Analysis of chromosome segregation in cytokinesis-blocked human lymphocytes: non-disjunction is the prevalent damage resulting from low dose exposure to spindle poisons.

Abstract

The chromosome malsegregation pattern produced by the spindle poisons vinblastine (VBL) and colchicine (COL) in human lymphocytes was investigated. For this purpose, the fluorescence in situ hybridization with centromeric DNA probes for chromosomes X and 1 was applied to cell cultures treated with cytochalasin B, a cytokinesis-blocking agent. With this method, chromosome segregation in daughter nuclei - retained in the same envelope - can be easily analysed, simultaneously determining the loss and non-disjunction of specific chromosomes. Preliminary experiments demonstrated that the aneugenic effects elicited by low dose exposure to spindle poisons were effectively detected with treatments from the S/G2 phase (43 h) to harvest of cell cultures (60 or 72 h), with no drug-free medium recovery. This exposure protocol was used in subsequent experiments, where COL and VBL were applied at concentrations which had no effect on the cell cycle ranging to producing marked mitotic block. To account for sex differences in chromosome X instability, lymphocyte cultures from both male and female donors were used to study X chromosome malsegregation. Chromosome 1 malsegregation, however, was analysed in female lymphocytes only. VBL induced reproducible, significant increases of micronuclei in cytokinesis-blocked cells at 5 ng/ml and over. In female lymphocytes, chromosome X loss was observed at 5 ng/ml whereas chromosome 1 loss was only observed at 10 ng/ml. In male lymphocytes, no significant chromosome loss was observed. On the other hand, non-disjunction of both chromosomes X and 1 was effectively induced in female lymphocytes even at 1.25 ng/ml, the lowest dose tested. In male lymphocytes, non-disjunction of chromosome X was observed at 5 ng/ml. Treatments with COL produced a significant increase of micronucleated cells only at the highest dose tested (20 ng/ml). No significant increase in the incidence of either chromosome X or chromosome 1 loss was observed. With cell cultures from donors of both genders, a significant increase in non-disjunction of chromosome X was observed at 5 ng/ml. At the same dose, chromosome 1 non-disjunction significantly increased. These results suggest that in cytokinesis-blocked human lymphocytes, non-disjunction is the prevalent error in chromosome segregation induced by low dose exposure to spindle poisons. Interestingly, non-disjunction was effectively induced even at doses which did not produce detectable detrimental effects on the cell cycle.