The expression of tumor rejection antigen on rat fetus fibroblasts transformed by the ras oncogene

The expression of tumor rejection antigens (TRA) was analyzed on clones of rat fetus-derived fibroblasts, WFB, transformed or transfected by oncogenes. It was shown that a tumorigenic W14 clone, which is an activated H-ras transformant of parental WFB, expressed TRA in transplantation experiments using syngeneic WKA rats. The data also showed that W14 TRA was acquired in the event of cell transformation, since it was not detected on parental non-transformed WFB cells or Wmyc-4 clone which is a transfectant of WFB by mouse plasmacytoma-derived c-myc DNA. However, TRA was not expressed or at least was not detected on highly tumorigenic W31, another clone of H-ras transformants of parental WFB, in the transplantation experiments. We also assessed the level of expression of major histocompatibility antigens (MHC) class I molecules on these cells by using R4-8B1 Mab that reacts specifically with rat class I antigen. The data indicated that it was decreased on W14, W31, and Wmyc-4, but not on parental WFB. Although this molecule was weakly positive on W14 cells, W31 and Wmyc-4 showed even greater decreases. These data may indicate that the TRA expression and its recognition by syngeneic hosts are dependent upon the transformed clones, although their parental cell is the same. We discuss in detail this difference of expression and recognition of TRA in the context of the cell transforming process.     

Oncoprotein expression and morphological phenotypes of human breast epithelial cells transformed by the c-Ha-ras oncogene

Activated oncogenes have been detected in a variety of malignant tumors and altered expressions of certain genes are known to play a functional role in the cancer process. The chemical carcinogen, BP, and the insertion of c-Ha-ras, induced characteristics of transformed phenotypes in a suitable human breast epithelial cell line. Carcinogen-treated and Ha-ras-transfected cells showed a progression of changes in the morphology, anchorage independent growth, invasiveness and tumorigenicity in SCID mice. Tumor growth occurs after a series of molecular events that parallel morphological changes. The aim of this work was to determine the neoplastic phenotypes following treatment with benzo(a)pyrene (BP) and transfection with c-Ha-ras oncogene changes and PCNA, Neu, ErbB-3 and Cytokeratin 18 protein expression in MCF-10F cells, a spontaneously immortalized human breast epithelial cell line. Protein expression was determined by immunofluorescent staining coupled with confocal microscopy. An increased oncoprotein expression in comparison to MCF-10F cells was observed in PCNA, Neu, ErbB-3 and Cytokeratin 18 protein expression in breast epithelial cells transformed with a chemical carcinogen and/or oncogene transfected that are not present in the MCF-10F. This in vitro cancer model can be used as a valuable model in the study of breast carcinogenesis.     

Immortalization of rat embryo fibroblasts by the cellular p53 oncogene

Intact and rearranged p53 genes from Friend virus-induced erythroleukemic cell lines which code for proteins of 53- (p53) and 44-kDa (p44), respectively, were cloned into pUC18 and tested for their ability to immortalize rat embryo fibroblasts. The functional p53 gene from normal Balb/c mouse liver was also tested for immortalizing activity. Immortal cells were obtained with the three genes although the efficiency of immortalization by p44 was lower than that by p53. Expression of murine p53 and p44 in the established rat cell lines was confirmed by metabolic labeling and Western Blotting. Our results demonstrate that elevated expression of the mouse p53 gene, driven by its natural promoter and in the absence of strong heterologous promoters and/or enhancers, can efficiently immortalize early-passage rat embryo fibroblasts. p53-immortalized cells but not p44-immortalized cells could be morphologically transformed by secondary transfection with activated Ha-ras. Thus 5'-coding sequences of the p53 gene appear necessary for ras complementation but not for immortalization.     

Purification and characterisation of soluble tumour haemolytic factor isolated from oncogene transformed fibroblasts

Numerous studies have shown that intact cancer cells and cell extracts have the capacity to lyse erythrocytes in vitro. The transformation of NIH-3T3 fibroblasts by ras oncogenes has recently been demonstrated to result in tumour cells releasing a haemolytic factor. The purpose of this study has been to purify and further characterise the soluble tumour haemolytic factor (sTHF) produced by mouse fibroblasts transformed by T24 human bladder cancer DNA and by the cloned Harvey murine sarcoma viral oncogene. To this end, transformed fibroblasts were cultivated in serum-free medium. The cell-free supernatant was treated with ammonium sulphate and the precipitate achieved at 60-100% saturation was dialysed and applied to a gel filtration column. A haemolytic factor was eluted with an Mr between 65,000 and 75,000. Zinc chelate and strong anion exchange column chromatography resulted in greater than 3,000-fold enrichment of sTHF. SDS-PAGE of sTHF resulted in a single protein band of 66,000 Da. Soluble THF had no immunological cross-reactivity with known cytokines produced by lymphocytes and macrophages. The pathophysiological role of sTHF in cancer remains to be determined.     

Analysis of lectin receptors on rat embryo fibroblasts transformed by adenovirus 2

Transformation of rat embryo fibroblasts by human adenovirus type 2 (Ad2) results in the production of a series of cell lines that cover a spectrum of malignancy from nontumorigenic to highly tumorigenic in a single species. A panel of plant lectins was used to study surface characteristics of these cell lines that might correlate with tumorigenicity. Because of the complex nature of lectin-cell surface interactions, a number of parameters were determined; they included numbers and densities of lectin receptors, binding affinities, and receptor mobilities. The lectins from Lens culinaris, Lotus tetragonolobus, and Ricinus communis were found to be the most useful for differentiating among the various Ad2-transformed cell lines. In general, the more tumorigenic cell lines were characterized by high numbers of lectin receptors, high percentages of lectin-binding cells, and heterogeneous distributions of receptors from cell to cell. In contrast, the nontumorigenic and the weakly tumorigenic cell lines were characterized by low numbers of lectin receptors present on a minority of cells within each population and a more homogeneous distribution of these receptors from cell to cell. These data demonstrate that lectins can identify surface properties that appear to correlate with malignant potential in the Ad2-transformed cell lines.     

A nonviral, virus strain-specific antigen expressed on rat cells transformed by avian sarcoma virus

Five of six rat sarcomas, induced by the Schmidt-Ruppin (SR) strain of avian tumor virus, expressed a Mr 60,000 tumor cell surface antigen (TSA), immunoprecipitable from non-ionic detergent extracts. Expression of the antigen was exclusive to rat cells transformed by the SR virus strain. Moreover, expression of TSA appeared restricted by cell type. The five TSA-positive SR-transformed rat cell lines tested were apparently of fibroblastic origin, but cultured rat cerebral endothelial cells (RCE-T1), transformed by SR virus, showed no expression of TSA. However, the antigen emerged on cultured tumors obtained after histoincompatible transplantation of these cells into newborn rats of another strain (tumor digest cells). Investigation of TSA for immunological relationship to viral structural antigens and the src gene product indicated that the TSA is distinct from any of these and more probably derives from a virus-directed alteration in a host molecule.     

Different chemosensitivities of rat 3Y1 fibroblasts transformed by various agents in vitro

Alterations of chemosensitivities associated with transformation of rat 3Y1 fibroblasts were compared among various agents, including either adenovirus type 12 (Ad12-3Y1), the E1A region of adenovirus type 12 (E1A-3Y1), mouse polyoma virus (Py-3Y1), Simian virus 40 (SV-3Y1), Rous avian sarcoma virus (SR-3Y1), plasmid DNA carrying v-Ha-ras oncogene (HR-3Y1), or N-methyl-N'-nitro-N-nitrosoguanidine (NG-3Y1), with untransformed 3Y1 fibroblasts, using a semiautomated non-clonogenic MTT assay. IC50 values were compared for each drug in each cell line. Among the different cell lines, untransformed 3Y1 cells were the most resistant to cisplatin (CDDP), adriamycin (ADM), carboquone (CQ), bleomycin (BLM), mitomycin C (MMC) and 4-hydroperoxy cyclophosphamide (4-hp-CPA). On the other hand, E1A-3Y1 cells were more resistant to vincristine (VCR) and NG-3Y1 and Ad12-3Y1 were more resistant to 5-fluorouracil (5-FU) than untransformed 3Y1 cells. These transformed lines did not display any cross-resistance to other drugs. The dissociation of chemosensitivities between Ad12-3Y1 and E1A-3Y1 to ADM, VCR and 5-FU was recognized. These results suggested that some of these transforming agents may alter the chemosensitivities of anticancer drugs.     

Detergent extraction and characterization of tumor hemolytic factor from plasma membranes of oncogene transformed fibroblasts

Cancer cells have the capacity to lyse erythrocytes by a cell-contact-requiring phenomenon. Subcellular fractionation procedures have revealed that the hemolytic principle resides in the cancer cell plasma membrane. In this study we report the detergent extraction of a potent hemolytic factor from the plasma membranes of ras-oncogene-transformed fibroblasts. Ammonium-sulfate partitioning (60-100%) of detergent-extracted proteins was used to enrich hemolytic activity. Tumor membrane Hemolytic Factor (mTHF) was inactivated by treatment with papain, suggesting that it is a protein. mTHF was inhibited by serum, but was unaffected by extremes of temperature and pH, also by metal chelation with EDTA. Surface radio-iodination of tumor cells and isolation of cell organelles was used to characterize the outer plasma membrane localization of mTHF. mTHF retained hemolytic activity when reconstituted into stable phospholipid vesicles. Pre-incubation of mTHF with red cell ghosts led to an abrogation of hemolytic activity. mTHF-induced hemolysis consists of a 2-stage phenomenon: an early binding step, followed by hemolysis after 4 hr.     

Cellular oncogene expression in cell lines derived from tumors produced by transformed rat tracheal epithelial cells

Five tumor-derived cell lines established from transformed rat tracheal epithelial (RTE) cells were examined for activated oncogenes using the NIH 3T3 assay, and the expression of 11 cellular oncogenes in the transformed cells was quantitated and compared with expression in normal RTE cells. DNA from the tumor-derived cell lines lacked transforming activity, but expression of several oncogenes (fos, abl, Ki-ras, Ha-ras, and p53) was higher in the transformed cells than in normal RTE cells.     

Induction of sister chromatid exchange by polyoma large viral tumor antigen in transformed rat fibroblasts

The frequency of sister chromatid exchange (SCE) was determined in rat fibroblasts transformed by wild-type polyoma virus or by a mutant temperature sensitive for viral large tumor antigen function (ts-a). Elevated SCE frequencies were observed in two wild-type transformed cell lines growing at 37 degrees and in four ts-a-transformed lines upon growth at the permissive temperature for large viral tumor antigen (33 degrees). The increase in SCE frequency in ts-a-transformed cells at 33 degrees was reversed by growth at 39 degrees (nonpermissive for T-antigen function). An increase in SCE at 33 degrees was not observed in untransformed cells or in a ts-a-transformed cell line which makes a defective large viral tumor antigen. These results suggest that large viral tumor antigen can induce SCEs. Since large viral tumor antigen is also responsible for amplification of integrated viral DNA sequences (4), we tried to correlate this phenomenon with the increased SCE frequency. However, increasing SCE artificially by growing cells in the presence of 12-O-tetradecanoylphorbol-13-acetate did not result in amplification of integrated viral DNA in the absence of large viral tumor antigen function. Thus, there is no simple causal relationship between increased SCE and amplification.     

Modulation of the transformed and neoplastic phenotype of rat fibroblasts by MHC-I gene expression

Transfection of the activated ras oncogene (Ha-ras) into second passage rat embryo fibroblasts can induce the metastatic phenotype, while cotransfection of Ha-ras with the adenovirus type 2 E1a gene (Ad2-E1a) yields cells which are tumorigenic but nonmetastatic in nude mice. Because of the presence in nude mice of natural killer cells and B-lymphocytes, which might account for the different metastatic behavior of single versus double transfectants, we used triple deficient mutants as recipient animals in tumorigenicity assays. These mice carry two additional mutations resulting in the deficiency of natural killer cells and activated B-lymphocytes. We observed that the rat embryo fibroblast transfectants exhibit the same metastatic behavior in nude as well as in triple deficient mice, indicating that natural killer and B-cells are not responsible for the observed difference in metastatic phenotype between Ha-ras and Ha-ras plus Ad2-E1a transfectants. Double transfectants were found to express higher levels of major histocompatibility complex class I genes and the degree of expression appeared to correlate inversely with in vitro and in vivo parameters such as the ability to grow in agar-containing semisolid media and rate of tumor formation in triple deficient mice. Our observations are consistent with the concept that expression of major histocompatibility class I genes may be involved in regulating and modifying cell behavior by mechanisms independent of their role in immune recognition.     

Effect of oncogene transfection or passage in vivo on malignant phenotypes of rat2 cells

Rat2 cells are thymidine kinase-deficient derivatives from the immortalized rat embryo cell line Rat1. They show no phenotypic correlates of malignancy in vitro and produce tumors in syngeneic Fischer rats after long latency periods. We have investigated how transfection with oncogenes would alter the in vitro and in vivo behavior of Rat2 cells. Thus we have manipulated Rat2 cultures in various ways. The cell lines obtained were categorized as parental, in vitro subclones, untransfected in vivo derivatives, non-oncogene (neor and tk) transfectants, oncogene (mutated c-Ha-ras, polyoma middle-T, FBR v-gag-fos-fox) transfectants, and in vivo derivatives of transfectants. They were tested in vitro for morphotype, colony formation in soft agar, growth in organ culture, invasion in organ culture, and in vivo for latency period of tumor formation, tumor growth rate, invasiveness, and metastasis. Differences between the consequences of various manipulations were found in the number of malignancy-related phenotypic alterations. The following trend could be deduced from our data: induction of invasiveness in organ culture by all manipulations; morphotypic transformation and shortening of tumor-latency period by all oncogene transfections and by passage with tumor formation in vivo; growth in organ culture and increased tumor growth rate in vivo by transfection with ras-, or fos-oncogenes and by passage in vivo. Metastatic capability (present in parental Rat2 cell tumors) and colony formation in soft agar (absent in Rat2 cells) were not affected by the present manipulations. We concluded that differences between the oncogene-transfectants and the untransfected in vivo derivatives do not lie in the expression of malignancy-related phenotypes but in the time needed to acquire them.     

Effects of various chemical agents on the transformation of rat fibroblasts by an activated c-Ha-ras oncogene

We have previously reported that the potent tumor-promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA) and a factor from fetal calf serum (FCS) markedly enhance the transformation of mouse C3H 10T1/2 and Rat 6 fibroblasts, when added to cultures following transfection with plasmid pT24 DNA that contains an activated c-Ha-ras oncogene. In the present study, we examined possible enhancing or inhibiting effects of various chemicals on the transformation of Rat 6 fibroblasts by T24 DNA when tested in the presence of calf serum, calf serum plus TPA or FCS. We found that, like TPA, the chemicals mezerein, 1-oleoyl-2-acetylglycerol, and phospholipase C increased the yield of T24-induced foci, thus further implicating protein kinase C as a critical constituent in this process. Low concentrations (10(-6)-10(-7)M) of retinoic acid (both trans and 13-cis) also stimulated cell transformation. Several compounds inhibited T24-induced transformation. These included nontoxic concentrations of the calcium ionophore A23187, indomethacin, and epsilon-amino-n-caproic acid. Compounds that failed to exert a significant reproducible effect included vasopressin, vitamin D3, selenium, antipain, Bowman-Birk inhibitor, vitamin B12, epidermal growth factor, platelet-derived growth factor, insulin, and transferrin. These findings suggest that this simple in vitro system might be useful for detecting enhancers and inhibitors of ras oncogene-induced cell transformation and also elucidating their mechanisms of action.     

Alterations in protein and glycoprotein composition of rat embryo fibroblasts transformed by herpes simplex virus type 2

Total and superficially exposed plasma membrane components of tumorigenic herpes-simplex-virus (HSV)-transformed fibroblasts were studied. As a result of oncogenic HSV transformation, a significant decrease of polypeptides 230,000, 180,000, 56,000 and 43,000 daltons was found. These changes were accompanied by a significant increase in superficial exposition of several sialopeptides and two peptides of 30,000 and 15,000 daltons. It was suggested that these two peptides may represent virus-coded components of HSV-transformed fibroblasts. The present results indicate that the HSV-transformed tumorigenic cell clone meets all previously described criteria for oncogenically transformed cells.     

Enhanced invasive properties of rat embryo fibroblasts transformed by adenovirus E1A mutants with deletions in the carboxy-terminal exon.

E1A genes deficient in the carboxy-terminal exon can cooperate with activated ras oncogenes to induce transformation of rat embryo fibroblasts. However, the resulting transformed foci show a distinct appearance characterized by a decreased adhesion of the cells to the substrate. Here, we demonstrate that cell lines derived from foci showing the variant morphology are defective in down-regulation of stromelysin 1 metalloprotease expression and show an increased invasive propensity compared with cells transformed by wild-type E1A. The altered focus morphology, the high invasive propensity and the elevated stromelysin 1 expression were abrogated by glucocorticoid treatment. Our results show that E1A functions necessary for transformation and inhibition of invasive properties may be separated, and indicate that a 23 amino acid serine/threonine-rich region within the E1A carboxy-terminal exon is required for efficient repression of metalloprotease expression in transformed cells.     

NIH3T3 fibroblasts transformed by the dbl oncogene show altered expression of bradykinin receptors: effect on inositol lipid turnover

We have examined polyphosphoinositide turnover in mouse fibroblasts (NIH3T3) transformed by the dbl oncogene as compared to normal cells. The dbl-transformed fibroblasts did not show alterations of the basal level of inositol polyphosphates, polyphosphoinositides, diacylglycerol or phosphatidic acid. This indicates that the activity of C-type phospholipases, inositol lipid kinases and diacylglycerol kinase is not altered in dbl-induced transformation. However, dbl-transformed NIH3T3 cells exhibited increased inositol lipid turnover in response to bradykinin. Further analysis revealed significantly higher number of bradykinin receptors in dbl transfectants as compared to control NIH3T3. When several clonally-derived dbl NIH3T3 transfectants were analyzed, we observed a large variation of their bradykinin receptor number. Cell lines exhibiting increased bradykinin binding, however, failed to show augmented mitogenic response to the peptide agonist. Among other oncogenes, only ras showed a similar effect. We conclude that increased bradykinin receptor number is a phenomenon observed with several cell lines transformed by different oncogenes, and it does not correlate with either enhanced mitogenic responsiveness of transformed cells to the peptide, or with the presence of a specific oncogene in the transformant.     

Clonal analysis of tumorigenicity and paratumorigenic phenotypes in rat liver epithelial cells chemically transformed in vitro

Clonal subpopulations of a chemically induced tumorigenic rat liver epithelial cell line were analyzed for their cellular, biochemical, and in vitro growth properties and their tumorigenicity after injection into day-old newborn isogeneic rats. The phenotypic properties studied included DNA content; growth rate in culture; activities of gamma-glutamyl transpeptidase, NADH diaphorase, pyruvate kinase, glucose-6-phosphate dehydrogenase, and lactate dehydrogenase; ability to grow in calcium-poor medium; and ability to form colonies in soft agar. The results show that none of these phenotypes cosegregates with tumorigenicity and therefore is not reliable as a "marker" phenotype for neoplastic transformation in cultured rat liver epithelial cells. The poor correlations, either qualitatively or quantitatively, between paratumorigenic phenotypes and tumorigenicity suggest that neoplastic transformation in these cells involves a specific transforming gene locus or loci and that in vitro paratumorigenic phenotypes are merely epiphenomena of neoplastic transformation and progression. This study further reveals that the efficiency of the tumorigenicity assay of cultured rat liver epithelial cells in isogeneic newborn rats can be considerably improved by incubating the cells in medium containing only trace amounts of serum prior to transplantation into the host animals.     

Malignant transformation of human fibroblasts by a transfected N-ras oncogene

The only ras oncogene as yet identified in cells from human fibrosarcomas is N-ras, but the relationship between N-ras oncogene expression and the malignant state of these cell lines is not known. To determine if expression of an N-ras oncogene causes human cells to become malignant, we transfected the N-ras oncogene from human leukemia cell line 8402, cloned into a high expression vector pSV N-ras, into MSU-1.1 cells, a nontumorigenic, infinite life span fibroblast cell strain with a normal morphology and a stable near-diploid karyotype. The transformants formed distinct foci composed of morphologically transformed cells. Cells from such foci expressed higher than normal levels of N-ras protein, exhibited growth factor independence, and formed large colonies in soft agar at a high frequency. Injection of progeny of these focus-derived cells s.c. into athymic mice resulted in progressively growing, invasive malignant tumors (round cell, spindle cell, or giant cell sarcomas) which reached a diameter of 6 mm in 3 to 4 weeks. Injection of focus-derived or tumor-derived cells i.v. resulted in tumors in various organs of the mice. The focus-derived cell strain tested, as well as the majority of the cells derived from the tumor it produced, exhibited the same near-diploid karyotype as the parental MSU-1.1 cells. Cells transfected with an N-ras oncogene that was expressed at a normal level formed only a single, indistinct focus, and cells from that focus were not malignant.