Suppression of tumorigenicity with continued expression of the c-Ha-ras oncogene in EJ bladder carcinoma-human fibroblast hybrid cells.

A human tumor cell line (EJ) expressing an activated c-Ha-ras oncogene was fused with a normal human fibroblast cell line. This fusion resulted in hybrids that behaved as transformed cells in culture but failed to form tumors in nude (athymic) mice. After repeated cell passage, two tumorigenic segregants of the hybrids arose in culture. The levels of expression of activated c-Ha-ras mRNA and its protein product, p21, were similar in the EJ cell line, the nontumorigenic hybrids, and the tumorigenic segregants. DNA transfections of the hybrids were performed with activated c-Ha-ras plasmid constructs, and transfectants expressing a 2-fold level of c-Ha-ras relative to the hybrid cells were found to maintain the nontumorigenic phenotype. We suggest that expression of the active c-Ha-ras oncogene is insufficient for the malignant transformation of these human cells.     

Transfection with plasmid pSV2gptEJ induces chromosome rearrangements in CHEF cells.

In previous cytogenetic studies, trisomy for 3q was found to be the most frequent chromosome change associated with induced tumorigenicity by a variety of agents in Chinese hamster cells. Here we describe similar chromosome changes in 11 lines of CHEF/18 cells transfected with the mutant c-Ha-ras containing plasmid pSV2gptEJ. All 11 lines contained the transfected EJ gene and expressed increased levels of p21, the EJ gene product. Ten of the 11 lines were tumorigenic and all but 2 of these were trisomic for all or part of 3q. One line remained diploid and was nontumorigenic despite expressing elevated p21. Two tumorigenic lines from "hit-and-run" transfection with pSV2gpt were shown to express only control levels of p21, but they were trisomic for 3q. These results show that increased p21 expression is neither necessary nor sufficient for inducing tumorigenicity of CHEF cells. We propose that tumorigenicity in the transfected CHEF/18 cells of this study was induced by chromosome rearrangements, especially trisomy for 3q, that occurred at increased frequencies following transfection with pSV2gptEJ.     

Resistance of human cells to tumorigenesis induced by cloned transforming genes.

The transformation of human cells was examined by transfection of cloned oncogenic DNAs derived from the tumor virus simian virus 40 and from the human bladder carcinoma cell line EJ into diploid fibroblasts derived from foreskin (FS-2 cells). The simian virus 40 DNA was found to induce a morphologically transformed phenotype, leading to easily detectable focus formation. Tumor antigen was produced, but the transformed cells were not tumorigenic in the nude mouse. The EJ gene, a mutant form of the cellular c-Ha-ras gene, actively transforms NIH/3T3 mouse cells and CHEF/18 hamster cells but is inactive in FS-2 cells. Morphological transformation, focus formation, and tumorigenicity in nude mice were not induced when EJ DNA was transfected into FS-2 cells by using the selectable vector pSVgptEJ. The intactness of the transfected EJ DNA was established by restriction fragment analysis. This result raises the question of what role, if any, the mutated gene derived from the EJ cells played in the origin of the EJ bladder carcinoma.     

Plasmid-induced "hit-and-run" tumorigenesis in Chinese hamster embryo fibroblast (CHEF) cells.

The Chinese hamster embryo fibroblast cell line CHEF/18 is readily transfected by plasmid DNA. In the present transfection studies with CHEF/18 cells, focus formation induced by plasmids containing the mutant human c-Ha-ras gene EJ was compared with that of control plasmids without the EJ insert. The focus-forming activity of the transfected plasmid J132, a recombinant of the Harvey murine sarcoma virus LTR and the normal human c-Ha-ras1 in pBR322, also was assessed. Foci were recovered after transfection with either pSV2gpt or pSV2neo at about 10% the frequency obtained with the EJ-containing plasmids, and J132 gave a similar frequency, all well above background obtained with salmon sperm DNA. Whereas foci from transfection with EJ-containing plasmids contained the EJ DNA, no plasmid DNA was detected in either tumorigenic or tumor-derived cells from foci transfected with pSVgpt, pSVneo, or J132. Evidence that genomic changes were induced by plasmid transfection is based on finding chromosomal aberrations in all expanded foci and tumor-derived cells examined. The results suggest the occurrence of "hit-and-run" tumorigenesis induced by transient plasmid transfection.     

Experimental metastasis in nude mice of NIH 3T3 cells containing various ras genes.

These studies have compared the ability of NIH 3T3 cells containing different ras oncogenes to form tumor nodules in the lungs of nude mice after tail vein injection. The genes studied include the normal cellular and bladder tumor ras genes, recombinant viral/cellular ras genes, recombinant yeast/mammalian ras genes, and a constructed gene with yeast RAS1 sequences significantly modified by deletions and an oncogenic mutation. The results show that NIH 3T3 cells containing these genes readily form lethal tumor nodules in the lungs of nude mice after tail vein injection. No control NIH 3T3 cells formed lung tumors within 66 days. Although there were some quantitative differences in the potencies of the various lines, the striking conclusion is that NIH 3T3 cells transformed by either normal or activated mammalian ras genes form approximately equal numbers of experimental lung metastases. In addition, cells transformed by a significantly modified yeast RAS1 gene containing a purposefully introduced oncogenic mutation were also equally active in this assay. The amount of p21 (the 21-kDa protein encoded by ras), as measured by immunoprecipitation, was approximately the same in the parent lines before injection as in the tumors recovered after injection. This result indicates that there is no selection for metastatic sublines containing larger quantities of p21. Transfection of EJ bladder tumor ras DNA into NIH 3T3 cells followed by injection 3 days later into the tail veins of nude/beige mice indicated that the EJ ras gene can confer a metastatic phenotype within 3.5 cell generations without selection or clonal growth in vitro. Thus, the biochemical changes initiated after introduction of the c-Ha-ras gene into NIH 3T3 cells result in the almost immediate acquisition of phenotypes necessary for experimental metastasis.     

DNA transfer of focus- and tumor-forming ability into nontumorigenic CHEF cells.

CHEF/18 fibroblastic cells derived from a Chinese hamster embryo are diploid and nontumorigenic and require multiple steps of chemical treatment and selection to produce tumorigenic derivatives. In this report, CHEF/18 cells and a mutant capable of growing in medium with a low concentration of serum, LS1-1, were recipients in DNA transfer experiments using the calcium phosphate coprecipitation method. Focus formation with donor DNAs from tumor-derived CHEF cells and from human bladder carcinoma cell line EJ gave yields of 0.02-0.59 focus per microgram of DNA per 10(6) recipients. In one experiment in which CHEF/18 cells were transfected with EJ DNA, the presence of human DNA was detected in five of seven foci by using a cloned Alu sequence. Cells from one of these foci gave rise to tumors in nude mice, and the DNA produced secondary CHEF/18 transfectants. Because normal human cells as well as CHEF/18 cells require multiple stages to become tumorigenic, these findings suggest that EJ cells contain tumor-inducing DNA as the result of prior changes that occurred during the development of this carcinoma.     

Azacytidine-induced tumorigenesis of CHEF/18 cells: correlated DNA methylation and chromosome changes.

5-Azacytidine (azaC), a drug that induces decreased methylation of DNA in mammalian cells, was shown previously to induce differentiation of mesenchymal cell types in CHEF/18 cells (Chinese hamster embryo fibroblasts). This paper describes the effectiveness of azaC in inducing tumorigenicity in CHEF/18 cells, previously shown to be nontumorigenic stable diploids. A short exposure of growing cells to 3 microM azaC induced tumor-forming ability in CHEF/18 stem cells. Pre-adipocyte clones and subclones derived from CHEF/18 by prior treatment with azaC were also found to be tumorigenic. Pre-adipocytes previously induced by insulin in the absence of azaC were mostly nontumorigenic, but one clone produced tumors and gave rise to both tumorigenic and nontumorigenic subclones. Karyotype analysis of 41 clones and subclones from azaC-induced and insulin-induced pre-adipocytes revealed a complete correlation between tumor-forming ability and the presence of trisomy for chromosome 3q. In addition, the tumorigenic and tumor-derived lines were demethylated at specific C-C-G-G sites in the preproinsulin, Ha-ras, and Ki-ras genes as revealed by blot hybridization to Msp I- and Hpa II-digested DNAs, whereas the nontumorigenic lines resembled the CHEF/18 controls. This three-way correlation between tumorigenicity, trisomy for 3q, and specific demethylation suggests that decreased DNA methylation may be involved both in differentiation and in tumorigenicity, and that azaC may induce chromosomal aberrations as well as altering DNA methylation.     

Tumorigenicity and its suppression in cybrids of mouse and Chinese hamster cell lines.

The effect of cytoplasm upon the expression of tumorigenicity was examined with a pair of mouse and a pair of Chinese hamster cell lines, in intraspecies cybrids formed by reciprocal fusions between either tumorigenic or nontumorigenic cells and cytoplasms derived from them. With the mouse cells, 3T3 and the simian virus 40-transformed line SVT2, the cybrid clones were tumorigenic when SVT2 cells were fused with 3T3 cytoplasts, but not in the reciprocal fusion. With the hamster cells, CHEF/18 and the spontaneous transformant CHEF/16, however, tumorigenicity was partially suppressed in cybrid clones formed by fusion of tumorigenic CHEF/16 cells with CHEF/18 cytoplasts; cybrids were nontumorigenic in the reciprocal fusion. Thus, cybrid analysis has shown that tumorigenicity is not cytoplasmically transmitted in these two cell pairs, but suppression of tumor-forming ability may be cytoplasmically transmitted in the hamster cybrids.     

Detection of genes with a potential for suppressing the transformed phenotype associated with activated ras genes.

Seven morphologically nontransformed (flat) revertants with reduced tumorigenicity in vivo have been isolated from populations of Kirsten sarcoma virus-transformed NIH 3T3 cells transfected with a cDNA expression library of normal human fibroblasts. Each revertant harbors 1-10 recombinant plasmids per cell and retains a rescuable transforming virus as well as high level expression of v-Ki-ras-specific RNA and the viral oncogene product, p21v-Ki-ras. Transformed phenotypes are suppressed in cell hybrids generated by fusing each revertant to v-Ki-ras-transformed NIH 3T3 cells. From two of the revertant lines, plasmids capable of giving rise to flat secondary transfectants have been recovered. Thus, in some, if not all, of the revertants, transfected cDNAs seem to be responsible for the suppression of specific transformed phenotypes.     

Tumor suppression involves down-regulation of interleukin 3 expression in hybrids between autocrine mastocytoma and interleukin 3-dependent parental mast cells.

Interleukin 3 (IL-3)-dependent PB-3c mouse mastocytes can be transformed by the v-Ha-ras oncogene to generate autocrine IL-3-producing mastocytomas. Hybrid cell lines were constructed by fusing an IL-3-producing mastocytoma cell line with its IL-3-dependent normal parental cell. Unlike the mastocytoma parent cell line, hybrid cell lines required growth factor for in vitro proliferation, indicating that the IL-3-dependent phenotype is dominant. IL-3 mRNA, expressed at high levels in the tumor cells, appeared down-regulated in the cell hybrids. In contrast, p21v-Ha-ras levels were not reduced in the hybrids. The hybrid lines generated tumors in vivo with drastically prolonged latency times when compared to the tumor parent (10 versus 2 weeks). We propose that down-regulation of IL-3 mRNA production after cell fusion is responsible for the loss of growth autonomy in the hybrids and is likely to play a role in the partial suppression of tumor formation in vivo. Our data are consistent with the hypothesis that a tumor suppressor, present in PB-3c cells, acts as a negative regulator of IL-3 expression.     

Lysis of Chinese hamster embryo fibroblast mutants by human natural cytotoxic (NK) cells.

The nontransformed, nontumorigenic CHEF/18 Chinese hamster embryo fibroblast line, as well as nontumorigenic CHEF/18 mutants that had become anchorage independent or acquired a reduced serum requirement for growth, and fully transformed, tumorigenic CHEF cell lines were analyzed for their sensitivity to killing in vitro by human natural killer (NK) cells. Nontumorigenic but transformed anchorage-independent and low-serum-requiring mutants remained insensitive to NK-mediated lysis like the parent CHEF/18 line. Only fully tumorigenic CHEF lines were found to be sensitive to NK-mediated lysis, although a few tumorigenic lines were resistant to NK lysis. These results indicate that NK sensitivity is not the result of any cellular changes associated with acquisition of an anchorage-independent or low-serum-requiring phenotype but is the result of some additional change(s) found only in fully tumorigenic CHEF cells. Our studies also show that, whatever the NK target structure is, it is evolutionarily conserved so that human NK cells are able to distinguish between Chinese hamster tumorigenic and nontumorigenic cells.     

Flat revertants isolated from Kirsten sarcoma virus-transformed cells are resistant to the action of specific oncogenes.

Two flat revertants have been isolated from mutagen-treated populations of Kirsten murine sarcoma virus (Ki-MuSV)-transformed NIH/3T3 cells. These revertants, which appear to be cellular variants resistant to transformation by the Ki-MuSV oncogene v-Ki-ras, contain Ki-MuSV-specific DNA, elevated levels of the v-Ki-ras gene product p21, and rescuable transforming virus. Cell hybridization studies indicated that the revertant phenotype is dominant in hybrids between revertant cells and cells transformed by Ki-MuSV or the closely related Harvey MuSV and BALB MuSV. Analysis of hybrid cells resulting from the fusion of these revertants to cell lines transformed by other retroviruses showed that the action of certain oncogenes structurally unrelated to v-Ki-ras also could be suppressed. Thus, there appear to be functional relationships and diversities among transforming genes (oncogenes) not readily apparent from their structural characteristics.     

Autophosphorylation of v-Ha-ras p21 is modulated by amino acid residue 12.

The 21,000-dalton protein (p21) encoded by the ras oncogene of Harvey murine sarcoma virus (v-Ha-ras) becomes phosphorylated (pp21) in vivo and in vitro on threonine residue 59. p21 molecules encoded by cellular ras genes (c-Ha-ras-1) contain an alanine at position 59, and thus these p21 molecules are not phosphorylated. In this investigation, recombinant ras genes have been constructed between the 5' p21 coding region of normal (EC) or oncogenically activated (EJ) human c-Ha-ras-1 and the 3' p21 coding region of v-Ha-ras to generate p21 molecules containing a threonine phosphoacceptor site at position 59 and a glycine (EC/v-Ha) or valine (EJ/v-Ha) at residue 12. In transformed NIH 3T3 mouse fibroblast cells labeled with [35S]methionine, the ratio of pp21 to p21 was strikingly modulated by the amino acid at residue 12. v-Ha-ras p21 has an arginine at position 12, and 24% of total p21 was in the phosphorylated form. A glycine at residue 12 decreased the amount of pp21 to 14% of total p21, and a valine at residue 12 dramatically increased this value to 50%. In vitro, the valine form of p21 had 2.4- and 2.7-fold greater autophosphorylating activity than the glycine and arginine forms of p21, respectively, using [gamma-32P]GTP as phosphate donor, but the three p21 species had similar Km values for GTP (0.20-0.27 microM). These results indicate that a biochemical activity of p21 distinguishes between previously observed biological differences of normal and activated human ras genes.     

Reduced hormone-stimulated adenylate cyclase activity in NIH-3T3 cells expressing the EJ human bladder ras oncogene.

Recent studies have shown that the 21-kilodalton protein (p21) Ha-ras gene product shares sequence homology with and may exhibit biochemical properties similar to the mammalian guanine nucleotide-binding proteins. These data suggested that one of the biochemical functions of p21 in the vertebrate cell may be to regulate adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1]. We determined both in intact NIH-3T3 murine cells and in membranes isolated from these cells that the hormone-stimulated adenylate cyclase activity of cells expressing the EJ human bladder carcinoma oncogene (EJ-ras) is significantly reduced compared with control cells. Thus, the levels of cAMP measured in the EJ-ras-transformed cells by radioimmunoassay are reduced 78% and 93% after prostaglandin and isoproterenol stimulation, respectively, compared with the levels in control cells. Treatment of the EJ-ras-transformed cells with pertussis toxin or cholera toxin did not correct the alterations in adenylate cyclase activity. Cells expressing the normal human Ha-ras gene displayed intermediate levels of adenylate cyclase hormone sensitivity; these levels of adenylate cyclase activity were greater than those in the EJ-ras-transformed cells but lower than in control cells. Hormone-stimulated adenylate cyclase activities in cells transfected with Rous sarcoma virus DNA were similar to those in control cells. These data support the hypothesis that both the normal and mutated Ha-ras p21s are related to guanine nucleotide-binding proteins.     

Gene amplification: an example of accelerated evolution in tumorigenic cells.

During selection for methotrexate resistance, tumorigenic CHEF/16 cells and derivatives from CHEF/16 tumors underwent amplification of the dihydrofolate reductase gene (DHFR) at accelerated rates compared with closely related nontumorigenic CHEF/18 cells. "Dot blot" analysis showed that the CHEF/16 cells contained many more copies of the DHFR gene than did the CHEF/18 cells, when assayed at similar elevated levels of methotrexate resistance. Chromosome analysis of cell samples taken at several time points during amplification revealed large differences between the nontumorigenic CHEF/18 cells and the two tumorigenic cell lines. The tumorigenic cells developed few chromosome rearrangements over a 4-log increase in methotrexate resistance, other than increased length of a single chromosome, which was shown by in situ hybridization to contain most or all of the amplified DHFR gene copies. In contrast, the CHEF/18 cells underwent complex, progressive changes in almost every chromosome, and in situ hybridization suggested a dispersed pattern of gene amplification. The data support the hypothesis that unregulated amplification is a pathological process, occurring readily in neoplastic but rarely in normal cells, that together with other chromosomal disturbances contributes to the rapid evolution and progression of cancer.     

An ets-related gene, ERG, is rearranged in human myeloid leukemia with t(16;21) chromosomal translocation.

The t(16;21)(p11;q22) translocation is a nonrandom chromosomal abnormality found in several types of myeloid leukemia, which show variable cytomorphological features. We constructed rodent-human somatic cell hybrids containing the der(16) chromosome from leukemic cells of a patient with t(16;21). Using these hybrids, we mapped the translocation breakpoint on the Not I restriction map of chromosome 21 which we had previously constructed. The result showed the proximity of the breakpoint to the ERG gene, a member of the ets oncogene superfamily. Polymerase chain reaction and Southern blot analyses of genomic DNA from the hybrids and from peripheral blood cells and bone marrow cells of patients with t(16;21) showed that the breakpoints were clustered within a single intron in the coding region of the ERG gene. This finding and the results obtained by Northern blot analysis suggested the formation of a chimeric product(s) by fusion of the ERG gene and an unknown counterpart gene on chromosome 16.     

Identification of an activated c-Ki-ras oncogene in rat liver tumors induced by aflatoxin B1.

Weanling male Fischer rats were administered 40 intraperitoneal injections of aflatoxin B1 (25 micrograms per animal per day) over a 2-month period. This chronic dosing regimen resulted in the sequential formation of hyperplastic foci, preneoplastic nodules, and hepatocellular carcinomas in all of the animals treated. The presence of transforming DNA sequences was detected by formation of anchorage-independent foci after transfection of tumor-derived DNA in NIH 3T3 mouse fibroblasts. Transfection of genomic DNA isolated from individual tumors from eight animals resulted in specific transforming activities ranging from 0.05 to 0.2 foci per micrograms of DNA. Primary transfectant DNAs were analyzed by Southern blot hybridization with DNA probes homologous to c-Ha-ras, c-Ki-ras, and N-ras oncogenes. A highly amplified c-Ki-ras oncogene of rat origin was detected in transformants derived from tumors in two of the eight animals tested. There was no evidence to suggest the presence of c-Ha-ras or N-ras sequences in any of the transformants. Analysis of primary liver tumor DNA showed no Ki-ras DNA amplification when compared to control liver DNA samples. Increased levels of c-Ki-ras p21 proteins were detected in 3T3 transformants containing activated rat c-Ki-ras genes. The presence of c-Ki-ras sequences of rat origin capable of inducing transformed foci can be taken as evidence that the c-Ki-ras gene has been activated in the primary liver tumors.     

Prediction of the three-dimensional structure of the transforming region of the EJ/T24 human bladder oncogene product and its normal cellular homologue.

The three-dimensional structures of the transforming region of the product of the EJ/T24 human bladder oncogene and of the c-Ha ras-1 gene product have been calculated by using conformational energy calculations. These two genes, representing a transforming oncogene and its normal cellular homologue, encode 21,000-dalton peptides that differ by one amino acid at position 12. We therefore examined the energetically allowed conformations of the hydrophobic decapeptide surrounding this substitution site. The calculations show that the most favorable form of the c-Ha ras-1 gene product exists when glycine-12 is in a left-handed bend conformation. No other amino acid can adopt this conformation and thus the bladder oncogene peptide containing valine at position 12 has a markedly different three-dimensional structure. A simple model is proposed to account for the consequences of a position 12 mutation.