Histone deacetylase inhibitors induce G2-checkpoint arrest and apoptosis in cisplatinum-resistant ovarian cancer cells associated with overexpression of the Bcl-2-related protein Bad.

Trichostatin A produces predominantly G(1) cell-cycle blockade and differentiation of the cisplatinum-sensitive A2780 ovarian cancer cell line. Given the propensity of ovarian tumors to become resistant to cisplatinum, often leading to cross-resistance to other agents, we have extended these observations by examining how the emergence of resistant phenotypes in A2780 cells affects the actions of histone deacetylase (HDAC) inhibitors. Trichostatin A exposure (100 ng/mL, 24 hours) induced ultrastructural differentiation of the "intrinsically" cisplatinum-resistant A2780-9M subline, with the reappearance of intercellular junctions and lumina containing primitive microvilli. Similar trichostatin A exposure in the acquired resistance A2780CP cells produced minimal differentiation consisting of occasional weak intercellular junctions. Independent of the differences in trichostatin A-induced differentiation, in both resistant sublines trichostatin A produced a similar reduction in cell viability, by >90%, within 5 days of treatment. Diminished viability in both A2780-9M and CP cells was associated with the absence of cell cycle arrest in G1, resulting in predominant G2-checkpoint arrest accompanied by a 10- to 20-fold increase in Annexin V binding and the reemergence of apoptosis. Similar cell cycle arrests and apoptosis were also observed using other HDAC inhibitors and in other resistant ovarian cancer cell lines (OVCAR-3 and SK-OV-3). Trichostatin A-induced apoptosis in resistant cells is in sharp contrast to its effects on the parental cisplatinum-sensitive A2780 and normal MRC-5 fibroblast cell lines (predominant cycle arrest in G1 with no detectable apoptosis). Western immunoblot analysis indicated trichostatin A triggers apoptosis in resistant ovarian cancer cells via p53-independent activation of the intrinsic "mitochondrial" pathway, commensurate with induction of the Bcl-2-related protein Bad. These results suggest cisplatinum resistance alters the effects of HDAC inhibition through a shift in cell cycle arrest from the G1 to the G2 checkpoint and reactivation of the intrinsic mitochondrial apoptotic cascade.

Cell type-dependent regulation of hMLH1 promoter activity is influenced by the presence of multiple redundant elements.

Immunohistochemical analysis confirmed the presence of MLH1 protein in A2780 ovarian cancer cells and its absence in this same cell line on acquired resistance to cisplatinum (A2780/CP). Transfection of a -1781-bp hMLH1 promoter construct into either A2780 or A2780/CP cells produced similar (30-fold) induction of luciferase, an indication that the transcriptional machinery for hMLH1 expression remains intact. hMLH1-luciferase activity was also unaffected by re-expression of hMLH1 following treatment of A2780/CP cells with the methylase inhibitor 2'-deoxy-5-azacytidine. Serial 5'-deletion studies of the hMLH1 promoter region in ovarian cancer cells localized transcriptional enhancers to a region (-250 to -151 bp) that excludes the previously identified CCAAT element (-282) active in HeLa cells. When these same deletion constructs were transfected into HeLa cells, deletion of the CCAAT-containing region caused a significant loss of promoter activity, an indication of cell-specific use of enhancer elements. Finally, a series of internal deletion and linker mutation studies of the -250 to -151 bp ovarian enhancer region revealed that the hMLH1 promoter contains multiple redundant enhancer elements capable of independent promoter activation and may explain the association of this region with methylation silencing of hMLH1.

Cell cycle blockade and differentiation of ovarian cancer cells by the histone deacetylase inhibitor trichostatin A are associated with changes in p21, Rb, and Id proteins.

Inhibitors of histone deacetylase activity are emerging as a potentially important new class of anticancer agents. In the current studies, exposing A2780 ovarian cancer cells to the histone deacetylase inhibitor trichostatin A (TSA) produced a marked change in cellular morphology, proliferation, and differentiation. Within 24 h of TSA treatment, there was a morphological transformation of the cells, with increased cytoplasm, a more epithelial-like columnar appearance, and the emergence of distinct cellular boundaries. Commensurate with the morphological transformation, TSA also inhibited cell proliferation; cells treated with TSA for 72 h increased to 110% of the initial cell numbers versus control cell numbers of 622%, with a corresponding reduction in mitotic activity and a flow cytometry S-phase fraction of 3.9% in TSA-treated cells versus 28.8% for control. TSA also induced epithelial-like differentiation with increased cytokeratin expression from 2% of controls to 22-25% of TSA-treated cells and the reappearance of intercellular plasma membrane junctions and primitive microvilli. Immunocytochemical analyses indicate the molecular mechanism underlying the actions of TSA on A2780 cell cycle progression and differentiation involves reexpression of the CDK inhibitor p21. Elevated levels of p21, in TSA-treated cells, were associated with a reduction in the phosphorylation of the cell cycle regulator retinoblastoma protein (Rb). TSA also caused a decrease in the helix-loop-helix inhibitor of differentiation/DNA binding protein Id1, with no change in Id2 levels. In conclusion, the observed TSA-induced changes in p21, Rb, and Id1 are consistent with cell cycle senescence and differentiation of A2780 ovarian cancer cells.