Perturbation of mitosis through inhibition of histone acetyltransferases: the key to ochratoxin a toxicity and carcinogenicity?

Abstract

Ochratoxin A (OTA) is one of the most potent rodent renal carcinogens studied to date. Although controversial results regarding OTA genotoxicity have been published, it is now widely accepted that OTA is not a mutagenic, DNA-reactive carcinogen. Instead, increasing evidence from both in vivo and in vitro studies suggests that OTA may promote genomic instability and tumorigenesis through interference with cell division. The aim of the present study was to provide further support for disruption of mitosis as a key event in OTA toxicity and to understand how OTA mediates these effects. Immortalized human kidney epithelial cells (IHKE) were treated with OTA and monitored by differential interference contrast microscopy for 15 h. Image analysis confirmed that OTA at concentrations ≥ 5 μM, which correlate with plasma concentrations in rats under conditions of carcinogenesis, causes sustained mitotic arrest and exit from mitosis without nuclear or cellular division. Mitotic chromosomes were characterized by aberrant condensation and premature sister chromatid separation associated with altered phosphorylation and acetylation of core histones. To test if OTA directly interferes with histone acetyltransferases (HATs) which regulate lysine acetylation of histones and nonhistone proteins, a cell-free HAT activity assay was conducted using total nuclear extracts of IHKE cells. In this assay, OTA significantly blocked HAT activity in a concentration-dependent manner Overall, results from this study provide further support for a mechanism of OTA carcinogenicity involving interference with the mitotic machinery and suggest HATs as a primary cellular target of OTA.

FIG. 1.

(a) Cell growth of IHKE cells in the presence of OTA and OTB as assessed by image analysis of movies created by live-cell imaging, demonstrating a growth inhibitory effect of OTA and OTB at concentrations ≥ 5μM. (b) In line with the cytostatic effect, prolongation of mitosis is observed in IHKE cells treated with OTA and OTB at concentrations ≥ 5μM. (c) DIC and fluorescence microscopy film sequences demonstrating mitotic aberrations in IHKE cells treated with OTA. In the absence of OTA, normal mitosis of IHKE cells, that is, mitotic roundup, chromatid condensation, chromosome segregation, and cytokinesis, is completed within 75–100 min. In contrast, sustained block of mitosis, aberrant exit from mitosis without cell division, and repeated exits/entries into mitosis are evident in OTA-treated cells. Full film sequences are provided online (Supplementary data). (d) Overall frequency of mitotic aberrations in OTA- and OTB-treated IHKE cells and frequency of mitotic arrest and mitotic exit as the most prominent aberrations. (e) Time-related incidence of mitotic aberrations in OTA- and OTB-treated IHKE cells. Continued on subsequent pages.

FIG. 1.

(a) Cell growth of IHKE cells in the presence of OTA and OTB as assessed by image analysis of movies created by live-cell imaging, demonstrating a growth inhibitory effect of OTA and OTB at concentrations ≥ 5μM. (b) In line with the cytostatic effect, prolongation of mitosis is observed in IHKE cells treated with OTA and OTB at concentrations ≥ 5μM. (c) DIC and fluorescence microscopy film sequences demonstrating mitotic aberrations in IHKE cells treated with OTA. In the absence of OTA, normal mitosis of IHKE cells, that is, mitotic roundup, chromatid condensation, chromosome segregation, and cytokinesis, is completed within 75–100 min. In contrast, sustained block of mitosis, aberrant exit from mitosis without cell division, and repeated exits/entries into mitosis are evident in OTA-treated cells. Full film sequences are provided online (Supplementary data). (d) Overall frequency of mitotic aberrations in OTA- and OTB-treated IHKE cells and frequency of mitotic arrest and mitotic exit as the most prominent aberrations. (e) Time-related incidence of mitotic aberrations in OTA- and OTB-treated IHKE cells. Continued on subsequent pages.

FIG. 1.

(a) Cell growth of IHKE cells in the presence of OTA and OTB as assessed by image analysis of movies created by live-cell imaging, demonstrating a growth inhibitory effect of OTA and OTB at concentrations ≥ 5μM. (b) In line with the cytostatic effect, prolongation of mitosis is observed in IHKE cells treated with OTA and OTB at concentrations ≥ 5μM. (c) DIC and fluorescence microscopy film sequences demonstrating mitotic aberrations in IHKE cells treated with OTA. In the absence of OTA, normal mitosis of IHKE cells, that is, mitotic roundup, chromatid condensation, chromosome segregation, and cytokinesis, is completed within 75–100 min. In contrast, sustained block of mitosis, aberrant exit from mitosis without cell division, and repeated exits/entries into mitosis are evident in OTA-treated cells. Full film sequences are provided online (Supplementary data). (d) Overall frequency of mitotic aberrations in OTA- and OTB-treated IHKE cells and frequency of mitotic arrest and mitotic exit as the most prominent aberrations. (e) Time-related incidence of mitotic aberrations in OTA- and OTB-treated IHKE cells. Continued on subsequent pages.

FIG. 2.

Metaphase spreads of IHKE treated with OTA, demonstrating overcondensation and aberrant separation of sister chromatids. Table: Frequency of OTA-induced metaphase aberrations in IHKE cells treated with OTA for 24 h (data are shown as % of total metaphases). Data are presented as mean ± SD of four independent experiments. (Statistical analysis was performed by ANOVA + Dunnett post hoc test. Statistically significant changes are indicated by *p < 0.05, **p < 0.01, ***p < 0.001.)

FIG. 3.

Western blot analysis of posttranslational histone modifications in IHKE cells exposed to OTA for 24 h, demonstrating loss of H3T3 phosphorylation and hypoacetylation of core histones. Sodium butyrate (NaB), a known histone deacetylase inhibitor, and colchicine were used as controls.

FIG. 4.

TR-FRET Haspin kinase assay demonstrating lack of effects of OTA and OTB on Haspin kinase activity.

FIG. 5.

Cell-free HAT activity assay using nuclear extracts of IHKE cells and isolated histones as substrates. A clear concentration-dependent decrease in total HAT activity of the nuclear extract is seen in the presence of OTA. The degree of inhibition by OTA was similar to 15μM AA (indicated by the dashed line), a known inhibitor of HATs, which was used as positive control.

FIG. 6.

Western blot analysis of global changes in protein lysine acetylation in response to OTA. A clear dose-related decrease in the overall level of protein acetylation is seen in both cytoplasmic and nuclear fractions of cells treated with OTA. In contrast, no clear changes are observed with sodium butyrate (NaB) and AA, known inhibitors of HDACs and HATs, respectively.