Transformation Of Cells In Culture Research Articles

Oncogenic transformation of cells in culture: Pragmatic comparisons of oncogenicity, cellular and molecular mechanisms

Oncogenic transformation of cells in culture: Pragmatic comparisons of oncogenicity, cellular and molecular mechanisms

Regulation of fibronectin receptor distribution by transformation, exogenous fibronectin, and synthetic peptides.

Recent studies have shown that fibronectin and its 140K membrane receptor complex are spatially associated with microfilaments to form cell surface linkage complexes which are thought to mediate adhesive interactions between fibroblasts and their substrata. We examined the regulation of the organization of these cell surface structures in transformed and fibronectin-reconstituted cells as well as in cells treated with a competitive synthetic peptide inhibitor of fibronectin binding to its receptor. Correlative localization experiments with interference reflection microscopy and double-label or triple-label immunofluorescence revealed a concomitant loss of fibronectin, 140K receptor, and alpha-actinin colocalization at cell substratum extracellular matrix contact sites after transformation of chick fibroblasts by wild-type or temperature-sensitive Rous sarcoma viruses (RSV). Western and dot immunoblot analyses established that although similar total quantities of intact 140K molecules were present in the transformed cell cultures, significantly more was released into the culture medium of transformed cells. The 140K molecules on transformed cells were available for interaction with exogenously added fibronectin, which could reconstitute fibronectin-140K linkage complexes. In such fibronectin reconstitution experiments, many cells expressed both fibronectin-140K-actin linkage complexes and RSV pp60src, indicating that the morphological reversion could occur even in the continued presence of RSV transformation. The synthetic peptide Gly-Arg-Gly-Asp-Ser derived from the sequence of the cell-binding region of fibronectin could also prevent the organization of fibronectin-140K linkage complexes. Our results suggest that fibronectin interaction with cells regulates the organization of fibronectin receptor complexes and cytoskeletal components at the cell surface.

Alteration in diethylstilbestrol-induced mutagenicity and cell transformation by exogenous metabolic activation.

It was shown previously that diethylstilbestrol (DES) can induce morphological and neoplastic transformation of Syrian hamster embryo cells in culture in the absence of detectable gene mutations or DNA damage. However, in the presence of exogenous metabolic activation with rat liver post-mitochondrial supernatant, DES can induce unscheduled DNA synthesis. In this report we have examined whether with exogenous metabolic activation DES can also induce biological effects possibly related to its carcinogenicity, i.e. specific locus mutations in Syrian hamster embryo cells and cell transformation. We observed that DES was mutagenic only in the presence of exogenous metabolic activation. DES induced morphological transformation both in the absence and presence of exogenous metabolic activation. Enhanced cell transformation was observed in the presence of exogenous metabolic activation. These results indicate that two pathways may exist for the induction of cell transformation by DES. One does not apparently involve direct DNA damage, and the other, which requires rat liver post-mitochondrial supernatant-mediated exogenous metabolic activation, is associated with DNA damage and mutagenicity. These results may provide an experimental model to elucidate the biological properties of DES metabolites.

Influence of added catalase on chromosome stability and neoplastic transformation of mouse cells in culture

The generation of hydrogen peroxide (H2O2) and the derivative free hydroxyl radical (. OH) in cultures of mouse cells grown in the presence of visible light and ambient oxygen was shown previously to be implicated in chromatid damage. Furthermore, chromosome alterations appear to be associated with the spontaneous neoplastic transformation of mouse cells in culture. An attempt was made in this study to reduce the incidence of chromosomal aberrations and delay or prevent the onset of spontaneous neoplastic transformation of freshly isolated mouse cells, both fibroblasts and epidermal keratinocytes, by adding catalase to the culture medium, shielding the cultures from wavelengths less than 500 nm and providing a gas phase of 0-1% O2. These conditions significantly decreased the incidence of chromosomal aberrations in both cell types, and in fibroblasts prevented tumourigenicity in non-irradiated syngeneic mice, and increased latent periods for tumour development in X-irradiated mice. The epidermal keratinocytes were particularly resistant to spontaneous neoplastic transformation under all conditions tested. These observations on the protective effect of extracellular catalase suggest that H2O2, a normal metabolite, and/or the derivative .OH can directly or indirectly produce genetic damage and neoplastic transformation in mouse fibroblasts.

Differentiation markers (S-100, GFAP, NSE and D2) in fetal rat brain cells during malignant transformation in cell culture.

Malignant cell lines obtained by ethylnitrosourea (EtNU)-induced transformation of fetal rat brain cells in culture express protein markers of different types of neural cells. These are the nervous system-characteristic S-100 protein; glial fibrillary acidic protein (GFAP); neuron-specific-enolase (NSE), and the D2-cell adhesion molecule. S-100 protein was absent in fetal brain cells in culture, but gradually appeared in the later stages of malignant transformation and further increased at onset of rapid growth of atypical cells (stage IV). GFAP and D2 were weakly expressed in primary fetal brain cells and did not change throughout malignant transformation. NSE was present in both normal and carcinogen-treated fetal brain cells, and increased at later stages of malignant transformation. From stage III (40-100 days) some cultures were strongly positive and some negative, and the same was seen in the resulting tumorigenic cells about 100 days later. In conclusion the stepwise process of malignant transformation of brain cells in culture ended with a stable phenotype of cells capable of expressing varying types of differentiation markers. The presence of these markers in rat brain cells undergoing malignant transformation may indicate that EtNU given at 18th days of gestation is acting on multipotent neuroectodermal cells.

A rat tracheal cell culture transformation system for assessment of environmental agents as carcinogens and promoters

A tracheal cell culture system which can be used for detection of hazardous environmental agents is described. The culture system makes use of primary tracheal cells that are isolated from rats by protease digestion of the tracheal epithelium. The epithelial cells are plated on a film of collagen or onto a layer of gamma-ray- or mitomicin-C-inactivated mouse 3T3 cells. One day after the rat tracheal cells are plated they are exposed to toxicants, and the effect on colony formation is tabulated after 1 week. By altering the culture conditions it has become possible to construct an assay for carcinogen-induced transformation of rat tracheal cells, which takes advantage of the observation that normal rat tracheal cells have a finite lifetime in culture. Rat tracheal cell transformants are visualized as discrete colonies of proliferating epithelial cells that arise and survive after the normal population has died out. Their number is proportional to concentration of carcinogen to which the cells were initially exposed. Agents that have been shown to induce tracheal cell transformation in culture include polycyclic hydrocarbons, metal salts, nitroso compounds, and cigarette smoke condensate. Tracheal cells in culture also respond to tumor promoters in a number of ways. The phorbol ester tumor promoters dramatically increas the colony-forming ability of primary rat tracheal cells in a concentration dependent fashion. This class of agents also induces permanent cell line formation in rat tracheal cells derived from explants exposed to phorbol esters. In two-stage exposure experiments in culture it has also been shown that phorbol esters enhance the number of cell lines initiated by carcinogen exposure in vitro. Because rat tracheal cell culture has evolved to the state where the above studies are readily performed, these limited results suggest that the inclusion of rat tracheal cells in environmental research may be useful.

Construction of rat cell lines that contain potential morphologically transforming regions of the herpes simplex virus type 2 genome.

Hybrid recombinant plasmids were constructed; they were composed of the herpes simplex virus type 2 (HSV2) thymidine kinase (tk) gene and DNA sequences of HSV2 that have been reported to induce morphological and/or oncogenic transformation of rodent cells in culture. Several plasmids were made in two versions, with or without the simian virus 40 enhancer sequences. These plasmids were employed to transfect contact-inhibited Rat-2 tk- cells. It was demonstrated that cell lines with stably integrated, morphologically transforming regions (MTRs) of HSV2 could be isolated at a low frequency. In addition, many cell lines were isolated that had lost the MTR moiety of the transforming plasmids. None of the isolated tk-positive cell lines exhibited significantly altered growth properties when compared to control cell lines. Transient expression of transfecting enhancer plasmids was detectable 40 h posttransfection, but no transformed cell lines were obtained from these experiments. This conflicted with some versions of the 'hit-and-run' hypothesis for HSV-mediated cell transformation.

The product of ras is a GTPase and the T24 oncogenic mutant is deficient in this activity.

Ha-ras is a member of a multigene family in man which encode highly related proteins of 189 amino acids (p21). In vitro, ras proteins bind GTP, and p21 mutants with treonine at position 59 autophosphorylate at that residue. Mutation (at amino acids 12 or 61) and elevated expression of ras genes result in cell transformation in culture, and are also observed in many types of human tumours. Normal and mutant transforming ras proteins show no differences in localization, lipidation or GTP binding. However, mutations at position 12 in recombinant (Thr 59) p21 molecules were observed to affect autophosphorylation. We have expressed the full-length normal and T24 transforming (Gly----Val at position 12) Ha-ras proteins in Escherichia coli and have purified them to homogeneity (ref. 19 and M.G. et al., in preparation); these proteins bound GTP with approximately molar stoichiometry and with an affinity comparable to partially purified mammalian proteins. Microinjection of the T24 protein into quiescent rodent fibroblasts resulted in a rapid alteration in cell morphology, stimulation of DNA synthesis and cell division; in contrast, little response was observed with the normal protein. We now report that the normal ras protein has an intrinsic GTPase activity, yielding GDP and Pi. In contrast, the T24 transforming protein is reduced 10-fold in this activity. We suggest that this deficiency in GTPase is the probable cause for the transforming phenotype of the T24 protein.

The 1984 Walter Hubert lecture. Activation of transforming genes in neoplasms.

Cellular oncogenes have been identified by the biological activity of tumour DNAs in transfection assays and/or by homology to the transforming genes of retroviruses. In some tumours, the biological activity, organization or expression of these genes is altered, suggesting that such alterations contribute to the development of neoplastic disease. Experiments leading to the identification of cellular oncogenes are reviewed and our current understanding of the mechanisms by which they induce transformation of cells in culture and may contribute to the pathogenesis of neoplasms in vivo is discussed.

Structurally and functionally modified forms of pp60v-src in Rous sarcoma virus-transformed cell lysates.

When analyzed from transformed cell lysates, pp60v-src, the product of the Rous sarcoma virus src gene, typically appears as a single polypeptide of 60,000 molecular weight, phosphorylated at two major sites, an amino-terminal region serine residue and carboxy-terminal region tyrosine residue. We describe here the identification of variant forms of pp60v-src present in transformed cell lysates that exhibited an altered electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels. This change in migration appeared to be the result of some alteration in the amino-terminal portion of the molecule and paralleled the appearance of extensive amino-terminal region tyrosine phosphorylation on the pp60v-src molecule. These structural modifications were further correlated with a dramatic increase in the protein kinase-specific activity of pp60v-src. The detection of these variant forms of pp60v-src depended on the prior treatment of the transformed cell cultures with vanadium ions or the inclusion in the cell disruption buffer of Mg2+ or ATP-Mg2+. The implications is that modified, highly active forms of the pp60v-src protein exist in transformed cells, but are transient and rapidly converted to stable forms, possibly by specific dephosphorylation. We suggest that amino-terminal region tyrosine phosphorylation of pp60v-src, presumably the result of autophosphorylation, serves to greatly enhance src protein enzymatic activity, but that much of the regulation of this transforming protein's function may involve a phosphotyrosyl protein phosphatase.

Structurally and functionally modified forms of pp60v-src in Rous sarcoma virus-transformed cell lysates.

When analyzed from transformed cell lysates, pp60v-src, the product of the Rous sarcoma virus src gene, typically appears as a single polypeptide of 60,000 molecular weight, phosphorylated at two major sites, an amino-terminal region serine residue and carboxy-terminal region tyrosine residue. We describe here the identification of variant forms of pp60v-src present in transformed cell lysates that exhibited an altered electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels. This change in migration appeared to be the result of some alteration in the amino-terminal portion of the molecule and paralleled the appearance of extensive amino-terminal region tyrosine phosphorylation on the pp60v-src molecule. These structural modifications were further correlated with a dramatic increase in the protein kinase-specific activity of pp60v-src. The detection of these variant forms of pp60v-src depended on the prior treatment of the transformed cell cultures with vanadium ions or the inclusion in the cell disruption buffer of Mg2+ or ATP-Mg2+. The implications is that modified, highly active forms of the pp60v-src protein exist in transformed cells, but are transient and rapidly converted to stable forms, possibly by specific dephosphorylation. We suggest that amino-terminal region tyrosine phosphorylation of pp60v-src, presumably the result of autophosphorylation, serves to greatly enhance src protein enzymatic activity, but that much of the regulation of this transforming protein's function may involve a phosphotyrosyl protein phosphatase.

Hepatitis B virus and hepatocellular carcinoma: molecular biology and mechanistic considerations.

The question of whether HBV causes HCC has to date best been answered by epidemiologic studies, in particular prospective analysis such as the ongoing study in Taiwan. The mechanism whereby HBV may cause HCC by genetic or epigenetic means, acting alone or in concert with other agents, is still moot . To date, studies of HBV gene expression have not shown the presence of a transforming gene or an oncogene associated with this virus. The finding of integrated HBV DNA in most HCC of carriers is highly suggestive of a causal role of the virus in the pathogenesis of neoplasia. In order to be more certain, it will be necessary to show that expression of integrated HBV genes or of cellular genes, as a result of HBV infection, causes neoplastic transformation of cells. Failure to date to demonstrate that HBV DNA induces neoplastic transformation of eukaryotic cells in culture may be simply due to an inadequate experimental model. The majority of these experiments have used fibroblasts or monkey kidney cells as the recipient cell, whereas cultured human hepatocytes might be more suitable. Whatever role HBV eventually is shown to have in the pathogenesis of HCC, molecular biologic techniques will have in great measure contributed to that knowledge. The use of these powerful new methods will permit studies of the replication of HBV and possible elucidation of how this virus affects the hepatocyte during persistent infection and will in time lead to a better understanding of cell and molecular events leading to development of hepatic malignancy.

Timing of the steps in transformation of C3H 10T 1/2 cells by X-irradiation.

Transformation of cells in culture by chemical carcinogens or X rays seems to require at least two steps. The initial step is a frequent event; for example, after transient exposure to either methylcholanthrene or X rays, almost every cell of established lines of mouse embryo fibroblasts proved capable of yielding transformed, tumorigenic descendants. Although results were interpreted as indicating that 100% of the progeny of methylcholanthrene-treated cells were potentially transformed, later experiments showed that only a very small minority of the progeny of cells initiated by X rays or methylcholanthrene actually produced transformed colonies. We thus concluded that there must be a second step in transformation that is a very rare event. We assumed that this event occurred after the cultures became confluent, a time when transformed cells have a selective growth advantage. Since then, however, others have shown that transformation can occur soon after initiation and that clones of transformed cells may already be present by the time initiated cultures become confluent. It has been hypothesized that the second step behaves like a spontaneous mutation in having a constant but small probability of occurring each time an initiated cell divides. We show here that the clone size distribution of transformed cells in growing cultures initiated by X rays is, indeed, exactly what would be expected on that hypothesis.

Does the large T protein of polyoma virus regulate the expression of the cellular myc gene?

Upon expression of the large T protein of polyoma virus from genes encoding only this protein (plt genes: intron-less gene in pPyLT1 plasmid) (Tyndall et al., 1981), hrt mutant NG18 (Schaffhausen et al., 1978)), changes were observed in the behaviour in culture of rodent embryo fibroblast cells in primary cultures (REF) (Rassoulzadegan et al., 1982, 1983): the cells grew in culture for an unlimited number of generations (“immortalization”) and grew in the presence of low concentrations of serum (below 1% newborn calf serum). They maintained, however, the extended morphology, the low saturation density and the anchorage dependency characteristic of the normal fibroblast. Unlike the original REF- cells, these “immortalized” lines could be transformed by transfer of the polyoma virus gene encoding only the middle T protein (pmt gene: plasmid pPyMT1, Treisman et al., 1981), thus demonstrating a two-step process in tumorigenic transformation. A similar two-step mechanism was suggested by Land et al. (1983) for the myc and ras families of cellular oncogenes: expression either of the polyoma plt or of v-myc and rearranged c-myc genes allowed transformation of REF cells with activated forms of the ras genes. These results suggest that both genes exert similar function (s) at an early step of the transformation of REF cells in culture. This class of oncogenes also includes the E1a genes of adenovirus 2, required for transformation by the E1b genes of the same virus and which can also cooperate with a ras oncogene in REF transformation (Van den Elsen et al., 1982, Ruley, 1983).

Differentiation markers (S-100, GFA, 14.3.2 and D2) in foetal rat brain cells during malignant transformation in cell culture : O.D. Laerum 1, S.J. Mørk 1, Å. Haugen 1, E. Block 2 and K. Haglid 3. 1The Gade Institute, Department of Pathology, University of Bergen, Norway; 2The Protein Laboratory, University of Copenhagen, Denmark; 3Institute of Neurology, Medical Faculty, University of Goteborg,

Differentiation markers (S-100, GFA, 14.3.2 and D2) in foetal rat brain cells during malignant transformation in cell culture : O.D. Laerum 1, S.J. Mørk 1, Å. Haugen 1, E. Block 2 and K. Haglid 3. 1The Gade Institute, Department of Pathology, University of Bergen, Norway; 2The Protein Laboratory, University of Copenhagen, Denmark; 3Institute of Neurology, Medical Faculty, University of Goteborg,

Sequential events in the induction of transformation in cell culture by specific nickel compounds

Crystalline metal sulfides (i.e., Ni3S2, NiS, CoS, CuS) are potent inducers of transformation in Syrian Hamster Embryo cells while the respective amorphous compounds exhibit low transforming activity. Both crystalline and amorphous compounds are relatively water insoluble and when these particulate compounds (2-4 µm) are added to various types of cultured fibroblasts, they exhibit striking differences in their phagocytosis. These differences in uptake were directly related to their surface charge, since crystalline NiS had a negative surface charge (-27 mV, zeta potential) while amorphous NiS had a positively charged surface (+9 mV, zeta potential). Chemical reduction of amorphous NiS with LiA1H4 resulting in acquisition of a negative surface charge, enhanced phagocytosis and caused an incidence of transformation equalling that of crystalline NiS. Following phagocytosis of crystalline NiS, video time lapse microscopy and studies using(63)NiS show that the particles undergo cytoplasmic dissolution that appears to be mediated by lysosomal interaction. Later, the phagocytized NiS particles aggregate in the perinuclear region, their movement by saltation decreases and in some instances vacuoles form around the particles. Particle dissolution products then enter the nucleus and induce strand breaks in the DNA as determined by alkaline sucrose gradient and alkaline elution techniques. Cesium chloride gradient analysis also indicates that phagocytized NiS particles induce repair of DNA. Damage to DNA appears to be a very selective and sensitive action of all metal compounds with potential carcinogenic activity.

Avian sarcoma viruses, protein kinases and cell transformation.

The first RNA tumour virus to be isolated and identified as such was the Rous sarcoma virus (RSV), which causes the transformation of cells in culture as well as fibrosarcomas when injected into suitable host animals (for reviews see Hanafusa (1977) and Bishop (1978)). The genome of RSV has been studied intensively for the past 10-12 years, and it has been shown that the virus itself carries a gene responsible for malignant transformation. This gene, denotedsrcfor sarcoma, was identified genetically through the isolation of temperature-sensitive mutants that were conditional for cell transformation in culture. These mutants are able to transform cells at a temperature of 35 °C, the permissive temperature, but are unable to transform cells morphologically at 41 °C, the non-permissive temperature. The existence of such temperature sensitive mutants implied that the product of the viral transforming gene, in RSV, was a protein (Kawai & Hanafusa 1971). In addition to temperature-sensitive mutants, non-conditional mutants were isolated that had deletions of the src gene. These mutants are unable to transform cells in culture or to cause fibrosarcomas under most conditions. About 4 years ago, the product of the src gene was identified as a phosphoprotein (Mt= 60000); this protein was denoted pp60src(Purchioet al.1978). The RSV genome and the expression of the src gene is illustrated in figure 1.

Transformed mouse mammary epithelial cells synthesize undersulfated basement membrane proteoglycan.

Proteoglycans deposited in the basal lamina of [14C] glucosamine-labeled normal and [3H]glucosamine-labeled transformed mouse mammary epithelial cells grown on type I-collagen gels, were extracted in 4 M guanidinium chloride and cofractionated over Sepharose CL 4B. The heparan sulfate chains carried by these proteoglycans were isolated by treatment with alkaline borohydride, protease K, chondroitinase ABC, and cetylpyridinium chloride precipitation. Heparan sulfate isolated from transformed cell cultures consistently eluted from DEAE-cellulose at lower salt concentrations and was of smaller apparent Mr when chromatographed over Sepharose CL 6B, than heparan sulfate of normal cell cultures. Experiments using doubly labeled cultures ([3H]glucosamine and [35S]sulfate) demonstrated an approximately 30% reduction in the sulfate/hexosamine ratio in heparan sulfate derived from transformed cultures. Both N- and O-sulfate were decreased. The decreased Mr and decreased sulfation of heparan sulfate upon transformation appear sufficient to explain the altered heparan sulfate/chondroitin sulfate ratios previously observed in these cells. These changes may have implications for the molecular interactions in which these proteoglycans are normally engaged during basal lamina assembly, and cause the poor basal lamina formation displayed by these transformed cells.

Role of Polyoma T Antigens in Malignant Cell Transformation

Publisher Summary This chapter discusses the nature of polyoma T antigens, mutations affecting them, and the way these mutations affect cell transformation. There are three polyoma T antigens, called small T (sm-T), medium or middle T (m-T), and large T (1-T). The T antigens are translated independently from individual mRNAs. The coding regions for the T antigens show several interesting and unusual features. First, the coding regions are partially overlapping. All three T antigens have a common N-terminal region of 79 amino acids. Mutations affecting the polyoma T antigens fall into four classes: (1) conditional-lethal (temperature-sensitive); (2) host-range nontransforming (hr-t); (3) viable-deletion; and (4) chain-terminating (nonsense). The overlapping coding regions for the T antigens make it difficult to deduce the effects of mutations on individual proteins. A direct assessment of the functions of individual proteins in transformation has recently been made possible by the construction of cloned cDNA copies of viral mRNAs. A DNA molecule capable of encoding only the polyoma m-T antigen causes the transformation of cells in culture. The transformed cells are capable of causing tumors in animals. Therefore, the m-T antigen alone is sufficient to induce most of the growth changes associated with cell transformation. The effects of the viable-deletion and chain-terminating mutations are also consistent with a primary role of the m-T antigen in transformation.

Loss of different pericellular matrix components of rat cells transformed with a T-Class ts mutant of rous sarcoma virus

Rat cells transformed by the T-class ts mutant LA339 derived from RSV B77 (2R cells) and the parental nontransformed Rat-1 cells were analyzed for the production and deposition of pericellular matrix components as a function of growth temperature. The cells were incubated at the permissive (35°) and nonpermissive (39.5°) temperatures for transformation and the collagenous and noncollagenous (fibronectin and laminin) matrix proteins were studied by polypeptide analysis of the metabolically labeled cells and their culture media. Immunofluorescence analysis of the pericellular matrix structures was performed using specific antibodies against laminin, fibronectin, and heparan sulfate proteoglycan. At 39.5° dense pericellular matrices were detected in both 211 and Rat-1 cell layers, whereas at 35° the 2R cells lost their pericellular matrix structures. The cells continued to synthesize and secrete fibronectin, laminin, and heparan sulfate proteoglycan as well as slightly reduced amounts of procollagen into the culture medium but failed to deposit them in a matrix form. Another major temperature-sensitive difference in the secreted polypeptides was the production by the 211 cells at 35° of a 56,000-dalton protein, similar to that reported in other transformed cell cultures. The present findings indicate that the transformed rat 2R cells lose, upon activity of the pp60 src, their capacity to deposit the synthesized matrix components into pericellular structures.