No abstract

The effects of deprivation of oxygen on proliferation kinetics of in vitro grown Ehrlich ascites tumor cells were studied by means of flow cytometry. The flow cytometric results obtained by applying the BUdR-H 33 258 technique showed that 6 -8 hours after establishment of anaerobiosis cells lose their capacity for proliferation and accumulate in the late G1-phase; during this time late S cells enter the G2 compartment but do not divide while cells, which are in G2+M at the beginning of anaerobiosis divide and enter G1. Cell cycle distribution remained constant up to about 20 hours, thereafter G1-cells enter the S-phase. Up to 24 hours of anaerobiosis cell number does not change and viability of the cells was not severely affected. Recultivation under aerobic conditions of 12 hours anaerobically treated cells revealed, that the cytokinetic properties of G1 cells were not impaired, while S-phase cells after reaeration did not enter G2 but began again to synthesize DNA forming cells with a DNA content greater than 4c. S-phase cells are most sensitive to deprivation of oxygen.

Centrifugal elutriation was applied to separate into the cell compartments asynchronous Ehrlich ascites cells grown under different culture conditions. The cytokinetic properties of the recultivated fractions were studied by flow cytometry. The present experiments prove that G 1- cohorts grown 12 h under exclusion of oxygen accumulate in the late G 1-compartment. The cyto­kinetic properties of these cells are not changed. In contrast the cytokinetic properties of anaerobically treated S-phase cells are changed; most of S-cells leave the cell cycle after they have attained the DNA content of G 2-cells and continue to synthesize DNA without preceeding division. Cells with a DNA content up to the fourfold of normal values are found.

The substrate specificity and the effects of nucleotides and SH-blocking agents on the p-nitro- phenylphosphatase activity of intact Ehrlich ascites tumor cells (EAT) cells were studied, ᴅʟ-β- Glycerophosphate, o-phosphoethanolamine, cholinephosphate, glucose-6-phosphate, o-carboxyphenyl- phosphate,, phosphoenolpyruvate and AMP were not attacked by intact cells. ATP > GTP > UTP > PPi > pNPP were cleaved with decreasing velocity. A stimulation of the cleavage of p-NPP by the following nucleotides was observed with decreasing effectivity: ATP > ADP > GTP > UTP; AMP was ineffective. The phosphatase activity was not affected by malate, tartrate and glutathion disulfide. The SH blocking agents diamide and thimerosal were more effective in­hibitors of the pNPPase than of the ATPase activity, whereas the hydrolysis of ATP is more affected by the ATP analog adenylylimidodiphosphate. The present data are best compatible with a double headed enzyme: Both active sites interact with ATP, only one is active against p-NPP and sensitive against SH-blocking agents.