Malignant transformation of an infinite life span human fibroblast cell strain by transfection with v-Ki-ras

To determine if human fibroblasts can be transformed into malignant cells by transfection of a K-ras oncogene, we transfected the provirus of Kirsten murine sarcoma virus (v-Ki-ras) into an infinite life span human cell strain, MSU-1.1, which has a normal morphology, is not anchorage independent, and has a stable, near-diploid karyotype. The transfected populations gave rise to distinct foci composed of morphologically-altered cells. The cells from several independent foci were isolated, propagated, and assayed for anchorage independence and/or tumorigenicity. They formed large-sized colonies in soft agar at a high frequency. Cell strains derived from colonies isolated from agar as well as focus-derived cell strains were injected subcutaneously into athymic mice to test for tumorigenicity. One cell strain yielded myxoid fibromas, the rest produced well-differentiated, progressively-growing, invasive, myxoid or spindle cell sarcomas. The karyotype of each of the cell strains tested, including cell strains derived from tumors, was identical to that of non-transfected MSU-1.1 cells. Two focus-derived strains, and two cell strains derived from sarcomas produced from them, were tested and shown by DNA and RNA hybridization to contain and express the v-Ki-ras oncogene. Radioimmunoprecipitation analysis showed that these strains expressed ras-specific p21 products not found in non-transfected MSU.1.1 cells. When injected intraperitoneally, a cell strain derived from a myxoid tumor gave rise to invasive myxoid tumors at various sites in the body. The same cell strain gave rise to invasive spindle cell sarcomas when injected into the tail vein of the animals.     

Malignant transformation of human fibroblast cell strain MSU-1.1 by N-methyl-N-nitrosourea: evidence of elimination of p53 by homologous recombination

To determine whether N-methyl-N-nitrosourea (MNU) can induce malignant transformation of human fibroblasts and whether O6-methylguanine (O6-MeG) is involved, two populations of infinite life span cell strain MISU-1.1, differing only in level of O6-alkylguanine-DNA alkyltransferase, were treated with MNU and assayed for focus formation. MNU caused a dose-dependent increase in the frequency of foci in both groups, but the dose required was significantly lower in the cells lacking O6-alkylguanine-DNA alkyltransferase, indicating that O6-MeG was causally involved. Of 35 independent focus-derived strains assayed for p53 transactivating abilily, one was heterozygous, and 15 had lost all activity, 1 of 7 from untreated cells and 14 of 27 from MNU-treated cells. These results indicate that loss of p53 is not required for focus formation but may permit cells to form foci. Of 35 strains assayed for tumorigenicity, 10 formed malignant tumors with a short latency, all 10 lacked wild-type p53. The p53 heterozygous strain also formed tumors after a long latency, and the cells from those tumors lacked p53 transactivating ability. None of the 19 strains with wild-type p53 formed tumors. These results indicate that although loss of p53 is not sufficient for malignant transformation of MSU-1.1 cells, it may be necessary. Analysis of the p53 cDNA from several focus-derived strains lacking p53 activity revealed that each contained the same mutation, an A to G transition at codon 215, resulting in a change from serine to glycine. Because p53 can be inactivated by mutations at any one of a large number of sites, finding the same mutation in each strain assayed strongly suggests that the target population included a subpopulation of cells with this codon 215 mutation in one allele. Further analysis showed that all 15 focus-derived cells strains that lacked p53 transactivating activity contained two alleles, each with the same codon 215 mutation, and that the mutant allele in the heterozygous strain also had that mutatation. Analysis of the p arm of chromosome 17 of the focus-derived cell strains containing the codon 215 mutation revealed seven patterns of loss of heterozygosity, evidence of mitotic homologous recombination. Similar analysis of a separate series of cell strains, derived from foci induced by cobalt-60, revealed four patterns of loss of heterozygosity, only two of which had been found with those induced by MNU. These data suggest that homologous mitotic recombination, induced by O6-MeG in a subpopulation of cells heterozygous for p53 mutation, rendered the cells homozygous for loss of p53 activity, that this allowed the cells to form foci, and that although loss of p53 is not sufficient for malignant transformation, it predisposes cells to acquire the additional changes needed for such transformation.     

Malignant transformation of human fibroblast strain MSU-1.1 by v-fes requires an additional genetic change

To determine whether the v-fes oncogenc can malignantly transform human fibroblasts that have acquired an infinite life span and are partially growth-factor-independent, we trans-fected cell strain MSU-1.1 with a plasmid containing the v-fes oncogene and a bacterial histidinol dehydrogenase gene. Of the 60 independent histidinol-resistant clones isolated and assayed for v-fes expression using a fes-specific monoclonal antibody, 6 were found to express the v-fes protein at a detectable level. When progeny cells from these 6 clones were further characterized, 3 of the 6 clonal populations exhibited a significant increase in the ability to form medium-sized colonies in agarose, but none were tumorigenic in athymic mice. However, when the 6 populations were propagated for many generations, the same 3 populations acquired the ability to form very large colonies in agarose (± 100 μm in diameter) at a frequency of 2% to 17%, and formed malignant tumors in athymic mice. This suggests that an additional genetic change required for malignant transformation had been spontaneously acquired in 3 of the v-fes -transformed cell strains. To determine whether the change or changes were the equivalent of an activated sis or ras proto-oncogene, we transfected the v-fes oncogene into derivative strains of MSU-1.1 that express a transfected v-sis, c-H-ras or c-N-ras oncogene, but that do not form tumors, and assayed the v-fes-expressing transfectants for tumorlgenicity. The results showed that when complemented either by a ras oncogene expressed at a somewhat enhanced level or by the v-sis oncogene, v-fes can supply the additional change required for malignant transformation.     

Tumorigenicity of human HT1080 fibrosarcoma X normal fibroblast hybrids: chromosome dosage dependency

The tumorigenic capacity of hybrids formed by fusion of the highly tumorigenic HT1080 human fibrosarcoma cell line with nontumorigenic normal fibroblasts was examined. The HT1080 also contains an activated N-ras oncogene. Near-tetraploid hybrids which contained an approximately complete chromosomal complement from both parental cells were nontumorigenic when 1 X 10(7) cells were injected s.c. into athymic (nude) mice, whereas the parental HT1080 cells produced tumors in 100% of the animals with no latency period following injection of 2 X 10(6) cells. Tumorigenic variants were obtained from these hybrids which had lost only a few chromosomes compared to cells from the nontumorigenic mass cultures. In addition, several near-hexaploid hybrids were obtained which contained approximately a double chromosomal complement from the HT1080 parental line and a single chromosomal complement from the normal fibroblasts. All of these near-hexaploid hybrids produce tumors in 100% of nude mice with no latency period. Our results indicate that tumorigenicity of these particular human malignant cells of mesenchymal origin can be suppressed when fused with normal diploid fibroblasts. In addition, the results suggest that tumorigenicity in this system is chromosomal dosage dependent, since a diploid chromosomal complement from normal fibroblasts is capable of suppressing the tumorigenicity of a near-diploid but not a near-tetraploid chromosomal complement from the tumorigenic HT1080 parent. Finally, the loss of chromosome 1 (the chromosome to which the N-ras oncogene has been assigned) as well as chromosome 4 was correlated with the reappearance of tumorigenicity in the rare variant populations from otherwise nontumorigenic near-tetraploid hybrid cultures. Our results also suggest the possibility that tumorigenicity in these hybrids may be a gene dosage effect involving the number of activated N-ras genes in the hybrids compared to the gene(s) controlling the suppression of the activated N-ras genes.     

Morphological transformation, focus formation, and anchorage independence induced in diploid human fibroblasts by expression of a transfected H-ras oncogene

In an attempt to determine how normal human fibroblasts respond to high expression of the T24 H-ras oncogene, we tranfected such cells with the plasmid vector pHO6T1 (D. A. Spandidos and N. M. Wilkie, Nature (Lond.), 310:469-475, 1984), containing the T24 H-ras oncogene with 5' and 3' enhancer sequences, and the aminoglycoside phosphotransferase gene which confers resistance to the drug, G418. Approximately 1.5% of the G418-resistant colonies obtained after transfection and selection consisted of cells exhibiting obvious morphological transformation; i.e., they were highly refractile and more rounded than normal fibroblasts. DNA hybridization analysis showed that the morphologically transformed cells contained the transfected T24 H-ras oncogene, and radioimmunoprecipitation analysis showed that they were expressing the T24 H-ras protein product, M, 21,000 protein. Morphologically transformed cells formed colonies in soft agar at a frequency at least 60 times higher than that of cells that had been transfected with the control plasmid containing the normal cellular H-ras gene. Cells transfected with plasmid pHO6T1 could also be identified by their ability to form distinct foci when grown to confluence in nonselective medium following transfection. This study demonstrates that normal diploid human fibroblasts in culture can be transformed by transfection with a H-ras oncogene, and that such transformation correlates with expression of the mutant Mr 21,000 protein.     

The role of wild-type and mutated N-ras in the malignant transformation of liver cells

In order to determine the role of N-ras overexpression and mutation in malignant liver cell transformation, wild-type and mutated N-ras were transfected into the rat liver epithelial cell line OC/CDE 22, and N-ras expression, growth kinetics, growth in soft agar, and tumorigenicity in vivo as well as the involvement of the mitogen-activated protein kinase (MAPK) signal transduction pathway in the expression of the malignant phenotype were analyzed. Although OC/CDE 22 cells transfected with wild-type N-ras showed a high expression of N-ras at the mRNA and protein levels, the cells did not grow in soft agar and were not tumorigenic in vivo. In contrast, OC/CDE 22 cells transfected with mutated N-ras showed anchorage-independent growth and were tumorigenic. When cultured in fetal bovine serum-supplemented medium, OC/CDE 22 cells expressing mutant N-ras showed a higher proliferation rate than nontransfected OC/CDE 22 cells or OC/CDE 22 cells transfected with wild-type N-ras. When held in serum-free medium, untreated OC/CDE 22 cells did not grow at all, while OC/CDE 22 cells transfected with wild-type or mutant N-ras proliferated at a similar rate, which can be explained by the high MAPK activity in these cells. Selective inhibition of the MAPK cascade abolished the growth of OC/CDE 22 cells carrying mutant N-ras in soft agar; furthermore, these cells ceased pile up and formed monolayers on Petri dishes. Thus, activation of the MAPK signaling pathway, though alone not sufficient to malignantly transform liver cells (as shown in liver cells overexpressing wild-type N-ras), is not only essential for growth control but also for the expression of the malignant phenotype (as demonstrated in liver cells transformed by mutated N-ras).     

Localization of oncoprotein P21ras in the human liver cancer

Using the human liver cancer DNA transfected NIH/3T3 cell line, the human N-ras oncogene and the over expression of the oncoprotein P21ras was demonstrated, BALB/C mice were immunized. The spleen cells from the immunized mice were fused with SP2/0 myeloma cells. After the HAT medium selection and screening, two hybridoma cell lines, SCI-Oncogema 1 and 2, were established. In the immunoprecipitation test, the molecular weight of the protein reacting to Oncogema 1 was 21,000. This M.W 21,000 protein possessed the capability to bind with GTP, i.e. the character of P21ras. These data indicate that the Oncogema 1 is the monoclonal antibody against P21ras. Using Oncogema 1, specimens from 6 liver cancer patients were studied by immunopathology. With ABC stain, it was observed that the malignant cells in all the samples showed dark staining; the P21ras revealed over expression. Although the staining was heterogeneous, it implied that the ras oncogene was involved in the carcinogenesis of these six samples. No over expression was seen in the normal liver cells even in those around the cancerous lesion. However, dysplastic cells were moderately stained which means that the ras oncogene was activated and P21ras over expressed in these cells. The results suggest that the ras oncogene and P21ras play an important role in the early stage of liver cancer carcinogenesis.     

Single-steep transformation of human breast epithelial cells by SV40 large T oncogene.

Normal human mammary epithelial cell (HMEC) cultures originating from 2 mammoplasty reduction surgical samples were transfected with replication-defective SV 40 DNA. Two independent cell lines designated as S2T2 and S1T3, selected for their increased proliferation potential and lifespan, were propagated for greater than 22 months in culture. They maintained a near-diploid karyotype with few chromosomal markers such as trisomy 1q (S1T3) and trisomy 8q (S2T2), which are most common in breast cancer in vivo. Immortalized S1T3 cells were not tumorigenic, whereas S2T2 cells produced slowly growing tumors in nude mice. One tumor was propagated in vitro and the transformed NS2T2 cell line subsequently raised 100% large tumors in the nude mouse. Rearrangement of the SV40 genome was observed in NS2T2 cells, which was not associated with increased expression of large T antigen. S1T3, S2T2 and transformed NS2T2 cell lines expressed cytokeratins CK18, CK19, the mammary-specific antigen DF3, and functional EGF receptors. Single-step immortalization and malignant transformation of human breast epithelial cells can thus occur upon transfection with SV40 large T oncogene. The chromosomal abnormalities observed in these cell lines suggest that they could offer a model for the study of breast-tumor progression in vitro.     

The influence of the oncogene N-ras on cell cycle delay in a human melanoma cell line with reduced radioresistance

In a previous study we found that transfection of a human melanoma cell line with the oncogene N-ras led to increased radiosensitivity as measured by clonogenic assays. Since a shift in radiosensitivity is often correlated with altered G2/M delay, we investigated whether this was also the case in this oncogene containing melanoma cell line (IGRras). A human melanoma cell line, stably transfected with mutated N-ras, and its parental cell line transfected with the neomycin phosphotransferase gene only (IGRneo), were irradiated with 5 Gy and cell cycle distribution was measured at hourly time intervals by DNA staining with propidium iodide. Next, the effect of ionising radiation on the duration of the S-phase was determined by pulse labelling cells with BrdUrd before irradiation. Both cell lines showed a radiation induced G2/M delay, which was most prolonged for the ras transfected cell line. After 5 Gy, the S-phase duration was unaltered, although the shape of the relative movement (RM) curves was slightly different. No G1 delay was observed in either cell line. Ras transfection in a melanoma cell line leads to prolonged G2/M delay after radiotherapy. This prolongation is associated with increased radiosensitivity and not with radioresistance. These data throw doubt on the use of oncogene expression or G2/M delay as predictors of radiosensitivity.     

Characterization of activated proto-oncogenes in chemically transformed syrian hamster embryo cells

The Syrian hamster embryo (SHE) cell transformation model has been used by many investigators to study the multistep process of neoplastic transformation induced by chemical carcinogens. In this study we have attempted to determine if activated proto-oncogenes are present in the transformed cells induced by a variety of chemical carcinogens. Twelve carcinogen-induced hamster cell lines, established by treatment of normal SHE cells with benzo[a]pyrene, diethylstilbestrol, or asbestos, were examined. One spontaneously transformed cell line (BHK-A) was also studied. Some of the cell lines were also tested for oncogene activation at the preneoplastic stage, before they acquired tumorigenic potential. DNAs from normal, preneoplastic, and neoplastic cells were tested by transfection into mouse NIH 3T3 cells, and morphologically transformed foci were scored on the contact-inhibited monolayer of 3T3 cells. The frequency of focus formation for normal SHE cell DNA was <0.0008 foci/μg DNA, while approximately 40% (5 of 12) of the DNAs from carcinogen-induced, tumorigenic hamster cell lines induced foci at a frequency of ? 0.012 foci/μg DNA. The other seven carcinogen-induced cell lines and the BHK-A cells were negative (<0.002 foci/μg DNA). When the DNAs from transformed foci induced by the five positive cell lines were retransfected into NIH 3T3 cells, the frequency of secondary foci of 3T3 cells was as much as 50-fold higher (1.34 foci/μg DNA) than with the primary transfectants. DNAs from transformed foci or tumors derived from transformed foci were screened by Southern blot analyses with known oncogenes and with a hamster repetitive DNA probe for the presence of transfected hamster oncogenes. Newly acquired hamster Ha-ras sequences were detected in transformed 3T3 cells induced by four of the five hamster tumor DNAs. Immunoprecipitation of lysates of several secondary transformants with a ras monoclonal antibody (Y13–259) showed altered gel mobility of the p21^ras protein consistent with a mutation at codon 12. These activated ras genes were detected by the NIH 3T3 assay in the tumorigenic hamster cells but not in the preneoplastic, immortal cell from which they were derived. The activated Ha-ras proto-oncogene was detected in cell lines induced by each of the three different carcinogens studied. Cells from transformed foci inauced by DNA from one of the hamster tumor cell lines (BP6T) contained hamster sequences but did not show newly acquired Haras, Ki-ras, or N-ras genes on Southern analysis or altered p21^ras protein. The transforming gene in this cell line appears to be a non-^ras oncogene. These observations indicate that ～40% of the chemically transformed Syrian hamster tumor cell lines have activated Ha-ras oncogenes. The activation of Ha-ras proto-oncogene is a late, postimmortalization step in the neoplastic progression of SHE cells. Only one cell line with a non-^ras oncogene was detected in the NIH 3T3 focus assay, and ～60% of the cell lines were inactive in this assay, indicating the need to develop alternative assay systems for oncogene activation. Some of the preneoplastic Syrian hamster cell lines may be useful for this purpose.     

Introduction of the activated N-ras oncogene into human fibroblasts by retroviral vector induces morphological transformation and tumorigenicity

The introduction of activated N-ras cDNA into normal diploid human skin fibroblast cell cultures using the retroviral vector pZIPneo results in a spectrum of morphologies ranging from near normal to, in rare instances, dense piled-up colonies of morphologically transformed cells. However, none of the clones isolated were transformed as assessed by growth on agar or tumorigenicity in nude mice. Introduction of both c-myc and N-ras oncogene cDNAs into normal skin fibroblasts failed to produce transformation as assessed by growth on agar and tumorigenicity in nude mice, although c-myc infection alone conferred immortality and the resultant doubly infected cell line was immortal. Using the same construct, activated N-ras cDNA was shown to transform immortalized human fibroblasts to tumorigenicity. However, immortalization per se was shown not to guarantee 'co-operation' with an activated N-ras gene to give malignant transformation. Although numerical and structural chromosome aberrations (clonal and non-clonal) were observed in some of the cell strains isolated after retroviral infection, these were not directly associated with viral infection, the presence of the oncogenes or with the morphologically transformed phenotype.     

Multiple effects on drug-sensitivity, genome stability and malignant potential by combinations of h-ras, C-myc and mutant p53 gene overexpression

Activation of specific oncogenes and inactivation of tumor suppressor genes play major roles in mechanisms leading to neoplastic transformation. The potential involvement of these genes in determining genome stability is an important issue. To examine the relationships between altered oncogene expression and the effects on genome stability, we have investigated the drug sensitivity properties of mouse 10T1/2 fibroblasts transfected with combinations of H-ras, c-myc and the proline 193 mutant form of p53. The relative colony forming efficiencies of these cells were investigated in the absence or presence of various concentrations of the chemotherapeutic agents, methotrexate, N-(phosphonacetyl)-L-aspartate (PALA) or hydroxyurea. The effects of altered oncogene expression were found to be drug and locus specific, and to lead to increased drug resistance (e.g. H-ras transfectants were significantly resistant to methotrexate or PALA), decreased drug resistance (e.g. H-ras/-myc transfectants were significantly less resistant to PALA or hydroxyurea than H-ras transfected cells), or to no significant change in drug sensitivity (e.g. H-ras transfected cells were not significantly different in sensitivity to hydroxyurea than non-transfected cells). Gene amplification was an important but not the only mechanism for drug resistance. Cells that were transfected with p53 (H-ras/p53 or H-ras/c-myc/p53) exhibited the greatest drug resistance properties with all three chemotherapeutic agents, in keeping with the important role of p53 in DNA repair and DNA amplification mechanisms. Although both H-ras/p53 and H-ras/c-myc/p53 groups exhibited very similar genome stability characteristics as determined by drug sensitivity results, they were significantly different in their abilities to produce transformed foci in vitro and lung metastases in vivo. The H-ras/c-myc/p53 transfected cells formed significantly higher numbers of transformed foci and exhibited a greater malignant potential. These results are consistent with observations that H-ras expression directly correlates with malignant potential, and that H-ras/c-myc/p53 transfected cells have higher H-ras expression than H-ras/p53 transfected cells. Alterations in genomic integrity through changes in onocogene expression play important roles in mechanisms determining drug sensitivity; in addition to genome destabilization, other events are critically involved in regulating transformed and malignant characteristics.     

Expression of hepatocyte and oval cell antigens in hepatocellular carcinomas produced by oncogene-transfected liver epithelial cells

We have established an in vivo/in vitro system in which epithelial cells ("oval cells") isolated from livers of rats fed a carcinogenic diet for a very brief period are placed in culture and transfected with an oncogene. Injection s.c. into nude mice of oval cells transfected with the activated c-Ha-ras (EJ oncogene) produces tumors with morphological features of differentiated hepatocellular carcinomas. Using monoclonal antibodies that can recognize hepatocyte, oval cell, and tumor antigens, we investigated the expression of these antigens in oval cells in culture, transfected with either the EJ oncogene or the normal c-Ha-ras allele and in tumors derived from the oncogene-transfected cells. We show that EJ-transfected cells and most particularly the tumors they produce expressed hepatocyte and oval cell antigens not detectable in untransfected cells or cells transfected with the normal c-Ha-ras gene. Furthermore, we found that in cloned tumor cells, the expression of hepatocyte antigens could be induced by changes in culture conditions and was accompanied by a decrease in the expression of oval cell markers. Trabecular hepatocellular carcinomas had higher reactivity toward monoclonal antibodies recognizing hepatocyte antigens while tumors with glandular architecture reacted predominantly with monoclonal antibodies against oval cells. We conclude that, in addition to its tumorigenic effect, the EJ oncogene induced the differentiation of tumor cells toward the hepatocyte lineage. In addition, the data provide further confirmation that oval cells can serve as progenitors of differentiated hepatocellular carcinomas.     

Rodent fibroblast tumours expressing human myc and ras genes: growth, metastasis and endogenous oncogene expression

The effects of expression of human c-myc and both mutated (T24) and normal forms of human Ha-ras-1 were studied in an aneuploid rat fibroblast line (208F). Mutated T24 Ha-ras was also studied in a near-diploid cell derived from early passage Chinese hamster lung fibroblasts (CHL). In contrast to the parental fibroblasts, cells expressing any of the human oncogenes engendered rapidly growing tumours in immune-suppressed animals. Blood- and lymph-borne metastases were observed from both ras- and myc-expressing cells. In general ras-expressing cells were more aggressive than those expressing myc. In the 208F background, expression of c-myc was associated with an incidence of mitosis similar to that in tumours expressing T24 Ha-ras, but incidence of single cell death by apoptosis was higher. Quantitatively, expression of human oncogene mRNA was constant during growth in vivo, and similar to that sometimes observed in human neoplasms. Of 9 endogenous proto-oncogenes, 7 showed no change in expression from the parental fibroblasts, but c-abl and c-fos were strongly expressed in all cells expressing human ras or myc. Thus these tumorigenic cells, although transfected with single human oncogenes, all expressed oncogenes with both nuclear- and membrane-associated products.     

The v-abl, c-fms, or v-myc oncogene induces gamma radiation resistance of hematopoietic progenitor cell line 32d cl 3 at clinical low dose rate

A variety of viral and cellular oncogenes have been described with differing mechanisms of action but with the common property of inducing morphologic alteration of cells in culture. Subclonal lines of oncogene expressing cells have been shown to produce tumors in vivo. Expression of the N-ras oncogene in embryo fibroblast NIH/3T3 cells has been demonstrated to increase radioresistance in vitro, and these results have been confirmed and extended to human cell lines expressing the c-raf oncogene. In the present report, we have examined the effects of expression of the c-fms, v-abl, or v-myc oncogene in a clonal hematopoietic progenitor cell line 32D cl 3. The 32D cell line is nonmalignant in vivo and is dependent upon a source of Interleukin-3 (IL-3) for growth in vitro. The radiation survival of 32D cl 3 cells transfected and expressed in the c-fms oncogene showed significant increase in the radioresistance at both 5 cGy/min and 116 cGy/min. A clone of 32D cl 3 transfected and expressing the v-myc oncogene demonstrated increased radioresistance at both dose rates. Results of split dose experiments suggested significant repair of sublethal irradiation damage of 32D-v-abl cells. Results were compared with expression of the same v-abl oncogene in the NIH/3T3 embryo fibroblast cell line. The data demonstrate that gamma irradiation resistance is significantly increased by each oncogene expressed in 32D cl 3 cells. The data on cell line 32D cl 3 may correlate with the radioresistance of v-abl expressing human hematopoietic cell malignancies treated by irradiation therapy.     

Tumorigenicity of human mesothelial cell line transfected with EJ-ras oncogene

We performed this study to determine whether human mesothelial cells are capable of undergoing neoplastic change in vitro and to observe their interaction with the activated c-Ha-ras (HRAS1) oncogene EJ-ras, which has a role in the development of many malignant human tumors. Mesothelial cells are presumed to be the progenitor cells of malignant mesothelioma, a cancer strongly correlated with asbestos exposure. Previously, we established a non-tumorigenic cell line, MeT-5A, from normal human mesothelial cells after transfection with a plasmid containing the simian virus 40 (SV40) early-region genes. In the present study, we performed transfection of a plasmid containing the EJ-ras gene and the neomycin-resistance gene into these cells and selected a population resistant to G418, a neomycin analogue. Cells from this cell line formed rapidly growing sc tumors in NIH Swiss athymic nude mice, but untransfected with the vector DNA and selected for G418 resistance formed no tumors. The tumors formed by EJ-ras-transfected cells were established in vitro, and cells from these tumor cell lines exhibited a characteristic altered morphology. The cells had the same isoenzyme phenotype as the parent cells, and they expressed the mutant EJ-ras p21 protein. This first demonstration of malignant transformation of human mesothelial cells in vitro may permit molecular analysis of mesothelial carcinogenesis.     

c-Ha-ras oncogene expression in immortalized human keratinocytes (HaCaT) alters growth potential in vivo but lacks correlation with malignancy

Spontaneously immortalized human skin keratinocytes (HaCaT) were transfected with the c-Ha-ras (EJ) oncogene via a plasmid construct which also contained the selectable neomycin gene. Clones were selected on the basis of G418 resistance. Those clones that had stable integrants of Ha-ras fell into 3 classes with respect to tumorigenicity. Class I clones were nontumorigenic, i.e., formed nodules which rapidly regressed. This phenotype is identical to that seen with parental HaCaT cells. Class II clones formed slowly growing, highly differentiated cystic or papillomatous-type benign tumors, and class III clones formed highly differentiated, locally invasive squamous cell carcinomas. The clones of all three classes exhibited similar morphology and growth potential in culture and retained the ability to reconstitute an epidermis-like stratified epithelium in transplantation experiments. Only the malignant clones showed locally invasive growth. Both the benign and the malignant clones exhibited higher levels of ras integration and variable levels of mutated p21 protein product. Thus, expression of the cellular Ha-ras oncogene in these human epithelial cells significantly altered growth regulation, resulting in varying degrees of growth potential in vivo, ranging from benign to malignant tumors. However, no direct correlation was seen between high levels of p21 expression and malignant growth.