Fast Control of DNA Replication in Response to Hypoxia and to Inhibited Protein Synthesis in CCRF-CEM and HeLa Cells

Abstract

In order to elucidate whether data about the fast regulation of DNA replication in dependence on oxygen supply and on a functioning protein synthesis, previously elaborated with Ehrlich ascites cells, are valid for human cells too, we repeated key experiments with CCRF-CEM and HeLa cells. The most important techniques employed were DNA fibre autoradiography and alkaline sedimentation analyses of growing (pulse-labeled) daughter strand DNA. It was found that CCRF-CEM and HeLa cells responded to transient hypoxia and to transient inhibition of protein synthesis in an almost identical fashion. Scheduled replicon initiations were reversibly suppressed and the progress rates of replication forks, which were already active before the respective inhibitory conditions were established, were reversibly slowed down. The inclusion of the fork progress rate in the response differs from Ehrlich ascites cells, which respond only by suppressing initiation. Further circumstances of the fast oxygen dependent response, concerning the behaviour of ribonucleotide reductase and of the dNTP pools, revealed no significant differences among the three cell lines. The striking identity of the response of each of the cell lines to hypoxia and to inhibited protein synthesis prompts the suspicion that converging fast regulatory pathways act on the cellular replication machinery. The phenomena as such seem to be rather widespread among mammalian cells.

Ribonucleotide Reductase, a Possible Agent in Deoxyribonucleotide Pool Asymmetries Induced by Hypoxia

“…Probst et al (5) observed a rapid inhibition of DNA replication during transient hypoxia in Ehrlich ascites cells, and Brischwein et al (6) observed a specific decline in the dCTP pool under these conditions and also a decline in the ribonucleotide reductase free radical content of the cells. Similar results have been noted for cultured human cell lines (19). O 2 is required for formation of the oxygen-bridged iron center that participates in forming the catalytically essential tyrosine free radical for ribonucleotide reductase (7)(8)(9).…”

Replication and Cytopathic Effect of Oncolytic Vesicular Stomatitis Virus in Hypoxic Tumor Cells In Vitro and In Vivo

“…On the cellular level, hypoxia induces several adaptive stress responses, including an inhibition of DNA synthesis (31), RNA synthesis, and a general inhibition of protein translation (22,27). At the same time, hypoxia selectively promotes the transcription and translation of hypoxia-adaptive genes such as vascular endothelial growth factor and hypoxia-inducible fac-tor 1 (HIF1) (10).…”

p21Cip1 and p27Kip1Regulate Cell Cycle Reentry after Hypoxic Stress but Are Not Necessary for Hypoxia-Induced Arrest

“…Studies using alkaline sedimentation analysis and fiber autoradiography have revealed that replicon initiation is suppressed by severe oxygen deprivation, but chain elongation and maturation continue normally for at least a few hours (11,29,32). While the persistence of chain elongation under hypoxic conditions implies that the lack of precursors is not directly responsible for the failure to synthesize DNA, it has been proposed that the nucleotide imbalance caused by hypoxia can act as a signal leading to cessation of replication (32).…”