Cells prepare for S phase during the G1 phase of the cell cycle. Cell biological methods have provided knowledge of cycle kinetics and of substages of G1 that are determined by extracellular signals. Through the use of biochemical and molecular biological techniques to study effects of growth factors, oncogenes, and inhibitors, intracellular events during G1 that lead to DNA synthesis are rapidly being discovered. Many cells in vivo are in a quiescent state (G0), with unduplicated DNA. Cells can be activated to reenter the cycle during G1. Similarly, cells in culture can be shifted between G0 and G1. These switches in and out of G1 are the main determinants of post-embryonic cell proliferation rate and are defectively controlled in cancer cells.

https://science.sciencemag.org/content/sci/246/4930/603.full.pdf

G1 events and regulation of cell proliferation.

The product of the retinoblastoma susceptibility gene has properties of a cell cycle regulatory element.

Synchronization of mammalian cells is essential for investigations involving cell proliferation. A simple method for obtaining synchrony in all types of cells, through several cycles and with minimal overall metabolic perturbations, has not yet been available. We describe a procedure for synchronizing normal as well as tumor cells reversibly in the G 1 phase of the cell cycle using Lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase. This method of synchronization was successful with all cell lines tested, including normal and tumor cells of mouse, hamster, and human origins. For example, when MCF-7 human breast cancer cells were synchronized with Lovastatin and released by the addition of mevalonic acid (the product of the reaction catalyzed by 3-hydroxy-3-methylglutaryl-coenzyme A reductase), 3 phases of accelerated thymidine incorporation into DNA corresponding to 3 S phases of the cell cycle occurred during a 90-h period of cell replication. Thymidine incorporation was decreased to ≤4% during the initial lag of 18 h before the first S phase, and maximum incorporation was then achieved after only 6 h. The antibody Ki-67, which detects a nuclear antigen associated with proliferation, was present in cells arrested with Lovastatin. This fact, together with the lack of thymidine incorporation during the initial lag time, indicates that the cells were arrested in the G 1 and not in the G 0 phase of the cell cycle. Furthermore, in synchronized tumor-derived human breast epithelial cells, histone H4 RNA was low after Lovastatin release and increased with the onset of DNA synthesis. Concomitant synthesis of DNA and histone H4 RNA expression could be observed for 2 cycles. Minimal perturbations of general metabolic functions occurred since the rate of RNA, protein, and initial DNA synthesis were unaffected by Lovastatin, as evidenced by [ 3 H]uridine, [ 3 H]leucine, and initial [ 3 H]thymidine incorporation. Finally, while the Lovastatin-induced synchronization was overcome by mevalonic acid, addition of squalene or cholesterol-ethanol had no such effect. Thus, Lovastatin appears to prevent formation of an early intermediate in the cholesterol pathway that is essential for progression of cells through early G 1 phase.

https://cancerres.aacrjournals.org/content/canres/51/13/3602.full.pdf

Synchronization of tumor and normal cells from G1 to multiple cell cycles by lovastatin.

Adult rat parenchymal hepatocytes in primary culture can be induced to enter into DNA synthesis and mitosis. The optimal conditions for hepatocyte replication are low plating density (less than 10,000 cells/sq cm) and 50% serum from two-thirds partially hepatectomized rats (48 hr after hepatectomy). Approximately 80% of the hepatocytes enter the cell cycle, and most of these cells go through mitosis. The replicating hepatocytes remain positive for glucose-6-phosphatase and negative for gamma-glutamyl transpeptidase, and they accumulate fat, in analogy to regenerating liver. Most of the replicating hepatocytes enter into multiple consecutive rounds of DNA synthesis. Dose-response studies between control animal serum and hepatocyte labeling index indicate that in unoperated animals the serum contains substances stimulatory as well as inhibitory for hepatic growth, with the inhibitory effect prevailing at high concentrations. After partial hepatectomy, the inhibitory activity disappears whereas the hepatopoietin activity reaches almost 90% of maximal biological effectiveness at 25% serum concentration. Addition of hormones to the system shows that the hepatopoietin activity is not identical to epidermal growth factor, platelet-derived growth factor, thyroxine, glucagon, or hydrocortisone. Norepinephrine abolishes the difference between control and hepatectomized serum but does not restore hepatopoietin activity when added to heat-inactivated serum. The results show that this system of replicating hepatocytes can be used to investigate the trophic factors that control growth of normal and neoplastic hepatocytes.

https://cancerres.aacrjournals.org/content/canres/42/11/4673.full.pdf

Liver Regeneration Studies with Rat Hepatocytes in Primary Culture

Gene expression during the mammalian cell cycle

Baserga1 has summarised evidence that there are two growth states in which cells have a diploid content of DNA; G1—the interval between mitosis and S phase in exponentially growing cultures—and G0, a quiescent state entered only when conditions for growth are suboptimal. G0 can be distinguished from G1 by temporal measurements; for BALB/c 3T3 cells, the lag time between G0 and S phase is never shorter than 12 h (ref. 2), whereas in exponentially growing cells the mean lag time between mitosis and S (G1 by definition) lasts 5–6 h (ref. 3). G0 can also be distinguished from G1 by some biochemical parameters including G0- and G1-specific intracellular proteins4,5. SV40- or polyoma virus-transformed cells cannot enter G0 to become quiescent6–9. Serum induces the growth of BALB/c 3T3 cells in tissue culture. It sustains the growth of exponentially replicating populations, and causes density-inhibited cells to leave G0 and replicate10–12. Serum contains several sets of hormonal growth factors which have recently been defined2,13,14. One of these hormones, the platelet-derived growth factor (PDGF), is released into serum during the clotting process; PDGF is absent in platelet-poor plasma, the liquid portion of unclotted blood15–19. It promotes the growth of G0-arrested cells2,16 by stimulating cells to become ‘competent’ to enter the S phase2; plasma allows these competent cells to progress through G0/G1, synthesise DNA2,20 and divide13. We now show that PDGF has a second function. It prevents replicating cells from entering G0.

Marilyn E. Stone

Papers

Platelet-derived growth factor prevents G0 growth arrest