A Proteomic approach for protein-profiling the oncogenic ras induced transformation (H-, K-, and N-Ras) in NIH/3T3 mouse embryonic fibroblasts

Point mutations in three kinds of Ras protein (H-, K-, and N-Ras) that specifically occur in codons 12, 13, and 61 facilitate virtually all of the malignant phenotype of the cancer cells, including cellular proliferation, transformation, invasion, and metastasis. In order to elucidate an understanding into the oncogenic ras networks by H-, K-, and N-Ras/G12V, we have established various oncogenic ras expressing NIH/3T3 mouse embryonic fibroblast clones using the tetracycline-induction system, which are expressing Ras/G12V proteins under the tight control of expression by an antibiotics, doxycycline. Here we provide a catalog of proteome profiles in total cell lysates derived from three oncogenic ras expressing NIH/3T3 cells and a good in vitro model system for dissecting the protein networks due to these oncogenic Ras proteins. In this biological context, we compared total proteome changes by the combined methods of 2-DE, quantitative image analysis, and MALDI-TOF MS analysis using the unique Tet-on inducible expression system. There were a large number of common targets for oncogenic ras, which were identified in all three cell lines and consisted of 204 proteins (61 in the pH range of 4-7, 63 in 4.5-5.5, and 80 in 5.5-6.7). Differentially regulated expression was further confirmed for some subsets of candidates by Western blot analysis using specific antibodies. Taken together, we implemented a 2-DE-based proteomics approach to the systematical analysis of the dysregulations in the cellular proteome of NIH/3T3 cells transformed by three kinds of oncogenic ras. Our results obtained and presented here show that the comparative analysis of proteome from oncogenic ras expressing cells has yielded interpretable data to elucidate the differential protein expression directly and/or indirectly, and contributed to evaluate the possibilities for physiological, and therapeutic targets. Further studies are in progress to elucidate the implications of these findings in the regulation of Ras induced transformation.     

Transfer of ‘immortalizing’ oncogenes into rat fibroblasts induces both high rates of sister chromatid exchange and appearance of abnormal karyotypes

Cell lines established after transfer into FR3T3 rat fibroblast cells of 'immortalizing' oncogenes (plt gene (large T protein) of polyoma virus, v-myc gene of MC29 virus, rearranged forms of c-myc) exhibited increased rates of sister chromatid exchange (SCE). This was observed neither in cells which expressed one of the oncogenes responsible for the terminal stages of tumorigenic transformation (polyoma virus pmt (middle T protein), mutated ras genes), nor in cell lines carrying oncogenes of both types. Abnormal chromosome numbers were observed in cell lines expressing plt or myc, but not after transformation by pmt or ras oncogenes.     

Alpha B-crystallin expression in mouse NIH 3T3 fibroblasts: glucocorticoid responsiveness and involvement in thermal protection.

alpha B-crystallin, a major soluble protein of vertebrate eye lenses, is a small heat shock protein which transiently accumulates in response to heat shock and other kinds of stress in mouse NIH 3T3 fibroblasts. Ectopic expression of an alpha B-crystallin cDNA clone renders NIH 3T3 cells thermoresistant. alpha B-crystallin accumulates in response to the synthetic glucocorticoid hormone dexamethasone. Dexamethasone-treated NIH 3T3 cells become thermoresistant to the same extent as they accumulate alpha B-crystallin. A cell clone in which alpha B-crystallin is superinduced upon heat shock acquires augmented thermotolerance. Expression of the ras oncogene causes a rapid but transient accumulation of alpha B-crystallin within 1 day. Later, sustained ras oncogene expression suppresses the dexamethasone-mediated alpha B-crystallin accumulation. Thus, oncogenic transformation triggered by the ras oncogene interferes with hormone-mediated accumulation of alpha B-crystallin and concomitant acquisition of thermoresistance. Other known heat shock proteins do not accumulate in response to ectopic alpha B-crystallin expression or to dexamethasone treatment. These results indicate that alpha B-crystallin can protect NIH 3T3 fibroblasts from thermal shock.     

Regulation of transformed state by calpastatin via PKCepsilon in NIH3T3 mouse fibroblasts.

Ca(2+)-activated neutral protease calpain is ubiquitously expressed and may have pleiotropic biological functions. We have previously reported that repeated treatment of NIH3T3 mouse fibroblasts with the calpain inhibitor N-acetyl-Leu-Leu-norleucinal (ALLN) resulted in the induction of transformed foci [T. Hiwasa, T. Sawada, and S. Sakiyama (1990) Carcinogenesis 11, 75-80]. To elucidate further the effects of calpain in malignant transformation of NIH3T3 cells, calpastatin, an endogenous specific inhibitor of calpain, was expressed in NIH3T3 cells by transfection with cDNA. G418-selected calpastatin-expressing clones showed a significant increase in the anchorage-independent growth ability. A similar increase in cloning efficiency in soft agar medium was also observed in calpain small-subunit-transfected clones. On the other hand, reduced expression of calpastatin achieved by transfection with calpastatin antisense cDNA in Ha-ras-transformed NIH3T3 (ras-NIH) cells caused morphological reversion as well as a decrease in anchorage-independent growth. When NIH3T3 cells were treated with ALLN for 3 days, cell growth was stimulated by approximately 10%. This growth stimulation by ALLN was not observed in ras-NIH cells, but recovered by expression of a dominant negative form of protein kinase C (PKC)epsilon but not by that of PKCalpha. Western blotting analysis showed that an increase in PKCepsilon was much more prominent than that of PKCalpha in NIH3T3 cells after treatment with ALLN. These results are concordant with the notion that calpain suppresses malignant transformation by predominant degradation of PKCepsilon.     

Mechanisms of oncogene-mediated alterations in metastatic ability.

Transfected ras oncogenes have been shown to induce metastatic properties in some cells. This altered behavior is likely due to changes in ras-mediated signal transduction pathways, resulting in altered expression of genes important to metastasis. Clarification of the mechanisms by which ras is able to induce metastatic ability in model systems will improve our understanding of tumor progression, even in those cells in which ras activation has not been implicated. Many of the consequences of ras expression also have been detected in cells that have become metastatic in the absence of altered ras, suggesting that there is a set of common changes that can lead to metastasis, with multiple signals capable of eliciting these changes. We have identified several changes in metastatic, ras-transformed NIH 3T3 cells that may contribute to their increased malignancy, including expression of proteolytic enzymes and their inhibitors, and adhesive and calcium-binding proteins. Not all cells, however, respond in this way to expression of oncogenic ras. We have found that murine LTA cells, which are tumorigenic but nonmetastatic, are ras resistant and remain nonmetastatic when expressing high levels of transfected ras, in contrast to NIH 3T3 cells, which are ras sensitive and become both tumorigenic and metastatic in response to comparable levels of ras. LTA cells differ in their patterns of gene expression in response to ras when compared with NIH 3T3 cells, suggesting that the two cell lines process the ras signal differently. Here we review our results with ras-transfected NIH 3T3 and LTA cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulated and constitutive activity by CDC25Mm (GRF), a Ras-specific exchange factor.

Serum stimulates cells to increase their proportion of Ras protein in the active GTP-bound state. We have recently identified four types (I to IV) of apparently full-length cDNAs from a single mammalian gene, called CDC25Mm or GRF, which is homologous to the Ras-specific exchange factor CDC25 of S. cerevisiae. The largest cDNA (type IV) is brain specific, with the other three classes, although they have distinct 5' ends, essentially representing progressive N-terminal deletions of this cDNA. When placed in a retroviral expression vector, all four types of cDNAs induced morphologic transformation of NIH 3T3 cells and an increase in the basal level of GTP.Ras. Serum stimulation of these transformants lead to a further increase in GTP.Ras only in cells expressing the type IV cDNA. Each type of GRF protein was found in cytosolic and membrane fractions, and the protein in each fraction could stimulate guanine nucleotide release from GDP.Ras in vitro. When NIH 3T3 cells and cells expressing the type IV protein were transfected with two versions of a mutant ras gene, one encoding membrane-associated Ras protein and the other encoding a cytosolic Ras protein, the basal levels of GTP bound to both forms of the mutant Ras protein were significantly higher in the cells expressing the type IV protein. However, serum increased the level of GTP bound to the membrane-associated mutant Ras protein in NIH 3T3 cells and in cells expressing the type IV protein but not in cells expressing the cytosolic version of the Ras protein. We conclude that each type of CDC25Mm induces cell transformation via the ability of its C terminus to stimulate guanine nucleotide exchange on Ras, the presence of N-terminal sequences is associated with a serum-dependent change in GTP.Ras, and the serum-dependent increase in GTP.Ras by exogenous CDC25Mm or by endogenous exchange factors probably requires membrane association of both Ras and the exchange factor.     

Antisense-fos RNA causes partial reversion of the transformed phenotypes induced by the c-Ha-ras oncogene.

Several lines of evidence have suggested that c-fos may act downstream from c-Ha-ras in a growth-regulatory signal transduction pathway. We used antisense RNA to inhibit c-fos gene expression and investigated the effects of diminished c-fos expression on the phenotypes induced by the EJ c-Ha-ras oncogene in NIH 3T3 cells. Immunofluorescent staining demonstrated that the antisense RNA caused a marked reduction in the amount of c-fos protein expressed following serum stimulation. EJ cells containing antisense-fos RNA continued to overexpress ras and remained capable of proliferating in vitro. However, the antisense-fos RNA caused a partial reversion of the major transformed phenotypes of EJ cells, including a restoration of both density-dependent growth arrest and the ability to be rendered quiescent by serum deprivation, a reversion to a flat morphology, inhibition of anchorage-independent growth, and inhibition of tumorigenicity in nude mice. Our results indicate that inhibition of c-fos expression, to a level still supporting in vitro proliferation, prevents the transforming effects of the ras oncogene; they thus provide additional evidence for the participation of c-fos in ras-regulated signal transduction pathways.     

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)     

Transformation of NIH 3T3 cells by enhanced PAR expression

Prostate androgen regulated (PAR) is a 1038bp novel gene located on chromosome 1 in epidermal differentiation complex. The gene is ubiquitously expressed in normal tissues and is overexpressed in most of their malignant counterparts. PAR cellular function is unknown. Here we report the effect of increased PAR expression induced by transfection of PAR cDNA on NIH3T3 cell phenotype. PAR-NIH3T3 transfectants expressing 3- to 4-fold higher PAR levels compared to controls grew faster in tissue cultures, formed colonies in soft agar, and exhibited a shortening of G1 and S phases of cell cycle and formed tumors in SCID mice. Transfection of NIH3T3 cells with increased ectopic PAR expression with a 22 mer oligonucleotide in antisense orientation with PAR mRNA abrogated their ability to form colonies in soft agar. The data presented here along with our previously reported results on DU145 cells transfected with antisense PAR cDNA suggest that PAR gene behaves like a proto-oncogene.     

Platelet-derived growth factor stimulation of GTPase-activating protein tyrosine phosphorylation in control and c-H-ras-expressing NIH 3T3 cells correlates with p21ras activation.

Platelet-derived growth factor (PDGF) stimulation of NIH 3T3 cells leads to the rapid tyrosine phosphorylation of the GTPase-activating protein (GAP) and an associated 64- to 62-kDa tyrosine-phosphorylated protein (p64/62). To assess the functions of these proteins, we evaluated their phosphorylation state in normal NIH 3T3 cells as well as in cells transformed by oncogenically activated v-H-ras or overexpression of c-H-ras genes. No significant GAP tyrosine phosphorylation was observed in unstimulated cultures, while PDGF-BB induced rapid tyrosine phosphorylation of GAP in all cell lines analyzed. In NIH 3T3 cells, we found that PDGF stimulation led to the recovery of between 37 and 52% of GAP molecules by immunoprecipitation with monoclonal antiphosphotyrosine antibodies. Furthermore, PDGF exposure led to a rapid and sustained increase in the levels of p21ras bound to GTP, with kinetics similar to those observed for GAP tyrosine phosphorylation. The PDGF-induced increases in GTP-bound p21ras in NIH 3T3 cells were comparable to the steady-state level observed in serum-starved c-H-ras-overexpressing transformants, conditions in which these cells maintained high rates of DNA synthesis. These results imply that the level of p21ras activation following PDGF stimulation of NIH 3T3 cells is sufficient to support mitogenic stimulation. Addition of PDGF to c-H-ras-overexpressing cells also resulted in a rapid and sustained increase in GTP-bound p21ras. In these cells GAP, but not p64/62, showed increased tyrosine phosphorylation, with kinetics similar to those observed for increased GTP-bound p21ras. All of these findings support a role for GAP tyrosine phosphorylation in p21ras activation and mitogenic signaling.     

Adenylate cyclase activity of NIH 3T3 cells morphologically transformed by ras genes

The observed homology between G-proteins which regulate adenylate cyclase and ras proteins and the suggested role of ras in the regulation of adenylate cyclase in yeast prompted us to examine the regulation of adenylate cyclase in three cell lines: (i) NIH 3T3 cells, (ii) NIH 3T3 cells transformed by high levels of the normal rasH gene product and (iii) NIH 3T3 cells transformed by a mutated rasH gene product. We found that the regulation of adenylate cyclase by G-proteins is identical in the three cell lines, although the response of the transformed NIH 3T3 cells to agonists is strongly attenuated. Our data suggest that mammalian ras products do not interact directly with adenylate cyclase, although their increased expression may indirectly inhibit the interaction of adenylate cyclase stimulatory receptors with G-proteins.     

The effects of ras gene expression on glucocorticoid receptors in mouse fibroblasts

Analysis of induction of glutamine synthetase activity by dexamethasone showed a 2-fold increase in NIH3T3 but no change in NIH3T3 ras (EJ-ras) cells. The observed increase could be abolished by the antagonist RU486. The lack of response in ras transformed cells might reflect oncoprotein effects on the glucocorticoid receptor (GR). Several GR parameters were studied in order to clarify this point. Total GR level was the same for both cells; cytoplasmic receptor level however, was 3 times lower in NIH3T3 ras than in NIH3T3 cells. Hormone-receptor binding affinity, specificity, thermostability, sedimentation coefficient, molecular weight as well as the cytoplasmic GR transformation ratio were similar for the two cell lines. On the other hand, the fraction of the total receptor pool involved with the recycling process was approximately 20% lower in NIH3T3 ras than in NIH3T3 cells. After 24 h of dexamethasone treatment, no GR down regulation was observed in NIH3T3 ras cells, whereas normal NIH3T3 cells exhibited a decrease of GR binding capacity around 80%. Further studies are necessary to define the mechanisms underlying the association between glucocorticoid insensitivity, and modifications in the GR nuclear/cytoplasmic ratio, in the recycling GR fraction and in the down-regulation process observed in ras transformed cells.     

Reversal of transformed phenotype by monoclonal antibodies against Ha-ras p21 proteins

The transforming activities of p21 ras proteins have been determined by micro-injection of these proteins into NIH3T3 cells. In order to facilitate functional studies on the effect of ras proteins on malignant transformation and normal cellular growth, analysis has been made with three monoclonal antibodies (YA6-172, Y13-238 and Y13-259) as originally reported by Furth et al. (J virol 43 (1982) 294). Purified immunoglobulin of Y13-259 has the highest titer of binding to bacterially synthesized p21 ras proteins. Experimental analyses indicate that only Y13-259 antibody will neutralize the transforming activity of the co-injected bacterially synthesized ras protein and the neutralization effect was blocked by co-injection of excess ras protein. In addition, micro-injection of Y13-259 immunoglobulin into transformed NIH3T3 cells (obtained by DNA transfection of NIH3T3 cells with molecularly cloned ras gene) reversed their transformed phenotypes. These results indicate that both bacterially synthesized p21 ras proteins and the natural ras proteins produced in NIH3T3 cells were neutralized by Y13-259 antibody.     

Ras (proto)oncogene induces N-linked carbohydrate modification: temporal relationship with induction of invasive potential

The effect of expression of the ras oncogene on protein glycosylation was studied. VSV G-protein and class I histocompatibility antigens were analysed to monitor ras-mediated changes in glycosylation. Transient expression of the c-Ha-ras oncogene, introduced into NIH 3T3 cells by the DEAE-dextran method, altered protein glycosylation within 25 h of transfection. The same result was obtained after dexamethasone-induced expression of p21-ras in stable NIH 3T3 transfectants containing either an activated Ha-ras oncogene or a normal N-ras proto-oncogene under control of the glucocorticoid-inducible MMTV promoter. The alteration of cell surface carbohydrates, induced by the ras (proto)oncogene and the subsequent acquisition of invasive potential, occurred prior to morphological transformation.     

Heterogeneity in transformation efficiency of NIH 3T3 cells as determined by transfection with oncogenes

Clones of cells have been isolated from a culture of NIH 3T3 cells and characterized. A high degree of variation between the clones was observed in their efficiency of transformation following transfection with the ras and myc oncogenes. No correlation was found between this characteristic and either the growth rate of these cells in vitro or the efficiency of transfection, as judged by the acquisition of geneticin resistance and chloramphenicol acetyl transferase activity. Continuous maintenance of NIH 3T3 cells in culture resulted in a significant decrease in susceptibility to transformation. It is suggested that the NIH 3T3 cell line comprises a heterogeneous population of cells, and it is the balance between the various types of cells which determines the phenotype of the culture. This balance can be spontaneously disrupted while the culture is grown continuously in vitro.     

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.     

Lipid signalling pathways in normal and ras-transfected NIH/3T3 cells

The role of ras oncogenes in cellular signalling pathways involving phospholipid breakdown was studied in untransfected and proto-H-ras and mutated H-, K- and N-ras transfected NIH/3T3 cells. When the cells were grown at low cell densities, all of the ras transfected cells had 2-4 fold higher diacylglycerol (DAG) levels compared to growing NIH/3T3 cells. At high cell densities, DAG levels decreased in the former and increased in contact inhibited NIH/3T3 cells. In this regard, only cells transformed by mutated cellular and viral H-ras oncogenes (but not by the H-ras proto-oncogene) had elevated DAG levels compared to contact inhibited NIH/3T3 cells. The basal levels of inositol phosphates in ras transfected cells were not significantly different from NIH/3T3 cells and did not vary with cell density. Thus, the elevated DAG levels are not a consequence of increased phosphoinositide hydrolysis. The latter was stimulated by serum and bombesin only in normal and proto-H-ras transfected cells. In contrast, stimulation by bradykinin was observed only in cells transformed by mutated cellular ras oncogenes. Furthermore, aluminum fluoride stimulated phosphoinositide breakdown in the latter cells indicating that there was no uncoupling of the G protein from phospholipase C. Treatment of ras transfected cells with dibutyryl cyclic AMP (DB-cAMP), which causes an inhibition of growth and a reversal of the transformed morphology, did not alter the basal levels of inositol phosphates, DB-cAMP, however, did lower DAG levels in some of the transformed cell lines, but elevated DAG levels in low density NIH/3T3 cells. These findings indicate that the ras gene product p21 is not involved in phosphoinositide hydrolysis and that DAG levels do not correlate with cell growth in either normal or ras transfected NIH/3T3 cells. Thus, p21 appears to alter cell growth through mechanism(s) independent of lipid signalling pathways.