Inhibition of Myc-dependent apoptosis by eukaryotic translation initiation factor 4E requires cyclin D1

Ectopically expressed eukaryotic translation initiation factor 4E (eIF4E) stimulates cell proliferation, suppresses apoptosis in growth factor restricted cells, and induces malignant transformation in primary rodent fibroblasts when coexpressed with protooncogene myc. We report here that eIF4E rescued rat embryo fibroblasts ectopically expressing c-Myc (REF/Myc) from genotoxic and non-genotoxic cytostatic drugs and identify cyclin D1 as a downstream effector in the antiapoptotic mechanism. In clones of REF/Myc ectopically expressing eIF4E, resistance to apoptosis paralleled steady state levels of cyclin D1. Stable expression of cyclin D1 in REF/Myc inhibited apoptosis in response to a broad range of cell cycle specific cytostatic agents. Partial loss-of-cyclin D1 function in REF/Myc ectopically expressing eIF4E (REF/Myc/4E) significantly increased chemosensitivity; either soluble antisense cyclin D1 oligomers or transfection with a dominant negative cyclin D1 mutant that prevents translocation of cyclin D-dependent kinases to the nucleus, significantly blunted the antiapoptotic effect of eIF4E. These data directly link eIF4E rescue from cytostatic drugs to cyclin D1. Since overexpression of eIF4E and cyclin D1 is observed in many aggressive forms of chemoresistant cancers, these findings provide insight into possible mechanisms responsible for this biological behavior.     

Efficient malignant transformation of rat embryo fibroblasts by genomic DNA from Walker carcinoma cells

DNA isolated from Walker carcinoma ascites cells was transfected into primary rat embryo fibroblasts (REF), selecting transformed cells by growth in soft agar after prolonged propagation in monolayer. Both high molecular weight genomic DNA and a partially purified mitochondrial DNA fraction were able to transform REF with high efficiency, whereas pure mitochondrial DNA failed to elicit a transformed phenotype. Hybridization experiments showed that the mitochondrial DNA fraction contained DNA species of presumably extramitochondrial origin. Colonies were cloned into morphologically transformed, foci-forming, immortalized cell lines, showing different degrees of chromosomal alterations, tumorigenicity, and production of cell growth factors. These results indicate that although REF are refractory to genomic neoplastic DNA or to single cloned oncogenes in the absence of enhancers, they can be efficiently transformed by chromosomal DNA from a highly malignant tumor under conditions selecting against the remaining normal cells.     

Cloning and characterization of human papillomavirus type 52 from cervical carcinoma in Indonesia.

Human papillomavirus (HPV) type 52 from a cervical carcinoma in Indonesia was molecularly cloned and characterized. By hybridization with cervical carcinoma DNAs from Indonesian patients, HPV 52 was detected in 3 of 52 cases (6%), whereas HPV 16 and 18 were detected in 8 and 7 cases, respectively (15% and 13%). Sequence analysis revealed that the E6-E7 ORFs contained several DNA binding motifs (Cys-X-X-Cys) like previously sequenced HPVs. The E6 ORF also contained splice donor and acceptor signals, which may allow the expression of E6* protein. The E7 ORF encoded an amino acid sequence that is conserved in some DNA tumor viruses and is involved in binding to Rb protein and in cellular transformation. Transfection of a subgenomic fragment of HPV 52 under the control of a heterologous promoter showed that the E7 ORF alone induced anchorage-independent growth of established rodent cells and immortalized primary rat embryo fibroblasts (REF), and that in cooperation with activated ras, it induced malignant transformation of REF. The E6 ORF also induced, less efficiently, anchorage-independent growth. These results strongly suggest that HPV 52, like HPV 16 and 18, has oncogenic potential.     

Search for metastasis suppressor genes

Cell fusion experiments have predicted the existence of cancer metastasis suppressor genes. The E1a gene of Adenovirus 2 has been demonstrated to suppress c-Ha-ras induction of experimental metastatic potential in rat embryo fibroblasts. Another approach to the identification of candidate metastasis suppressor genes has utilized differential or subtraction hybridizations to clone genes which are downregulated as cells become highly metastatic. To date, three such genes have been identified: nm23, WDNM1, and fibronectin. With regard to nm23, downregulation of nm23 RNA levels in high metastatic potential cells has been demonstrated in a wide variety of rodent metastasis systems, including K-1735 murine melanoma cell lines, nitrosomethylurea-induced rat mammary tumors, MMTV-induced mouse mammary tumors, and ras +/- E1a transfected rat embryo fibroblasts. Whether the expression of the nm23 gene, and other down-regulated genes in tumor metastasis, correlates with changes in metastatic potential, or actually has suppressive activity, will require transfection experiments.     

LMP1 of Epstein-Barr virus suppresses cellular senescence associated with the inhibition of p16INK4a expression

Epstein-Barr virus is associated with a number of human proliferative and malignant diseases. It is capable of immortalizing human primary B-lymphocytes in vitro. Studies indicate that latent membrane protein LMP1 is one of the viral proteins essential for this process. In this report, LMP1 was shown to prevent primary mouse embryonic fibroblasts from entering into replicative senescence in vitro. It further suppresses the senescence-associated induction of p16INK4a, commonly believed to be a key regulator of replicative senescence. In addition, LMP1 was shown to prevent premature senescence provoked by oncogenic ras in mouse embryonic fibroblasts, and to inhibit the oncogene ras-mediated induction of p16INK4a and p21WAF1. In parallel, LMP1 also prevents ras-induced premature senescence in rat embryonic fibroblasts REF52 and human diploid fibroblasts IMR90. Moreover, LMP1 is capable of suppressing the p16INK4a promoter in REF52 and Saos-2 cells in a promoter reporter assay. Our findings suggest that with the expression of p16INK4a and replicative senescence being suppressed, LMP1 may play a key role in Epstein-Barr virus-associated proliferative diseases, and it may further contribute to cancer development by preventing premature senescence induced by mitogenic oncogenes.     

Immunologic identification of fetal calf serum-derived proteins on the surfaces of cultured transformed and untransformed rat cells.

The antigens of rat embryo fibroblasts (REF) and of rat Rous sarcoma cells (derived by in vivo passage of oncogenically transformed REF) were studied using the technique of non-ionic detergent solubilization of radiolabelled cells. Solubilized antigens were complexed with rat immune IgG, and precipitation of the complexes was accomplished with rabbit anti-rat IgG. The precipitated radiolabelled antigens were then dissolved in sodium dodecyl sulfate and separated by polyacrylamide gel electrophoresis. This investigation disclosed the existence of cell surface antigenic proteins which are derived from the fetal calf serum (FCS) used in the cell-culture medium. These FCS-dependent antigens include at least three molecular species of approximate molecular weights 95,000, 80,000 and 98,000 daltons. They are probably derived from simple adsorption of FCS proteins to the cell surface, although more complex interactions are possible. One of these proteins (95,000 daltons) is of particular interest. It tenaciously adheres to the cell surface so that a trace amount remains even after subculture in the absence of FCS. Rat Rous sarcomas which are morphologically highly transformed appear to bind very little or none of this protein to their surfaces, whereas untransformed rat embryo fibroblasts bind large quantities. A rat Rous sarcoma line which is intermediate in morphological transformation binds an intermediate amount of this antigen. These findings invite speculation that the interaction of certain serum components with the cell surface may be related to plasma membrane properties which distinguish untransformed and transformed cells.     

Altered expression of NM23, a gene associated with low tumor metastatic potential, during adenovirus 2 Ela inhibition of experimental metastasis

NM23, a novel gene associated with low tumor metastatic potential, has been investigated in an experimental system in which metastasis is inhibited by the transfection of viral and cellular oncogenes. The experimental system utilizes transfection of the Adenovirus 2 Ela gene to inhibit metastasis: rat embryo fibroblasts (REF) transfected with c-Ha-ras were highly metastatic, while REF cotransfected with ras and Ela were virtually nonmetastatic. NM23 RNA levels were higher in three independently ras + Ela-cotransfected, low metastatic REF lines than in three independently ras-transfected, highly metastatic REF line. Differences in hybridizable NM23 RNA levels between the two groups of transfected cell lines ranged from 2- to 8-fold. In situ hybridization demonstrated that the relatively high NM23 RNA levels in low metastatic ras + Ela-cotransfected REF cells were not due to overexpression of the NM23 gene by a subpopulation of cells. Thus, the metastasis-inhibitory effect of the exogenously added Ela gene has been associated with increased activation of the cellular NM23 gene. This associated is particularly significant in light of the very few changes observed in translatable steady-state RNA levels between ras- and ras + Ela-transfected REF lines. The data identify NM23 as a candidate for a gene that suppresses the malignant state.     

The characteristics of the regulation of the cellular proliferation of embryonic rat fibroblasts transformed by E1A+c-Ha-ras oncogenes

The ability of growth factors (GF) to stimulate proliferation of rat embryo fibroblast (REF) lines immortalized by E 1A oncogene and transformed by complementing E 1A and c-Ha-ras oncogenes (mutant form) was studied. Unlike untreated REF, those immortalized by E 1A oncogene required less serum and GF to proliferate. Proliferation could be stimulated by specific GF alone. Serum appeared to be capable of stimulating proliferation of cells transformed by E 1A + c-Ha-ras oncogenes; however, both GF and proteinases C and A activators failed to exert such effect. Transferrin, usually required by normal cells in late G1-phase, shared the stimulating effect on E 1A+c-Ha-ras-transformed cells. To summarize, E 1A+c-Ha-ras oncogene-transformed REF can grow independently of GF but still require transferrin.     

Invasiveness and metastatic capability of rat fibroblast-like cells before and after transfection with immortalizing and transforming genes

Invasion in vitro and in vivo and spontaneous metastasis was investigated in cell lines before and after introduction of immortalizing (polyoma large-T and activated myc) genes and of transforming (polyoma middle-T and activated ras) genes in Fischer rat cells. Invasion in vitro was tested by confrontation of rat cells with embryonic chick heart fragments in organ culture. Invasion in vivo and metastasis was evaluated in nude mice and in syngeneic rats after injection of cells i.p. or s.c. in the flank and after implantation of cell aggregates s.c. in the tail. Rat cells were also analyzed for the presence of myc oncogenes, and for the expression of ras oncogenes. Cells from primary or low passage rat embryo (REF) cells were not invasive in vitro and did not produce tumors in vivo. Cell lines (LTRAT1, LTaRAT1) derived from REF cultures after transfection with plasmids encoding polyoma large-T antigens, behaved like REF cells. Cell lines (REFpEJgpt4, REFpEJmycN7) established from REF cultures after transfection with either a plasmid encoding an activated human ras protein or with the latter plasmid plus one containing an activated myc gene, were invasive in vitro and in vivo and produced invasive and metastatic tumors in syngeneic rats. Cell lines (FR3T3) established in an apparently spontaneous way were invasive in vitro and produced invasive tumors in vivo without metastasis. Derivatives of FR3T3 (FRLT1, MTT4, MMC1, and PyT21) transfected with plasmids encoding one or more of the polyoma antigens, differed from FR3T3 cells by a shorter latency period of tumor formation (less than 1 versus 1 to 3 weeks). Like FR3T3 tumors, FRLT1, MTT4, MMC1, and PyT21 tumors were invasive but not metastatic. Other spontaneously established lines (Rat1) were invasive and metastatic. Cells (Rat1pEJ6.6) derived from Rat1 cultures after transfection with a plasmid encoding an activated ras protein, showed shorter tumor latency periods (less than 1 versus 7 weeks). A thymidine kinase deficient Rat1 derivative (Rat2) was not invasive in vitro but produced invasive and metastatic tumors in vivo with long (9 to 21 weeks) latency periods. Rat2pT24B4 cells derived by us from Rat2 cells after transfection with a plasmid containing a mutated human ras gene (pT24), were invasive in vitro and in vivo as were cells derived from Rat2 tumors. We conclude from our experiments that invasiveness and metastatic capability are often acquired by established REF-derived cell lines in an apparently spontaneous way.(ABSTRACT TRUNCATED AT 400 WORDS)     

Neoplastic transformation of rat embryo cells with herpes simplex virus.

Sprague Dawley rat embryo cells (REF) were transformed by inoculation with herpes simplex virus (HSV) and incubation at 42 degrees C for 8 days. The infected cultures were subsequently returned to 37 degrees C and two types of cell clone were isolated from foci of growing cells after 4 weeks. One of the clones consisted of epithelial-like cells and did not produce HSV (REF-Tep-NP). The second consisted of spindle-shaped cells and cultures of these cells persistently developed small areas of degeneration where production of infectious HSV (REF-Tsp-P) took place. An additional clone which did not produce any more HSV (REF-Tsp-NP) was isolated from REF-Tsp-P in the presence of HSV-antiserum. REF-Tsp-P and REF-Tsp-NP grew more rapidly than REF and also formed foci in soft agar. REF-Tep-NP had a growth rate between that of normal rat embryo cells and that of both REF-Tsp-NP and REF-Tsp-P and did not form foci in soft agar. REF-Tsp-NP cells, in contrast to REF-Tep-NP cells, were resistant to superinfection with HSV types 1 and 2. REF-Tsp-P and REF-Tsp-NP produced metastasizing sarcomas in rats. After inoculation of 10(3) REF-Tsp-NP cells into 1-day-old rats tumours developed rapidly. REF-Tep-NP cells did not induce tumours in rats. The parental REF cells produced no tumours, even when 10(8) cells were inoculated into the rats. Positive immunofluorescence was observed in all three transformed cells only with the hyperimmune rabbit sera but not with human anti-HSV reconvalescence immune sera.