The organization of replicon clusters is not affected by polyamine depletion

Earlier investigations have shown that polyamine depletion affects DNA replication negatively. DNA is synthesized in replicons which are gathered in replicon clusters. DNA replication is initiated simultaneously in every replicon of a replicon cluster. By pulse labeling cells with the thymidine analog bromodeoxyuridine and then detecting bromodeoxyuridine in situ with immunofluorescence, replicon clusters can be studied. We have used this method to investigate the effects of 2-difluoromethylornithine (DFMO)- and 4-amidinoindan-1-one 2'-amidinohydrazone (CGP 48664)-mediated polyamine depletion on the organization of replicon clusters. The cells were studied by fluorescence microscopy and confocal laser scanning microscopy. Our studies give at hand that neither the number nor the distribution of replicon clusters were affected even after 4 days of treatment with 5 mM DFMO or 20 microM CGP 48664, indicating that polyamine depletion did not affect the organization of replicon clusters. However, the fluorescence intensity of the replicon clusters was much lower in inhibitor-treated cells. The results indicate that the impaired DNA replication observed in polyamine-depleted cells is not due to an effect on the initiation step of DNA replication, but rather on the elongation process. To confirm that it is possible to observe changes in the organization of replicon clusters using bromodeoxyuridine, we treated the cells with various drugs that affect DNA replication. Aphidicolin, which inhibits DNA elongation, gave results similar to those of DFMO and CGP 48664.