Examination continued of the cellular effects of FR901228, an anti-cancer agent currently in Phase 1 clinical trials. With each of three human tumor cell lines, the drug was found to block a significant portion of the cell population in mitosis, specifically, in prometaphase. An increase in mitotic index was seen within 3 hours of drug addition, peaking at 12-16 hours, then declining due to apoptosis. In prometaphase-blocked cells the nuclear membrane was absent, chromosomes were condensed, and a bipolar spindle was evident. Following brief treatment with a microtubule depolymerizing agent, drug-treated prometaphase cells, unlike control cells, did not reform a mitotic spindle although microtubules repolymerized, suggesting defects in centrosomes and/or kinetochores. Immunofluorescent staining showed no effects on pericentrin or gamma-tubulin, structural components of centrosomes; however, Plk1, a mitotic kinase, disappeared from the centrosomes. Kinetochores, the chromosomal attachment sites for microtubules, assemble on centromeres; FR901228 had no effect on CENP-C, a resident centromeric protein; however, association of CENP-E, a mitotic motor protein, with kinetochores decreased markedly. Recently FR901228 was shown by others to be an inhibitor of histone deacetylation (HDI). The experiments described above were repeated using two other structurally unrelated HDI, with results identical to those obtained using FR901228. In addition each HDI was shown to cause a dramatic increase in the association of acetylated histone H3 with mitotic chromosomes. Cellular effects of HDI generally reflect changes in gene regulation; regarding loss of CENP-E from kinetochores, an alternate possibility is that the presence of hyperacetylated histones at the centromere, a region where histones are normally under-acetylated, interferes with normal kinetochore assembly. We continued our analysis of the mechanism by which a single amino acid substitution in 15-25% of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) polypeptides in CHO cells alters the morphology, mobility and distribution of late endocytic organelles. Increased binding of GAPDH to microtubules in cells with mutant enzyme was demonstrated by quantitation of immunogold labeling in cytoskeletal whole mount preparations. Cells with mutant GAPDH contain a subpopulation of microtubules of increased stability, shown by both immunofluorescence and Western blotting. This cadre of stable microtubules could account for the increased levels and altered organization of intermediate filaments we previously reported for cells with mutant GAPDH. However, the stable subpopulation of microtubules does not of itself support the changes in endocytosis characteristic of these cells.
