Virus Deoxyribonucleic Acid Sequences in Subdiploid and Subtetraploid Revertants of Polyoma- Transformed Cells

Polyoma-transformed cells can revert in the properties characteristic of transformation, although they maintain the polyoma-specific T antigen. Transformed cells contain the same number of copies of polyoma virus deoxyribonucleic acid (DNA) per cell (eight) as revertants with a subdiploid or a subtetraploid chromosome number. The results indicate that the duplication of chromosomes in the subtetraploid revertants did not include the chromosomes that carry the viral genome. The virus DNA in both transformed and revertant cells was associated with high-molecular-weight cell DNA. Reversion of the properties of transformed cells was, therefore, not associated either with a decrease in number of virus DNA copies per cell or with a lack of association of the virus DNA with cell DNA.     

Recessive (mediator-) revertants from c-H-ras oncogene-transformed NIH 3T3 cells: tumorigenicity in nude mice and transient anchorage and serum independence of the recovered tumor cells in culture.

We have reported earlier the isolation of two recessive, serum- and anchorage-dependent revertants (R116 and R260) from a c-H-ras oncogene-transformed NIH 3T3 line. In both revertants, the oncogene was fully expressed and fusion of either revertant with (untransformed) NIH 3T3 cells, or of the two revertants with one another, resulted in transformed progeny. These, and other data, indicated that the transforming activity of the oncogene was impaired in the two revertants in consequence of defects in distinct genes needed to mediate this activity. We report here that neither revertant could be re-transformed by the K-ras or N-ras oncogene (though they could be re-transformed by several other oncogenes). The two revertants turned out to be tumorigenic in nude mice (though less so than the parental transformed cells). The tumor cells, as recovered, formed foci and had a transformed morphology and a greatly diminished serum and anchorage dependence. Growth of the cells in culture (for 20 passages) resulted in their regaining the characteristics (i.e., anchorage and serum dependence) of cultured R116 and R260 cells. Proliferation of the cells in nude mice was not accompanied by a change in the level of ras oncogene expression or in gene amplification, at least as manifested in the lack of appearance of double-minute chromosomes. The addition of the growth factors TGF alpha and beta to the medium of either revertant did not support anchorage-independent growth.     

Characterization of flat revertant cells isolated from simian virus 40-transformed mouse and rat cells which contain multiple copies of viral genomes.

Eight clones of flat revertants were isolated by negative selection from simian virus 40 (SV40)-transformed mouse and rat cell lines in which two and six viral genome equivalents per cell were integrated, respectively. These revertants showed either a normal cell phenotype or a phenotype intermediate between normal and transformed cells as to cellular morphology and saturation density and were unable to grow in soft agar medium. One revertant derived from SV40-transformed mouse cells was T antigen positive, whereas the other seven revertants were T antigen negative. SV40 could be rescued only from the T-antigen-positive revertant by fusion with permissive monkey cells. The susceptibility of the revertants to retransformation by wild-type SV40 was variable among these revertants. T-antigen-negative revertants from SV40-transformed mouse cells were retransformed at a frequency of 3 to 10 times higher than their grandparental untransformed cells. In contrast, T-antigen-negative revertants from SV40-transformed rat cells could not be retransformed. The arrangement of viral genomes was analyzed by digestion of cellular DNA with restriction enzymes of different specificity, followed by detection of DNA fragments containing a viral sequence and rat cells were serially arranged within the length of about 30 kilobases, with at least two intervening cellular sequences. A head-to-tail tandem array of unit length viral genomes was present in at least one insertion site in the transformed rat cells. All of the revertants had undergone a deletion(s), and only a part of the viral genome was retained in T-antigen-negative revertants. A relatively high frequency of reversion in the transformed rat cells suggests that reversion occurs by homologous recombination between the integrated viral genomes.     

Flat revertants derived from Kirsten murine sarcoma virus-transformed cells produce transforming growth factors

Two flat cellular revertant cell lines, F-2 and C-11, which were originally selected from the DT line of Kirsten murine sarcoma virus (Ki-MuSV)-transformed NIH/3T3 cells, were examined for the production of transforming growth factors (TGFs). The revertant cells fail to grow in semisolid medium as colonies and exhibit a markedly reduced level of tumorigenicity in nude mice, although they are known to express high levels of p21ras, the product of the Kirsten sarcoma virus oncogene, ras, and they contain a rescuable transforming virus. TGF activity associated with the transformed, revertant, and non-transformed cell lines was measured by the ability of concentrated conditioned medium (CM) from these cells to induce normal rat kidney (NRK) and NIH/3T3 cells to form colonies in semisolid agar suspension cultures and to inhibit the binding of 125I epidermal growth factor (EGF) to specific cell surface receptors. CM from the transformed DT cells and from both the F-2 and C-11 revertants contains TGF activity, in contrast to CM obtained from normal NIH/3T3 cells. Furthermore, unlike NIH/3T3 cells, neither the DT nor the revertant cells were able to bind 125I EGF. All four cell lines were able to proliferate in serum-free medium supplemented with transferrin, insulin, EGF, and Pedersen fetuin. However, in basal medium lacking these growth factors, only DT cells and, to a lesser extent, the revertant cells were able to grow. These results suggest that the F-2 and C-11 revertants fail to exhibit all of the properties associated with transformation because the series of events leading to the transformed phenotype is blocked at a point(s) distal both to the expression of the p21 ras gene product and also to the production of TGFs and that the production of TGFs may be necessary but not sufficient for maintaining the transformed state.     

Suppression of the transformed phenotype of hepatoma cells after hybridization with normal diploid fibroblasts

Hybrids were generated between mouse hepatoma cells which exhibit a transformed phenotype, and rat normal diploid fibroblasts. Most isolated hybrid clones contain a single set of chromosomes from each parent. Such clones grow to low saturation densities and are unable to grow or to form colonies in soft agar. The transformed phenotype of the parental hepatoma cells is thus suppressed in these hybrids. Suppression is very stable; however, subclones which have regained a transformed phenotype could be selected; these subclones show a significant reduction of their chromosome number. Amongst the hybrid clones isolated after fusion, a few are characterized by an excess of mouse chromosomes and a reduced number of rat chromosomes. Such clones exhibit a transformed phenotype. Our results show that, provided the hybrids contain an almost complete single set of chromosomes of each parent, spontaneous transformation behaves as a recessive trait in hybrids formed with normal diploid cells.     

Chromosome balance and the control of malignancy.

The Giemsa banding pattern of the chromosomes has been analyzed in a line of transformed golden hamster cells, revertant and re-revertant cells and their tumors. The transformed and re-revertant cells were malignant in vivo and had gained an additional chromosome 5(7). Revertants with a suppression of malignancy lost this additional chromosome 5(7) and gained an additional chromosome 7(2). The tumors produced by segregants from the revertant cells were malignant, although to a lower degree than transformed and re-revertant cells. These tumors had lost the additional chromosome 7(2) found in revertants and gained one or two 5(12) chromosomes. The results support the hypothesis that the balance between genes for expression and suppression controls malignancy. The data indicate that chromosome 7(2) carries genes for suppression and that chromosomes 5(7) and 5(12) carry genes for expression of malignancy. The genes on chromosome 5(7) seem to result in a greater degree of expression than the genes on chromosome 5(12). The chromosome balance that controlled malignancy in these cells, also controlled the expression and suppression of transformed properties in vitro.     

Loss of tumorigenicity correlates with a reduction in pp60src kinase activity in a revertant subclone of avian sarcoma virus-infected field vole cells

We have recently isolated an interesting revertant subclone (revertant 866-4) of ESV-infected field vole cells that is indistinguishable from uninfected vole cells with respect to its lack of transformed cell properties. These revertants are not only normal morphologically, but they do not grow in soft agar and are nontumorigenic in athymic nude mice. Despite this lack of transformed cell properties, we have found that this cell line still contains pp60src at concentrations (0.30 microgram/mg cell protein) similar to those (0.13-0.42 microgram/mg cell protein) found in transformed and morphologically reverted, but tumorigenic vole cells (partial revertants). However, the most interesting aspect of this newly isolated subclone is the marked reduction in its pp60src kinase activity (2--3%) when compared with the specific activity of pp60src immunoprecipitated from transformed and partially revertant vole cell lines. Since the reduction in pp60src kinase activity strongly correlates with the loss of tumorigenicity in this particular revertant cell line, these data support the contention that this enzymatic activity is a crucial factor in the tumorigenic conversion of cells by avian sarcoma virus. Proteolytic peptide analysis of the structure of pp60src from revertant 866-4 indicates that it is similar to pp60src obtained from avian sarcoma virus-transformed chick embryo fibroblasts. Moreover, the reduction in kinase activity does not appear to be due to a lack of phosphorylation of the tyrosine residue in pp60src. Thus neither an obvious structural alteration nor a reduction in phosphorylation of pp60src appears responsible for the reduced kinase activity observed, suggesting that some as of yet undetermined feature of pp60src can influence the pp60src phosphorylating event.     

Isolation and characterization of revertant cell lines. VII. DNA synthesis and mitotic rate of serum-sensitive revertants in non-permissive growth conditions.

The ability to synthesize DNA and enter mitosis was studied in Balb/c and Swiss 3T3 cells, SV40 and MSV-transformed 3T3 cells and revertants of these transformed cells in cultures of different serum concentrations and cell densities. Three ways were found by which cells were able to maintain a constant cell number in non-permissive growth conditions: cessation of DNA synthesis, synthesis of DNA coupled with failure to enter mitosis, and the slow traverse of the cell cycle coupled with cell shedding. Growth control of the revertant of an MSV-transformed Balb/3T3 cell most closely resembled that of Balb or Swiss 3T3. This line did not grow in 1% serum and did not synthesize DNA in either non-permissive condition. Serum-sensitive revertants of SV40-transformed 3T3 cells are also unable to grow in 1% serum and also do not grow beyond confluence in 10% serum, but these cells differ from 3T3 in the manner in which this growth arrest is accomplished. In 1% serum, revertants synthesize DNA but do not enter mitosis. At confluence in 10% serum, they slowly traverse the cell cycle, with dividing cells replacing cells that are shed into the medium.     

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.     

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.     

Density dependent inhibition of both growth and T-antigen expression in revertants isolated from simian virus 40-transformed mouse SVT2 cells.

Phenotypic revertants were isolated from simian virus 40-transformed cells in order to examine the relationship between simian virus 40 T-antigen expression and G1 arrest of growth. Revertant clones with increased adherence were selected from cultures of SVT2, a simian virus 40-transformed BALB/c mouse cell line, and screened to find arrestable revertant clones which inhibited DNA synthesis when crowded. The clones selected from untreated SVT2 were unstable and showed little or no inhibition of DNA synthesis when crowded. Stable revertants were found after treatment of SVT2 with Colcemid to increase ploidy. The stable revertants all lost most transformed growth properties tested, including tumorigenicity, but only a few showed the same degree of inhibition of DNA synthesis at high cell density as BALB/3T3. All revertant clones expressed T antigen at low cell density. Three revertants showed coordinate inhibition of DNA synthesis and apparent loss of T antigen at high cell density. We suggest that changes in gene dosage rather than mutations caused the altered properties of the new revertants and that continued DNA synthesis in confluent cultures may be the transformed phenotype that requires the least simian virus 40 T antigen.     

Arrangement of Integrated Avian Sarcoma Virus DNA Sequences Within the Cellular Genomes of Transformed and Revertant Mammalian Cells

We have examined the arrangement of integrated avian sarcoma virus (ASV) DNA sequences in several different avian sarcoma virus transformed mammalian cell lines, in independently isolated clones of avian sarcoma virus transformed rat liver cells, and in morphologically normal revertants of avian sarcoma virus transformed rat embryo cells. By using restriction endonuclease digestion, agarose gel electrophoresis, Southern blotting, and hybridization with labeled avian sarcoma virus complementary DNA probes, we have compared the restriction enzyme cleavage maps of integrated viral DNA and adjacent cellular DNA sequences in four different mouse and rat cell lines transformed with either Bratislava 77 or Schmidt-Ruppin strains of avian sarcoma virus. The results of these experiments indicated that the integrated viral DNA resided at a different site within the host cell genome in each transformed cell line. A similar analysis of several independently derived clones of Schmidt-Ruppin transformed rat liver cells also revealed that each clone contained a unique cellular site for the integration of proviral DNA. Examination of several morphologically normal revertants and spontaneous retransformants of Schmidt-Ruppin transformed rat embryo cells revealed that the internal arrangement and cellular integration site of viral DNA sequences was identical with that of the transformed parent cell line. The loss of the transformed phenotype in these revertant cell lines, therefore, does not appear to be the result of rearrangement or deletions either within the viral genome or in adjacent cellular DNA sequences. The data presented support a model for ASV proviral DNA integration in which recombination can occur at multiple sites within the mammalian cell genome. The integration and maintenance of at least one complete copy of the viral genome appear to be required for continuous expression of the transformed phenotype in mammalian cells.     

Suppression of tumorigenicity in transformed cells after transfection with vinculin cDNA

Transfection of chicken vinculin cDNA into two tumor cell lines expressing diminished levels of the endogenous protein, brought about a drastic suppression of their tumorigenic ability. The SV-40-transformed Balb/c 3T3 line (SVT2) contains four times less vinculin than the parental 3T3 cells, and the rat adenocarcinoma BSp73ASML has no detectable vinculin. Restoration of vinculin in these cells, up to the levels found in 3T3 cells, resulted in an apparent increase in substrate adhesiveness, a decrease in the ability to grow in soft agar, and suppression of their capacity to develop tumors after injection into syngeneic hosts or nude mice. These results suggest that vinculin, a cytoplasmic component of cell-matrix and cell-cell adhesions, may have a major suppressive effect on the transformed phenotype.     

A specific protein, p92, detected in flat revertants derived from NIH/3T3 transformed by human activated c-Ha-ras oncogene

Total proteins from a mouse embryo fibroblast cell line NIH/3T3, NIH/3T3 cells transformed by human activated c-Ha-ras (EJ-ras) oncogene (EJ-NIH/3T3), and the two flat revertant cell lines, R1 and R2, were analyzed by two-dimensional gel electrophoresis (IEF and NEPHGE). Several hundred polypeptides were resolved as seen by silver staining. Common alterations in four polypeptide spots were observed in the revertants when compared with NIH/3T3 and EJ-NIH/3T3 cells. In these alterations, a new polypeptide spot p92-5.7 (designated by molecular weight x 10(-3) and pI) was detected only in the revertants and not in NIH/3T3 and EJ-NIH/3T3 cells. Furthermore, the expression level of p92-5.7 seemed to be associated with the flat morphology and the reduced tumorigenicity of the revertants. Polypeptide p92-5.7 was also not detected in the total proteins extracted from BALB/3T3 cells, NIH Swiss mouse primary embryo fibroblasts, NRK (normal rat kidney) cells, and L6 (rat myoblast). Subcellular fractionation of total protein from R1 cells revealed that the p92-5.7 was present in the cytosol. Western blot analysis using an anti-gelsolin antibody demonstrated that the p92-5.7 might be a variant form of gelsolin which is thought to be an actin regulatory protein or a gelsolin-like polypeptide. These results may suggest that the expression of p92-5.7 detected only in the revertants is associated, at least in part, with the reversion. This may be the first demonstration of specific protein expression in the flat revertants.     

Cellular tumorigenicity in nude mice. Role of susceptibility to natural killer cells

The athymic nude mouse is a useful animal model for assaying the neoplastic growth potential in vivo of animal cells transformed in vitro. Despite the demonstrated absence of thymus-dependent immunological functions, however, the nude mouse has now been shown to reject transplants of certain highly malignant heterologous tumors. In addition, a few transformed mammalian cell lines that exhibit all or most of the cellular phenotypes usually associated with malignancy fail to grow as tumors when injected into nude mice. In a continuing study to identify the in vitro phenotypes associated with tumor-forming ability in vivo, we investigated the role of cellular susceptibility to the naturally occurring, thymus-independent lymphocytes (natural killer or NK cells) in determining tumor induction by animal cells in nude mice. A representative collection of animal cells (ranging from normal human diploid cell strains to highly tumorigenic clonal cell lines, either transformed in vitro or derived from experimental tumors) was tested to see if the ability of cells to form tumors is consistently correlated with their susceptibility to NK cell-mediated lysis measured in vitro with splenic leukocytes from nude mice. If the physiological role of the NK cells in vivo were to recognize, and possibly to destroy, incipient tumor cells in situ, a direct association between cellular tumorigenicity and susceptibility to NK activity, might be expected. If, on the other hand, the formation of growing tumors by animal cells in nude mice depended on their ability to escape the cytolytic activity of NK cells, cellular tumorigenicity would be associated with cellular resistance to NK cells. Results obtained in this study failed to confirm either of these associations. Thus, cellular suscepbibility to NK cells, at least as determined by direct cytotoxicity assay in vitro, is not a useful predictive indicator of cellular tumorigenicity in nude mice.     

Overexpression of human UDP-glucose pyrophosphorylase rescues galactose-1-phosphate uridyltransferase-deficient yeast

To better understand the pathophysiology of galactose-1-phosphate uridyltransferase (GALT) deficiency in humans, we studied the mechanisms by which a GALT-deficient yeast survived on galactose medium. Under normal conditions, GALT-deficient yeast cannot grow in medium that contains 0.2% galactose as the sole carbohydrate, a phenotype of Gal(-). We isolated revertants from a GALT-deficient yeast by direct selection for growth in galactose, a phenotype of Gal(+). Comparison of gene expression profiles among wild-type and revertant strains on galactose medium revealed that the revertant down-regulated genes encoding enzymes including galactokinase, galactose permease, and UDP-galactose-4-epimerase (the GAL regulon). By contrast, the revertant strain up-regulated the gene for UDP-glucose pyrophosphorylase, UGP1. There was reduced accumulation of galactose-1-phosphate in the galactose-grown revertant cells when compared to the GALT-deficient parent cells. In vitro biochemical analysis showed that UDP-glucose pyrophosphorylase had bifunctional properties and could catalyze the conversion of galactose-1-phosphate to UDP-galactose in the presence of UTP. To test if augmented expression of this gene could produce a Gal(+) phenotype in the GALT-deficient parent cells, we overexpressed the yeast UGP1 and the human homolog, hUGP2 in the mutant strain. The Gal(-) yeast transformed with either UGP1 or hUGP2 regained their ability to grow on galactose. We conclude that revertant can grow on galactose medium by reducing the accumulation of toxic precursors through down-regulation of the GAL regulon and up-regulation of the UGP1 gene. We speculate that increased expression of hUGP2 in humans could alleviate poor outcomes in humans with classic galactosemia.     

Novel revertants of H-ras oncogene-transformed R6-PKC3 cells.

Rat 6 fibroblasts that overproduce protein kinase C beta 1 (R6-PKC3 cells) are hypersensitive to complete transformation by the T24 H-ras oncogene; yet T24 H-ras-transformed R6-PKC3 cells are killed when exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA) (W.-L. W. Hsiao, G. M. Housey, M. D. Johnson, and I. B. Weinstein, Mol. Cell. Biol. 9:2641-2647, 1989). Treatment of an R6-PKC3 subclone that harbors a T24 H-ras gene under the control of an inducible mouse metallothionein I promoter with ZnSO4 and TPA is extremely cytocidal. This procedure was used to isolate rare revertants that are resistant to this toxicity. Two revertant lines, R-1a and ER-1-2, continue to express very high levels of protein kinase C enzyme activity but, unlike the parental cells, do not grow in soft agar. Furthermore, these revertants are resistant to the induction of anchorage-independent growth by the v-src, v-H-ras, v-raf, and, in the case of the R-1a line, v-fos oncogenes. Both revertant lines, however, retain the ability to undergo morphological alterations when either treated with TPA or infected with a v-H-ras virus, thus dissociating anchorage independence from morphological transformation. The revertant phenotype of both R-1a and ER-1-2 cells is dominant over the transformed phenotype in somatic cell hybridizations. Interestingly, the revertant lines no longer induce the metallothionein I-T24 H-ras construct or the endogenous metallothionein I and II genes in response to three distinct agents: ZnSO4, TPA, and dexamethasone. The reduction in activity of metallothionein promoters seen in these revertants may reflect defects in signal transduction pathways that control the expression of genes mediating specific effects of protein kinase C and certain oncogenes in cell transformation.     

Revertants of v-fos-transformed rat fibroblasts: suppression of transformation is dominant.

Phenotypic revertants of Finkel-Biskis-Riley (FBR)-murine sarcoma virus-transformed rat fibroblasts were isolated on the basis of their adherence to plastic tissue culture dishes in the absence of divalent cations. Some revertants had sustained deletions or inactivating mutations of the v-fos gene. However, two revertants expressed a functional v-fos gene at levels equal to that in the transformed parental cells, and therefore phenotypic reversion was due to mutations in nonviral genes. These revertants were considered nontransformed according to four criteria: (i) they were flat and had a nontransformed morphology, (ii) they were contact inhibited when grown to confluence, (iii) they did not display anchorage-independent growth in soft agar, and (iv) they did not form tumors in nude mice. Somatic-cell hybrids between the revertants and the transformed parental cells were nontransformed, suggesting that the revertants had sustained an activating mutation of a gene capable of suppressing transformation. The expression of c-jun, junB, and junD was not altered in the revertants, and they could not be transformed by transfection with a c-jun expression vector. The revertants were resistant to transformation by an activated c-Ha-ras gene but were susceptible to transformation by simian virus 40. Our results demonstrate the existence of a class of revertants that harbor genes capable of suppressing transformation by v-fos and some other oncogenes. This contrasts with previously described revertants of transformation by v-fos that contain recessive mutations.