Increased susceptibility to SV40 transformation with development and in vitro aging

The incidence of most cancers increases with aging. To examine whether this increased risk might be related to a higher susceptibility of older cells to neoplastic transformation, we transfected rat fibroblasts aged in vivo and in vitro with origin-defective SV40 DNA and measured the number of transformed foci. Substantial increases in the number of transformed foci were observed in cells from adult rats when compared with those of cells from embryos or weanlings. Much higher numbers of foci were also obtained at late passage, when 68% or more of the in vitro lifespan had been completed, while no foci were produced from cells at early or middle passage. To control for changes with aging in uptake, integration, or expression of exogenous DNA, parallel cultures were transfected with a G418 resistance gene. The number of G418-resistant colonies did not increase with aging and, in fact, decreased in late passage embryonic cell cultures. Therefore, increased susceptibility to SV40 transformation appears to be a feature of development and in vitro aging in rat cells.     

Karyologically identified homozygous tw18 embryos: extrauterine growth properties and transformation by SV40.

A method for identifying individual embryos from crosses segregating homozygous t lethals using the marker chromosome Rb7 is described and applied to the tw18 haplotype. In contrast to wild-type and heterozygous littermates, homozygous tw18/tw18 6 and 8 day embryos have very limited growth potentialities in vitro. When transplanted under the testis capsule, homozygous tw18 embryos (3, 6 and 7 day) produce teratomas at a much lower efficiency than heterozygous and wild-type embryos, and the rare teratomas thus obtained have limited growth potentialities when cultured in vitro. Upon transformation with SV40, however, permanent lines of cells of mesodermal origin, capable of myoblastic or adipocytic differentiation, have been obtained. This shows that the effect of the mutation on cell growth can be overcome by SV40 transformation.     

Anchorage-independent growth of normal calf lens epithelial cells and effect of SV40 transformation on their growth properties

Calf lens epithelial cells cultured in vitro show growth properties usually associated with virally transformed fibroblasts. The lens cells are anchorage independent, forming colonies in agar, and show a low requirement for added mitogens. In dense culture they form multilayers and maintain a constant cell number by proliferation and shedding. Strains of lens cells transformed by SV40 virus have been obtained that show similar growth properties to the normal lens epithelium. The major effect of SV40 transformation is to increase the growth rate, final cell density and in vitro life-span of the lens cells and to inhibit the increase in size that occurs after 2-3 weeks of culturing the untransformed cells.     

Covalent affinity labeling by periodate-oxidized [alpha-32P]ATP of the large-T proteins of polyoma and SV40 viruses

Incubation of crude extracts from cells lytically infected with polyoma virus in the presence of periodate-oxidized [alpha-32P]ATP led to the radioactive labeling of one main polypeptide immunoprecipitated by anti-T antigen antibodies. It was absent from extracts of mock-infected cells and exhibited an apparent Mr value of 105,000, identical with that of the large-T viral protein. This polypeptide was unambiguously identified as large-T on the basis of the heat sensitivity of the in vitro labeling in extracts from cells infected with a tsa mutant. The amount of incorporated radioactivity was found to increase linearly with that of infected cell extract and with the incubation time. Labeling resulted from the specific binding of an oxidized ATP molecule to a nucleotide binding site of the large-T protein, since it was efficiently completed by unlabeled ATP. Study of the dependence on the concentration of oxidized ATP evidenced a first order kinetic for the labeling reaction. Similar results were obtained for the large-T protein of SV40 virus.     

Membrane antigens associated with infection,transformation, and tumorigenesis by polyoma virus

Conclusion Infection, transformation, or tumorigenesis by Py virus leads inter alia to modifications in the membrane antigens of the affected cells. The modifications include antigenic gains or losses or quantitative changes in both directions. Although there is a pronounced common denominator in the antigenic alterations in the three distinct Py-induced biological processes, it is nontheless possible that each of them is characterized by specific antigenic modifications. This possibility has yet to be analyzed. Specific antigenic modifications, if they occur, are probably the result of different selective processes and adaptions to these pressures.In this brief review, we have attempted to survey the literature pertinent to this aspect. While doing so, we discovered that most researchers have not considered the possibility that differences could exist between antigens of cells infected by Py, cells transformed by this virus, and Py-induced tumor cells. We feel that a comprehensive antigenic comparison between these cells utilizing well-defined reagents is an essential prerequisite to understanding of the successful immunological surveillance against Py-induced malignancy.Incumbent: The David Furman Chair of Cancer Immunobiology     

Transformation of human cystinotic fibroblasts by SV40: characteristics of transformed cells with limited and unlimited growth potential.

Human skin fibroblasts derived from patients with nephropathic cystinosis were transformed with SV40 virions, cloned and permitted to enter the degenerative stage of growth termed "crisis," characteristic of SV40 transformed human cells. Nephropathic cystinosis is an autosomal recessively inherited metabolic disorder resulting in the intracellular accumulation of the amino acid cystine. A transformed cystinotic cell line which was recovered from the crisis stage was indistinguishable from its transformed precrisis parental cell strain in growth rate in media containing either 1% or 10% serum, cloning efficiency on plastic, in semisolid media, or upon confluent monolayers of normal skin fibroblasts, expression of SV40 T antigen, or production of virus. However, the modal DNA content of the recovered postcrisis transformed cystinotic cell line was different from that of the cloned parental precrisis transformed cell strain, suggesting that the postcrisis line was derived from a small subpopulation of the precrisis strain. The DNA content of the established cystinotic cell line continued to be unstable during subsequent subculturing and gave rise to subclones with both more and less DNA per cell. This line now has an apparently infinite growth potential and still has the hallmark of the cystinotic parental line, the storage of abnormally large amounts of intracellular nonprotein cystine.     

A high speed, high capacity homology matrix: zooming through SV40 and polyoma.

We present a new homology matrix program which owes its basic conception to the two-dimensional dot matrices previously described (1,2), but has important improvements and new features. It scores sequence homology over an adjustable range and plots the scores which are above an operator-determined filtration level. Its powerful noise-filtration system, capacity for compression without much loss of information, and speed of execution make this program a valuable tool in the analysis of homologies, internal direct repeats and reverse repeats, including palindromic sequences. The properties of the program are exemplified by analysis of SV40 and polyoma DNA sequences.     

Quantitative estimation of nuclear buds and micronuclei in bovine cells transformed by Rous sarcoma and SV 40 viruses

Summary A study of nuclear-budding and micronuclei formation has been performed on bovine fibroblastic cells in serial tissue culture. A comparison was made between control cells and cells morphologically transformed by Rous sarcoma virus and SV 40 virus. It was found that no obvious differences in the frequency of nuclear buds and micronuclei existed between RSV transformed and control cells whereas SV 40 transformed cells showed a high frequency of nuclear buds and micronuclei. There are indications that the frequency of nuclear buds and micronuclei is correlated to chromosomal abnormalities.Supported by grants from the Medical Faculty of the University of Uppsala.     

Morphological and cytochemical characteristics of human cells transformed and made malignant by Rous and polyoma viruses]

In cells of human embryo skin--muscle tissue transformed by the Rouse sarcoma virus (23rd cell line) and polyoma virus (P-2 cell line), the mitotic activity was 48 0/00 for 23rd line, 51 0/00 for P-2 line as against 28 0/00 in the control cells. The transformed cells possessed greater amounts of RNA and DNA and protein--bound SH-groups, different forms of glycogen deposits, as well as higher acid phosphatase enzyme activities; there was practically no difference in acid mucopolysaccharide content or NAD-H2-diaphorase and succinate dehydrogenase activities.     

Decreased actin and tubulin synthesis in 3T3 cells after transformation by SV40 virus.

Actin and tubulin are major protein constituents of 3T3 and SV40 virus-transformed 3T3 cells. We have fractionated growing, confluent and SV403T3 cells into particulate and soluble fractions using conditions designed to sediment microtubules, actin filaments or membrane associated actin or tubulin. The ratio of particulate to soluble actin synthesized in growing or confluent 3T3 cells is 2 to 1, while the ratio is reversed in transformed cells. There is also a 60% decrease in particulate tubulin synthesis in SV403T3 cells when compared with that in normal cells. Similar results are obtained when total actin and tubulin amounts are determined. The half-lives of actin, tubulin and total protein are over 3 days in growing 3T3 and SV40 cells and decrease over two-fold in confluent 3T3 cells. The significance of these results with respect to loss of contact inhibition and development of malignancy by these cells after transformation is discussed.     

Tumor formation by SV40-transformed human cells in nude mice: the role of SV40 T antigens

Human cells transformed in vitro by SV40 rarely form tumors in nude mice. We examined whether these cells as a group are inherently nontumorigenic or whether they are potentially tumorigenic but rejected by the athymic host, possibly by nonspecific immune mechanisms. SV80 and NG8 are SV40-transformed human cell lines that express all of the transformed properties, including anchorage-independent growth, but do not form tumors in adult nude mice after injection of as many as 10(8) cells. Both the SV80 and NG8 cell lines have SV40-specific transplantation antigens which crossreact with those present on SV40-transformed (but tumorigenic) rodent cells. We found that SV80 cells, though not NG8 cells, induced progressively growing lethal tumors if the cells are injected repeatedly into neonatal nude mice. Somatic cell hybrids between SV80 or NG8 cells and a highly tumorigenic cell line derived from a human tumor continue to express the virus-induced antigens and fail to form tumors in adult nude mice. These results strongly suggest that at least for some SV40-transformed human cells, the failure to form tumors in nude mice may be due to their expression of virus-induced transplantation antigens rather than the absence of tumorigenic potential.