The 0,-dependent regulation of replication in Ehrlich ascites cells, characterized by a reversible shutdown of replicon initiation during hypoxia, was scrutinized with respect to the involvement of gene expression. Synchronous and asynchronous cells were subjected to transient hypoxia and examined for expression of selected 'late' growth-regulated mRNA and for the influence of inhibitors of transcription and translation on DNA replication. Trrespective of whether replicon initiation was suppressed by hypoxia or retriggered by reoxygenation, the levels of thymidine kinase mRNA and of proliferating cell-nuclear antigen/cyclin mRNA were as high as in untreated replicating cells. The level of histone H3.1 mRNA followed, with a distinct delay, the replicative activity of the cells governed by the imposed changes of p02. The response of replication to inhibition of transcription and translation was virtually the same as to hypoxia, i.e. a selective suppression of replicon initiation. It was demonstrated that replicon initiation depends on one or several short-lived protein(s) (lifetime about 5 min) which is (are) formed under hypoxic conditions as well. The lifetime of the corresponding RNA message(s) is in the range of several hours. It is suggested that the expression of genes conditioning resting cells for DNA replication remains unaffected by hypoxia or by restoring the normal PO,. Hypoxic cells appear to rest in a state fully prepared for entering DNA replication, but a yet unknown event essential for replicon initiation is blocked. This event depends on a critical oxygen tension as well as on short-lived protein(s).In previous communications, we demonstrated that DNA replication in Ehrlich ascites cells is subject to a regulation which depends on the O2 tension in the cellular environment [l -41. This regulation operates at 0, tensions distinctly above the minimum required to support mitochondria1 respiration [5] and to establish a normal adenylate energy charge [3].Furthermore, we have shown that this regulation takes place during ascites tumor growth in vivo [3, 41. There, it may be a major determinant of tumor-cell propagation by adapting the tumor cell to the supply of nutrients (not only of 0,) which is increasingly impairing when the tumor mass in the peritoneal cavity grows.Work with cultured cells under controlled O2 tension [3,4, 61 revealed the responses of the cellular replication machinery. When the PO, is reduced to values of 200 -2000 ppm (relative to lo5 Pa total pressure), scheduled replicon initiations are specifically, reversibly and coordinately suppressed, whereas DNA chain growth and maturation in replicons already initiated before the reduction of the p0, continue essentially normally. Re-elevating the p 0 2 triggers, within a few minutes, a burst of initiations. Thereby, no detectable over-replication, i.e. repeated initiations of the same replicons within a single [7]. When the pOz is reduced below 150 ppm, cell damage occurs and the reversibility is lost [3, 41. The reversible shutdown ...

Simian virus 40 (SV40)-infected CV1 cells transiently exposed to hypoxia show a burst of viral replication immediately after reoxygenation. DNA precursor incorporation and analysis of growing daughter strands by alkaline sedimentation demonstrated that SV40 DNA synthesis began with a lag of about 3 to 5 min after reoxygenation followed by a largely synchronous viral replication round. Viral RNA-DNA primers complementary to the SV40 origin region were not detectable before 3 min upon reoxygenation. A distinct form of circular closed, supercoiled SV40 DNA was detectable as soon as 3 min after reoxygenation but not under hypoxia. Sensitivity to the DNA nucleaseBal 31 and migration behavior in chloroquine-containing agarose gels suggested that this DNA species was highly underwound compared to other SV40 topoisomers and was probably related to the highly underwound form U DNA first described by Dean et al. (F. B. Dean, P. Bullock, Y. Murakami, C. R. Wobbe, L. Weissbach, and J. Hurwitz, Proc. Natl. Acad. Sci. USA 84:16–20, 1987), in vitro. 3′-OH ends of presumed RNA-DNA primers could be detected in form U by 3′ end labeling with T7 polymerase. Addition of aphidicolin to the cells before reoxygenation led to a pronounced accumulation of form U DNA containing RNA-DNA primers. In vivo pulse-chase kinetic studies performed with aphidicolin-treated SV40-infected cells showed that form U is an initial intermediate of SV40 DNA replication which matures into higher-molecular-weight replication intermediates and into SV40 form I DNA after removal of the inhibitor. These results suggest that in vivo initiation of SV40 replication is arrested by hypoxia before origin unwinding and primer synthesis.

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...

Simian virus 40 (SV40)-infected CV1 cells exposed to hypoxia show an inhibition of viral replication. Reoxygenation after several hours of hypoxia results in new initiations followed by a nearly synchronous round of SV40 replication. In this communication, we examined the effect of glucose on inhibition of viral DNA replication under hypoxia. We found that glucose stimulated SV40 DNA replication under hypoxia in two different ways. First, the rate of DNA synthesis, i.e. the fork propagation rate, increased. This effect seemed to be mediated by inhibition of mitochondrial respiration by glucose (Crabtree effect). Inhibition of mitochondrial respiration probably resulted in a higher intracellular oxygen concentration and an activation of oxygen-dependent ribonucleotide reductase, which provides the precursors for DNA synthesis. This glucose effect was consequently strongly dependent on the strength of hypoxia and the extent of intracellular respiration; hypoxic gassing with 10 ppm instead of 200 -400 ppm O 2 or treatment of hypoxic cells with a mitochondrial uncoupler (carbonyl cyanide m-chlorophenylhydrazone) reduced the glucose effect on replication, whereas antimycin A, an inhibitor of respiration, increased it. The second effect of glucose concerned initiation, i.e. stimulation of unwinding of the viral origin. This effect was not influenced by the strength of hypoxia or the extent of cellular respiration and seemed, therefore, not to be mediated through a Crabtree effect. No evidence for a direct correlation between the cellular ATP concentration and the extent of SV40 replication under hypoxia was found. The effect of glucose on replication under hypoxia was not restricted to SV40-infected CV1 cells but was also detectable in HeLa cells. This suggests it to be a mechanism of more general validity.DNA replication in mammalian cells is subject to a fast acting regulation that depends on the O 2 tension in the cellular environment. This regulation results in inhibition of cellular replication when the concentration of O 2 falls below 0.1%. Regulation of cellular replication by O 2 has first been demonstrated for Ehrlich ascites cells (1-4). Further studies revealed that it is also valid for HeLa and CCRF cells (5), suggesting it to be a mechanism of general importance that adapts the cellular DNA replication to the supply of O 2 and other nutrients. This seems to be of particular significance during embryonic growth, wound healing, or tumor cell propagation. Inhibition of replication under hypoxia primarily affects replicon initiation. Additionally, the rate of DNA chain growth is frequently reduced. Readmission of O 2 after several hours of hypoxia reverses the suppression of DNA replication within a few minutes. This remarkably fast response suggests that the O 2 -dependent replication control acts very directly on the replication apparatus.The molecular mechanism of the oxygen-dependent replication control is largely obscure. A reduction of the intracellular concentrations of deoxynucleoside triphosphates, e...