Isolation of recessive (mediator-) revertants from NIH 3T3 cells transformed with a c-H-ras oncogene.

We have generated two serum- and anchorage-dependent revertants from NIH 3T3 cells transformed with multiple copies of the human c-H-ras oncogene. In both revertants, the c-H-ras oncogene was fully expressed. Fusion of either revertant with untransformed cells or of the two revertants with one another resulted in transformed progeny. These results indicated that the two revertants were recessive and in different complementation groups. We believe that in our two revertants some of the genes mediating the transforming activity of the c-H-ras oncogene are defective; we are attempting to identify these mediator genes.     

Short-term treatment with gamma interferon induces stable reversion of ras-transformed mouse fibroblasts.

Persistent revertants have been generated from NIH 3T3 cells transformed by an activated human Ha-ras gene after short-term gamma interferon treatment in the presence of the cardiac aminoglycoside ouabain. Normal fibroblastlike morphology and anchorage dependence are restored in revertants. Tumorigenicity in nude mice is abolished. The revertants continue to express high steady-state levels of the ras oncogene. Partial retransformation of reverted cells is induced after 5-azacytidine treatment or after infection with retrovirus vectors carrying the v-abl, v-fes, v-myc, or v-src oncogene. The revertants resist the transforming activities of the v-Ha-ras and v-mos oncogenes.     

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.     

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.     

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.     

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.     

Resistance to oncogenic transformation in revertant R1 of human ras-transformed NIH 3T3 cells.

A flat revertant, R1, was isolated from human activated c-Ha-ras-1 (hu-ac-Ha-ras) gene-transformed NIH 3T3 cells (EJ-NIH 3T3) treated with mutagens. R1 contained unchanged transfected hu-ac-Ha-ras DNA and expressed high levels of hu-ac-Ha-ras-specific mRNA and p21 protein. Transfection experiments revealed that NIH 3T3 cells could be transformed by DNA from R1 cells but R1 cells could not be retransformed by Kirsten sarcoma virus, DNA from EJ-NIH 3T3 cells, hu-ac-Ha-ras, v-src, v-mos, simian virus 40 large T antigen, or polyomavirus middle T antigen. Somatic cell hybridization studies showed that R1 was not retransformed by fusion with NIH 3T3 cells and suppressed anchorage independence of EJ-NIH 3T3 and hu-ac-Ha-ras gene-transformed rat W31 cells in soft agar. These results suggest that the reversion and resistance to several oncogenes in R1 is due not to cellular defects in the production of the transformed phenotype but rather to enhancement of cellular mechanisms that suppress oncogenic transformation.     

Expression of retrovirus-related, cytotoxic T lymphocyte- and transplantation-defined antigens in NIH/3T3 transfectants after a single passage in nude mice

Transfection of T24c-Ha-ras oncogene into NIH/3T3 fibroblasts resulted in the establishment of a transformed cell line (pT) that was tumorigenic when injected s.c. both into Swiss outbred nude mice and normal NIH inbred mice. The passage into nude mice, however, led to the development of a tumor variant (pT-nude) able to subsequently grow into sublethally x-irradiated but not into immunocompetent NIH mice. NIH mice immunized with this tumor variant developed a strong specific CTL response against the immunizing cell line, whereas the parental transformed pT cell line was not lysed. Clones were derived by limiting dilution from anti-pT-nude bulk population and were tested on a panel of transformed NIH/3T3 lines before and after their growth as tumor into nude mice. All of these lines were lysed by the Lyt-2+ CTL clones as a sole consequence of one in vivo passage into nude mice. The cross-reactive Ag were shown to be related to endogenous retroviral products as assessed by 1) immunoprecipitations of gp70, p15E, and p30 viral proteins in the nude variants but not in parental lines, and 2) by the ability of retroviruses from irradiated pT-nude cells to infect NIH/3T3 or pT lines making them susceptible to lysis by anti-pT-nude CTL clones. These results show that a single passage in nude mice can induce retrovirus-related, cell-surface Ag in transplanted neoplastic cells.     

Human recipient cell for oncogene transfection studies.

We used human oncogene DNA to transform the nontumorigenic, revertant, human osteosarcoma cell line HOS TE-85 clone 5 (ATCC CRL 1543) to tumorigenicity in athymic nude mice with latency periods as short as 3 weeks. These cells were also transformed by genetic markers in genomic DNA samples. Because of their low rate of spontaneous tumor formation and the simplicity of culturing them, HOS cells provide a human cell alternative to NIH 3T3 murine fibroblasts for oncogene transfection studies.     

Activated N-ras controls the transformed phenotype of HT1080 human fibrosarcoma cells

To investigate whether the activated N-ras oncogene of HT1080 human fibrosarcoma cells contributes to the expression of the transformed phenotype, we have isolated flat revertants. In two independent revertant lines, an increase in chromosomal ploidy occurred without a concomitant increase in the number of copies of the N-ras transforming allele. Immunoprecipitation confirms that the level of the mutant N-ras p21 gene product in the revertants is correspondingly lower than in HT1080. Analysis of sporadic tumors derived from the revertant cells reveals an increased dosage of the transforming allele. The revertants also retransform after transfection of cloned activated ras oncogenes. These results imply direct participation of an N-ras oncogene in maintaining the transformed phenotype of a human tumor cell line.     

Interferon-induced revertants of ras-transformed cells: resistance to transformation by specific oncogenes and retransformation by 5-azacytidine.

Prolonged alpha/beta interferon (IFN-alpha/beta) treatment of NIH 3T3 cells transformed by a long terminal repeat-activated Ha-ras proto-oncogene resulted in revertants that maintained a nontransformed phenotype long after IFN treatment had been discontinued. Cloned persistent revertants (PRs) produced large amounts of the ras-encoded p21 and were refractile to transformation by EJras DNA and by transforming retroviruses which carried the v-Ha-ras, v-Ki-ras, v-abl, or v-fes oncogene. Transient treatment either in vitro or in vivo with cytidine analogs that alter gene expression by inhibiting DNA methylation resulted in transformation of PR, but not of NIH 3T3, cells. The PR retransformants reverted again with IFN, suggesting that DNA methylation is involved in IFN-induced persistent reversion.     

v-myb transformation of Xeroderma pigmentosum human fibroblasts: overexpression of the c-Ha-ras oncogene in the transformed cells

Human Xeroderma pigmentosum "normal" fibroblasts AS16 (XP4 VI) were transformed after transfection with a recombinant v-myb clone. In this clone (pKXA 3457) derived from avian myeloblastosis virus (AMV), the expression of the oncogene sequences is driven by the AMV U-5 LTR promoter. The transformed cells (ASKXA), which have integrated a rearranged v-myb oncogene, grow in agar, are not tumorigenic in nude mice, and express a 45-kDa v-myb protein. The HMW DNA of these cells transform chicken embryo fibroblasts. The c-Ha-ras oncogene is overexpressed in the ASKXA cells but not in the parental "normal" AS16 cells and a revertant clone (ASKXA Cl 1.1 G). Our results lead to the conclusion that the XP fibroblasts are phenotypically transformed by the presence of the transfected v-myb oncogene, which is able to induce an overexpression of the c-Ha-ras gene.     

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.     

Some immunological properties of high and low tumorigenic cellular variants of c-H-ras transformed 3T3 cells

NIH 3T3 cells transformed in vitro with the c-H-ras oncogene were subcloned. The resulting subclones were assayed for in vivo tumorigenicity in nude and in immunocompetent mice. The response of two high tumorigenic and two low tumorigenic clones to mediators of natural immunity was analyzed. The clones did not differ in sensitivity to NK cell-mediated lysis. However, compared to low tumorigenic clones, the high tumorigenic ones had a down-regulated expression of a membrane determinant recognized by a certain monoclonal naturally occurring antibody. The determinants recognized by other monoclonal naturally occurring antibodies available in the laboratory were equally expressed on the high and low tumorigenic clones. The high tumorigenic cells showed an increased resistance to cytotoxicity mediated by lymphotoxin. These results suggest that naturally occurring antibodies and lymphotoxin may participate in controlling the tumorigenicity of transformed cells. The high tumorigenic clones but not the low tumorigenic ones contained a novel 3.5-kb ras mRNA.     

Tumorigenicity of morphologically distinct transformed foci induced by 3-methylcholanthrene in BALB/c-3T3 cells

4 mm in diameter), invasiveness (smooth vs. invading margins) and other properties (piling vs. spread). In our previous report, we showed that cells from all five types grew in soft agar, transformed normal NIH 3T3 cells and formed foci on normal layer of BALB/c-3T3 cells. In this study, the neoplastic/tumorigenic potential of cells from the five different types of transformed foci was investigated in nude mice. About two million cells from each transformed focus were injected into 4-week-old nude mice. Non-transformed BALB/c-3T3 cells were used as control. The results of this study indicate that all the 45 athymic mice injected with different transformants developed tumors between 2 and 4 weeks after injection. Tumors were not observed in eight mice injected with non-transformed BALB/c-3T3 cells. All tumors were histopathologically confirmed fibrosarcomas. These findings indicate that all five morphologically different foci show tumorigenicity and that any foci of size > or =2 mm regardless of invasiveness and piling could be scored as positive during the cell transformation assay.     

Tumorigenic transformation of NIH 3T3 cells by the autocrine synthesis of transforming growth factor alpha

A variety of cancer cells overexpress transforming growth factor alpha (TGF alpha), a mitogenic peptide. A cDNA sequence coding for the full-length human TGF alpha precursor protein was subcloned into a retroviral expression vector and introduced into clone 7 NIH 3T3 cells, which have low numbers of endogenous epidermal growth factor receptors (EGFRs). The autocrine synthesis of TGF alpha by these cells resulted in their focal transformation. In contrast, control NIH 3T3 cells treated in a paracrine manner with exogenous, saturating concentrations of the mature form of TGF alpha, though stimulated to divide, remained morphologically untransformed. The addition of saturating quantities of soluble, mature TGF alpha to NIH 3T3 cells expressing the transferred TGF alpha gene actually suppressed their growth and focal transformation. The transformation induced by the TGF alpha gene remained an EGFR-dependent process, since the degree of transformation was correlated with EGFR expression in NIH 3T3 cells and since NR6 cells, which are Swiss 3T3 cells devoid of endogenous EGFRs, were transformed by the TGF alpha vector only when exogenous EGFR genes were also introduced. When inoculated into nude mice, the TGF alpha-expressing cells rapidly gave rise to tumors that grew progressively, whereas control cells did not form tumors. We conclude that in certain circumstances autocrine TGF alpha can be more oncogenic than paracrine and that paracrine TGF alpha can suppress this effect.