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.

Cultured Ehrlich ascites cells were exposed to different oxygen tensions (ranging from nearly complete anoxia to 95 % 0, at 1 O5 Pa) and to transient (5 -10 h) hypoxia (0.02 % 0, at 1 O5 Pa). Treated cells were examined with respect to the intracellular concentration of the M2-specific tyrosyl free radical of ribonucleotide reductase by EPR spectroscopy, and with respect to the pool sizes of all four deoxynucleoside triphosphates by an enzymatic assay employing DNA polymerase I of Escherichia coli. From 2% to 0.02% O,, the free radical level decreased continually from a normal value to just above detectability by the EPR measurement employed, and quickly recovered when hypoxic cells were resupplied with atmospheric 0,. Concurrently, analogous changes of the size of the dCTP pool occurred, whereas the pool sizes dATP and dGTP underwent no changes, and the size of the dTTP pool only moderate changes. The changes of the free radical concentration and of the dCTP pool correlated well with the suppression or reactivation of DNA replication under the respective 0, conditions. The results consistently support the hypothesis of a fast-acting regulatory pathway that controls the rate of DNA replication in proliferating cells according to sufficient availability of 0,. Therefore, ribonucleotide reductase may serve, in addition to providing DNA building blocks, as a PO, sensor, which transmits the signal in the form of an altered intracellular dCTP concentration, directly or indirectly, to the nuclear-replication machinery.Keywords: DNA replication ; regulation of DNA replication ; mammalian cells ; ribonucleotide reductase; deoxynucleotide pool.DNA replication in Ehrlich ascites cells has been demonstrated to be subject to a rapidly acting regulation that depends on the 0, in the cellular environment [1-61. This regulation responds at 0, tensions that are distinctly above that which significantly diminishes mitochondria1 respiration [3, 71 and at a normal adenylate energy charge [3].Work with cultured cells under controlled 0, tension (controlled hypoxia [l -61) revealed distinct changes in cellular DNA replication. When, in a typical transient-hypoxia experiment, the PO, is reduced 0.02-0.2% (relative to. 10' Pa total pressure), scheduled replicon initiations are specifically, reversibly and coordinately suppressed, whereas DNA-chain growth and maturation in replicons initiated before reduction of PO, continue normally. Reelevation of the PO, causes a subsequent burst of initiations. When the PO, is reduced to values below 0.01 5 % the fast reversibility of the depression of replication is lost [3]. By controlled hypoxia, scheduled replicon initiation can be reversibly suppressed in any stage of the S phase [3, 51. A constant high expression of genes directly related to the replicative state (e.g. thymidine kinase, proliferating cell nuclear antigen) is maintained when logarithmically growing Ehrlich ascites cells are subjected to several hours of controlled hypoxia [5]. Under Reoxygenation of such hypoxically accumula...

Replicationin cellular rephcons of mouse Ehrlich ascites. human CCRF-CEM and hamster BHK-71 cells was analyzed. after exposition of the cells to staurosporme, by measuring the overall DNA synthesis rate, by alkahne sedimentation analysis of length distributions of growing daughter strand DNA and by DNA fibre autoradiographyThe results consistently Indicated that micromolar concentrations of staurosporme caused. m all three cell hnes. a fast suppression of rephcon initiation which was reversible if the drug treatment did not exceed about 2 h. The mhibition of initiation was accompanied by a slight reduction of rates of propagation of replication forks. The data are Interpreted m terms of the existence of a so far unknown factor which seems to be involved relatively directly in the imtiation process of cellular replicons and has to be activated. hke the large T antigen of SV 40 for the replication nntiation m the viral genome. by a specific phosphorylation event. Unhhe several other protem phosphorylations of cellular regulation. the kmase concerned here seems to be Inhibited only by relatively high stauroaporme concentrations.

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