Mitomycin-induced chromatid breaks in HeLa cells: a consequence of incomplete DNA replication.

The formation of chromosome aberrations induced by alkylating agents such as mitomycin C has been shown to require the passage of the treated cell through S phase. However, the exact mechanisms by which mitomycin C-induced DNA lesions are translated into chromosome aberrations during S phase are not known. The purpose of these studies was to better understand the molecular basis of chromosome aberration formation after mitomycin C treatment. The morphology of metaphases of cells treated in G1 phase with mitomycin C resembled that of prematurely condensed chromosomes of S-phase cells. Consequently we postulated that chromosome aberrations resulted from cells reaching mitosis without completing DNA replication. This was tested by treating HeLa cells in G1 phase with mitomycin C and then analyzing these cells at mitosis for residual DNA damage and DNA content. Utilizing the DNA alkaline elution assay for DNA damage, we showed that HeLa cells progress through S phase into mitosis with intact DNA-DNA interstrand cross-links. These cross-links, originally induced into parental DNA, were associated equally with parental and newly replicated DNA at the time the cells reached mitosis. This suggests that recombinational events had taken place during the DNA replication process. Cells that were treated in G1 phase and allowed to proceed to mitosis in the presence of bromodeoxyuridine to density label newly replicated DNA were analyzed with cesium chloride density sedimentation. Unreplicated DNA was present in the mitotic cells of the treated populations but not in the untreated control cells. Further, flow cytometric measurements, made under hypotonic conditions in order to reduce chromatin condensation effects, demonstrated that the mitotic cells from the mitomycin C-treated populations contained 10-20% less DNA than untreated mitotic controls. These results indicate that chromosome breaks induced by mitomycin C are the result of cells reaching mitosis without having fully completed DNA replication.