Mechanism for differential sensitivity of the chromosome and growth cycles of mammalian cells to the rate of protein synthesis.

Abstract

It has been docuiliented widely that when the generation times of eucaryotic cells are lengthened by slowing the rate of protein synthesis, the duration of the chromosotne cycle (S, G2, and M phases) remains relatively invariant. Paradoxically, when the growth of exponentially growing cultures of CHO cells is partially inhibited with inhibitors of protein synthesis, the immediate effect is a proportionate reduction in the rate of total protein, histone protein, and DNA synthesis. However, on -further investigation it was found that over the next 2 h the rates of histone protein and DNA synthesis recover, in some cases completely to the uninhibited rate, while the synthesis rates of other proteins do. not recover. We called this process chromosome cycle compensation. The amount of compensation seen in CHO cell cultures can account quantitatively for the relative invariance in the length of the chromosome cycle (S, G2, and M phases) reported for these cells. The mechanism for this compensation involves a specific increase in the levels of histone mRNAs. An invariant chromosome cycle coupled with a lengthening growth cycle must result in a disproportionate lengthening of the GI phase. Thus, these results suggest that chromosome cycle invariance may be due more to specific cellular compensation mechanisms rather than to the more usual interpretation involving a rate-limiting step for cell cycle progression in the Gl phase.When inhibitors of protein synthesis are added to cultures of exponentially growing cells, their doubling time lengthens approximately in proportion to the decreased rate of protein synthesis. With moderate inhibition of protein synthesis, almost all of the lengthening occurs in the G1 interval while the S, G2, and M intervals, named collectively by Mitchison (17) as the DNA division or chromosome cycle, are relatively unaffected (14,15,19,20,21). Rossow et al. (21) studied a Swiss 3T3 cell line which grew with a doubling time of about 16 h, 4 h in the G1 phase and 12 h in the chromosome cycle (S, G2, and M phases). When the rate of protein synthesis in this culture was inhibited to about 35% of the control rate by the addition of cycloheximide, the doubling time increased to about 63 h with a greatly increased G1 phase of about 47 h but only a slightly increased chromosome cycle of 16 h. Liskay et al. (14) have studied the Chinese hamster cell line V79-8 which had no measurable G1 interval; the chromosome cycle of 9.2 h accounted for the doubling time. When the rate of protein synthesis in these cultures was inhibited to 60% that of the control, their doubling times increased to 14.2 h with a G1 phase of 4.2 h but only a slightly increased chromosome cycle of 10.2 h. A relative invariance for the chromosome cycle has also been reported for yeasts (11) and other fungi (16). These results are commonly interpreted in terms of a step just before the S phase, usually in the G1 phase, which is rate limiting for cell cycle progression and which is particularly sensitive to the rate of protein synthesis ...

Histone H2A.X gene transcription is regulated differently than transcription of other replication-linked histone genes.

“…The second experiment is based on the finding that ongoing protein synthesis is necessary for the replication-linked degradation of cytoplasmic histone mRNAs (3,11,27,33,39). We examined whether this constraint also applied to the H2A.X 0.6-kb mRNA.…”

“…In addition to decreasing when DNA replication is inhibited, replication-linked histone mRNA levels increase in response to the inhibition of protein synthesis (11,33,39). A detailed study of the increases in mRNA levels in Jurkat cell cultures with inhibited protein synthesis is shown in Fig.…”

“…The transcripts for these replication-linked histones are derived from genes without introns and are processed at a site between two conserved motifs to yield mature mRNAs which lack polyadenylate tracts and which are processed at a highly conserved stem-loop or hairpin structure (reviewed in references 18 and 26). Replication-linked histone mRNAs, among the most abundant mRNAs in proliferating cells, are accumulated and degraded rapidly in concert with changes in the rate of DNA replication during the S phase of the cell cycle (2,19,32) as well as in accordance with the rate of protein synthesis (3,11,33,39).The linkage of replication-linked histone mRNA levels to the rate of DNA replication throughout the cell cycle may involve regulation of transcription, transcript processing, and mRNA stability (15). When DNA replication is inhibited, these histone mRNA levels decrease 10-to 20-fold within 30 min, due in part to a decrease in the rates of histone gene transcription and in part to a decrease in histone mRNA stability (19,32).…”

“…The transcripts for these replication-linked histones are derived from genes without introns and are processed at a site between two conserved motifs to yield mature mRNAs which lack polyadenylate tracts and which are processed at a highly conserved stem-loop or hairpin structure (reviewed in references 18 and 26). Replication-linked histone mRNAs, among the most abundant mRNAs in proliferating cells, are accumulated and degraded rapidly in concert with changes in the rate of DNA replication during the S phase of the cell cycle (2,19,32) as well as in accordance with the rate of protein synthesis (3,11,33,39).…”

“…When DNA replication is inhibited, these histone mRNA levels decrease 10-to 20-fold within 30 min, due in part to a decrease in the rates of histone gene transcription and in part to a decrease in histone mRNA stability (19,32). This latter process requires the 3'-terminal stem-loop (23,27) and may involve an exonuclease that becomes more active or accessible to the mRNA as the rate of DNA synthesis decreases (29) relative to the rate of protein synthesis (3,11,39). There is evidence that soluble histone may play an autoregulatory role (4,7).…”