RESUMEN
BACKGROUND: We have previously used the ATAD5-luciferase high-throughput screening assay to identify genotoxic compounds with potential chemotherapeutic capabilities. The successful identification of known genotoxic agents, including the histone deacetylase inhibitor (HDACi) trichostatin A (TSA), confirmed the specificity of the screen since TSA has been widely studied for its ability to cause apoptosis in cancer cells. Because many cancers have acquired mutations in DNA damage checkpoints or repair pathways, we hypothesized that these cancers may be susceptible to treatments that target compensatory pathways. Here, we used a panel of isogenic chicken DT40 B lymphocyte mutant and human cell lines to investigate the ability of TSA to define selective pathways that promote HDACi toxicity. RESULTS: HDACi induced a DNA damage response and reduced viability in all repair deficient DT40 mutants although ATM-nulls were least affected. The most dramatic sensitivity was observed in mutants lacking the homology dependent repair (HDR) factor BLM or the non-homologous end-joining (NHEJ) and HDR factors, KU/RAD54, suggesting an involvement of either HDR or NHEJ in HDACi-induced cell death. To extend these findings, we measured the frequencies of HDR and NHEJ after HDACi treatment and monitored viability in human cell lines comparably deficient in HDR or NHEJ. Although no difference in HDR frequency was observed between HDACi treated and untreated cells, HDR-defective human cell lines were clearly more sensitive than wild type. Unexpectedly, cells treated with HDACis showed a significantly elevated NHEJ frequency. CONCLUSIONS: HDACi targeting drugs induced significant increases in NHEJ activity in human cell lines but did not alter HDR frequency. Moreover, HDR is required for cellular resistance to HDACi therapy; therefore, NHEJ does not appear to be a critical axis for HDACi resistance. Rather, HDACi compounds induced DNA damage, most likely double strand breaks (DSBs), and HDR proficiency is correlated with cell survival.
Asunto(s)
Linfocitos B/metabolismo , Reparación del ADN por Unión de Extremidades/genética , Reparación del ADN/genética , Inhibidores de Histona Desacetilasas/farmacología , Mutación/genética , Neoplasias/genética , Animales , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Linfocitos B/citología , Linfocitos B/efectos de los fármacos , Western Blotting , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Pollos , Daño del ADN/efectos de los fármacos , Daño del ADN/genética , Reparación del ADN por Unión de Extremidades/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Electroforesis en Gel de Campo Pulsado , Técnica del Anticuerpo Fluorescente , Humanos , Ácidos Hidroxámicos/farmacología , Autoantígeno Ku , Luciferasas/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/patología , ARN Interferente Pequeño/genética , Recombinasa Rad51/antagonistas & inhibidores , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , RecQ Helicasas/genética , RecQ Helicasas/metabolismo , VorinostatRESUMEN
The structures, spectroscopy, and cytotoxicity of four novel nominally square-planar gold(III) chelates 1-4 with the general formula cis-AuCl2(X), where the ligand X is an anionic bidentate pyridyl- or isoquinolylamido chelating agent, are described. The Au-N(amido), Au-N(pyridyl), and Au-N(isoquinolyl) distances are 2.002(9)-2.016(3), 2.01(1)-2.037(3), and 2.037(3) Å, respectively. Density functional theory simulations afforded accurate gold(III) coordination geometries for 1-4 (bond distances and angles to within 5% of the X-ray values), while accurate transition energies were limited to those calculated in the UV spectral region. The complexes had variable stability in dimethyl sulfoxide: compound 3 (relatively rigid) was indefinitely stable, compounds 1 and 2 (conformationally flexible) slowly demetalated over 30 days, and 4 (extensively aromatic) formed an insoluble precipitate after 10 days (72 h in an aqueous buffer). The isoquinolylamido derivative 4 was sufficiently cytotoxic in the NCI-60 screen to undergo full five-dose testing. Notably low GI50 (1.8, 2.3, and 3.2 µM) and IC50 (4.0, 9.8, and 15 µM) values were recorded for the OVCAR-3, IGROV1, and SW-620 cell lines, respectively. Hierarchical cluster analysis employing the National Cancer Institute (NCI) data for known anticancer drugs and 4 revealed that compound 4 is mechanistically identical with the topoisomerase IIα (Top2) poison zorubicin and statistically similar to the topoisomerase IB (Top1) poisons camptothecin and 9-methoxycamptothecin. The Top2-catalyzed decatenation reaction of kinetoplast DNA was studied as a function of the concentration of 4: the compound acts as an interfacial poison of Top2 at low concentrations (<1 µM) and a catalytic inhibitor of the enzyme above 5 µM. Gel mobility shift assays (plasmid DNA substrate) showed that the catalytic inhibition of Top2 likely correlates with DNA binding by 4 at concentrations >5 µM. Compound 4 is also a catalytic inhibitor of Top1 at higher concentrations, consistent with DNA binding by the complex.