Prolonged Particulate Hexavalent Chromium Exposure Suppresses Homologous Recombination Repair in Human Lung Cells

Abstract

Genomic instability is one of the primary models of carcinogenesis and a feature of almost all cancers. Homologous recombination (HR) repair protects against genomic instability by maintaining high genomic fidelity during the repair of DNA double strand breaks. The defining step of HR repair is the formation of the Rad51 nucleofilament, which facilitates the search for a homologous sequence and invasion of the template DNA strand. Particulate hexavalent chromium (Cr(VI)), a human lung carcinogen, induces DNA double strand breaks and chromosome instability. Since the loss of HR repair increases Cr(VI)-induced chromosome instability, we investigated the effect of extended Cr(VI) exposure on HR repair. We show acute (24 h) Cr(VI) exposure induces a normal HR repair response. In contrast, prolonged (120 h) exposure to particulate Cr(VI) inhibited HR repair and Rad51 nucleofilament formation. Prolonged Cr(VI) exposure had a profound effect on Rad51, evidenced by reduced protein levels and Rad51 mislocalization to the cytoplasm. The response of proteins involved in Rad51 nuclear import and nucleofilament formation displayed varying responses to prolonged Cr(VI) exposure. BRCA2 formed nuclear foci after prolonged Cr(VI) exposure, while Rad51C foci formation was suppressed. These results suggest that particulate Cr(VI), a major chemical carcinogen, inhibits HR repair by targeting Rad51, causing DNA double strand breaks to be repaired by a low fidelity, Rad51-independent repair pathway. These results further enhance our understanding of the underlying mechanism of Cr(VI)-induced chromosome instability and thus, carcinogenesis.

Keywords: Rad51 nucleofilament; chromosome instability; homologous recombination repair; particulate chromium (VI).

FIG. 1

Prolonged exposure to zinc chromate decreases whole cell and nuclear Rad51 protein expression in a time and dose dependent manner. A, This figure shows zinc chromate induced a dose-dependent decrease in whole cell Rad51 at all time points. Zinc chromate exposure caused an increase in nuclear Rad51 protein at 24h exposure, but a dose-dependent decrease in nuclear Rad51 protein at 72 and 120 h exposure. Whole cell protein data represent an average of 3 experiments. Error bars = SEM. All doses except for 0.1 μg/cm² at 72 h exposure were significantly different from controls (*P < .05; # P < .005; ## P < .001). Nuclear protein data represent an average of 3 experiments. Nuclear protein levels were significantly lower than controls after 0.2 μg/cm² zinc chromate at 72 and 120 h and 0.3 μg/cm² at all time points (P < .01). B, Representative images of whole cell Rad51 western blots. C, Representative images of nuclear Rad51 western blots.

FIG. 2

Prolonged Cr(VI) exposure induces the mislocalization of Rad51 in response to DNA damage. This figure shows prolonged exposure to zinc chromate caused an increase in cytoplasmic Rad51 localization. Data represent an average of at least 3 experiments. Error bars = standard error of the mean. A, Representative images of Rad51 subcellular localization after 24 and 120 h exposure to 0.2 μg/cm² zinc chromate. B, Rad51 cytoplasmic to nuclear ratio determined by fluorescent intensity. The cytoplasmic/nuclear ratio after exposure to 0.2 and 0.3 μg/cm² zinc chromate at 120 h were significantly higher than control (P < .05). C, Percent of cells with cytoplasmic accumulation of Rad51 and cytoplasmic Rad51 aggregates. The percent of cells with cytoplasmic accumulation of Rad51 after 0.1 μg/cm² zinc chromate at 72 and 120 h and 0.3 μg/cm² zinc chromate at 120 h were significantly higher than control (P <.05). The percent of cells with cytoplasmic Rad51 accumulation and cytoplasmic Rad51 aggregate formation correlate.

FIG. 3

Prolonged Cr(VI) exposure inhibits Rad51 nucleofilament formation. This figure shows prolonged exposure to zinc chromate decreased the number of Rad51 nucleofilaments and resulted in the formation of primarily simple Rad51 filament structures. Data represent an average of 3 experiments. A, Representative TEM images of Rad51 nucleofilaments. B, Number of Rad51 nucleofilaments in 37 μm². The number of Rad51 nucleofilaments significantly increased (P < .05) after 24 h 0.2 μg/cm² zinc chromate exposure and significantly decreased (P < .05) after 120 h exposure to the same concentration. C, Quantification of ‘simple’ or ‘complex’ Rad51 filament structures. 120 h to 0.2 μg/cm² zinc chromate significantly shifted the filament composition towards simple filaments (P < .05).

FIG. 4

Prolonged Cr(VI) exposure induces BRCA2 foci formation. This figure shows both acute and prolonged zinc chromate exposure induced BRCA2 foci formation. Data represent an average of 4 experiments. Error bars = SEM. A, Percent of cells with BRCA2 foci. The percent of cells with BRCA2 foci increased in a concentration-dependent manner (*P < .05; # P < .005). B, Representative images of BRCA2 foci.

FIG. 5

Prolonged Cr(VI) exposure inhibits Rad51C localization. This figure shows prolonged exposure to zinc chromate inhibited Rad51C foci formation but not RAD51C protein levels. A, The percent of cells with Rad51C foci (relative to control). The percent of cells with Rad51C foci was significantly higher than control after 24 h exposure to 0.2 and 0.3 μg/cm² zinc chromate. However, the percent of cells with Rad51C foci decreased in a concentration and time-dependent manner after 72 and 120 h exposure and was significantly lower than control after 120 h exposure to 0.3 μg/cm² zinc chromate (*P < .05). B, Representative images of Rad51C foci. C, Rad51C whole cell protein levels. No time or concentration related change in Rad51C protein levels were observed after zinc chromate exposure. D, Representative images of whole cell Rad51C western blots.

FIG. 6

Prolonged Cr(VI) exposure inhibits homology directed repair in human lung cells. This figure shows prolonged exposure to zinc chromate inhibited homology directed repair. Data represent an average of 3 experiments. Error bars = SEM. A, hygro-DR-GFP reporter construct. The construct was stably inserted into WTHBF-6 cells. Cleavage of the I-SceI site in vitro and repair by homology directed repair using the downstream iGFP repeat results in GFP+ cells. B, Flow cytometry analysis of Cr(VI)-treated cells after integration of the I-SceI expression vector. The control vector (NZE) demonstrates equal transfection efficiency between conditions. C, Percent of cells with homology directed repair after zinc chromate exposure and integration of the I-SceI expression vector. The percent of cells with homology directed repair after 120 h exposure to 0.2 μg/cm² zinc chromate was significantly less then control (P < .05), while there was no significant difference after 24 h exposure.