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.     

Growth-Inhibitory Functions of a Mutated Gelsolin (His321) in NIH/3T3 Mouse Fibroblasts

We have previously established a murine flat revertant cell line R1 from an activated H-ras transformant EJ-NIH/3T3 by subjecting it to ethyl methanesulfonate. From the R1 cells, we cloned a mutated gelsolin gene His321 and have shown the inhibitory activity of His321 against EJ-NIH/3T3 tumors. Our present experiments were conducted to find out whether the His321 gene has any effects on untransformed NIH/3T3 fibroblasts. Rhodamine-phalloidin staining revealed that two NIH/3T3 clones expressing His321 (NIH/λ2S-3 and NIH/λ2S-6) form organized actin stress fibers as two clones transfected with the vector alone (NIH/neo-3 and NIH/neo-5). We also found that in a liquid medium, NIH/λ2S-3 and NIH/λ2S-6 grew more slowly than NIH/neo-3 and NIH/neo-5 and that the doubling times of the former were about 10 h slower than those of the latter. To investigate the effects of His321 on the signal transduction pathway necessary for cell growth, we stimulated the cell lines by prostaglandin E1 (PGE1), a platelet-derived growth factor (PDGF), or the epidermal growth factor (EGF). Although stimulation by PGE1 increased intercellular cyclic AMP in R1 cells, it did not do so in NIH/λ2S-3 and NIH/λ2S-6 cells. On the other hand, stimulation by PDGF or EGF induced far less DNA synthesis in NIH/λ2S-3 and NIH/λ2S-6 than in NIH/neo-3 and NIH/neo5. These results suggest that through the effects on the signal transduction pathway of PDGF and/or EGF His321-mutated gelsolin inhibits the growth of NIH/3T3.     

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.     

Structure of the provirus within NIH 3T3 cells transfected with Harvey sarcoma virus DNA.

NIH 3T3 cells transformed with unintegrated Harvey sarcoma virus (HSV) linear DNA generally acquired a complete HSV provirus. Infection of these transformed cells with Moloney murine leukemia helper virus was followed by release of infectious particles. The HSV provirus within these transfected cells was convalently joined to nonviral DNA sequences and was termed "cell-linked" HSV DNA. The association of this cell-virus DNA sequence with the chromosomal DNA of a transfected cell was unclear. NIH 3T3 cells could also become transformed by transfection with this cell-linked HSV DNA. In this case, the recipient cells generally acquired a donor DNA fragment containing both the HSV provirus and its flanking nonviral sequences. After cells acquired either unintegrated or cell-linked HSV DNA, the newly established provirus and flanking cellular sequences underwent amplifications to between 5 and 100 copies per diploid cell. NIH 3T3 cells transfected with HSV DNA may acquire deleted proviral DNA lacking at least 1.3 kilobase pairs from the right end of full-length HSV 6-kilobase-pair DNA (corresponding to the 3'-proximal portion of wild-type HSV RNA). Cells bearing such deleted HSV genomes were transformed, indicating that the viral transformation gene lies in the middle or 5'-proximal portion of the HSV RNA genome. However, when these cells were infected with Moloney murine leukemia helper virus, only low levels of biologically active sarcoma virus particles were released. Therefore, the 3' end of full-length HSV RNA was required for efficient transmission of the viral genome.     

Isolation and characterization of NIH 3T3 cells expressing polyomavirus small T antigen.

The polyomavirus small T-antigen gene, together with the polyomavirus promoter, was inserted into a retrovirus vector pGV16 which contains the Moloney sarcoma virus long terminal repeat and neomycin resistance gene driven by the simian virus 40 promoter. This expression vector, pGVST, was packaged into retrovirus particles by transfection of psi 2 cells which harbor packaging-defective murine retrovirus genome. NIH 3T3 cells were infected by this replication-defective retrovirus containing pGVST. Of the 15 G418-resistant cell clones, 8 express small T antigen at various levels as revealed by immunoprecipitation. A cellular protein with an apparent molecular weight of about 32,000 coprecipitates with small T antigen. Immunofluorescent staining shows that small T antigen is mainly present in the nuclei. Morphologically, cells expressing small T antigen are indistinguishable from parental NIH 3T3 cells and have a microfilament pattern similar to that in parental NIH 3T3 cells. Cells expressing small T antigen form a flat monolayer but continue to grow beyond the saturation density observed for parental NIH 3T3 cells and eventually come off the culture plate as a result of overconfluency. There is some correlation between the level of expression of small T antigen and the growth rate of the cells. Small T-antigen-expressing cells form small colonies in soft agar. However, the proportion of cells which form these small colonies is rather small. A clone of these cells tested did not form tumors in nude mice within 3 months after inoculation of 10(6) cells per animal. Thus, present studies establish that the small T antigen of polyomavirus is a second nucleus-localized transforming gene product of the virus (the first one being large T antigen) and by itself has a function which is to stimulate the growth of NIH 3T3 cells beyond their saturation density in monolayer culture.     

Isolation of a simian virus 40 T-antigen-positive, transformation-resistant cell line by indirect selection.

In an attempt to identify cellular genes that might be involved in simian virus 40 (SV40) transformation, we have set out to isolate cells which express T antigen but are not transformed. SV40 DNA and the herpes simplex virus thymidine kinase gene were cotransfected into tk- 3T3 fibroblasts. Of 72 colonies screened that were resistant to hypoxanthine-aminopterin-thymidine, 57 were T antigen positive as judged by immunofluorescence. One of these lines, A27, had a normal growth phenotype in monolayer overgrowth and soft agar assays. It contained intact SV40 sequences that could be rescued by fusion to permissive cells. This rescued virus was fully capable of transforming nonpermissive cells to the same extent as did wild-type virus. The A27 cells, however, were not transformable by infection with SV40 or by transfection of SV40 DNA. It is likely that these cells were altered in a cellular function required for the establishment of the transformed state.     

Image analysis of Feulgen-stained NIH/3T3 cells transformed with DNA of 4-nitroquinoline 1-oxide treated human breast epithelial cells.

The nuclear phenotypes of Feulgen-stained NIH/3T3 cells transformed with 4-nitroquinoline 1-oxide (4NQO) treated, human breast epithelial cell (HBEC) DNA were studied by scanning microspectrophotometry and image analysis and compared with data obtained for nontransformed cells and for NIH/3T3 cells under ras oncogene transfecting situations. The Feulgen-DNA content of the individual nuclei (NQ1, NQ2, and NQ3 phenotypes) of the transformed cells was found not to be deeply affected, although presence of chromatin structures resembling double minutes could be verified in part of the metaphases of the transformed cells. On the other hand, the chromatin supraorganization of these cells showed some changes involving increased (NQ2, NQ3) or decreased (NQ1) levels of condensation. The changes in chromatin packing states, however, were of small magnitude compared with those reported for NIH/3T3 cells transfected with a c-H-ras oncogene or an N-ras-containing MCF-7 cell DNA. It was assumed that the transformation of the NIH/3T3 cells is not always necessarily accompanied by high levels of chromatin condensation. The transformation of the NIH/3T3 cells induced by the 4NQO-treated HBEC DNA and particularly the changes in chromatin condensation in these transformed cells could not be attributed merely to a ras activation elicited by the carcinogen. It is suggested that a more complex transforming mechanism is involved, probably owing to the fact that a whole genomic DNA of the 4NQO-treated HBEC has been used for transfection.     

Reduced induction of c-fos but not of c-myc expressions in a nontumorigenic revertant R1 of Ej-ras-transformed NIH/3T3 cells treated with 12-O-tetradecanoylphorbol-13-acetate (TPA)

It has been reported that both c-fos and c-myc mRNAs are induced in NIH/3T3 cells after 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. We have studied the effect of TPA on the expression of c-fos and c-myc in EJ-ras-transformed NIH/3T3 and its nontumorigenic flat revertant R1 cells. Although TPA treatment induces c-myc mRNA, as in the case of NIH/3T3 cells, the induced level of c-fos mRNA is greatly reduced not only in slow-growing EJ-ras-transformed NIH/3T3 but also in quiescent R1 cells. In addition, serum-induced c-fos expression is also reduced in EJ-ras-transformed NIH/3T3 and R1 cells. These observations suggest that the pathway from TPA to c-fos gene is different from that to c-myc gene and that the former pathway is down-regulated in association not with the transformed phenotype, but with EJ-ras expression, and it is possible that this reduced induction of c-fos is not specific to TPA.     

Lipid signalling pathways in normal and ras-transfected NIH/3T3 cells

The role of ras oncogenes in cellular signalling pathways involving phospholipid breakdown was studied in untransfected and proto-H-ras and mutated H-, K- and N-ras transfected NIH/3T3 cells. When the cells were grown at low cell densities, all of the ras transfected cells had 2-4 fold higher diacylglycerol (DAG) levels compared to growing NIH/3T3 cells. At high cell densities, DAG levels decreased in the former and increased in contact inhibited NIH/3T3 cells. In this regard, only cells transformed by mutated cellular and viral H-ras oncogenes (but not by the H-ras proto-oncogene) had elevated DAG levels compared to contact inhibited NIH/3T3 cells. The basal levels of inositol phosphates in ras transfected cells were not significantly different from NIH/3T3 cells and did not vary with cell density. Thus, the elevated DAG levels are not a consequence of increased phosphoinositide hydrolysis. The latter was stimulated by serum and bombesin only in normal and proto-H-ras transfected cells. In contrast, stimulation by bradykinin was observed only in cells transformed by mutated cellular ras oncogenes. Furthermore, aluminum fluoride stimulated phosphoinositide breakdown in the latter cells indicating that there was no uncoupling of the G protein from phospholipase C. Treatment of ras transfected cells with dibutyryl cyclic AMP (DB-cAMP), which causes an inhibition of growth and a reversal of the transformed morphology, did not alter the basal levels of inositol phosphates, DB-cAMP, however, did lower DAG levels in some of the transformed cell lines, but elevated DAG levels in low density NIH/3T3 cells. These findings indicate that the ras gene product p21 is not involved in phosphoinositide hydrolysis and that DAG levels do not correlate with cell growth in either normal or ras transfected NIH/3T3 cells. Thus, p21 appears to alter cell growth through mechanism(s) independent of lipid signalling pathways.     

Enhanced production of hepatitis B surface antigen in NIH 3T3 mouse fibroblasts by using extrachromosomally replicating bovine papillomavirus vector.

We have constructed a recombinant pBR322 plasmid composed of a subgenomic transforming fragment of bovine papillomavirus DNA and the hepatitis B surface antigen gene from cloned hepatitis B virus DNA and used it for transfection of NIH 3T3 mouse fibroblasts. The transformed cells retain the plasmids in extrachromosomal form with a copy number of about 50 to 100 per cell. Expression of the hepatitis B surface antigen gene linked to bovine papillomavirus DNA is independent of its orientation relative to the bovine papillomavirus vector. Cell lines continuously secreting high amounts of hepatitis B surface antigen into the medium could be established. The antigen is released into the culture medium as 22-nm particles, having the same physical properties and constituent polypeptides as those found in the serum of hepatitis B virus-infected patients.     

Integration of Rous sarcoma virus DNA during transfection

We have investigated the organization and integration sites of Rous sarcoma virus (RSV) DNA in NIH 3T3 mouse cells transformed by transfection with unintegrated and integrated donor RSV DNAs. RSV DNAs of different cell lines transformed by unintegrated donor DNA were flanked by different cellular DNA sequences, indicating that RSV DNA integrates at multiple sites during transfection. The RSV genomes of cells transformed by transfection were colinear with unintegrated RSV DNA, except that deletions within the terminal repeat units of RSV DNA were detected in some cell lines. These results suggested that the terminal repeat sequences of RSV DNA did not necessarily provide a specific integration site for viral DNA during transfection. In addition, cell lines transformed by integrated RSV DNAs contained both the RSV genomes and flanking cellular sequences of the parental cell lines, indicating that integration of integrated viral DNA during transfection occurred by recombinational events within flanking cellular DNA sequences rather than at the terminal of viral DNA. Integration of RSV DNA during transfection thus appears to differ from integration of RSV DNA in virus-infected cells, where the terminal repeat units of viral DNA provide a highly specific integration site. Integration of donor DNA during transfection of NIH 3T3 cells instead appears to proceed by a pathway which is nonspecific for both donor and recipient DNA sequences.     

Platelet-derived growth factor does not induce c-fos in NIH 3T3 cells expressing the EJ-ras oncogene.

Platelet-derived growth factor (PDGF), the calcium ionophore A23187, and the tumor promoter phorbol myristate acetate stimulated c-fos mRNA levels in control NIH 3T3 cells. However, NIH 3T3 cells transformed by EJ-ras DNA transfection, which have diminished PDGF-stimulated phospholipase C activity, showed a 95% reduction in PDGF-stimulated c-fos mRNA levels. The responses to A23187 and phorbol myristate acetate were also attenuated, but not as severely as the PDGF-mediated induction. The reduction in PDGF-stimulated c-fos induction did not appear to be a general result of cellular transformation, since src-transformed NIH 3T3 cells displayed a strong PDGF-stimulated c-fos induction. Despite the reduction in PDGF-stimulated c-fos induction, EJ-ras-transformed cells still responded mitogenically to PDGF. These data suggest that the magnitude of c-fos induction cannot be directly correlated with PDGF-stimulated mitogenesis in EJ-ras-transformed NIH 3T3 cells.     

Alteration in cyclic adenosine 3':5'-monophosphate binding proteins during the phenotypic change of mouse macrophages transformed by a temperature-sensitive mutant (tsA640) of SV40

By using a photoaffinity ligand, cell extracts from transformed macrophages that were established by infection with temperature-sensitive mutants (tsA640) of simian virus 40 (SV40) were examined for cyclic adenosine 3':5'-monophosphate (cAMP)-binding proteins. At the nonpermissive temperature for SV40 large T antigen, 39.0 degrees C, no significant cAMP-binding proteins could be detected, such as primary mouse macrophages. At the permissive temperature of 33.0 degrees C, cAMP-binding proteins appeared later than SV40 T antigen expression and cellular DNA synthesis. The profile of cAMP-binding proteins was similar to that of resting, but not proliferating, mouse clonal fibroblasts (BALB/c 3T3). These and previous results suggest that SV40 T antigen influences the expression of cAMP-binding proteins in tsA640-transformed macrophages; the large/small T antigen converts the profile of cAMP-binding proteins from macrophage to fibroblastic cells.     

Effect of genistein on topoisomerase activity and on the growth of [Val 12]Ha-ras-transformed NIH 3T3 cells

Genistein inhibited topoisomerase II and I; it increased the enzyme-DNA complex in L1210 cells at 1 micrograms/ml, and interfered with pBR322 DNA relaxation by the enzymes. To test the role of topoisomerase in the transformation by oncogenes, the effect of genistein on the transformation of NIH 3T3 cells by transfection with [Val 12]Ha-ras was compared with that of N-alpha-tosyl-L-lysyl-chloromethyl ketone (TLCK), since genistein inhibits tyrosine kinase as well as TLCK. Genistein reduced the number of foci of the transformed cells, and suppressed selectively the growth of ras-transformed NIH 3T3 cells but not normal NIH 3T3 cells. In contrast, TLCK did not affect the transformation. It inhibited the growth of the normal cells but not the transformed cells.     

NIH/3T3 cells transfected with human tumor DNA containing activated ras oncogenes express the metastatic phenotype in nude mice.

NIH/3T3 cells transfected with DNA from malignant human tumors produced experimental and spontaneous metastases in nude mice. In contrast, parent or spontaneously transformed NIH/3T3 cells failed to metastasize. The transfected clones contained either activated c-Harvey-ras or N-ras oncogenes. A representative clone (T71-17SA2) which was used to assess selected cellular and host factors relevant to the metastatic process produced lung metastases in 100% of the NIH nude mice recipients, secreted augmented levels of type IV collagenase, and invaded human amnion basement membrane in vitro. Expression of the metastatic phenotype was not related to decreased sensitivity to natural killer cells or macrophage-mediated cytotoxicity. Analysis of the cellular DNA from the T71-17SA2 transfectant and its corresponding metastases, both of which contained activated N-ras oncogenes, revealed a twofold increase in the N-ras-specific DNA sequences in the metastatic cells. Thus, transfection with human tumor DNA containing activated ras oncogenes can induce the complete metastatic phenotype in NIH/3T3 cells by a mechanism apparently unrelated to immune cell killing.     

Genetic transformation of somatic cells. XI. The autonomic replication of the cloned DNA of the bovine papilloma virus in NIH/3T3 mouse fibroblasts and the changes in the growth characteristics of the transformed cells

Mouse fibroblasts NIH 3T3 were transfected with the plasmid pBPV (142-6) containing full genome of bovine papilloma virus 1, and focuses of morphological transformation were selected 2-3 weeks later. DNA molecules, containing BPV-1 sequences, were isolated from extrachromosomal fraction of transformed clones suggesting stable autonomous replication of BPV in 3T3 NIH cells. In some rescued plasmids deletions spanning E6, 7 genes of BPV were found. It is suggested that these genes are not essential for morphological transformation and autonomous replication in 3T3 NIH cells. BPV-transformed clones are able to grow in the medium containing low concentration (0.5%) of serum.     

Polyomavirus transforms rat F111 and mouse NIH 3T3 cells by different mechanisms.

Polyomavirus middle tumor antigen (mT) was expressed in a line of mouse NIH 3T3 cells under control of the dexamethasone-regulatable mouse mammary tumor virus promotor. Contrary to rat F111 cells which were rendered anchorage independent by mT expression alone (L. Raptis, H. Lamfrom, and T.L. Benjamin, Mol. Cell. Biol. 5:2476-2487, 1985), mT-producing NIH 3T3 cells were unable to grow in agar even after full mT induction. The mT:pp60c-src-associated phosphatidylinositol kinase was activated in these cells to a degree similar to that in fully transformed cells expressing the small and large T antigens, in addition to mT. We therefore propose that the stimulation of this phosphatidylinositol kinase, although apparently necessary, is not sufficient for transformation of NIH 3T3 cells by polyomavirus.