Time of c-fos and c-myc expression in human diploid fibroblasts stimulated to proliferate after prolonged periods in quiescence.
When human diploid fibroblasts such as WI-38 cells become crowded, they enter a viable state of quiescence (G0) in which they can remain for prolonged periods of time. These quiescent cells can be induced to re-enter the cell cycle by addition of fresh serum. However, cells held in G0 for long periods before stimulation require more time to enter DNA synthesis as compared to cells held in a quiescent state for short periods. We have used this model system to determine if a close temporal coupling exists between the time of expression of two proto-oncogenes associated with cell growth, c-fos and c-myc, and the time of entry into DNA synthesis. WI-38 cells were stimulated to enter DNA synthesis by the addition of fresh culture medium and serum at various lengths of time after plating, ranging from 7 to 34 days. At hourly intervals thereafter, cells were harvested and total RNA was isolated. These samples were then analyzed by RNase protection assay to determine the levels of c-fos and c-myc mRNA. Our results show that the time and pattern of c-fos and c-myc mRNA accumulation after stimulation is determined only by the time which the cells are treated with serum even when they exhibit a 19-h delay in the entry into DNA synthesis. In all of our experiments, c-fos could be detected 0.5 h after stimulation and remained detectable for approximately 2 h. Likewise, the peak of c-myc accumulation occurred at about 3 h after serum addition, regardless of how long it took to initiate DNA synthesis. These results suggest that the time of c-fos and c-myc induction clearly is not the only factor which determines the length of the prereplicative period and thus the ultimate time of initiation of DNA synthesis.
Owen
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TA
Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.
Cosenza
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SC
Soprano
D R
DR
Soprano
K J
KJ
D002453:Cell Cycle; D002455:Cell Division; D002460:Cell Line; D005347:Fibroblasts / Q000166:cytology; D006801:Humans; D007700:Kinetics; D010957:Plasmids; D011519:Proto-Oncogenes; D013997:Time Factors; D014158:Transcription, Genetic