Effects of ouabain on NIH/3T3 cells transformed with retroviral oncogenes and on human tumor cell lines

Both murine and human cell lines transformed by the v-Ki-ras gene have been shown to be much more sensitive to the toxic effects of the cardiac glycoside ouabain than their respective controls. This differential toxicity has previously been used in the isolation of flat revertant clones from populations of Kirsten murine sarcoma virus transformed NIH/3T3 cells. Here, we have undertaken a further characterization of this phenomenon in murine and human tumor cells. Two different techniques, a 51Cr-release assay and a quantitative Crystal violet elution assay, have been employed to compare the sensitivities to ouabain of normal and v-Ki-ras-transformed NIH/3T3 cells. In each assay, ras-transformed NIH/3T3 cell lines displayed an increased sensitivity to ouabain as compared to the parental NIH/3T3 cell line, both in dose-response and in time-course experiments. In a separate study, ouabain was also able to inhibit the growth in semi-solid medium of 2 v-Ki-ras-transformed NIH/3T3 cell lines (DT and K-NIH) in a dose-dependent fashion. The same concentrations of ouabain were effective in both the 51Cr-release and Crystal violet assays. To address the question of whether increased sensitivity to ouabain is a specific result of transformation with the ras oncogene or is a common event which accompanies transformation by other oncogenes, we have screened a variety of transformed NIH/3T3 derivatives. All of these lines displayed an increased sensitivity to ouabain when compared to the parental NIH/3T3 cell line.     

Effect of adeno-associated virus on transformation of NIH 3T3 cells by ras gene and on tumorigenicity of an NIH 3T3 transformed cell line

Transfection of NIH-3T3 cells with the plasmid pJ234, containing DNA from the human bladder carcinoma T24 cell line (ras gene), results in their transformation. Adeno-associated virus did not affect significantly the number of the transformed foci when different multiplicities of infection were used and when the virus was added to the cultures at different time intervals before or after transfection. A transformed cell line was derived following transfection of NIH 3T3 cells by the ras gene. Infection of these cells with adeno-associated virus resulted in a decrease in their growth rate and cloning efficiency. These infected cells showed a dose-dependent reduction in the frequency and an increase in the latent period for tumor appearance in nude mice.     

Constitutive overexpression of a 89 kDa heat shock protein gene in the HBL100 human mammary cell line converted to a tumorigenic phenotype by the EJ/T24 Harvey-ras oncogene

The non tumorigenic human mammary cell line HBL100 has been transformed by the EJ/T24 human bladder carcinoma Harvey(Ha)-ras oncogene. Six cell lines were established from transformed colonies. They all expressed a high level of the ras oncogene and were tumorigenic in athymic nude mice. During an in vivo passage in animals, tumour cells presenting a growth advantage were selected, and some of the tumours revealed an amplification of the transfected ras sequences. Using this model of human cell transformation, we have isolated a cDNA clone corresponding to a heat shock protein gene (hsp89 alpha). This gene, normally transcribed at a higher rate in response to serum stimulation, was found to be constitutively overexpressed in ras-transformed HBL100 cells. In contrast, a closely related hsp gene (hsp89 beta), remained sensitive to serum stimulation, in both untransformed and ras-transformed HBL100 cells. Thus, the regulation of the expression of the hsp89 genes, upon serum stimulation, involves ras-dependent and ras-independent pathways. Constitutive overexpression of the murine homolog of the hsp89 alpha was observed in NIH3T3 cells transformed by the three ras oncogenes, but not with some other oncogenes. Therefore, alteration of the hsp89 alpha gene expression is not a general characteristic of transformed cells, but seems to be linked to ras transformation.     

Sensitivity pattern of normal and Ha-ras transformed NIH3T3 fibroblasts to antineoplastic drugs

Ha-ras-transformed NIH3T3 fibroblasts were compared with the parental cell line to investigate the influence of the Ha-ras oncogene on cellular chemosensitivity to antineoplastic drugs. Four NIH3T3 cell clones independently transformed by the Ha-ras oncogene, activated by mutation or overexpression, were analyzed: 3 clones were obtained by transfection of NIH3T3 cells with a mutation-activated Ha-ras gene and 1 clone by transfection of a large copy number of the normal Ha-ras proto-oncogene. Chemosensitivity of the transformed clones and of the parental cell line was analyzed when cells were in the same condition of proliferative activity and cell cycle phase distribution. No significant differences in chemosensitivity were observed between transformed and untransformed cell lines to doxorubicin, VP-16, cis-platinum or mitomycin C. Therefore, data suggest that activated Ha-ras oncogenes have no role in sensitivity to these antineoplastic agents.     

A mutant cell line derived from NIH/3T3 cells: two oncogenes required for in vitro transformation

EK-3, a cell line derived from NIH/3T3 cells, was isolated. These cells are nontumorigenic to NIH Swiss nude mice. They required both myc and ras genes for in vitro transformation in contrast to NIH/3T3 cells, which are efficiently transformed following transfection by ras alone. Two other genes, chloramphenicol acetyl transferase and geneticin resistance, could be efficiently transfected and expressed in both EK-3 cells and the parental NIH/3T3 cells. Thus the possibility that the requirement of myc in EK3 cells is due to low efficiency of transfection could be ruled out. The present study suggests that myc plays a significant role in the overall process of transformation rather than simply immortalization of the cells. The EK-3 line can be very helpful in elucidating this function.     

Selection for spontaneous metastasis after calcium phosphate-mediated transfer of DNA

The ras oncogene has been shown to induce metastatic potential in a wide variety of cells. However, one cell line, C127, when transformed by ras (HC127), proved to be an exception in that the transformed cells gained the ability to form tumors in nude mice, but these tumors did not form spontaneous metastases. Because C127 cells do not metastasize after ras transfection, these cells can be used to identify other factors which contribute to the development of metastatic potential. Specifically, these cells can be used as recipients in DNA transfer experiments utilizing DNA from other cells in which H-ras has been used to induce the metastatic phenotype, thus allowing the search for genes in addition to ras itself which may be necessary for metastasis. A system utilizing genomic DNA transfer into C127 cells transformed by ras has been developed. These cells were used as the recipient for genomic DNA in cotransfection with pSV2neo followed by selection both in the experimental i.v. assay and in the spontaneous metastasis assay in nude mice. DNA from two lines with metastatic potential (both transformed by ras genes) gave rise to metastatic clones, whereas DNA from the recipient cells, HC127, NIH 3T3 cells, and two human tumor cell lines, CHP126 and A2058, failed to give rise to transfectants which could metastasize. The metastases after the first cycle were put into culture, and DNA was extracted from these cells and used in a second cycle of transfection. The capacity to metastasize was also transferred in the second cycle. Of seven metastatic clones examined, four showed no detectable rearrangements of the transfected v-H-ras gene, but in three of these clones, there were rearrangements of this gene, which was originally present in the recipient cell, HC127. This indicates that in a subset of the selected clones there may have been selection for rearrangements of the host genome rather than introduction of foreign DNA sequences, and that such effects must be considered in gene transfer experiments. It is also possible that the transfer of particular genomic DNAs are leading to genetic instability in these experiments. mRNA levels were compared in the metastatic variants and the parental cells; H-ras-specific RNA was raised from 4- to 22-fold in the metastatic cell lines, regardless of rearrangement of the v-H-ras gene.     

Mechanism of activation of human ras genes cloned from a gastric adenocarcinoma and a pancreatic carcinoma cell line

We have analyzed the mechanism of activation of two human ras oncogenes. We have also identified a rasN gene from a human gastric adenocarcinoma which efficiently induced both morphological transformation and tumorigenicity of NIH3T3 cells in a transfection assay. The rasN gene in tumor tissue DNA did not appear to be rearranged or amplified. A molecular clone, which contained an EcoRI fragment spanning the first and second rasN exons, was molecularly cloned directly from the human tumor DNA. Chimeric constructions and DNA sequencing defined the mechanism of activation of the gene as a mutation in the 61st amino acid codon substituting arginine for glutamine. Normal DNA isolated from Epstein-Barr virus immortalized lymphocytes derived from the same patient did not induce morphological transformation or tumorigenicity in NIH3T3 cells. A cloned cell line isolated from the human pancreatic carcinoma cell line Panc1 had previously been shown to contain an activated rasK-2. Sequence analysis of the cloned transfected gene reveals a G to A change within codon 12, which is presumably responsible for its biological activity. This represents the first identification of a codon 12 aspartic acid substitution of a c-rask oncogene from a human tumor-derived cell line.     

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.     

Characterization of human transforming genes from chemically transformed, teratocarcinoma, and pancreatic carcinoma cell lines

Dominant transforming genes that were transferred to mouse NIH3T3 cells by cellular DNAs prepared from a chemically transformed human cell line (MNNG-HOS), a human teratocarcinoma cell line (PA1), and a human pancreatic carcinoma cell line (A1165) were characterized (a) analyzing the repetitive human DNA sequences that were associated with the transforming gene and (b) determining their relationship to the oncogenes of the Harvey (rasH) and Kirsten (rasK) sarcoma viruses and to the human neuroblastoma transforming gene (rasN). The results show that the transforming gene activated in the teratocarcinoma cell line is identical to the neuroblastoma transforming gene and that the transforming gene of the pancreatic carcinoma cell line is a human homologue of rasK. In contrast, the transforming gene activated in the chemically transformed human cell line showed no detectable homology to rasK, rasH, and rasN.     

Sensitivities of NIH/3T3-derived clonal cell lines to ionizing radiation: significance for gene transfer studies

Rodent cells are frequently used as recipients in experiments involving gene transfer, isolation, and characterization. The present studies were designed to investigate the clonal responses to ionizing radiation of NIH/3T3 cells subjected to DNA-mediated gene transfer. Radiation sensitivity (D0) values were determined for the parental NIH/3T3 cell strain, six clonal cell lines transfected with DNA from radiation-resistant human tumor cells, and six nontransfected clonal cell lines. The radiation sensitivities of four transfected and two nontransfected clonal cell lines differed significantly from parental NIH/3T3 cells (P less than 0.05). Detailed karyotype analysis of two nontransfected clonal cell lines with differing radiation sensitivities showed variation in chromosomal composition. Specifically, a minute chromosome was observed to segregate consistently (in 49 of 50 metaphases) with the genome of one NIH/3T3 clone (D0 2.07 Gy) and was completely absent (from 50 metaphases) in another NIH/3T3 clone (D0 1.06 Gy). In the parental NIH/3T3 strain (D0 2.02 Gy) 10% of cells (3 of 30 metaphases) had such minute chromosomes. These findings demonstrate that the clonal cellular heterogeneity of NIH/3T3 cells is characterized by genotypic and phenotypic variations which must be considered in the experimental design involving gene transfer and expression.     

Human colon cancer cells surviving high doses of cisplatin or oxaliplatin in vitro are not defective in DNA mismatch repair proteins

PURPOSE: Alterations in the DNA mismatch repair (MMR) proteins have been associated with an increased resistance of many cancer cell lines to cisplatin. The aim of this work was to investigate whether defects in DNA MMR proteins are involved in the survival of human colorectal cancer cells in the presence of high concentrations of cisplatin and oxaliplatin, a diaminocyclohexane (DACH) platinum compound whose adducts are not recognized by the MMR system. METHODS: Six unselected human colon cancer cell lines (HT29, HCT15, HCT116, Caco2, SW480 and SW620) were treated with a single 3-h exposure to cisplatin or oxaliplatin at suprapharmacological concentrations, ranging from 50 to 200 microg/ml. The microsatellite stability and the expression of MMR proteins in the parental cell lines and in the drug-selected subpopulations were studied. RESULTS: Most cells underwent apoptosis in the days following the cisplatin or oxaliplatin treatment, but some colonies expanded 3 to 4 weeks after, suggesting the presence of innately resistant cells in the six parental cell lines. Microsatellite instability (MIN), which reflects genetic defects in the DNA MMR system, was detected only in the HCT116 parental cell line and its drug-selected counterparts, due to a known mutation in the hMLH1 gene. No acquired MIN was observed in the other cisplatin-selected sublines derived from the HT29, HCT15, Caco2, SW480 or SW620 parental cells. In the same way, Western blot analysis showed that expression of the DNA MMR proteins hMLH1, hPMS1, hPMS2, hMSH2 and hMSH6 did not differ between the parental and the drug-surviving cells. CONCLUSIONS: These results indicate that high-level resistance of human colon cancer cells to high doses of cisplatin and oxaliplatin does not seem to be related to acquired defects in the DNA MMR proteins.     

Human oncogenes detected by a defined medium culture assay

Oncogenes in DNAs from human tumor cell lines have been detected by a new transformation assay. Cellular DNAs are transfected into NIH3T3 murine fibroblasts, and transformed cells are selected by maintaining cell cultures in a defined medium lacking platelet-derived or fibroblast growth factors. DNAs from eight of 17 human tumor cell lines have yielded transformants by this method. Activated cellular ras genes account for three of the transforming activities. The SAOS2 osteosarcoma cell line contains an activated oncogene distinct from 18 known oncogenes. Another cellular oncogene was detected as the consequence of a fortuitous transfection-mediated DNA rearrangement.     

Frequent activation of non-ras transforming sequences in neoplastic Syrian hamster cells initiated with chemical carcinogens

Carcinogen-caused transformation of Syrian hamster embryo cells has been widely used as a model for experimental carcinogenesis. However, analysis of the molecular mechanisms of hamster cell transformation has been limited. To expand the understanding of the molecular basis of this system, 22 independently derived Syrian hamster neoplastic cell lines initiated with chemical carcinogens were screened for the presence of dominant transforming sequences by DNA transfection into mouse NIH3T3 cells. High molecular weight DNAs from 12 (55%) of these cell lines transformed NIH3T3 cells through serial transfection cycles. NIH3T3 transformants contained hamster-specific repetitive sequences, which co-segregated with the transformed phenotype in successive transfection rounds. Results from Southern hybridization analyses and p21ras mobility assays indicated the presence of N-ras oncogenes, presumably activated by point mutations at codon 61, in 3 of the 12 (25%) transfection positive lines, all initiated with sodium bisulfite; non-ras transforming sequences were apparently activated in the remaining 9 (75%) lines. DNA prepared from NIH3T3 transformants derived from cell line 81C39 was analysed by Southern hybridization with a battery of 38 probes including non-ras oncogenes known to score as positive in the NIH3T3 assay as well as other retroviral and mammalian oncogenes. Each probe hybridized to DNA fragments showing the mobility characteristic of NIH3T3 protooncogenes, but failed to detect homolog sequences of hamster origin, even under hybridization conditions which allowed their detection in hamster DNA. Results show that ras activation occurs at a low frequency in hamster neoplastic transformation and strongly suggest that novel transforming sequences are activated, thus validating the use of this system for investigating the role of non-ras transforming sequences in neoplasia.     

Correlation between H-ras p21TLeu61 protein content and tumorigenicity of NIH3T3 cells

We have prepared a number of NIH3T3 clonal cell lines that contain an H-ras transforming gene with an A----T transversion at the 61st codon. The clonal lines contain 1 to 3 cell equivalents of the transforming oncogene and some lines look more morphologically transformed than others. Using Y13-238, a rat monoclonal antibody that recognizes H-ras p21 but not Ki- or N-ras in rodent cells, we found that the degree of morphological change is correlated with the relative amount of transforming protein in the selected clonal lines. Nude mice were injected with cells from lines containing different amounts of the transforming protein, ranging from approximately 1 to 10 times the level of normal H-ras protein present in NIH3T3 cells. Tumors arose in all mice that received cells containing the transforming protein. Their time of appearance (tumor latency) was correlated with the number of cells injected and the amount of transforming protein present in each clonal line; however, the subsequent rate of growth and ultimate size of the tumors were similar. Thus, it appears that the transforming protein has a significant effect on some early step in tumor development. Our results also show that relatively low amounts of transforming ras protein are sufficient to cause tumorigenicity in NIH3T3 cells and that higher amounts of the transforming protein cause proportionately faster responses.     

Inhibition of cell growth by lovastatin is independent of ras function

We have investigated the inhibition of cell growth by lovastatin (previously known as mevinolin), an antagonist of hydroxymethylglutaryl coenzyme A reductase which blocks the processing and membrane localization of ras proteins via inhibition of polyisoprenylation. A series of NIH 3T3 cells transformed by oncogenes with activities that are dependent or independent of isoprenylated ras were studied, including cells transformed by myristylated ras protein that is isoprenylation independent. Treatment with lovastatin at concentrations ranging from 5 to 15 microM for up to 96 h resulted in a time- and dose-dependent inhibition of cell growth in all lines tested. The inhibition ranged from 25 to 50% when cells were treated with 5 microM lovastatin for 48 h, to 72-90% for cells treated with 15 microM lovastatin for 96 h. Cells transformed by c-ras, v-ras, v-src, v-raf, and the myristylated ras genes displayed similar sensitivities; the parental NIH 3T3 line was the most resistant of the lines tested. Metabolic labeling of control and lovastatin-treated cells with [35S]methionine or tritiated lipids revealed that 15 microM lovastatin blocked the processing of both endogenous ras and v-ras proteins yet had no effect on the lipidation of myristylated ras proteins. Addition of 300 microM mevalonic acid overcame the inhibition induced by 15 microM lovastatin. Thus the inhibition of cell growth in vitro by lovastatin did not show specificity for cells the transformation of which is dependent upon isoprenylated ras protein. It is therefore likely that the inhibition of other pathways affected by lovastatin, such as cholesterol biosynthesis or the processing of other cellular proteins, are responsible for the growth inhibition by lovastatin.     

Experimental Metastasis of Oncogene-transformed NIH 3T3 Cells in Chick Embryo

By means of a highly sensitive and quantitative assay for specific detection of metastasized tumor cells in chick embryonic organs using the polymerase chain reaction (PCR), we have examined the experimental metastatic ability of individual clones of NIH 3T3 cells, transformed with oncogenes: v-Ki- ras , v-Ha- ras , v- src , v- fos , and v- abl. Such a transformed clone had different metastatic abilities in different embryonic organs. Among them, two clones of NIH 3T3 cells transformed with ras -oncogenes (v-Ki- ras or v-Ha-ras) metastasized to liver and lungs of chick embryo, and grew there more rapidly than the other clones. The parental NIH 3T3 cells were detected as slight bands of PCR products after iv injection, indicating some cells were trapped in chick embryonic organs, but did not grow. These findings indicate that the transformed cells are able to invade the organ tissues and grow in embryonic chick organs, but non-metastatic cells such as the untransformed-NIH 3T3 cells are not able to grow in the secondary sites. These experiments clearly demonstrate the usefulness of this assay system to study genes involved in malignant transformation.