Mutant p53 tumor suppressor alleles release ras-induced cell cycle growth arrest.

Overexpression of an activated ras gene in the rat embryo fibroblast line REF52 results in growth arrest at either the G1/S or G2/M boundary of the cell cycle. Both the DNA tumor virus proteins simian virus 40 large T antigen and adenovirus 5 E1a are able to rescue ras induced lethality and cooperate with ras to fully transform REF52 cells. In this report, we present evidence that the wild-type activity of the tumor suppressor gene p53 is involved in the negative growth regulation of this model system. p53 genes encoding either a p53Val-135 or p53Pro-193 mutation express a highly stable p53 protein with a conformation-dependent loss of wild-type activity and the ability to eliminate any endogenous wild-type p53 activity in a dominant negative manner. In cotransfection assays, these mutant p53 genes are able to rescue REF52 cells from ras-induced growth arrest, resulting in established cell lines which express elevated levels of the ras oncoprotein and show morphological transformation. Full transformation, as assayed by tumor formation in nude mice, is found only in the p53Pro-193-plus-ras transfectants. These cells express higher levels of the ras protein than do the p53Val-135-plus-ras-transfected cells. Transfection of REF52 cells with ras alone or a full-length genomic wild-type p53 plus ras results in growth arrest and lethality. Therefore, the selective event for p53 inactivation or loss during tumor progression may be to overcome a cell cycle restriction induced by oncogene overexpression (ras). These results suggest that a normal function of p53 may be to mediate negative growth regulation in response to ras or other proliferative inducing signals.     

Glucocorticoid hormones may partially substitute for adenovirus E1A in cooperation with ras

The effects of hormonal promotion of T24-ras oncogene-transfected rat embryo fibroblasts (REF) were compared to cotransformation of these cells with adenovirus E1A and ras. Cotransfection of E1A + ras resulted in the appearance of morphologically transformed cells which were very efficiently established into cell lines. Addition of glucocorticoid hormones to T24-ras-transfected REF cells resulted in cells with a transformed morphology and a capacity to form foci. These foci were, however, inefficiently established into stable cell lines. Removal of hormone from growing cells resulted in retarded growth, suggesting that the hormone acted as a growth factor on these cells. Both E1A-transformed cells and hormone-treated ras-transformed cells showed a reduction in synthesis of high molecular weight tropomyosin isoforms and a decreased expression of surface fibronectin. Control experiments demonstrated that the effects of hormone were mediated through the glucocorticoid receptor. Our findings suggest that glucocorticoid hormones may promote the in vitro growth of ras-initiated REF cells into stably transformed cell lines, but that this ability is limited compared to that of adenovirus E1A.     

Differential induction of cytolytic susceptibility by E1A, myc, and ras oncogenes in immortalized cells.

The E1A oncogene of adenovirus serotypes 2 and 5 induces susceptibility to the cytolytic effects of natural killer lymphocytes and activated macrophages when expressed in infected and transformed mammalian cells (cytolysis-susceptible phenotype). E1A and the oncogenes v-myc, long-terminal-repeat-promoted c-myc, and activated c-ras share the ability to immortalize transfected low-passage rodent cells. The cytolytic phenotypes of well-characterized rodent cell lines immortalized by these three oncogenes were defined. In contrast to target cells expressing the intact E1A gene, myc- and ras-expressing, immortalized primary transfectants were resistant to lysis by both types of killer cell populations. The same patterns of susceptibility (E1A) and resistance (myc and ras) to cytolysis were observed in oncogene-transfected continuous rat (REF52) and mouse (NIH 3T3) cell lines, indicating that differences in the cytolytic phenotypes associated with expression of these oncogenes are not due to cell selection during immortalization. The results suggest that the E1A oncogene may possess a functional domain that is different from those of other oncogenes, such as myc and ras, and that the activity linked to this postulated domain is dissociable from the process of immortalization.     

Model systems of carcinogenesis in vitro: the interaction of the ras oncogene with immortalized cells

Gene transfer experiments have shown that ras effector functions are sufficient to transform cells from a variety of established lines (e. g., mouse NIH3T3 cells). In contrast, primary cells and early passage rodent cells can be transformed by ras oncogenes only at low frequencies, unless cotransfected with collaborating genes such as adenovirus early region IA (EIA) or myc retroviral oncogene homologue. Primary rat embryo fibroblasts (REF) were chosen as a model for the analysis of multistep cellular transformation. Transfection of REF, immortalized by early region of simian adenovirus SA7 with c-Ha-ras oncogene cannot induce their morphological transformation. This phenomenon is observed only after second transfection with the same oncogene. These different cell lines can be used for further analysis of the mechanisms of carcinogenesis.     

Adenovirus type 5 E1A immortalizes primary rat cells expressing wild-type p53

Adenovirus (Ad) E1A induces apoptosis in cells expressing wild-type p53, and stable transformation by Ad E1A requires the co-introduction of an anti-apoptotic gene such as Ad E1B 19K. Thus, cells immortalized by Ad E1A alone might have lost functional p53. In order to analyze the p53 in rat cells expressing Ad E1A, we established rat cell lines by transfecting primary rat embryo fibroblast (REF) and baby rat kidney (BRK) cells with cloned Ad5 E1A. By using a yeast functional assay, we analyzed p53 in six primary REF and three BRK cell lines immortalized by Ad5 E1A as well as five spontaneously immortalized rat cell lines (REF52, NRK, WFB, Rat-1 and 3Y1). The yeast functional assay revealed that all of the spontaneously and Ad5 ElA-immortalized rat cell lines except for 3Y1 expressed wild-type p53. All of the Ad5 E1A-immortalized rat cell lines contained p53 detectable by immunoprecipitation. Recombinant adenovirus expressing rat p53 cloned from a REF cell line immortalized by Ad5 E1A, as well as that expressing murine wild-type p53, induced apoptosis in p53-null cells in collaboration with E1A. Thus, it is suggested that the mutation of p53 appears to be not frequent in the spontaneous immortalization of primary rat cells, and that the functional loss of wild-type p53 is not a prerequisite of E1A-mediated immortalization.     

Functional genetic screen for genes involved in senescence: role of Tid1, a homologue of the Drosophila tumor suppressor l(2)tid, in senescence and cell survival

We performed a genetic suppressor element screen to identify genes whose inhibition bypasses cellular senescence. A normalized library of fragmented cDNAs was used to select for elements that promote immortalization of rat embryo fibroblasts. Fragments isolated by the screen include those with homology to genes that function in intracellular signaling, cellular adhesion and contact, protein degradation, and apoptosis. They include mouse Tid1, a homologue of the Drosophila tumor suppressor gene l(2)tid, recently implicated in modulation of apoptosis as well as gamma interferon and NF-kappaB signaling. We show that GSE-Tid1 enhances immortalization by human papillomavirus E7 and simian virus 40 T antigen and cooperates with activated ras for transformation. Expression of Tid1 is upregulated upon cellular senescence in rat and mouse embryo fibroblasts and premature senescence of REF52 cells triggered by activated ras. In accordance with this, spontaneous immortalization of rat embryo fibroblasts is suppressed upon ectopic expression of Tid1. Modulation of endogenous Tid1 activity by GSE-Tid1 or Tid1-specific RNA interference alleviates the suppression of tumor necrosis factor alpha-induced NF-kappaB activity by Tid1. We also show that NF-kappaB sequence-specific binding is strongly downregulated upon senescence in rat embryo fibroblasts. We therefore propose that Tid1 contributes to senescence by acting as a repressor of NF-kappaB signaling.     

Tumorigenicity of fibroblast lines expressing the adenovirus E1a, cellular p53, or normal c-myc genes.

Cellular and viral oncogenes have been linked to the transformation of established cell lines in vitro, to the induction of tumors in vivo, and to the partial transformation or immortalization of primary cells. Based on the ability to cooperate with mutated ras oncogenes in the transformation of primary cells, the adenovirus E1a and cellular p53 genes have been assigned an immortalizing activity. It is demonstrated in this paper that the adenovirus type 5 E1a gene and simian virus 40 promoter-linked p53 cDNA are able to transform previously immortalized cells to a tumorigenic phenotype without a significant change in cell morphology. It is also shown that, when linked to a constitutive promoter, the normal mouse and human c-myc genes have the same transforming activity. Cells transformed by each of these oncogenes have an increased capacity to grow in the absence of growth factors and a limited anchorage-independent growth capability.     

Rat embryo cells immortalized with transfected oncogenes are transformed by gamma irradiation.

Cesium-137 gamma rays were used to transform rat embryo cells (REC) which were first transfected with activated c-myc or c-Ha-ras oncogenes to produce immortal cell lines (REC:myc and REC:ras). When exposed to 6 Gy of 137Cs gamma rays, some cells became morphologically transformed with focus formation frequencies of approximately 3 x 10(-4) for REC:myc and approximately 1 x 10(-4) for REC:ras, respectively. Cells isolated from foci of gamma-ray-transformed REC:myc (REC:myc:gamma) formed anchorage-independent colonies and were tumorigenic in nude mice, but foci from gamma-ray-transformed REC:ras (REC:ras:gamma) did not exhibit either of these criteria of transformation. Similar to the results with gamma irradiation, we observed a sequence-dependent phenomenon when myc and ras were transfected into REC, one at a time. REC immortalized by ras transfection were not converted to a tumorigenic phenotype by secondary transfection with myc, but REC transfected with myc were very susceptible to transformation by subsequent ras transfection. This suggests that myc-immortalized cells are more permissive to transformation via secondary treatments. In sequentially transfected REC, myc expression was high whether it was transfected first or second, whereas ras expression was highest when the ras gene was transfected secondarily into myc-containing REC. Molecular analysis of REC:ras:gamma transformants showed no alterations in structure of the transfected ras or of the endogenous ras, myc, p53, or fos genes. The expression of ras and p53 was increased in some isolates of REC:ras:gamma, but myc and fos expression were not affected. Similarly, REC:myc:gamma transformants did not demonstrate rearrangement or amplification of the transfected or the endogenous myc genes, or of the potentially cooperating Ha-, Ki-, or N-ras genes. Northern hybridization analysis revealed increased expression of N-ras in two isolates, REC:myc:gamma 33 and gamma 41, but no alterations in the expression of myc, raf, Ha-ras, or Ki-ras genes in any REC:myc transformant. DNA from several transformed REC:myc:gamma cell lines induced focus formation in recipient C3H 10T1/2 and NIH 3T3 cells. The NIH 3T3 foci tested positive when hybridized to a probe for rat repetitive DNA. A detailed analysis of the NIH 3T3 transformants generated from REC:myc:gamma 33 and gamma 41 DNA failed to detect Ha-ras, Ki-ras, raf, neu, trk, abl, fms, or src oncogenes of rat origin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Changes in surface relief of suspended cells are morphological signs of the initial stage of neoplastic transformation in fibroblastic monolayer cultures.

The percentages of cells with different types of cell surface relief were determined in cell suspensions derived from monolayer cultures. Primary cultures of rat embryo fibroblasts (REF) and cell lines REF (LT) and REF-1, immortalized cells of which preserved normal phenotypic characteristics of the initial primary culture REF, as well as morphologically transformed tumorigenic lines REF (LT) ras and REF-2EJ were studied. In REF suspensions the cells with the blebbed type of surface relief were shown to be predominant as compared with those with microvillus relief whereas cell suspensions derived from both immortalized and fully transformed cultures display the reverse ratio of cells with those types of surface relief. Therefore, the pattern of cell surface relief in cell suspensions derived from fibroblastic monolayer cultures may serve as a morphological marker of the initial stage of neoplastic transformation-immortalization when typical morphological signs of cell transformation are not yet manifested in monolayer cultures.     

Ras-mediated cell cycle arrest is altered by nuclear oncogenes to induce Schwann cell transformation.

The cellular responses to ras and nuclear oncogenes were investigated in purified populations of rat Schwann cells. v-Ha-ras and SV40 large T cooperate to transform Schwann cells, inducing growth in soft agar and allowing proliferation in the absence of added mitogens. Expression of large T alone reduces their growth factor requirements but is insufficient to induce full transformation. In contrast, expression of v-Ha-ras leads to proliferation arrest in Schwann cells expressing a temperature-sensitive mutant of large T at the restrictive temperature. Cells arrest in either the G1 or G2/M phases of the cell cycle, and can re-enter cell division at the permissive temperature even after prolonged periods at the restrictive conditions. Oncogenic ras proteins also inhibit DNA synthesis when microinjected into Schwann cells. Adenovirus E1a and c-myc oncogenes behave similarly to SV40 large T. They cooperate with Ha-ras oncogenes to transform Schwann cells, and prevent ras-induced growth arrest. Thus nuclear oncogenes fundamentally alter the response of Schwann cells to a ras oncogene from cell cycle arrest to transformation.     

High intensity ras signaling induces premature senescence by activating p38 pathway in primary human fibroblasts

Although oncogenic ras plays a pivotal role in neoplastic transformation, it triggers an anti-oncogenic defense mechanism known as premature senescence in normal cells. In this study, we investigated the induction of cellular responses by different expression levels of oncogenic ras in primary human fibroblasts. We found that a moderate, severalfold increase in ras expression promoted cell growth. Further elevation of ras expression initially enhanced proliferation but eventually induced p16INK4A expression and senescence. The induction of these opposing cellular responses by ras signals of different intensity was achieved through differential activation of the MAPK pathways that mediated these responses. Whereas moderate ras activities only stimulated the mitogenic MEK-ERK pathway, high intensity ras signals induced MEK and ERK to higher levels, leading to stimulation of the MKK3/6-p38 pathway, which had been shown previously to act downstream of Ras-MEK to trigger the senescence response. Thus, these studies have revealed a mechanism for the differential effects of ras on cell proliferation. Furthermore, moderate ras activity mediated transformation in cooperation with E6E7 and hTERT, suggesting that a moderate intensity ras signal can provide sufficient oncogenic activities for tumorigenesis. This result also implies that the ability of ras to promote proliferation and oncogenic transformation can be uncoupled with that to induce senescence in cell culture and that the development of tumors with relatively low ras activities may not need to acquire genetic alterations that bypass premature senescence.     

Large E1B proteins of adenovirus types 5 and 12 have different effects on p53 and distinct roles in cell transformation.

The formation of complexes between oncoproteins of DNA tumor viruses and the cellular protein p53 is thought to result in inactivation of the growth suppressor function of p53. In cells transformed by nononcogenic human adenovirus type 5 (Ad5), the 55-kDa protein encoded by E1B forms a stable complex with p53 and sequesters it in the cytoplasm. However, the homologous 54-kDa protein of highly oncogenic Ad12 does not detectably associate with p53. Yet in Ad12-transformed cells, p53 is metabolically stable, is present at high levels in the nucleus, and contributes to the oncogenicity of the cells. Such properties have previously been described for mutant forms of p53. Here, we show that stable p53 in Ad12-transformed cells is wild type rather than mutant and that stabilization of p53 is a direct consequence of the expression of the Ad12 E1B protein. We also compared the effects of the E1B proteins on transformation of rodent cells by different combinations of oncogenes. A synergistic interaction was observed for the gene encoding the 54-kDa E1B protein of Ad12 with myc plus ras oncogenes, resembling the effect of mutant p53 on myc plus ras. In contrast, the Ad5 55-kDa E1B protein strongly inhibited transformation by myc plus ras but stimulated transformation by E1A plus ras. The data are explained in terms of different interactions of the two E1B proteins with endogenous p53. The results suggest that in cultured rat cells, endogenous wild-type p53 plays an essential role in cell proliferation, even in the presence of myc plus ras. The dependence on p53 is lost, however, when the adenovirus E1A oncogene is present.     

Glucocorticoid dexamethasone reversibly complements EJ-RAS oncogene to transform mouse embryo BALB-3T3 cells

EJ-A is a Balb-3T3 transfectant cell line that bears a small number of EJ-ras oncogene copies/cell, has low EJ-ras expression, and resembles the parental cell line in displaying a non-transformed phenotype. The glucocorticoid hormone dexamethasone reversibly induces transformation traits in EJ-A cells, namely: 1) morphological transformation; 2) increased growth rate and saturation density; 3) reduced G1 length; and 4) independence of the FGF requirement to initiate DNA synthesis. Western blot analysis revealed that dexamethasone does not increase the p21ras protein intracellular level. beta-IFN, added to the culture medium, does not suppress the dexamethasone-induced growth stimulation and morphological transformation. Therefore, glucocorticoid hormones can complement low EJ-ras expression to transform Balb-3T3 cells, by a mechanism that is likely to be independent of p21ras increase and beta-IFN decrease.     

The c-fos proto-oncogene promotor is not regulated by serum, epidermal growth factor, and phorbol ester in embryonal fibroblasts transformed by E1Aad5+cHa-ras-oncogenes]

Regulation of c-fos protooncogene activity in rat embryonal fibroblasts (REF), E1Aad5-immortalized REF cells, and E1Aad5 + cHa-ras transformed REF cells has been investigated. The analysis of regulation of fos-promoter activity was done by means of transient and stable transfection of fos-CAT plasmid into immortalized and transformed cells. In parallel, the regulation of cellular c-fos as well as c-jun and c-myc genes expression has been studied. It has been found that in E1Aad5 + cHa-ras-transformed cells the expression of c-fos promoter has a constitutive, non-inducible character while in REF cells and cells immortalized by E1Aad5 the fos-promoter can be regulated by serum growth factors, EGF, and TPA.     

MAP-kinase cascade analysis in transformed cell with different abilities to make G1/S block under serum starvation

We have studied the ability of ERK1,2, JNK1,2 and p38 kinases to be regulated after serum deprivation in E1A + E1B-19 kDa- and E1A + E1A + c-Ha-ras-transformed rat embryo fibroblasts. It was demonstrated that oncogene transformation resulted in an increase of total kinase content independently of the type of complementing oncogene. However, for ERK1,2 kinases phosphorylation was found to depend on the type of complementing oncogene. Besides, unusual biphasic character for ERK1,2 kinases phosphorylation was checked in control fibroblasts REF52 and in transformed E1A + E1B-19 kDa cells, which undergo G1/S arrest after a 24 h serum starvation. According to the immunoblotting data, phosphorylated forms of ERK1,2 kinases are not detected after 15-30 min of serum deprivation, but their content is restored up to the control level within several hours. At the same time, the level of ERK1,2 phosphorylation in E1A + c-Ha-ras cells did not change after serum withdrawal. Besides, serum deprivation did not lead to significant changes in the level of phosphorylation of both type stress kinases--JNK2 and p38 in all types of studied cells. We discuss possible mechanisms of biphasic alteration in ERK1,2 phosphorylation level under condition of serum deprivation of REF52 cells and E1A + E1B-19 kDa-transformed fibroblasts, able to be arrested in G1 phase.     

The effect of Ha-ras oncogene on cells immortalized by the gene for polyomavirus large T-antigen

Activated human Ha-ras oncogene cloned on the plasmid pEJras6,6 was transfected into REF (LT) cells immortalized by the gene for large T-antigen of the polyoma virus. The cells were shown to become completely transformed (in the terms of morphology and tumorogeneity) only after three cycles of transfection with the plasmid pEJras6,6. The integrated sequences of the plasmid pEJras6,6 and the ras oncogene product p21Ha-ras were detected in cells only after their selection in the nude mice (in the cell culture REF (LT) ras X 3tu obtained from the tumor and directly in the tumor cells). Thus, after sequential transfections with a c-Ha-ras oncogene we developed cell cultures on the different stages of transformation process.     

Transformation-sensitive and growth-related changes of protein synthesis in REF52 cells. A two-dimensional gel analysis of SV40-, adenovirus-, and Kirsten murine sarcoma virus-transformed rat cells using the REF52 protein database

Two-dimensional gels of normal and virally transformed REF52 cells have been quantified and compared using the QUEST system for construction and analysis of protein databases. The REF52 protein map is based on more than 1600 high quality spots, and the relative amounts of these proteins are studied in 79 gels representing 12 major experiments. REF52 cells transformed by SV40, adenovirus, and Kirsten murine sarcoma virus (KiMSV) are compared to normal REF52 cells at several stages of growth from low density to confluence and after refeeding confluent cells. In addition, early (1-4 h) and late (21-24 h) responses to serum stimulation were measured in normal, SV40-and adenovirus-transformed cells. The database has been analyzed with respect to 1) known marker proteins and protein sets, 2) global comparison of protein patterns, and 3) selection of unknown spots which have interesting patterns of regulation. For the marker proteins, which include the tropomyosin family and the proliferation-sensitive nuclear antigen, new aspects of regulation by growth and transformation have been revealed. Proliferation-sensitive nuclear antigen, a protein known to be involved in DNA synthesis, is growth-regulated in normal cells and overexpressed in some SV40- and adenovirus-transformed cells. Global comparisons reveal no overall correlation between growth-regulated changes and transformation-induced changes; however, a set of 26 coregulated proteins, including proliferation-sensitive nuclear antigen, was found to be overexpressed in REF52 cells transformed by SV40 or adenovirus. These proteins are synthesized at rates that correlate with the rate of cell proliferation in REF52 and Kirsten murine sarcoma virus-transformed cells but, in SV40- and adenovirus-transformed cells, these proteins are synthesized at high levels independent of the rate of growth. These data suggest that the transforming proteins of SV40 and adenovirus share a function that results in deregulation of the genes coding for a class of cell cycle-regulated proteins.