Cellular cultures, heat effects

An alternative explanation for the accumulation of DNA strand breaks with increasing time of incubation with MBA and the continued effects of MBA at times well after the minimum number of strand breaks was attained at 2 h of culture, would be a nonspecific effect of the inhibitor on DNA synthesis rather than that on ADP-ribosylation-mediated repair. One such possibiUty consistent with the data of Fig. 1, would be that MBA was itself directly or indirectly introducing breaks into the DNA. This hypothesis was excluded by experiments in which the effect of MBA was measured in cultures in which ADP-ribosylation had already been maximally inhibited by alternative inhibitors such as 3-aminobenzamide or high concentrations of nicotinamide, or heat shock [11] (data not shown). No additional breaks were introduced by MBA under these conditions. Furthermore, there was no effect of MBA on cellular levels of purine or pyrimidine nucleotides, and MBA treatment resulted in only 4% cell death. Table 2 shows that the inhibitory effects of MBA on ADP-ribosylation, and on cell proliferation were completely reversible. In addition, the data in Table 3 show a direct proportionality between concentration of MBA on the one hand and inhibition of ADP-ribosylation in permeabilized cells, increased number of DNA strand breaks and decreased cell proliferation at 48 h on the other hand. This, and the data cited above, permit us to conclude that MBA acts on this system by inhibiting the poly(ADP-ribosylation) reaction,... 

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