A comprehensive evaluation of the genotoxic potential of chemicals requires the assessment of the ability to induce gene mutations and structural chromosome (clastogenic activity) and numerical chromosome (aneugenic activity) aberrations. Aneuploidy is a major cause of human reproductive failure and an important contributor to cancer and it is therefore important that any increase in its frequency due to chemical exposures should be recognized and controlled. The in vitro binucleate cell micronucleus assay provides a powerful tool to determine the ability of a chemical to induce chromosome damage. The application of an anti-kinetochore antibody to micronuclei allows their classification into kinetochore-positive and kinetochorenegative, indicating their origin by aneugenic or clastogenic mechanisms, respectively. The availability of chromosomespecific centromere probes allows the analysis of the segregation of chromosomes into the daughter nuclei of binucleate cells to evaluate chromosome non-disjunction. Quantitative relationships between the two major causes of aneuploidy, chromosome loss and non-disjunction, can be determined. The mechanisms leading to chromosome loss and non-disjunction can be investigated by the analysis of morphological and structural changes in the cell division apparatus by the application of specific stains and antibodies for various cell division components. We illustrate such analyses by the demonstration of the interaction of the monomer bisphenol-A with the centrosome of the mitotic spindle and the folic acid antagonist pyrimethamine with the centromeres of chromosomes. Both types of modifications lead to the induction of aneuploidy in exposed cells. Our studies also implicate the products of the p53 and XPD genes in the regulation of the fidelity of chromosome segregation at mitosis.

The DNA mismatch repair (MMR) proteins are essential for the maintenance of genomic stability of human cells. Compared with hereditary or even sporadic carcinomas, MMR gene mutations are very uncommon in leukemia. However, genetic instability, attested by either loss of heterozygosity or microsatellite instability, has been extensively documented in chronic or acute malignant myeloid disorders. This observation suggests that in leukemia some internal or external signals may interfere with MMR protein expression and/or function. We investigated the effects of protein kinase C (PKC) stimulation by 12-O-tetradecanoylphorbol-13-acetate (TPA) on MMR protein expression and activity in human myeloid leukemia cell lines. First, we show here that unstimulated U937 cells displayed low level of PKC activity as well as MMR protein expression and activity compared with a panel of myeloid cell lines. Second, treatment of U937 cells with TPA significantly increased (3-5-fold) hMSH2 expression and, to a lesser extent, hMSH6 and hPMS2 expression, correlated to a restoration of MMR function. In addition, diacylglycerol, a physiological PKC agonist, induced a significant increase in hMSH2 expression, whereas chelerythrine or calphostin C, two PKC inhibitors, significantly decreased TPA-induced hMSH2 expression. Reciprocally, treatment of HEL and KG1a cells that exhibited a high level of PKC expression, with chelerythrine significantly decreased hMSH2 and hMSH6 expression. Moreover, the alteration of MMR protein expression paralleled the difference in microsatellite instability and cell sensitivity to 6-thioguanine. Our results suggest that PKC could play a role in regulating MMR protein expression and function in some myeloid leukemia cells.

A collaborative study of three laboratories compared the induction of aneuploidy by X-rays in human lymphocytes and fibroblasts. The induction of non-disjunction versus chromosome loss by X-rays was investigated using a variety of aneuploidy detection methods. Chromosome loss was determined by fluorescence in situ hybridization (FISH) with pan-centromeric probes in cytochalasin-B-blocked binucleated cells. Chromosome non-disjunction was estimated by FISH with chromosome-specific centromeric probes in binucleated interphase cells. Chromosomes were counted in parallel in lymphocyte metaphase cells; chromosome counts of the whole karyotype and counts of chromosomes 2 and 8 using chromosome paints. A major observation in spontaneous non-disjunction frequencies concerned the clear difference in frequencies observed between the two painted chromosomes in the same primary cells. When cells were irradiated elevated frequencies were observed for all the different cytogenetic endpoints. Although only a small number of the micronuclei were positive for the centromeric signal and presumably contained whole chromosomes, the absolute number %oC+ increased with dose. Higher rates of non-disjunction were found for irradiated cells; in fibroblasts a statistically significant increase was observed at a dose of 0.5 Gy. The detection of hyperdiploidy by means of chromosome counts and chromosome painting revealed an increase from doses of 1 Gy and higher. Comparison of the different methods detecting different endpoints indicates that non-disjunction may be an important mechanism leading to spontaneous and X-ray-induced aneuploidy. The relative radiosensitivity of aneuploidy induction was compared in two types of primary human cells - lymphocytes and fibroblasts. For chromosome loss both cell types showed similar results, whereas for non-disjunction fibroblasts seemed to be more sensitive. However, these differences may reflect a different sensitivity in the scoring methods used.

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