The structure m7GpppN (where N is any nucleotide), termed cap, is present at the 5' end of all eukaryotic cellular mRNAs (except organellar). The eukaryotic initiation factor 4E (eIF-4E) binds to the cap and facilitates the formation of translation initiation complexes. eIF-4E is implicated in control of cell growth, as its overexpression causes malignant transformation of rodent cells and deregulates HeLa cell growth. It was suggested that overexpression of eIF-4E results in the enhanced translation of poorly translated mRNAs that encode growth-promoting proteins. Indeed, enhanced expression of several proteins, including cyclin Dl and ornithine decarboxylase (ODC), was documented in eIF-4E-overexpressing NIH 3T3 cells. However, the mechanism underlying this increase has not been elucidated. Here, we studied the mode by which eIF-4E increases the expression of cyclin DI and ODC. We show that the increase in the amount of cyclin Dl and ODC is directly proportional to the degree of eIF-4E overexpression. Two mechanisms, which are not mutually exclusive, are responsible for the increase. In eIF-4E-overexpressing cells the rate of translation initiation of ODC mRNA was increased inasmuch as the mRNA sedimented with heavier polysomes. For cyclin Dl mRNA, translation initiation was not increased, but rather its amount in the cytoplasm increased, without a significant increase in total mRNA. Whereas, in the parental NIH 3T3 cell line, a large proportion of the cyclin Dl mRNA was confined to the nucleus, in eIF-4E-overexpressing cells the vast majority of the mRNA was present in the cytoplasm. These results indicate that eIF-4E affects directly or indirectly mRNA nucleocytoplasmic transport, in addition to its role in translation initiation.

Regulation of the cell cycle is orchestrated by cyclins and cyclin-dependent kinases. We have demonstrated previously that overexpression of eukaryotic translation initiation factor 4E (eIF-4E) in NIH 3T3 cells growing in 10% fetal calf serum leads to highly elevated levels of cyclin D1 protein without significant increase in cyclin D1 mRNA levels, suggesting that a post-transcriptional mechanism is involved. (Rosenwald, I. B., LazarisKaratzas, A., Sonenberg, N., and Schmidt, E. V. (1993) Mol. Cell. Biol. 13, 7358 -7363). In the present reseach, we did not find any significant effect of eIF-4E on polysomal distribution of cyclin D1 mRNA. However, the total amount of cyclin D1 mRNA associated with polysomes was significantly increased by eIF-4E overexpression. Further, we determined that the levels of both cyclin D1 protein and mRNA are increased in serum-deprived cells overexpressing eIF-4E. Nuclear run-on experiments demonstrated that the rate of the cyclin D1 transcription is not down-regulated in serum-deprived cells overexpressing eIF-4E. Thus, elevated levels of eIF-4E may lead to increased transcription of the cyclin D1 gene, and this effect becomes visible when serum deprivation down-regulates the rate of cyclin D1 mRNA synthesis in control cells. However, artificial overexpression of cyclin D1 mRNA in serum-deprived cells in the absence of eIF-4E overexpression did not cause the elevation of cyclin D1 protein, and this overexpressed cyclin D1 mRNA accumulated in the nucleus, suggesting that one post-transcriptional role of eIF-4E is to transport cyclin D1 mRNA from the nucleus to cytoplasmic polysomes.Mitogenic stimulation leads to increased rates of protein synthesis, which is required for entry of resting cells into the cell cycle (2-5). The increase in net protein synthesis after mitogenic stimulation of resting cells is connected with mitogen-induced expression of genes coding for translation initiation factors (6 -10). In addition to the total increase in protein synthesis, it is reasonable to expect that there should be selective increases in the synthesis of growth-promoting proteins. One of the translation initiation factors whose levels are increased after mitogenic stimulation of resting cells is the ratelimiting mRNA cap-binding protein eukaryotic translation initiation factor 4E (eIF-4E), 1 which may be involved in unwinding of mRNA 5Ј secondary structures, mRNA splicing, mRNA 3Ј processing, and mRNA nucleocytoplasmic transport (11-13). An important role for eIF-4E in cell growth has been demonstrated in experiments in which microinjection of this translation initiation factor into quiescent NIH 3T3 cells induced them to enter the S phase (14). Furthermore, overexpression of eIF-4E transforms both established and primary cells (15)(16)(17). It has also been demonstrated that the c-myc oncoprotein increases the expression of eIF-4E by a transcriptional mechanism (6), while ras and src oncoproteins increase the function of eIF-4E by increasing its phosphorylation (34,35).We have examined previo...

Cyclin D1 is a Gl-specific cyclin that has been linked to lymphoid, parathyroid, and breast tumors. Recent studies suggested that high protein levels of cyclin D1 are not always produced when cyclin D1 mRNA is overexpressed in transfected cells, suggesting that posttranscriptional events may be important in cyclin D1 product, c-Myc, actin, and eukaryotic initiation factor 2a. We also examined cyclin D1 protein in cells expressing an estrogen receptor-Myc fusion protein because we previously found that eIF-4E increases after induction of c-myc function. In these cells, increased levels of eIF-4E protein were closely followed by increases in levels of cyclin D1 protein, but the level of cyclin D1 mRNA was not increased. We conclude that increases in cyclin D1 levels may result from increased expression of eIF-4E, and this regulation may be one determinant of cyclin D1 levels in the cell.

Although activation of c-myc is a critical step in the development of lymphomas and other tumors, Its normal fumction(s) in cefl growth remain obscure because few mycregulated genes are known. myc expression normally ncrases in response to mitogens and peaks in GI when additional protein synthesis is required for cell-cycle progression. Protein synthesis jB controlled by the availability of translation initiation factors, including the mRNA cap binding protein (eIF-4E) and the a subunit of the eIF-2 complex that binds the initiator Met-tRNA. Consequently we examined eIF-4E and eIF-2a for evidence of regulation by c-myc. Expression of eIF-4E and eIF-2a correlated with c-myc expression in fibroblasts after growth stimulation. In addition, expression of eIF-4E and eIF-2a was increased in myc-transformed rat embryo fibroblasts but was not increased in iws-transformed cells. Transcription rates ofeIF-4E and eIF-2a mRNAs were regulated by c-myc in cells expressing an estrogen receptor-Myc fusion protein. Finally, electrophoretic mobiblty-shift assays Identified a sequence element in the eIF-2a promoter, TCCGCAL-GCGCG, which was speciflcally retarded by extracts of mycexpressing cells. c-myc is thought to deregulate the growth of cancer cells by activating btanwription, suggesting that specific genes regulated by c-myc should also function as oncogenes. In previous studies these translation initiation factors could induce neoplastic growth because overexpression of eIF-4E-transformed cells and inhibition of a suppressor of eIF-2a (eIF-2a kinase) also caused malignant transformation. Our studies suggest that one important biological function of c-myc may be to increase cell growth by increasing expression of eIF-4E and eIF-2a.

There is increasing evidence that cell cycle transit is potentially lethal, with survival depending on the activation of metabolic pathways which block apoptosis. However, the identities of those pathways coupling cell cycle transit to survival remain undefined. Here we show that the eukaryotic translation initiation factor 4E (eIF4E) can mediate both proliferative and survival signaling. Overexpression of eIF4E completely substituted for serum or individual growth factors in preserving the viability of established NIH 3T3 fibroblasts. An eIF4E mutant (Ser-53 changed to Ala) defective in mediating its growth-factor-regulated functions was also defective in its survival signaling. Survival signaling by enforced expression of eIF4E did not result from autocrine release of survival factors, nor did it lead to increased expression of the apoptosis antagonists Bcl-2 and Bcl-X L . In addition, the execution apparatus of the apoptotic response in eIF4E-overexpressing cells was found to be intact. Increased expression of eIF4E was sufficient to inhibit apoptosis in serum-restricted primary fibroblasts with enforced expression of Myc. In contrast, activation of Ha-Ras, which is required for eIF4E proliferative signaling, did not suppress Myc-induced apoptosis. These data suggest that the eIF4E-activated pathways leading to survival and cell cycle progression are distinct. This dual signaling of proliferation and survival might be the basis for the potency of eIF4E as an inducer of neoplastic transformation.Normal diploid fibroblasts deprived of serum or polypeptide growth factors exit the cell cycle and persist in a quiescent state. When quiescent cells are stimulated to enter the cell cycle by viral oncogenes (31) or by enforced expression of cellular growth-promoting proteins such as c-Myc (2, 8; reviewed in reference 38), E2F (1, 17, 68), or cyclin A (18, 35), they do not progress toward mitosis but undergo programmed cell death. Similarly, the relaxation of negative growth control that occurs during preneoplastic progression of fibroblasts toward immortalization (42, 62) or by decreased retinoblastoma protein expression (1, 57) leads to apoptosis in response to growth factor withdrawal. Along these lines, peptide growth factors such as platelet-derived growth factor (PDGF) and epidermal growth factor can signal both cell cycle entry and apoptosis in fibroblasts deprived of progression-type growth factors (24). These findings support the idea that cell cycle transit is a potentially lethal condition unless specific rescue factors prevent this fate (20,43,44). Therefore, a mitogenic stimulus must coordinately activate metabolic pathways directing both proliferation and survival in order to result in cell division without death. While recent advances in our understanding of the cell division cycle reveal how cells integrate proliferative signals with the cell cycle machinery, the molecular mechanisms and regulatory pathways involved in growth factor survival signaling remain uncertain.The quiescent state is characterize...