Exposure to caffeine and suppression of DNA replication combine to stabilize the proteins and RNA required for premature mitotic events

Abstract

Caffeine had been shown to induce mitotic events in Syrian hamster fibroblast (BHK) cells that were arrested during DNA replication (Schlegel and Pardee, Science 232:1264-1266, 1986). Inhibition of protein synthesis blocked these caffeine-induced events, while inhibition of RNA synthesis showed little effect. We now report that the protein(s) that are required for inducing mitosis in these cells were synthesized shortly after caffeine addition, the activity was very labile in the absence of caffeine, and the activity was lost through an ATP-dependent mechanism. Caffeine dramatically increased the stability of these putative proteins while having no effect on overall protein degradation. Experiments with an inhibitor of RNA synthesis indicated that mitosis-related RNA had accumulated during the suppression of DNA replication, and this RNA was unstable when replication was allowed to resume. These results suggest that the stability of RNA needed for mitosis is regulated by the DNA replicative state of the cell and that caffeine selectively stabilizes the protein product(s) of this RNA. Conditions can therefore be selected that permit mitotic factors to accumulate in cells at inappropriate times in the cell cycle. Two-dimensional gel electrophoresis has demonstrated several protein changes resulting from caffeine treatment; their relevance to mitosis-inducing activity remains to be determined.

Periodic mitotic events induced in the absence of DNA replication.

“…Both exposure to caffeine and suppression of DNA replication are required for this premature induction of mitosis in normal BHK cells (4). Experiments with inhibitors suggest that these conditions permit the accumulation of mitosis-inducing mRNA(s) and their protein(s) (5). The present report extends these observations by providing evidence that mitotic events can be made to occur periodically, independent of Gj-and S-phase events.…”

“…3. RNA needed for mitosis can be synthesized during S-phase arrest (4,5,11), and this RNA appears to become very labile if DNA replication is allowed to resume (5). Exposure to caffeine (and perhaps raising tsBN2 to 39°C) increases the stability of the mitosis-inducing proteins (5) and allows them to reach critical levels and trigger mitosis.…”

“…Chromosome condensation, nuclear envelope breakdown, and other mitotic events occur at the restrictive temperature in the BHK temperature-sensitive mutant tsBN2 (3) and in normal BHK cells that have been arrested in S phase and exposed to caffeine (4,5). These studies showed that one final mitosis can be uncoupled from the completion of DNA replication.…”

Chronic morphine induces premature mitosis of proliferating cells in the adult mouse subgranular zone

“…3a), a reasonable interpretation of premature mitosis is that morphine shortens the length of G 2 /M. Premature mitosis can occur in a variety of systems when checkpoint proteins are not regulated correctly (e.g., Schlegel et al, 1987). It is possible that chronic morphine acts on proteins, such as p34 cdc2 and cyclin B1, which can bring about premature mitosis (Steinmann et al, 1991;Tam et al, 1995) by shortening G 2 /M.…”

Extended access methamphetamine decreases immature neurons in the hippocampus which results from loss and altered development of neural progenitors without altered dynamics of the S-phase of the cell cycle

“…The lack of effect on S -phase length by methamphetamine by double S -phase labeling with IdU/CldU is further supported by co-labeling analysis of Ki-67 and CldU, where the proportion of Ki-67 cells in the S -phase (CldU) remain unchanged in all of the methamphetamine groups. Unchanged S -phase dynamics by methamphetamine was not surprising because insults such as chemicals, radiation, faulty replication, and drugs, including morphine and caffeine, alter other phases of the cell cycle, such as the G 2 /M checkpoint or G 2 /M phase (Arguello et al , 2008, Chappell and Dalton, 2010, Mandyam et al , 2004, Schlegel et al , 1987). Therefore, these results support the speculation that the duration of the S -phase of the cell cycle is most preserved and least sensitive to modifications (Eisch and Mandyam, 2007, Ford and Pardee, 1998, Katou et al , 2003).…”