Adenovirus 2 early gene expression promotes susceptibility to effector cell lysis of hybrids formed between hamster cells transformed by adenovirus 2 and simian virus 40.

Weakly oncogenic adenovirus 2 (Ad2)-transformed LSH hamster cells are sensitive to lysis by spontaneously cytolytic lymphoid cells and activated macrophages, whereas highly oncogenic simian virus 40 (SV40)-transformed LSH cells are relatively resistant to these nonspecific effector cells. Somatic cell hybrids formed between Ad2- and SV40-transformed hamster cells, which expressed Ad2 tumor (T) antigens, exhibited an increased cytolytic susceptibility compared to Ad2 T antigen-negative cell hybrids or nonhybrid SV40-transformed cells. No correlation was found between the expression of SV40 T antigen in hybrid cells and cytolytic susceptibility. The results suggest the existence of a novel function for early Ad2 genome-encoded polypeptides (T antigens) expressed in transformed hamster cells--the induction of susceptibility to destruction mediated by immunologically nonspecific effector cells.     

Somatic cell hybrids producing antibodies specific for the tumor antigen of simian virus 40.

We have produced somatic cell hybrids between mouse myeloma cells deficient in hypoxanthine phosphoribosyltransferase IMP: pyrophosphate phosphoribosyltransferase; EC 2.4.2.8) and spleen cells derived from mice primed with either syngeneic or allogeneic cells transformed by simian virus 40. Such hybrids produced antibodies specific for simian virus 40 tumor (T) antigen. Only four of twelve independent hybrid cell cultures produced antibodies against simian virus 40 T antigen that crossreacted with the T antigen induced by BK virus, a human papovavirus isolated from patients who had undergone immunosuppressive therapy.     

Modulation of adenovirus transformation by thyroid hormone.

We have examined the effect of triiodothyronine (T3) on de novo transformation of a cloned population of Fischer rat embryo fibroblasts (CREF) by a temperature-sensitive mutant (H5ts125) of type 5 adenovirus and on the expression of the transformed phenotype in these cells. When CREF cells were grown in medium lacking T3 before, during, and after infection with H5ts125, the yield of transformed foci was half that in the cultures supplemented with 1 nM T3. Selective addition or removal of T3 during various phases of the transformation process indicated that the hormone exerted its maximal effect within 72 hr after viral infection. T3 was also required for optimal growth in agar of two clones of CREF cells previously transformed by type 5 adenovirus, wt-3A and ts-7E. The tumor promoter 12-O-tetradecanoylphorbol 13-acetate could substitute for T3 in enhancing growth in agar of wt-3A but not of ts-7E, suggesting that the promoter and T3 modify anchorage-independent growth by different mechanisms. Normal CREF cells and both of the transformed CREF clones grew equally well in monolayer culture in medium containing or lacking T3. Both of the transformed CREF clones contained a lower number of nuclear T3 receptors than did CREF cells and they bound somewhat lower levels of phorbol dibutyrate. These results indicate that thyroid hormone modulates an early stage involved in adenovirus transformation and that it also enhances the expression of the transformed state in previously transformed cells.     

Helper factor(s) for growth of adeno-associated virus in cells transformed by adenovirus 12.

Evidence is presented that a helper factor(s) for growth of adeno-associated virus (AAV) is present in cells transformed by adenovirus type 12 (Ad12). The growth of AAV was observed in heterokaryons formed by fusion of human KB and Ad12-transformed rodent cells by using ultraviolet-inactivated Sendai virus without coinfection of cells with adenovirus. The presence of the helper factor(s) for AAV growth in rat cells transformed by the EcoRI-C fragment or the HindIII-G fragment of Ad12DNA suggests that the helper factor(s) induced by infection with adenovirus is the Ad12-specific T antigen.     

Neoplastic transformation of immortalized human epidermal keratinocytes by ionizing radiation.

Efforts to investigate the progression of events that cause human cells to become neoplastic in response to ionizing radiation have been aided by the development of tissue culture systems of epithelial cells. In the present study, nontumorigenic human epidermal keratinocytes immortalized by adenovirus type 12 and simian virus 40 have been transformed by exposure to x-ray irradiation. Such transformants showed morphological alterations, formed colonies in soft agar, and induced carcinomas when transplanted into nude mice, whereas primary human epidermal keratinocytes exposed to radiation in this manner failed to show any evidence of transformation. These findings demonstrate the malignant transformation of human primary epithelial cells in culture by the combined action of a DNA tumor virus and radiation, indicating a multistep process for radiation-induced neoplastic conversion. This in vitro system may be useful as a tool for dissecting the process of radiation-induced neoplastic transformation of human epithelial cells and for detecting previously unreported human oncogenes.     

Adenovirus type 12 early region 1A proteins repress class I HLA expression in transformed human cells

The adenovirus type 12 (Ad12) early region 1A (E1A) gene is thought to play a major role in repressing class I major histocompatibility complex expression in transformed rodent cells. However, since transformation by adenovirus requires both E1A and E1B genes, it has not been demonstrated whether the Ad12 E1A gene acts alone or synergistically with the E1B gene to accomplish this effect. Moreover, it is not known whether the repression of class I antigen synthesis by Ad12-transforming gene products occurs only in rodent cells. We show that the Ad12 E1A gene, in the absence of the E1B gene, is capable of greatly reducing the levels of class I HLA antigens and mRNAs in primary human cells transformed by the E1A gene of Ad12 and the large tumor antigen (T-antigen) gene of BK virus; control cells transformed by BK virus T-antigen gene alone or the highly related simian virus 40 T-antigen gene showed no apparent alteration in class I HLA expression. Human recombinant interferon gamma was able to restore synthesis of class I HLA antigens in transformed cells that produced Ad12 E1A proteins, indicating that these cells were not deficient for class I genes. These results strongly indicate that the Ad12 E1A proteins modulate class I gene expression by similar mechanisms in both transformed rodent and human cells.     

Characterization of murine sarcoma virus transformation of guinea pig cells and activation of an RNA tumor-like virus from nonproducer guinea pig cells.

Guinea pig embryo (GEP) cells were transformed in vitro by the Kirsten strain of mouse sarcoma virus (Ki-MSV). The transformed cells were found to release infectious virus continuously and produced high titers of group-specific (gs) complement-fixing (CF) antigen characteristics of the murine sarcoma-leukemia virus complex. Foci of transformed cells were similar in appearance to those obtained with Ki-MSV in mouse and rat cells. The transformed cells produced RNA dependent DNA polymerase and type C virus particles with a density of approximately 1.15 g/ml in sucrose gradients by 3H-uridine labeling. The transformed cells produced tumors when transplanted into newborn guinea pigs. A number of focus-derived clonal lines from Ki-MSV transformed cells were isolated and characterized. All the focus-derived lines were found to be either producers or nonproducers (NP). The NP guinea pig cells produced neither infectious virus nor viral antigens of the murine sarcoma-leukemia virus complex although they were morphologically indistinguishable from virus-releasing MSV transformed GPE lines and produced tumors when transplanted into newborn guinea pigs. However, the sarcoma virus genome could be rescued in these NP cells by cocultivation with "helper" murine leukemia virus (MuLV) releasing GPE cells. Particles resembling guinea pig leukemia virus were activated from guinea pig NP cells or cultured normal guinea pig cells following chemical treatment. These particles were approximately 100 nm in the mature form and had a density of 1.16-1.17 g/ml. They contained RNA dependent DNA polymerase activity.     

Assignment of the integration site for simian virus 40 to chromosome 17 in GM54VA, a human cell line transformed by simian virus 40.

GM54VA human cells transformed by simian virus 40 (SV40) were fused with peritoneal macrophages obtained from three different mouse strains. All 27 hybrid clones studied were positive for SV40 tumor antigen in 100% of their cells and contained human chromosome 17. Human chromosome 17 was the only human chromosome present in five of the hybrid clones. Fusion of GM54VA cells and either thymidine kinase (EC 2.7.1.75)-deficient mouse or Chinese hamster fibroblasts resulted in the growth in hypoxanthine-aminopterin-thymidine medium of hybrid clones positive and negative for SV40 tumor antigen. Counterselection of the hybrid clones positive for tumor antigen in medium containing 5-bromodeoxyuridine resulted in the growth of hybrid cells that were negative for tumor antigen. These experiments indicate that negative for tumor antigen. These experiments indicate that SV40 is integrated in only one of the two parental human chromosomes 17. Because the genome of SV40 has been assigned to human chromosome 7 in two other SV40-transformed human cell lines, at least two different integration sites for SV40 would seem to be present in human cells: one located in human chromosome 7 and the other located in human chromosome 17.     

Assignment of the T-antigen gene of simian virus 40 to human chromosome C-7

Hybrid cell clones between mouse cells deficient in thymidine kinase (EC 2.7.1.21) and two different human cell lines transformed by simian virus 40 (SV40) and deficient in hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) were examined for SV40 tumor (T) antigen(s). Concordant segregation of the gene(s) for SV40 T antigen and human chromosome C-7 was observed in these hybrids. The human chromosome C-7 which contains the gene(s) for SV40 T antigen is preferentially retained by the majority of the hybrid clones tested. When hybrid clones positive and negative for SV40 T antigen, derived from the fusion of SV40-transformed Lesch-Nyhan fibroblasts with mouse cells, were fused with CV-1 permissive cells, SV40-specific V antigen was observed only in the cultures derived from fusion of the hybrid clones positive for T antigen. This result indicates a linkage relationship between human chromosome C-7, SV40 T-antigen gene(s), and SV40 genome(s) integrated in the human transformed cells.     

Tumor-specific transplantation antigen: use of the Ad2+ND1 hybrid virus to identify the protein responsible for simian virus 40 tumor rejection and its genetic origin.

Cells transformed by simian virus 40 (SV40) possess a tumor-specific transplantation antigen (TSTA) that has the property of immunizing animals against syngeneic tumor challenge. We find that the early SV40 DNA segment present in the human adenovirus 2 (Ad2)-SV40 hybrid, Ad2+ND1, is sufficient to induce this SV40-specific TSTA in BALB/c mice. Moreover, studies on the intracellular distribution of TSTA activity in Ad2+ND1-infected cells, as determined by the ability of various subcellular fractions to immunize mice against syngeneic tumor challenge, have suggested a correlation between this biological activity and the presence of the SV40-specific 28,000Mr protein in coded by this hybrid virus. Both the TSTA activity and the 28,000 Mr protein are found in the plasma membrane fraction and in the perinuclear region of infected cells but are virtually undetectable in the cytoplasmic fraction. Using a hamster antitumor antiserum that can specifically immunoprecipitate the 28,000 Mr protein, we are able to demonstrate a loss of TSTA activity concomitant with the removal of this SV40-coded protein. Thus, it appears that antigenic determinants responsible for SV40-specific tumor rejection in mice are contained within the 28,000 Mr protein coded for by the early SV40 DNA segment that extends from 0.17 to 0.28 map unit.     

Host nuclear proteins expressed in simian virus 40-transformed and -infected cells.

Two new families of host proteins (Mr, 48,000 and 55,000), in additional to the viral large (T) and small tumor antigens, are precipitable, with anti-T antiserum, from cells transformed or infected by the DNA tumor virus simian virus 40 (SV40). Rabbit anti-mouse 48,000 protein antiserum reacts specifically with SV40-infected or -transformed mouse cells to give nuclear staining indistinguishable from T-antigen staining but does not react with SV40-transformed human cells which nevertheless have structurally analogous 48,000 proteins, nor does it give nuclear fluorescence with untransformed mouse cells. Comparison of the partial proteolytic digests of the 48,000 proteins from cultured cells of various mammalian species shows that they are structurally related but not related to the 55,000 or large T-antigen proteins. The 55,000 proteins from the various mammalian species were also structurally related.     

Altered pattern of growth and differentiation in human keratinocytes infected by simian virus 40.

Human epidermal keratinocytes were infected by simian virus 40 in vitro. The structure of the developing keratinocyte colony reflects the spatial separation of cell division and keratinization in intact skin; thymidine-incorporating cells were primarily localized at the colony periphery whereas nondividing, histologically differentiated cells accumulated in the interior. Viral infection produced a dramatic increase in the size of the proliferative population as, simultaneously, differentiation was reduced in the colony interior. These changes were manifest when simian virus 40 T-antigen synthesis was detectable in only a small percentage of the cells; differentiation became increasingly density dependent as the percentage of T-antigen-positive cells rose over serial passage. The disruption of the normal pattern of growth/differentiation localization coincided with a loss of dependence on serum for growth, but preceded the appearance of other virus-induced properties associated with transformation; i.e., the ability to form colonies in soft agar and independence of growth from fibroblasts.     

In vitro transformation of cells from human neoplasms.

The possible involvement of RNA tumor virus genomes in human cell transformation was investigated. Forty-nine cell cultures from neoplastic, normal, or embryo tissues were examined for transformation, following inoculation of murine leukemia virus (MuLV), feline leukemia virus (FeLV) grown in human cells, or bone marrow aspirates from leukemia patients. Five cultures exhibited transformation (1 after inoculation of MuLV grown in human cells; 4 after inoculation of human leukemic bone marrow), and 4 were established as cell lines. They were derived from giant cell tumor and fibrosarcomas. The established transformed cells formed colonies in soft agar, grew progressively in immunosuppressed mice, and carried antigens common to FeLV and MuLV. Although virus particles were not seen in these cultures, 68S RNA was detected in their media. Medium from nontransformed parent cultures also contained 68S RNA but in amounts about 15 times less than in transformed cultures. Transformed human cells passaged in mice produced both type C virus particles and 68S RNA. Antigens common to MuLV and FeLV were found in these particles. However, the results of biological and serological studies indicate their difference from conventional MuLV and FeLV. The relationship of this virus and 68S RNA found in transformed cultures remains to be determined.     

Lack of correlation between tumorigenicity and level of plasminogen activator in fibroblasts transformed by Rous sarcoma virus

We have previously isolated, from agar suspension culture, clones of chicken embryo fibroblasts transformed by B77 and Prague strains of Rous sarcoma virus (RSV) that varied in the expression of plasminogen activator activity [Wolf, B. A. & Goldberg, A. (1976) Proc. Natl. Acad. Sci. USA 73, 3613-3617]. All of the clones exhibited an altered cellular morphology, an increased rate of sugar transport, and a high efficiency of colony formation in agar suspension regardless of the level of plasminogen activator. Because B77 and Prague strains of RSV replicate as well as cause sarcomas in chickens, the tumorigenicity of the transformed cells could not be evaluated with clones of these cells. In order to determine the oncogenicity of clones with various levels of plasminogen activator, it was necessary to isolate cells transformed by the replication-defective Bryan strain of RSV, which release noninfectious virus. All of the agar suspension clones of transformed cells, derived by infection of chicken embryo cells with replication-defective Bryan RSV, fell within the continuum observed for B77- and Prague-transformed clones with respect to altered morphology, increased rate of sugar transport, efficiency of colony formation in agar suspension, and variations in plasminogen activator activity. All of the clones, regardless of the level of plasminogen activator, produced tumors when as few as 5 x 10(2) cells were injected into the wing web of 1-day-old chicks. The latency period for tumor formation after injection of cells was similar regardless of the level of plasminogen activator of the injected cell. Primary explants of tumors resulting from inoculation of clones having low, intermediate, or high activator activity displayed a spectrum of activator activity.     

Episomal simian virus 40 genomes in human brain tumors.

Eight out of 35 human intracranial tumors were shown by restriction enzyme analysis to contain unintegrated simian virus 40 (SV40) DNA molecules. The relative amount of viral DNA was estimated to be the equivalent of one viral genome within every 10th to 20th cell. No infectious virus was detected in tissue cultures established from the tumors. From only one tumor was it possible to rescue, by cell fusion, infectious SV40 displaying wild-type properties. In those cases that permitted a more detailed analysis, the restriction enzyme cleavage patterns appeared to correspond to the wild-type patterns with one exception, in which the SV40 episomes displayed a deletion of approximately 70 base pairs close to the origin of DNA replication. From one tumor, the SV40 genomes were transferred into permissive CV-1 monkey cells by transfection with the total tumor DNA. Despite their persistence as episomes no infectious virus was produced. Furthermore, no viral antigens were detectable, although the SV40 messengers for the small and the large tumor antigens were present. These cells had, however, acquired the ability to form colonies in low concentrations of serum. Thus this report provides, by restriction enzyme analysis, direct evidence for the presence of SV40 DNA in human tumors.     

Specific Aggregation of SV40-Transformed Cells by Ornithine, Leucine Copolymers

A basic copolymer of ornithine and leucine (1:1) was shown to rapidly agglutinate, in the absence of serum, normal cells and cells transformed by viral and nonviral carcinogens. This agglutination was inhibited by addition of serum. In presence of serum, the same copolymer and those of ornithine and valine (1:1) and arginine and leucine (1:1), produced a specific aggregation of simian virus 40 (SV40)-transformed cells cultured for about 24 hr after addition of the peptide. The rapid agglutination and SV40-specific aggregation could not be inhibited by a variety of individual amino acids or carbohydrates. The specific aggregation could be detected in mixtures of SV40-transformed and other cells, and it was not prevented by x-irradiating the cells with 4000 R. Aggregation of the SV40-transformed cells was inhibited by acidic polyamino acids provided these were added not later than about 5 hr after addition of the basic copolymer. The results indicate that the basic copolymer, in the presence of serum, produces a change in SV40-transformed cells, presumably in the surface membrane, that causes the cells to aggregate. In addition to the aggregation of cells transformed by SV40, cells transformed by adenovirus 12, which did not contain detectable SV40-specific nuclear tumor (T) antigen, were also aggregated by the basic copolymer in the presence of serum. This indicates that the ornithine, leucine copolymer is able to detect an SV40-like change in the surface membrane of cells transformed by adenovirus 12.     

Carcinogen-transformed human cells are inhibited from entry into S phase by fusion to senescent cells but cells transformed by DNA tumor viruses overcome the inhibition.

Senescent human diploid cells (HDC) were fused to replicative transformed cells of different types, and DNA synthesis was monitored in the resulting heterodikaryons. Human cells transformed by simian virus 40 or adenovirus serotype 5 were able to induce DNA synthesis in senescent HDC nuclei in heterodikaryons. In contrast, carcinogen-transformed cells were not able to induce DNA synthesis in senescent HDC nuclei; rather, the transformed nuclei in these heterodikaryons were inhibited from entering S phase. Cells transformed by Rous sarcoma virus and most human tumor cells tested are similarly inhibited by fusion to senescent HDC. These results suggest that the mechanism for transformation by DNA tumor viruses may be fundamentally different from that of other viruses and carcinogens and from that of most human tumor cells. A simple model to explain these results is that (i) senescent HDC contain an inhibitor of entry into S phase; (ii) cells transformed by DNA tumor viruses have gained a transforming factor, perhaps large tumor antigen, that is capable of overriding the normal inhibitor; and (iii) cells transformed by carcinogens or RNA viruses have lost or altered the mechanism for expression of the normal inhibitor yet are still sensitive to it. We propose that this inhibitor is produced in normal cells when they experience conditions that are inadequate for proliferation and that it plays a role in putting the cells into a distinct quiescent state with long-term viability. The override of this inhibitor function in simian virus 40-transformed HDC can explain why they have low viability in plateau-phase cultures and why they die during crisis.     

Deletion of 43 amino acids in the NH2-terminal half of the large tumor antigen of simian virus 40 results in a non-karyophilic protein capable of transforming established cells.

We have characterized a simian virus 40 (SV40) mutant, derived from the viral DNA insertion present in simian cell transformants, which carries a deletion affecting the NH2-terminal region of the SV40 large tumor antigen. This mutant protein is 6% smaller than normal, has lost the typical nuclear localization of the SV40 large tumor antigen, and accumulates in the cytoplasm. The deletion begins at nucleotide position 4490 of the SV40 DNA and ends in-frame at nucleotide position 4362. The missing 43 amino acids begin with proline-110 and end with serine-152 of the predicted sequence; they include a cluster of basic residues, presumably important for the viral origin-DNA binding, and most of the phosphorylation sites present in the NH2-terminal half of the molecule. The protein can still be phosphorylated considerably in vivo. This mutant viral genome is replication-defective but has conserved the competence to transform established cells, such as NIH/3T3 cells. Transfection of cloned mutant DNA into such cells resulted in the production of full transformants. Full transformants were not produced in similar transfections carried out in primary rat embryo fibroblasts, although some primary transfectants expressing the non-karyophilic large tumor antigen might be considered minimally transformed.