Complementation by BCL2 and C-HA-RAS oncogenes in malignant transformation of rat embryo fibroblasts.

The BCL2 (B cell lymphoma/leukemia-2) and C-HA-RAS oncogenes encode membrane-associated proteins of 26 and 21 kilodaltons, respectively. Although RAS proteins have long been known for their ability to bind and hydrolyze GTP, recent investigations suggest that BCL2 encodes a novel GTP-binding protein (S. Haldar, C. Beatty, Y. Tsujimoto, and C. M. Croce, Nature [London] 342:195-198, 1989). Cotransfection of BCL2 and HA-RAS oncogenes resulted in morphological transformation of early-passage rodent fibroblasts, rendering these cells tumorigenic in animals and enabling them to grow in semisolid medium. In contrast, cotransfection of BCL2 with oncogenes that encode nuclear proteins (E1A and C-MYC) did not produce malignant transformation, whereas HA-RAS did complement with these genes. These findings suggest that proteins encoded by oncogenes such as BCL2 and HA-RAS, although having similar subcellular locations and perhaps similar biochemical properties, can regulate distinct complementary pathways involved in cellular transformation.     

Inhibition of spermidine and spermine synthesis leads to growth arrest of rat embryo fibroblasts in G1

Following growth stimulation of rat embryo fibroblast (REF) cells previously arrested in G₁ by serum deprivation, there occurs a large increase in the synthesis of the polyamines putrescine, spermidine and spermine. Methylglyoxal bis(guanylhydrazone) (MGBG), a potent inhibitor of S-adenosylmethionine decarboxylase can block the accumulation of both spermidine and spermine over a period of several days. Under such conditions REF cells treated with MGBG will approximately double in number and then become growth-arrested again predominantly in the G₁ phase of the cell cycle. REF cells therefore appear to contain sufficient spermidine and spermine to progress through one cell cycle before the intracellular levels of these polyamines is reduced sufficiently to arrest growth in the absence of continued polyamine synthesis. Limitation of intracellular polyamine levels is therefore not the mechanism by which deprivation of serum growth factors arrests cell growth. While continued growth is nevertheless dependent on polyamine synthesis, this cell type is capable of limited proliferation in its absence. Addition of spermidine or spermine to MGBG-arrested REF cells results in a rapid resumption of proliferation demonstrating that either polyamine can fulfill the role played by these polyamines in the growth process. Low levels of spermidine and spermine therefore arrest this cell type at a resriction point in G₁ at which it is decided whether the intracellular level of these polyamines is sufficiently high to enable a cell to enter into and complete a new cell cycle. This polyamine-sensitive restriction point is considered to be analogous to the restriction point(s) in G₁ at which serum and nutrient limitation act.     

Behavior of myc and ras oncogenes in transformation of rat embryo fibroblasts.

The requirements for transformation of rat embryo fibroblasts (REFs) by transfected ras and myc oncogenes were explored. Under conditions of dense monolayer culture, neither oncogene was able to transform REFs on its own. However, the introduction of a ras oncogene together with a selectable neomycin resistance marker into REFs allowed killing of the normal nontransfected cells and the outgrowth of colonies of ras transformants, 10% of which survived crisis and became tumorigenic. These cells expressed greater than 10-fold-higher levels of ras p21 than tumorigenic cells cotransfected with ras and myc oncogenes. The myc oncogene similarly was unable to induce tumorigenic conversion of REFs unless especially refractile colonies of oncogene-bearing cells, produced by use of a cotransfected selectable marker, were picked and subcultured. Tumorigenic conversion of REFs by single transfected oncogenes appears to require special culture conditions and high levels of gene expression.     

Immortalization of rat embryo fibroblasts by an adenovirus 2 mutant expressing a single functional E1a protein.

Expression of the adenovirus E1a and E1b genes is required for transformation of nonpermissive rodent cells. Differential splicing of the E1a precursor RNA molecules results in the production of two early mRNAs, 13S and 12S, which encode a 289-amino-acid-residue (289R) and 243R protein, respectively. Previously we constructed a mutant virus, dl231, which can only produce normal 289R protein from the E1a gene. In this report we demonstrate that dl231 induced focal transformation of primary rat embryo fibroblasts at 20% of the level of wild-type virus. dl231 transformants were immortalized and produced normal levels of E1a 13S and E1b mRNAs but only minute levels of defective E1a 12S mRNA. These transformants only minimally expressed the transformation phenotype and were similar to untransformed cells. Unlike wild-type transformants, they had a more fibroblastic morphology, were contact inhibited, grew to only low saturation density, and were limited in their ability to grow in an anchorage-independent manner in soft agar. We conclude that the 289R E1a protein can mediate immortalization of primary cells and that the 243R E1a protein is required to elicit the full transformation phenotype.     

Overexpression of E2F-1 inhibits progression of gastric cancer in vitro

E2F-1 plays a critical role in cell cycle regulation and other biological processes in cells. E2F-1 mediates apoptosis and suppresses tumorigenesis in many tissue types, but there are few data available on E2F-1 expression and its relationship to tumor kinetics in gastric cancer. To gain better insight into the involvement of E2F-1 in the biological characteristics of gastric tumors, we investigated the effect of E2F-1 overexpression on the progression of gastric carcinoma cells. A gastric cancer cell line stably overexpressing E2F-1 (MGC-803/E2F-1) was established. The influence of E2F-1 overexpression on in vitro cell growth was assessed by measuring cell survival, colony formation, and cell cycle progression. The results clearly show that overexpression of E2F-1 significantly inhibits cell growth and proliferation, blocking entry into the S-phase of the cell cycle. MGC-803/E2F-1 cells also had a higher apoptotic rate than control cells. In addition, E2F-1 reduced the motility and invasion of gastric cancer cells.     

Viral DNA synthesis in nonpermissive rat F-111 cells and its role in neoplastic transformation by polyomavirus.

We have investigated the occurrence and role of polyomavirus DNA synthesis in neoplastic transformation by this virus. We show that after infection of Fischer rat F-111 cells at 37 degrees C, there is two- to threefold increase in the level of viral DNA as compared with the input signal, with a peak observed between 5 and 7 days postinfection. Viral DNA synthesis is about 10 times higher at 33 degrees C and increases up to 15 days postinfection. Most of the viral DNA produced is supercoiled (form I DNA). On the basis of in situ hybridization, it appears that viral replication is restricted to a small fraction of the population. At the lower temperature, more cells are permissive for viral DNA synthesis and the level of synthesis per permissive cell is higher. The DNA synthesis observed is large T-antigen dependent, and the increase in viral DNA synthesis at 33 degrees C is paralleled by an increase in the expression of this viral protein. When large T antigen is inactivated, the half-life of de novo-synthesized viral DNA is less than 12 h, suggesting that large T antigen may be responsible for the stability of the viral genomes as well as their synthesis. Surprisingly, at early times postinfection (0 to 48 h), when the essential function of large T antigen in transformation is expressed (as demonstrated in shift-up experiments with tsa mutants), the level of large T antigen is below the detection level and is at least 10-fold lower than the levels observed in permissive infections at the start of viral DNA synthesis. The difference in viral DNA at 37 and 33 degrees C allowed us to study its effect on transformation. Although an increase in transformation frequency is observed in wild-type A2 infections carried at 33 degrees C (frequencies two to three times higher than at 37 degrees C), this increase appears to be unrelated to the increase in viral DNA synthesis. Furthermore, the overall level of viral DNA and large T antigen in F-111 cells may not affect the integration of the viral genome, since the patterns of integration in cells transformed by wild-type A2 at 33 and 37 degrees C appear similar. The results are compatible with a role for large T antigen in integration-transformation which is not simply to amplify the viral genome to enhance the probability of its integration.     

In vivo interference with Skp1 function leads to genetic instability and neoplastic transformation

Skp1 is involved in a variety of crucial cellular functions, among which the best understood is the formation together with Cul1 of Skp1-cullin-F-box protein ubiquitin ligases. To investigate the role of Skp1, we generated transgenic (Tg) mice expressing a Cul1 deletion mutant (Cul1-N252) able to sequestrate and inactivate Skp1. In vivo interference with Skp1 function through expression of the Cul1-N252 mutant into the T-cell lineage results in lymphoid organ hypoplasia and reduced proliferation. Nonetheless, after a period of latency, Cul1-N252 Tg mice succumb to T-cell lymphomas with high penetrance (>80%). Both T-cell depletion and the neoplastic phenotype of Cul1-N252 Tg mice are largely rescued in Cul1-N252, Skp1 double-Tg mice, indicating that the effects of Cul1-N252 are due to a sequestration of the endogenous Skp1. Analysis of Cul1-N252 lymphomas demonstrates striking karyotype heterogeneity associated with c-myc amplification and c-Myc overexpression. We show that the in vitro expression of the Cul1-N252 mutant causes a pleiotrophic phenotype, which includes the formation of multinucleated cells, centrosome and mitotic spindle abnormalities, and impaired chromosome segregation. Our findings support a crucial role for Skp1 in proper chromosomal segregation, which is required for the maintenance of euploidy and suppression of transformation.     

v-src transformation of rat embryo fibroblasts. Inefficient conversion to anchorage-independent growth involves heterogeneity of primary cultures

To clarify whether a single oncogene can transform primary cells in culture, we compared the transforming effect of a recombinant retrovirus (ZSV) containing the v-src gene in rat embryo fibroblasts (REFs) to that in the rat cell line 3Y1. In the focus assay, REFs exhibited resistance to transformation as only six foci were observed in the primary cultures as opposed to 98 in 3Y1 cells. After G418 selection, efficiency of transformation was again somewhat lower with REFs compared to that with 3Y1 cells, but the number of G418-resistant REF colonies was much greater than the number of foci in REF cultures. Furthermore, while 98% of G418-resistant colonies of ZSV-infected REFs were morphologically transformed, only 25% were converted to anchorage- independent growth, as opposed to 100% conversion seen in ZSV-infected 3Y1 cells. The poor susceptibility of REFs to anchorage-independent transformation did not involve differences in expression and subcellular distribution of p60v-src, or its kinase activity in vitro and in vivo. It rather reflected a property of the primary cultures, as cloning of REFs before ZSV infection demonstrated that only 2 out of 6 REF clones tested were permissive for anchorage-independent growth. The nonpermissive phenotype was dominant over the permissive one in somatic hybrid cells, and associated with organized actin filament bundles and a lower growth rate, both before and after ZSV infection. These results indicate that the poor susceptibility of REFs to anchorage-independent transformation by p60v-src reflects the heterogeneity of the primary cultures. REFs can be morphologically transformed by p60v-src with high efficiency but only a small fraction is convertible to anchorage- independent growth. REF resistance seems to involve the presence of a suppressor factor which may emerge from REF differentiation during embryonic development.     

Alteration of cell cycle kinase complexes in human papillomavirus E6- and E7-expressing fibroblasts precedes neoplastic transformation.

Expression of viral oncoproteins results in the loss of cell cycle checkpoint control and the accumulation of chromosomal abnormalities. Expression of both human papillomavirus type 16 oncoproteins, E6 and E7, in normal human fibroblasts completely dissociates p21 and proliferating cell nuclear antigen from the quarternary cyclin-cyclin-dependent kinase (CDK) complexes present in normal cells, causes disruption of the cyclin D-CDK4 complex and replacement with a CDK4-p16 complex, and leaves binary complexes of cyclin B1-CDC2 and cyclin A-CDK2 intact. These results are identical to those observed in fully transformed cells. The expression of the individual oncoproteins dramatically affects the association of proliferating cell nuclear antigen into the complexes while leaving the total cellular levels unaltered. Expression of low-risk human papillomavirus has no effect on cyclin complexes. These findings provide evidence for the gross alteration of cyclin-CDK complexes in preneoplastic cells and links this alteration to the loss of genomic stability.     

Point mutations in c-Myc uncouple neoplastic transformation from multiple other phenotypes in rat fibroblasts

Deregulation of c-Myc (Myc) occurs in many cancers. In addition to transforming various cell types, Myc also influences additional transformation-associated cellular phenotypes including proliferation, survival, genomic instability, reactive oxygen species production, and metabolism. Although Myc is wild type in most cancers (wtMyc), it occasionally acquires point mutations in certain lymphomas. Some of these mutations confer a survival advantage despite partially attenuating proliferation and transformation. Here, we have evaluated four naturally-occurring or synthetic point mutations of Myc for their ability to affect these phenotypes, as well as to promote genomic instability, to generate reactive oxygen species and to up-regulate aerobic glycolysis and oxidative phosphorylation. Our findings indicate that many of these phenotypes are genetically and functionally independent of one another and are not necessary for transformation. Specifically, the higher rate of glucose metabolism known to be associated with wtMyc deregulation was found to be independent of transformation. One mutation (Q131R) was greatly impaired for nearly all of the studied Myc phenotypes, yet was able to retain some ability to transform. These findings indicate that, while the Myc phenotypes examined here make additive contributions to transformation, none, with the possible exception of increased reliance on extracellular glutamine for survival, are necessary for achieving this state.     

Deprival of nicotinamide leads to enhanced glucose transport in chick embryo fibroblasts

Chick embryo fibroblasts growing in medium free of pyridine ring precursors of NADH and NADPH replicate several times before multiplication ceases. The rate of glucose transport is progressively enhanced, finally reaching levels several times higher than those normally observed in cells severely depleted of NADH. Whereas normal cells respond to additional glucose by further reducing transport, the NADH-depleted cell is refractory to glucose even at five times the normal glucose concentration. Readdition of nicotinamide does little to restore normal transport within 24 h. On the other hand NAD+ or NADP+ provided simultaneously with glucose results in a sharp decline in measurable transport within 2-4 h. The role of the pyridine nucleotides in this reduction of transport function is for the moment unknown.     

Ras transformation of cloned rat embryo fibroblasts results in increased rates of protein synthesis and phosphorylation of eukaryotic initiation factor 4E.

Eukaryotic initiation factor 4E (eIF-4E) is a 25-kDa phosphoprotein that binds to the 7-methylguanosine cap of mRNA and acts, along with other eIF-4 polypeptides, to unwind mRNA secondary structure at the 5' terminus. Recent studies have indicated that eIF-4E acts as a protooncogene, but only in its phosphorylated state. In order to determine the role of eIF-4E in oncogenesis, we examined its regulation and expression in cloned rat embryo fibroblasts transformed with the Harvey ras (Ha-ras) oncogene. The expression of Ha-ras increased the rate of protein synthesis but did not increase the levels of eIF-4E mRNA or protein. However, a dramatic increase (7-fold) in phosphate incorporation into eIF-4E was observed. The percentage of eIF-4E in the phosphorylated state was the same in transfected and control cells, indicating that both phosphorylation and dephosphorylation of eIF-4E were increased. Phosphopeptide mapping of eIF-4E from transformed cells indicated a single site of phosphorylation at Ser-53, which is the same as that identified previously in eIF-4E from reticulocytes and HeLa cells. These results indicate that p21ras is part of the signal transduction pathway leading to phosphorylation of eIF-4E. These findings also provide a potential mechanism for cell transformation by p21ras which involves the preferential stimulation of translation of certain mRNAs.