Regulation of cell cycle duration by c-myc levels.
Early passage murine fibroblasts infected with retroviral vectors carrying human c-myc 'minigenes' express high levels of c-myc and have a dramatically shortened G1-phase of the cell cycle. Cells infected with viruses where c-myc is expressed from the viral LTR (MSN-4 virus) express more c-myc protein than cells infected with viruses where c-myc is expressed from the SV40 early promoter (NSM-7 virus). Populations of cells were infected with high titre viruses, selected for drug-resistance, pulse labelled with bromodeoxyuridine (BrdUrd) and chased in BrdUrd free media. This allows accurate, simultaneous, measurement of the rate of exit of unlabelled cells from G1 and progression of BrdUrd-labelled cells through S-phase. The length of the G1-phase in cell populations infected with the MSN-4 virus is 4.65 h, a reduction of nearly 30% compared to the G1-phase length of 6.50 h seen in cells infected with the VSN-2 control virus. Cells infected with NSM-7 virus show an intermediate phenotype and have a G1-phase of 5.25 h. The lengths of the S-phase (4.50 to 4.75 h) and G2 + M phases (2.75 h) were not significantly altered by exogenous c-myc expression. When chases are performed in growth-factor free media, the G1-phase of infected and non-infected cells is extended by approximately 2 h. Cells infected with the c-myc viruses continue to cycle more rapidly than uninfected cells. Growth factor-deprived cells, restimulated with serum, show similar alterations of the cell cycle kinetics. MSN-4 and NSM-7 infected cells, expressing high levels of c-myc, enter S-phase 2 to 4 h earlier, but less synchronously, than control cells, and sustain subsequent rounds of DNA synthesis, while control cells do not. However, cells carrying activated c-myc genes have nearly-normal morphologies and are not tumourgenic in syngenic mice. These results demonstrate that c-myc levels are rate limiting for events in G1, and the length of G1 varies proportionally with the level of exogenous c-myc expression.
Karn
J
J
MRC Laboratory of Molecular Biology, Medical Research Council Centre, Cambridge, UK.
Watson
J V
JV
Lowe
A D
AD
Green
S M
SM
Vedeckis
W
W
D000818:Animals; D001769:Blood; D001973:Bromodeoxyuridine; D002453:Cell Cycle; D002460:Cell Line; D002472:Cell Transformation, Viral; D003198:Computer Simulation; D003470:Culture Media / Q000494:pharmacology; D005347:Fibroblasts / Q000032:analysis / Q000166:cytology / Q000382:microbiology; D005434:Flow Cytometry; D005822:Genetic Vectors; D006801:Humans; D015151:Immunoblotting; D007399:Interphase; D007700:Kinetics; D051379:Mice; D011518:Proto-Oncogene Proteins / Q000032:analysis / Q000096:biosynthesis / Q000502:physiology*; D016271:Proto-Oncogene Proteins c-myc; D011519:Proto-Oncogenes; D012190:Retroviridae / Q000235:genetics
D003470:Culture Media; D011518:Proto-Oncogene Proteins; D016271:Proto-Oncogene Proteins c-myc; D001973:Bromodeoxyuridine