serum-treated, and normal mice.

Antisera prepared in syngeneic mice by hyperimmunization with intact SV40-transformed mouse cells or with somatic cell hybrids between SV40-transformed human and normal mouse cells exhibit anti-SV40 tumor (T) antigen reactivity. Athymic mice bearing tumors formed by SV40-transformed mouse, human or mouse-human hybrids were not reactive with SV40 T antigen. Anti-thymocyte serum (ATS)-treated mice also lacked T antigen reactivity during suppressive treatment but developed antibody to T antigen after discontinuing ATS treatment and tumor regression. We conclude that that presence of growing tumors in the mouse is not necessary for the production of anti-SV40 T antigen antibodies but that helper thymus-derived cells are essential for the humoral response.     

Generation of cytotoxic lymphocytes by SV40-induced antigens

In order to study the correlation of in vivo tumor transplantation immunity and in vitro immunologic assays, cell-mediated cytotoxicity against SV40-transformed cells was studied in AL/N strain mice by using 51Cr-release assay. Killing of SV40-transformed AL/N fibroblast cells was observed by spleen cells of AL/N mice immunized with syngeneic SV40-transformed cells. Immunization with the solubilized SV40 tumor-specific transplantation antigen (TSTA) that induced transplantation immunity in vivo did not elicit cytotoxic spleen cells in vitro. However, the spleen cells from mice immunized with solubilized TSTA and then sensitized in vitro with SV40-transformed cells became cytotoxic against SV40-transformed fibroblasts. Similarly, SV40 TSTA (T antigen) purified by immunoprecipitation was able to prime the lymphocytes in AL/N mice: the primed lymphocytes could differentiate into cytotoxic lymphocytes upon in vitro stimulation by SV40-transformed cells. These data indicate that SV40 TSTA (T antigen) plays a role in the induction of cytotoxic lymphocytes.     

Induction of SV40-related transplantation immunity in mice by BK virus-transformed cells.

The reactivity of tumor-specific transplantation antigens (TSTAs) induced by SV40 and BK virus (BKV) was studied in Balb/Ccr mice with SV40-transformed tumor cells (mKSA-4AC). The mice, which had received BKV-transformed mouse or hamster cells, were highly resistant to the challenge with mKSA-4AC. The mKSA-rejected animals, however, were as susceptible as non-immunized ones to the subsequent challenge with Moloney sarcoma virus (Kirsten)-transformed cells. The result shows that BKV-transformed cells have a TSTA immunologically related closely to that induced by SV40.     

Cell properties after repeated transplantation of spontaneously and of SV40 virus transformed mouse cell lines. I. Growth in culture.

"Spontaneously" or SV40 virus transformed AL/N mouse cell lines were passed repeatedly through syngeneic mice. Cell lines were re-established in culture from minced pieces of tumors in the presence of concentrated fetal calf serum or from tumor cells dispersed by trypsin. The aim of this study was to compare the two cell lines in regard to the selection processes which operate during such procedures by characterization of the resulting cell lines. Measurements of growth in tissue culture on substratum showed no significant difference between any of the transformed cell lines. The SV40 transformed cells and its derivative cells had a low anchorage requirement for growth. The greatest anchorage requirement for growth was in the normal untransformed cells and in the derivative cells from the "spontaneously" transformed cells which were established from minced tumors. The spontaneously transformed cells and all derivative cells had high tumorigenicity (TD50 is less than 10-2). The SV40 transformed cells had no observable tumorigenicity (TD50 is greater than 10-8), except when injected into irradiated mice (TD50 = 1-5 X 10-5 in the immunocompetent mice, 5 X 10-4 in the irradiated mice). The SV40 transformed derivative cells maintained their SV40 specific T antigen and their susceptibility to lysis by specific antiserum.     

Synergy between Apc min and an activated ras mutation is sufficient to induce colon carcinomas.

Colon carcinomas appear to arise from the cumulative effect of mutations to several genes (APC, DCC, p53, ras, hMLH1, and hMSH2). By using novel colonic epithelial cell lines derived from the Immorto mouse, named the YAMC (young adult mouse colon) cell line, and an Immorto-Min mouse hybrid, named the IMCE (Immorto-Min colonic epithelial) cell line, carrying the Apc min mutation, we investigated the effect of an activated v-Ha-ras gene on tumor progression. The YAMC and IMCE cell lines are normal colonic epithelial cell lines which are conditionally immortalized by virtue of expression of a temperature-sensitive simian virus 40 (SV40) large T antigen. Under conditions which permit expression of a functional SV40 large T antigen (33 degrees C plus gamma interferon), neither the YAMC nor the IMCE cell line grows in soft agar or is tumorigenic in nude mice. In vitro, when the SV40 large T antigen is inactivated (39 degrees C without gamma interferon), the cells stop proliferating and die. By infecting the YAMC and IMCE cell lines with a replication-defective psi2-v-Ha-ras virus, we derived cell lines which overexpress the v-Ha-ras gene (YAMC-Ras and IMCE-Ras). In contrast to the parental cell lines, under conditions in which the SV40 large T antigen is inactive, both the YAMC-Ras and IMCE-Ras cell lines continue to proliferate. Initally YAMC-Ras cells do not form tumors; however, tumors are visible after 90 days of incubation. IMCE-Ras cells form colonies in soft agar under both permissive and nonpermissive culture conditions. Furthermore, IMCE-Ras cells form tumors in nude mice within 3 weeks. The phenotype of the IMCE-Ras cell line thus clearly demonstrates that a defective Apc allele and an activated ras gene are sufficient to transform normal colonic epithelial cells and render them tumorigenic.     

Reversion to methionine independence by malignant rat and SV40-transformed human fibroblasts.

Although many lines of malignant and transformed cells are unable to grow in folate- and cobalamin-supplemented medium in which methionine is replaced by homocysteine its immediate metabolic precursor, rare cells from these lines regained the normal ability to grow under these conditions. Six revertant lines, one from Walker-256 rat breast carcinoma cells and five from SV40-transformed human fibroblasts, have been characterized with regard to growth and three measures of methionine biosynthetic capacity: methionine synthetase and methylenetetrahydrofolate reductase activities in cell extracts, and uptake of label from [5-¹⁴C]methyltetrahydrofolate by intact cells. When all three measures of methionine biosynthetic capacity were considered, two revertants isolated from SV40-transformed cells had regained the ability to grow like normal cells in homocysteine medium without substantial changes in these measures. Increased methionine biosynthesis thus is not a prerequisite to reversion of the methionine auxotrophy present in the transformed parental lines.     

Simian virus 40 (SV40) T-antigen mutations in tumorigenic transformation of SV40-immortalized human uroepithelial cells.

pSV2Neo, a plasmid that contains the wild-type simian virus 40 (SV40) origin of replication (ori), is widely used in mammalian cell transfection experiments. We observed that pSV2Neo transforms two nontumorigenic SV40-immortalized human uroepithelial cell lines (SV-HUC and CK/SV-HUC2) to G418 resistance (G418r) at a frequency lower than that at which it transforms SV-HUC tumorigenic derivatives (T-SV-HUC). Transient expression studies with the chloramphenicol transferase assay showed that these differences could not be explained by differences in Neo gene expression. However, when we replaced the SV40 ori in pSV2Neo with a replication-defective ori to generate G13.1Neo and G13.1'Neo, the G418r transformation frequency of the SV40-immortalized cell lines was elevated. Because SV40 T antigen stimulates replication at its ori, we tested plasmid replication in these transfected cell lines. The immortalized cell lines that showed low G418r transformation frequencies after transfection with pSV2Neo showed high levels of plasmid replication, while the T-SV-HUC that showed high G418r transformation frequencies failed to replicate pSV2Neo. To determine whether differences in the status of the T-antigen gene contributed to the phenomenon, we characterized the T-antigen gene in these cell lines. The results showed that the T-SV-HUC had sustained mutations in the T-antigen gene that would interfere with the ability of the T antigen to stimulate replication at its ori. Most T-SV-HUC contained a super-T-antigen replication-defective ori that apparently resulted from the partial duplication of SV40 early genes, but one T-SV-HUC had a point mutation in the ori DNA-binding domain of the T-antigen gene. These results correlate with the high G418r transformation frequencies with pSV2Neo in T-SV-HUC compared with SV-HUC and CK/SV-HUC2. Furthermore, these results suggest that alterations in SV40 T antigen may be important in stabilizing human cells immortalized by SV40 genes that contain the wild-type SV40 ori, thus contributing to tumorigenic transformation. This is the first report of a super T antigen occurring in human SV40-transformed cells.