SV40 transformation of mouse brain cells: Critical role of gene A in maintenance of the transformed phenotype

Anderson, Jeffrey L.; Martin, Robert G.

doi:10.1002/jcp.1040880109

Cited by 26 publications

(17 citation statements)

References 24 publications

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“…Single cell suspensions were fixed in 70% ethanol/30% 50 mM MgCl2, and stained with mithramycin, which binds stoichiometrically to DNA (16 Thus, the characteristics used to define the transformed phenotype (cell morphology, colony formation on plastic or on soft agar, and saturation density) were quite temperature sensitive in the tsA transformed cells. The WT transformants were also somewhat temperature sensitive, as has occasionally been observed in other WT SV40-transformed cell populations (2,3,5,18). The molecular mechanism of the thermal sensitivity of transformed cells is a well described, but as yet poorly understood, phenomenon (19,20).…”

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confidence: 69%

Simian virus 40 A gene function: DNA content analysis of Chinese hamster cells transformed by an early temperature-sensitive virus mutant.

Robinson¹,

Lehman²

1978

Proc. Natl. Acad. Sci. U.S.A.

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Replication of two Chinese hamster embryo cell lines transformed by an early temperature-sensitive mutant of simian virus 40, tsAS8, was examined by flow microfluorometry and autoradiography of [3H~thymidine-labeled cells in order to determine whether transformed cell DNA synthesis is initiated by the virus A gene. At the permissive temperature (370), cells transformed by the mutant were like the wild-type virus transformants in appearance, colony-forming ability, high saturation density, and rapid replication. At One approach to understanding neoplasia has been to study cells transformed by temperature-sensitive virus mutants at temperatures permissive and nonpermissive for the function of specific viral genes. In this way, the A gene of simian virus 40 (SV40) has been tentatively identified as the gene responsible for initiation and maintenance of transformation in mammalan cells (1-6). In the lytic system, the A gene initiates viral and perhaps cellular DNA synthesis (6-8). It has therefore been suggested that transformation of nonpermissive cells is maintained by the A gene product continuously initiating cell DNA synthesis, which would free cell replication from its normal control mechanisms (1-5, 9, 10).If this "initiator" hypothesis is correct, cells transformed by SV40 mutants that specify a temperature-sensitive A gene product (tsA mutants) should not only lose the transformed phenotype at the nonpermissive temperature, but also should return to normal growth control, and arrest in the G1 (Go) phase of the cell cycle at confluence as normal cells do. When grown at the permissive temperature, the same cells should resume cell cycle traverse in response to the functional A gene. This "initiator" hypothesis was tested by analyzing the cell cycle of Chinese hamster embryo (ChH) cells transformed by wild-type (WT) SV40 and the SV40 tsA mutant, tsA58. Several independent methods of cell cycle analysis, including flow microfluorometric determination of cell DNA content, were used. The results of our studies suggest that the loss of the A gene function represented by the tsA58 mutation does not allow SV40-transformed ChH cells to arrest in the G1 (Go) phase of the cell cycle.MATERIALS AND METHODS Viruses and Cell Cultures. WT SV40 and the temperature-sensitive group A SV40 mutant, tsA58, were obtained from Peter Tegtmeyer. Virus pools were prepared and were plaque assayed on CV-1 monkey kidney cells at 330 and 40.5°(11). The presence of SV40-induced T or V antigen in infected cells was demonstrated by indirect immunofluorescence (12).Confluent secondary cultures of normal ChH fibroblasts (12) were infected with 2-5 plaque-forming units of SV40 per cell at 370 and subcultured frequently until morphologic transformation was observed (five to six passages). This uncloned population, selected only for rapid growth on plastic, was designated ChH A58. Another embryo cell culture was similarly infected with WT or tsA58 SV40, but the cells were seeded into soft agar (13) after morphologic transformation was o...

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confidence: 69%

Simian virus 40 A gene function: DNA content analysis of Chinese hamster cells transformed by an early temperature-sensitive virus mutant.

Robinson¹,

Lehman²

1978

Proc. Natl. Acad. Sci. U.S.A.

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“…TA binds to DNA (7), and, after incuba-pacity of the early region of the SV40 genome tion at 440C, TA obtained from tsA-transformed (25,31). Second, all known tsA mutants synthe-As control, we demonstrate that a typical tsA MgCl2, 0.15 mM CaCl2, 0.1% [grams/100 milliliters] mutant-transformed cell line that continues to Difco gelatin, 0.05 M sodium barbital, pH 7.5) to express TA at the restrictive temperature (as give about 20% (vol/vol) suspensions, sonicated at 0 do most tsA-transformed cell lines [3,6,14,24, to 40C for 2 min, and stored at -70°C; these TA- 29,30]) also continues to express TSTA. In a containing lysates were used directly as TA or after separate communication we demonstrate that clarification by centrifugation at either 1,000 x g for several different cell lines transformed by tsA 10 min or 17,000 x g for 15 min.…”

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confidence: 75%

“…Genetic studies with simian virus 40 (SV40) now show in addition that the TA from tsAmutants indicate that the "early" gene A is transformed cells is more thermolabile by comessential for the initiation ofviral replication in plement fixation (CF). permissive infection and for the initiation and Tumor-specific transplantation antigen maintenance of transformation in nonpermis-(TSTA) is more difficult to assay and has been sive infection (3,6,9,14,22,23,25,26,29,30). less thoroughly investigated than TA, although The SV40-specific antigens T, TST, and U are its expression also requires part of the early expressions of this early region of the SV40 region ofthe SV40 genome (17,19).…”

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confidence: 99%

“…sublines were derived from a secondary line of astro-Heat inactivation of antigens. Cell lysates in cytes originating from National Institutes of Health buffer or detergent extracts obtained as described (NIH) Swiss mouse brain and have been previously above were heated in equal-volume aliquots in plasdescribed (3 (Table 1B), as originally antibody in the presence of rabbit complement at reported by Osborn and Weber (26). After 5 In vvo assays for TSTA.…”

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confidence: 99%

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Expression and thermal stability of simian virus 40 tumor-specific transplantation antigen and tumor antigen in wild type- and tsA mutant-transformed cells

Anderson¹,

Chang²,

Mora³

et al. 1977

J Virol

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We have explored aspects of a suggested relationship between the expression of simian virus 40 tumor-specific transplantation antigen (TSTA) and tumor antigen (TA). A unique rat embryo cell line transformed by a temperaturesensitive A mutant that loses TA during exposure to the nonpermissive temperature (A28-RE) was found to lose TSTA. On the other hand, a typical control tsA-transformed cell line (A239-MB) expressed both TA and TSTA at the nonpermissive temperature. TA in lysates obtained from A239-MB cells was found to be three to four times more thermolabile by complement fixation than TA obtained from wild-type-transformed cells (SVWT-MB) when incubated at either 33 or 400C. These data complement previous reports using TA from lytic infection and are consistent with the suggestion that TA is virus encoded. In contrast to TA, which even in wild-type-transformed cells was completely destroyed in <10 min at 500C, TSTA, assayed in vivo by tumor rejection, and tumor-specific surface antigen(s) (TSSA), defined by an in vitro cytolytic assay, were thermostabile. Even after 24 h of incubation of extracts at 500C, high levels of TSTA and TSSA activity were present. Since these surface antigens when obtained from cells transformed by tsA mutants were also thermostabile, they could not be distinguished from the wild-type antigens. These results (i) indicate a coordinate expression of TA and TSTA in transformed cells; (ii) confirm that TA is virus encoded; and (iii) confirm that the antigenic and immunogenic determinants that characterize TA and TSTA activities are distinct. However, the possibility that TSTA, like TA, is of viral rather than cellular origin is not excluded. cells loses binding activity more rapidly than size defective TAs (2, 15, 32), but contain ge-TA from wild-type-transformed cells (32). We netic lesions that map within the region re-459

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“…Robinson and Lehman (1978) reported that a n apparent growth arrest, assayed as number of cells, in tsA mutant-transformed Chinese hamster embryo fibroblasts at nonpermissive temperature is attributable to a balance of cell death and multiplication but not to arrest in the particular phases of the cell cycle. Arrest of growth in tsA mutant-transformed fibroblasts (Martin and Stein, 1976;Brockman, 1978;Butel and Soule, 1978;Hiscott and Defendi, 1979;O'Neill et al, 1980) and in tsA mutant-transformed cells that retain differentiated functions (Anderson and Martin, 1976;Chou, 1978a;Maltzman et al, 1979;Schlegel-Haueter et al, 1980) has been reported.…”

Section: Discussionmentioning

confidence: 98%

Cell growth and differentiation in vitro in mouse macrophages transformed by a tsA mutant of simian virus40. I. Cellular response in proliferative and phagocytic activities to the shift of temperature differs depending on the culture state in mouse bone marrow cells transformed by the tsA640 mutant of simian virus 40

Tanigawa

Takayama

Takagi

et al. 1983

Journal Cellular Physiology

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It was shown previously that mouse bone marrow cells transformed by simian virus 40 (SV40) show a reversible cell density-dependent phenotypic transition between the nonmacrophage (rapidly growing) and the macrophage (stationary) states; cells in low-density cultures are in the growing phase, express SV40 T antigen strongly as revealed by immunofluorescence, and lose typical macrophage properties such as immune phagocytosis; whereas cells in high-density cultures are in the stationary (nongrowing) phase, express SV40 T antigen weakly, and recover their macrophage properties (Takayama, 1980). In the hope of clarifying the relationship between T antigen, cell growth, and macrophage-specific cellular function, we examined the behavior at 33 and 39 degrees C of mouse bone marrow cells transformed by an SV40 gene A mutant (tsA640) whose mutation renders the molecular weight of 90K (large) T antigen temperature sensitive. The results presented in this paper suggest that functional large T antigen is required for cells in the stationary phase to initiate multiplication when transferred at lower density and is not necessary for a majority of them to maintain the nongrowing state (viability) at both high and lower cell densities, whereas it is required for cells in the growing phase to keep multiplying without losing their viability. The results also suggest that the functional large T antigen does not play a direct role in maintaining the cells as either phagocytic or nonphagocytic. It is also suggested that the physiological or tsA mutation-mediated arrest of growth may or may not be accompanied by induction and/or maintenance of cellular phagocytic activity depending on the culture state.

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SV40 transformation of mouse brain cells: Critical role of gene A in maintenance of the transformed phenotype

Cited by 26 publications

References 24 publications

Simian virus 40 A gene function: DNA content analysis of Chinese hamster cells transformed by an early temperature-sensitive virus mutant.

Simian virus 40 A gene function: DNA content analysis of Chinese hamster cells transformed by an early temperature-sensitive virus mutant.

Expression and thermal stability of simian virus 40 tumor-specific transplantation antigen and tumor antigen in wild type- and tsA mutant-transformed cells

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