Myc induces cyclin D1 expression in the absence of de novo protein synthesis and links mitogen-stimulated signal transduction to the cell cycle.

Mitogen-activated signal transduction frequently leads to the induction of the c-myc proto-oncogene, but the subsequent molecular events downstream of Myc protein expression which promote cell cycle progression remain unclear. To study Myc-specific effects, without the complexity of the broader proliferative response evoked by serum, we employed the MycER-inducible system in the non-transformed Rat-1 cell line. We demonstrate that activation of wild-type, but not mutant, MycER is sufficient to transiently induce cyclin D1 RNA as well as protein expression to physiological levels, and promote G0/G1 to S phase transition of the cell cycle. Stimulation of endogenous cyclin D1 RNA is rapid and clearly evident within 30 min of MycER-activation, reaching a peak at 3 h. Nuclear run-on analysis demonstrates that this induction occurs at the transcriptional level with a fivefold increase in the rate of transcription. Moreover, MycER induces cyclin D1 transcription with equal efficacy in the presence or absence of de novo protein synthesis. Our work shows that Myc and cyclin D1 lie consecutively in a major proliferation-control pathway, and together create a pivotal connection between signal transduction and cell cycle control.

Dysfunction of the Myc-induced apoptosis mechanism accompanies c-myc activation in the tumorigenic L929 cell line.

Activation of the c-myc protooncogene, resulting in deregulated, over-expression of the c-Myc protein, can induce both cell proliferation and programmed cell death (apoptosis) in nontransformed cells. Yet, c-myc activation is commonly tolerated in many tumors. This apparent paradox can be resolved if activation of c-myc in transformed cells is associated with loss of Myc-induced apoptosis. To examine this hypothesis, we characterized both the mechanisms of c-myc activation and programmed cell death in the tumorigenic L929 cell line. We showed that activation of c-myc in the L929 cell line involves several distinct mechanisms, including dysfunction of the Myc autosuppression pathway and alteration of c-Myc protein expression. In addition, we demonstrated that L929 cells do not undergo Myc-induced apoptosis. Analysis of somatic cell hybrids revealed that this abrogation of programmed cell death can be partially restored and is likely due to one or more genetic lesions. Our results support the hypothesis that the dysfunction of the Myc-induced apoptosis mechanism can accompany c-myc activation and provide an in vivo example illustrating two cooperative events which can contribute to tumor progression.

Domains of human c-myc protein required for autosuppression and cooperation with ras oncogenes are overlapping.

Amino acids 106 to 143 and 354 to 433 of the human c-myc protein (439 amino acids) were shown to be required for the protein to suppress c-myc gene transcription and were found to exactly overlap with those necessary for c-myc to cooperate with ras oncogenes in the transformation of rat embryo fibroblasts. The essential carboxyl-terminal region harbors structural motifs (a basic region, a helix-loop-helix motif, and a "leucine zipper"), which, in other proteins, can mediate dimerization and sequence-specific DNA binding.

Negative autoregulation of c-myc transcription.

The introduction of activated c-myc and v-myc genes into a variety of non-established and established cells results in the suppression of endogenous c-myc expression. As measured in Rat-1 fibroblasts, the suppression occurs at the level of transcriptional initiation. Moreover, the extent of the down-regulation is proportional to the cellular concentration of c-myc protein, and the critical concentration range in which the endogenous c-myc RNA is effectively suppressed corresponds to that found in non-transformed cells. In addition, the autoregulatory mechanism is not only dependent on c-myc protein, but also requires additional trans-acting factors. These results support a role for c-myc in the regulation of cellular gene transcription and suggest that a negative feedback mechanism can act as a homeostatic regulator of c-myc expression in vivo.

C-MYC: evidence for multiple regulatory functions.

The nuclear c-myc proto-oncogene promotes cell proliferation and can inhibit terminal differentiation as well as induce immortalisation in its oncogenic form. There is increasing evidence that c-myc exerts these biological activities by modulating transcription and by directly affecting the initiation of DNA replication. The regulation of these disparate activities may involve the carboxyl end of the c-myc protein, which is essential for transformation and autosuppression of c-myc transcription. Conserved motifs in this region of the c-myc protein may mediate complex formation and sequence-specific nucleic acid binding.

Production and characterization of a monoclonal antibody to a human interferon-induced double-stranded RNA-binding Mr 68,000 protein kinase.

One of the interferon-induced proteins thought to be involved in the antiviral effects of interferon is a double-stranded RNA-dependent protein kinase. This paper reports the development of a monoclonal antibody, 10A5, that recognizes a protein that co-migrates with the double-stranded RNA-dependent protein kinase at an approximate molecular weight of 68,000. Levels of this protein and of the protein kinase activity increase 3-fold on interferon treatment of T98G cells. The specificity of the monoclonal antibody was determined by ELISA, immunoblotting, and immunoprecipitation procedures. Furthermore, immunoaffinity chromatography of an interferon-induced T98G cell extract previously phosphorylated in the presence of double-stranded RNA and radiolabeled ATP resulted in the specific elution of a phosphorylated Mr 68,000 protein from the monoclonal antibody 10A5-Sepharose column. Monoclonal antibody 10A5 recognizes both native and denatured protein kinase, independent of double-stranded RNA binding or phosphorylation, and should therefore serve as a useful tool in analyzing the role of the double-stranded RNA-dependent protein kinase in the mechanism of interferon action.

Interferon-induced 2-5A synthetase activity in human peripheral blood mononuclear cells after immunization with influenza virus and rubella virus vaccines.

The interferon-induced enzyme 2-5A synthetase can be a sensitive indicator of activation of the human interferon system during viral infection or interferon therapy. To determine the response of the human interferon system to viral antigens, the level of 2-5A synthetase activity was monitored in peripheral blood mononuclear cells of healthy adults before and after immunization with influenza or rubella virus vaccine. The influenza virus-vaccinated individuals demonstrated increases in enzyme activity on days 1 and 11 in vivo, whereas those vaccinated with rubella virus vaccine showed an increase only on day 11. The difference in the day 1 in vivo 2-5A synthetase response in the two vaccinated groups could be demonstrated by in vitro incubations of peripheral blood mononuclear cells isolated approximately 90 days postvaccination with the two vaccines. The day 11 increase of enzyme activity in the rubella virus group showed a positive correlation with an increase in serum antibody titer, suggesting activation of the interferon system during antibody production in vivo after human exposure to virus antigens. The demonstration of increased 2-5A synthetase activity at specific times postimmunization in this investigation indicates that the interferon system is involved in the human in vivo response to virus vaccination.