Effects of spindle poisons in peripheral human lymphocytes by the in vitro cytokinesis-block micronucleus assay.

The search for micronuclei (MN) in binucleated cells is not always the best choice to recognize microtubule-perturbing agents, as they give rise to (micronucleated) mononucleated cells, mainly via mitotic slippage. We therefore treated peripheral lymphocytes with vincristine (VCR), nocodazole (NOC) and colcemid (COL): (i) to quantify the formation of MN in mononucleated cells and the occurrence of abnormal mitoses (c-anaphases, endoreduplicated or tetraploid metaphases); (ii) to investigate the role of cytokinesis inhibition in determining or modulating the cytogenetic effects induced by the spindle poisons (we used either cytochalasin B (cyt B) or latrunculin A, a cytokinesis inhibitor that acts differently as compared with cyt B); (iii) to assess the ploidy of cells bearing MN by fluorescence in situ hybridisation (FISH) analysis; and (iv) to evaluate the levels of the mitotic arrest deficient (MAD2) protein, that blocks the cell at the metaphase-anaphase transition, by immunoblotting. We observed the induction of numerous abnormal mitoses and tetraploid interphase nuclei, as well as of MN in mononucleated cells, a high percentage of which had a diploid complement. We also found that the effects were generally not dose but chemical dependent, where NOC was proven to be more effective than COL and VCR in inducing overall MN formation and, specifically, diploid micronucleated lymphocytes. Aneugens damaged cells to a greater extent in the presence of cytokinesis inhibitors rather than in their absence. MAD2 protein was expressed in controls to an extent reflecting the amount of lymphocytes which were initially in the G2/M transition phase. The same trend was seen in aneugen-treated cells where MAD2 levels decreased with increasing spindle poison concentration. Here, we demonstrate that micronucleated mononucleated cells and aberrant mitoses can be considered useful markers of exposure to aneugens-like spindle poisons causing preferentially, but not exclusively, mitotic slippage. Assessment of MAD2 levels can be used to confirm the cell-damaging activity of the compounds.