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.
We have prepared extracts from cycloheximide-treated cells for the study of simian-virus-40 (SV40)-DNA replication in vitro. When supplemented with the viral initiator protein (large T antigen), these extracts fully supported SV40-DNA replication. We also determined that SV40-DNA replication in vivo is much more resistant to cyclohenimide than cellular DNA replication. SV40 encodes its own initiator protein, T antigen, which also functions as a DNA helicase, but depends on cellular functions for all additional replication reactions. Therefore, it appears to be quite likely that cycloheximide affects cellular DNA replication by blocking the synthesis of (a) cellular function(s) that is(are) performed by T antigen in SV40-DNA replication. Indeed, DNA fiber autoradiography and alkaline sucrose gradient centrifugation of pulse-lal~l~ cellular DNA showed that eycloheximide treatment almost completely suppressed rcplicon initiation and reduced the rate of replication fork movement to about one third of the control.
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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