Skip to main content
NCBI home page
Toxicological Sciences logoLink to Toxicological Sciences
. 2021 Mar 2;181(1):35–46. doi: 10.1093/toxsci/kfab019

Particulate Hexavalent Chromium Inhibits E2F1 Leading to Reduced RAD51 Nuclear Foci Formation in Human Lung Cells

Rachel M Speer 1, Jennifer H Toyoda 1, Tayler J Croom-Perez 1, Ke Jian Liu 2, John Pierce Wise Sr 1,✉
PMCID: PMC8081024  PMID: 33677506

Abstract

Lung cancer is the leading cause of cancer death; however, the mechanisms of lung carcinogens are poorly understood. Metals, including hexavalent chromium [Cr(VI)], induce chromosome instability, an early event in lung cancer. Failure of homologous recombination repair is a key mechanism for chromosome instability. Particulate Cr(VI) causes DNA double-strand breaks and prolonged exposure impairs homologous recombination targeting a key effector protein in this pathway, RAD51. Reduced RAD51 protein is a key endpoint of particulate Cr(VI) exposure. It is currently unknown how Cr(VI) reduces RAD51 protein. E2F1 is the predominant transcription factor for RAD51. This study sought to identify if E2F1 modulates the RAD51 response to particulate Cr(VI). Particulate Cr(VI) reduced RAD51 protein and mRNA levels but had a minimal effect on RAD51 half-life. E2F1 protein and mRNA were also inhibited by particulate Cr(VI) exposure. To connect these two outcomes, we tested if modulating E2F1 affects RAD51 outcomes after particulate Cr(VI) exposure. E2F1 knockdown inhibited RAD51 nuclear foci formation after acute particulate Cr(VI) exposure. These data indicate reduced RAD51 protein levels after prolonged particulate Cr(VI) exposure are predominantly due to inhibited expression. Particulate Cr(VI) also inhibits E2F1 expression. However, although loss of E2F1 does not modulate RAD51 expression after particulate Cr(VI) exposure, RAD51 nuclear foci formation is inhibited. These findings suggest E2F1 is important for RAD51 localization to double-strand breaks, but not expression after particulate Cr(VI) exposure in human lung cells.

Keywords: RAD51, E2F1, hexavalent chromium, DNA repair, carcinogenesis

Highlights

  • Particulate Cr(VI) affects RAD51 expression not protein half-life

  • E2F1 expression is inhibited by prolonged particulate Cr(VI) exposure

  • Loss of E2F1 inhibits RAD51 nuclear foci formation

Particulate hexavalent chromium [Cr(VI)] is an established human lung carcinogen, however, the mechanisms by which it induces carcinogenesis are not well understood. Studies show Cr(VI) induces chromosome instability, a type of genomic instability, which arises as an early event in lung cancer (Albertson et al., 2003; Chen et al., 2019; Holmes et al., 2008; Maeng et al., 2004; Rager et al., 2019; Wise et al., 2018). Structural chromosome instability, including aberrations such as chromosome breaks and translocations, arise when DNA-strand breaks are either left unrepaired or are repaired using low fidelity repair pathways (Janssen et al., 2011).

DNA double-strand breaks are a detrimental form of DNA damage that must be efficiently repaired to maintain the integrity of the genome and prevent structural chromosome instability. In a normal cell, DNA double-strand breaks are repaired by homologous recombination repair when a homologous sequence of DNA is available resulting in high fidelity repair (San Filippo et al., 2008). Functional homologous recombination repair protects cells from structural chromosome instability thereby reducing carcinogenic risk.

Cr(VI) induces DNA double-strand breaks after acute and prolonged exposure (Qin et al., 2014; Wise et al., 2002; 2003; Xie et al., 2009). Homologous recombination repair is the preferred mechanism for repairing Cr(VI)-induced double-strand breaks (Tamblyn et al., 2009; Xie et al., 2008). For example, one study showed Chinese hamster ovary cells deficient in homologous recombination repair had increased chromosome instability following exposure to Cr(VI) (Stackpole et al., 2007). However, studies show Cr(VI) inhibits homologous recombination repair following prolonged Cr(VI) exposure (Browning et al., 2016; Qin et al., 2014). Because Cr(VI) induces structural chromosome instability and inhibits homologous recombination repair, we sought to investigate the homologous recombination repair pathway, which may contribute to structural chromosome instability.

Homologous recombination repair is characterized by 3 main steps: signaling, transducing, and finally an effecting step mediated by RAD51 (D’Amours and Jackson, 2002; Williams et al., 2007). RAD51 is a recombinase protein that is involved in coating ssDNA and strand exchange specific to homologous recombination repair and is essential for successful repair by this pathway (Baumann et al., 1996; Sung & Robberson, 1995). Previous studies in human lung cells report prolonged exposure to particulate Cr(VI) targets RAD51, whereas earlier steps in the homologous recombination pathway remain unaffected (Browning et al., 2016; Qin et al., 2014). RAD51 failure following particulate Cr(VI) exposure was demonstrated by inhibited protein expression, nuclear foci formation, and RAD51 nucleofilament formation. However, it is currently unknown how Cr(VI) impairs RAD51 function.

RAD51 expression is tightly regulated in normal cells, but it is unknown if RAD51 transcription is inhibited following Cr(VI) exposure. E2F1 is considered the primary transcription factor for RAD51 and is known to participate in the recruitment and stability of DNA repair proteins at double-strand breaks (Choi and Kim, 2019; Wu et al., 2014). Knockdown of E2F1 is shown to result in loss of RAD51 protein, mRNA, and nuclear foci (Chen et al., 2011; Choi and Kim, 2019; Wu et al., 2014). Studies show E2F1 is inhibited following exposure to other metals including arsenic and cadmium (Lam et al., 2014, 2015; Li et al., 2008; Wu et al., 2013). However, only 1 study investigated E2F1 after Cr(VI) exposure and found 24-h exposure increased E2F1 expression (Permenter et al., 2011) and another predicted E2F1 would be upregulated in mouse duodenum after a 91-day drinking water exposure (Kopec et al., 2012). The goal of this study was to determine if E2F1 mediates the RAD51 response after acute and prolonged particulate Cr(VI) exposure. We determined if particulate Cr(VI)-induced reduction of RAD51 protein is due to altered protein half-life or reduced expression. Because E2F1 is the predominant transcription factor for RAD51, we assessed the effects of particulate Cr(VI) exposure on E2F1. Finally, to further explore the role of E2F1 in the RAD51 response, we assessed if E2F1 could modulate RAD51 after particulate Cr(VI) exposure.

MATERIALS and METHODS

 

 

Chemicals and reagents

DMEM/F12 (1×), phosphate-buffered saline (PBS) 1× without calcium or magnesium, PBS with calcium and magnesium, penicillin/streptomycin, glutagro supplement (200 mM), tissue culture dishes, flasks, and plasticware were purchased from Corning (Corning, New York). Cosmic calf serum was purchased from Hyclone (Logan, Utah). Sodium pyruvate (100 mM) was purchased from Lonza (Allendale, New Jersey). Trypsin-EDTA (0.25%) was purchased from Gibco. Zinc chromate (CAS no. 13530–65-9, 99.7% purity) was purchased from Pfaltz and Bauer (lot Z00277, Waterbury, Connecticut). Blocking buffer (TBS), secondary IRDye antibodies, and 4× protein sample loading buffer were purchased from LI-COR (Lincoln, Nebraska). Nitrocellulose membranes, Nunc Lab Tek II glass chamber slides, DAPI Diamond, Pierce RIPA buffer, Pierce rapid gold BCA protein assay kit, High Capacity cDNA Reverse Transcription Kit, TaqMan Small RNA Assays, Universal Mastermix II, and MirVana miRNA isolation kit were purchased from Thermo Fisher Scientific Inc. (Waltham, Massachusetts). Protease and phosphatase inhibitors were purchased from Roche (Indianapolis, Indiana). Paraformaldehyde (4%) was purchased from Alfa Aesar (Ward Hill, Massachusetts). Triton-X 100 was purchased from Sigma-Aldrich (St Louis, Mississippi). Tween-20 and cycloheximide (CHX) were purchased from VWR (Radnor, Pennsylvania). FNC Coating Mix was purchased from AthenaES (Baltimore, Maryland). Bovine serum albumin standard was purchased from Bio-Rad (Hercules, California). DharmaFECT transfection reagent I, and siRNA were purchased from Horizon Discovery (Lafayette, Colorado).

Cell culture

This study used an hTERT immortalized clonal cell line derived from human bronchial fibroblasts (WTHBF-6) with normal growth parameters, a normal, stable karyotype, and a similar cytotoxic and clastogenic response to metals as its parent, primary cell line (Wise et al., 2004). Cells were cultured as sub-confluent monolayers in DMEM/F12 50:50 mixture supplemented with 15% cosmic calf serum, 0.1 mM sodium pyruvate, 1% glutagro, and 1% penicillin/streptomycin. Cells were maintained in a 5% CO2-humidified incubator at 37°C, fed every other day, and subcultured every 3–4 days. Cells were confirmed to be mycoplasma negative monthly and monitored for any growth or morphological changes. All cells were karyotyped when thawed for use and again after every 3 months of continuous culture to ensure authenticity. WTHBF-6 short tandem repeat analysis was confirmed by the American Type Culture Collection (Manassas, Virginia).

Zinc chromate preparation

For all experiments, particulate zinc chromate was administered as a suspension in cold, sterile water as described previously (Xie et al., 2009). Briefly, zinc chromate was added to ice-cold sterile water and spun overnight at 4°C. The next day dilutions were prepared and used to treat cells. Cells were treated with zinc chromate concentrations ranging from 0.1 to 0.3 μg/cm2 based on previous studies showing these doses are within the range of inducing genotoxicity and DNA damage with moderate cytotoxicity (Holmes et al., 2010; Qin et al., 2014; Wise et al., 2010).

siRNA transfections

WTHBF-6 cells were seeded and allowed to reenter logarithmic growth for 48 h before siRNA transfection. Transfections were carried out per the manufacturer’s suggestions with slight modifications. DharmaFECT transfection reagent 1 (cat no. T-2001-01) and E2F1 ON-TARGET plus siRNA 09 (J-003259-09-0005), E2F1 ON-TAREGET plus siRNA 10 (J-003259-10-0005), E2F1 ON-TARGET plus siRNA 11 (J-003259-11-0005), E2F1 ON-TARGET plus siRNA 12 (J-003259-12-0005), and ON-TARGET plus nontargeting control siRNA 1 (D-001810-01-05) were combined with serum-free and antibiotic-free media and incubated 5 min separately. The siRNAs were then combined with the Dharmafect for 20 min and added to the cells with antibiotic-free media. Final concentrations of Dharmafect and siRNAs were 2 μl/ml, and 25 nM, respectively. After 24 h, media was replaced and cells were treated with zinc chromate for 24 h (48 h total transfection time). Cells were harvested for total RNA, whole cell protein, and immunofluorescence staining.

Cell equivalent protein extractions

Cells were seeded and allowed to reenter logarithmic growth for 48 h before treating with zinc chromate for 24, 72, or 120 h. For protein half-life experiments, at the end of the zinc chromate treatment 10 μg/ml CHX was added to all dishes and cells were harvested immediately (0 h) then at 1, 2, 4, 6, 8, 10, and 12 h. At the end of treatment, media were aspirated and cells were rinsed once with 1X PBS without calcium and magnesium. Cells were trypsinized and the reaction was neutralized using fresh media. Cells were centrifuged for 5 min at 1000 rpm (4°C), the supernatant was aspirated, and cells were resuspended in cold PBS. Cells were counted using a Beckman Coulter Multisizer 4 and centrifuged. The PBS was aspirated to 500 μl and 1 ml of cold PBS was used to dislodge the pellet and transfer the cells to a microcentrifuge tube. Cells were centrifuged in a microcentrifuge 5 min at 3500 rpm (4°C). The PBS was gently aspirated and samples were placed on ice.

Whole-cell protein was extracted using ice-cold Pierce RIPA buffer with 10% phosphatase and protease inhibitors added immediately before use. The volume of extraction buffer added to each sample was calculated based on cell number resulting in the same number of cells per volume extraction buffer. Extraction buffer was added to the cell pellet and pipetted up and down to resuspend the pellet. Samples were placed on ice for 20 min, vortexing every 5 min at max speed for 5 s. Samples were then centrifuged at max speed (14 × g) for 10 min (4°C). The supernatant with the protein sample was transferred to a fresh tube. Protein was quantified using the Pierce Rapid Gold BCA kit and BSA standards on a Biotek microplate reader. Samples were boiled with 4× loading buffer + 10% 2-mercaptoethanol 5 min at 95°C and stored at −20°C.

Nuclear protein was extracted using the NE-PER Nuclear and Cytoplasmic Extraction Reagents kits (Thermo cat: 78833) using the manufacturer’s instructions with some modifications. After treatment, cells were collected using the methods above to collect a cell pellet. Then, ice-cold cytoplasmic extraction buffer I (plus 10% phosphatase and protease inhibitors) was added to each sample, vortexed 15 s and placed on ice 10 min. Ice-cold cytoplasmic extraction buffer II was added, vortexed 5 s and placed on ice 1 min. The samples were vortexed again for 5 s and centrifuged at maximum speed (14 × g) 5 min. The supernatant with cytoplasmic protein was transferred to a fresh tube. The pellet (nuclear fraction) was resuspended with ice-cold nuclear extraction buffer and vortexed every 10 min for 15 s for a total of 40 min. The samples were centrifuged at maximum speed (14 × g) 10 min and the supernatant (nuclear protein) was transferred to fresh tubes. Samples were boiled with 4× loading buffer + 10% 2-mercaptoethanol 5 min at 95°C and stored at −20°C.

Cell equivalent Western blot analysis

Protein was loaded using cell equivalents and resolved on 10% Bis-Tris SDS-PAGE gels (approximately 1 h) and transferred to 0.45 μM nitrocellulose membranes (approximately 1.5 h). Immunoblots were dried (approximately 1 h), rehydrated with 1× tris-buffered saline (TBS), and blocked with odyssey blocking buffer (TBS) diluted 1:1 with TBS 1 h. Immunoblots were probed with RAD51 (Santa Cruz sc-8349; 1:1000) and E2F1 (Santa Cruz sc-251; 1:500) antibodies in odyssey blocking buffer (TBS) diluted 1:1 with TBS + 0.2% tween-20 overnight. Equal loading was confirmed by GAPDH (Genetex GT293; 1:500) or H3 (Cell Signaling no. 9715, 1:500) in odyssey blocking buffer (TBS) diluted 1:1 with TBS + 0.2% tween-20. Immunoblots were incubated with IRDye secondary antibodies (LI-COR, 1:15,000) in odyssey blocking buffer (TBS) diluted 1:1 with TBS + 0.2% tween-20 1 h and imaged on a LI-COR Odyssey CLx. Results were normalized to GAPDH or H3 and then represented relative to the untreated control at each time point, respectively. For siRNA experiments, results were normalized to the untransfected 0 μg/cm2 zinc chromate control.

Total RNA isolation and qPCR

At the end of treatment, total RNA was isolated from WTHBF-6 cells using the mirVana miRNA Isolation Kit. Briefly, cells were lysed directly in the culture plates and homogenized. RNA was extracted using acid-phenol:chloroform and the aqueous phase was transferred to filter cartridges. Total RNA was washed several times using ethanol and eluted into a fresh tube. RNA quality and concentration were measured using a NanoDrop ND-1000 spectrophotometer.

cDNA synthesis was carried out using a High Capacity cDNA Reverse Transcription Kit per the manufacturer’s instructions with slight modifications. Briefly, 2× RT master mix was prepared using random primers, combined with 2 μg total RNA (per 20 μl reaction). A no reverse transcriptase control and no RNA control were included in each cDNA synthesis reaction.

qPCR analysis was carried out using the TaqMan RNA assays per the manufacturer’s instructions with slight modifications. Briefly, TaqMan RNA primers (E2F1- Hs00153451_m1; RAD51- Hs00947967_m1; GAPDH-Hs02786624_g1 or Hs02758991_g1) were combined with TaqMan Universal PCR Mastermix II (Thermo Fisher Inc.) and cDNA in triplicate. The no RNA and no reverse transcriptase controls from cDNA synthesis and a no cDNA control were included in all qPCR runs. qPCR was carried out using a StepOnePlus Real-Time PCR system (Applied Biosystems) using standard conditions. The CT threshold was set by the instrument’s calculations and results are displayed as ΔΔCt values relative to the untreated (0 μg/cm2 zinc chromate) control for each time point, respectively.

Immunofluorescence

Immunofluorescence staining was performed as described previously with minor adjustments (Xie et al., 2005). Briefly, cells were seeded on glass chamber slides precoated with FNC and allowed to reenter logarithmic growth for 48 h before treatment. At harvest, cells were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.2% triton-X-100 for 5 min and blocked with 10% goat serum and 1% BSA in PBS with calcium and magnesium for 1 h. Cells were incubated with RAD51 (Santa Cruz sc-8349; 1:200) primary antibody in 1% BSA overnight, washed with PBS, and incubated with secondary Alexa Fluor 488 rabbit 1:2000 in 1% BSA 1 h. Cells were washed with PBS and coverslips were mounted with Prolong Diamond Antifade Mountant with DAPI.

RAD51 nuclear foci were scored in 100 cells per concentration per time point using fluorescent microscopy. Results were expressed as the percentage of cells with >10 foci so that untreated controls had less than 5% of cells with this level of foci. For cytoplasmic accumulation analysis, images of 50 cells per concentration per time point were obtained with a Nikon A1 confocal laser microscope and analyzed using an automated program in NIS-Elements software. Z-stack images were taken with a 60× objective with a step size of 0.5 μM. All camera and laser settings were the same across all images per experiment. Images were processed using the Denoise.ai noise reduction technology in NIS-Elements software and maximum image projections (MaxIPs) were created. The MaxIP images were analyzed in NIS-Elements software using the autodetect ROI program to automatically detect nuclei. Then the outline of the cell was traced manually to compare nuclear and cytoplasmic RAD51 total intensity levels at the single cell level. Cells were considered positive for cytoplasmic accumulation if the cytoplasmic intensity was greater than 95% of control cells.

Statistics

Results are expressed as the mean ± SEM (standard error of the mean) of at least 3 independent experiments unless otherwise noted. Two-way ANOVA with Tukey post hoc analysis was used to determine the significance of Cr(VI) concentrations between exposure times (ie, 24 h 0.1 μg/cm2 vs 120 h 0.1 μg/cm2 zinc chromate) or between Cr(VI) concentrations within a single exposure time (ie, 24 h 0 μg/cm2 zinc chromate vs 0.1 μg/cm2 zinc chromate). When comparing between only 2 exposure concentrations Sidak’s post hoc analysis was used. Statistical significance was set at p < .05. Statistical analysis was performed using GraphPad Prism v8.4.2.

RESULTS

 

Particulate Cr(VI)-Induced Reduction of RAD51 Protein Is a Result of Inhibited Expression

We previously showed particulate Cr(VI) reduces RAD51 whole cell protein (Browning et al., 2016). We confirmed this effect using the cell-equivalence Western blot method. Figure 1A shows representative Western blots of whole cell RAD51 protein. RAD51 whole cell protein is unaffected after 24 h particulate Cr(VI) exposure, but decreases after prolonged 72 and 120 h exposures in a concentration- and time-dependent manner (Figure 1B) consistent with previous results. Specifically, 24 h exposure to 0.1, 0.2, and 0.3 μg/cm2 zinc chromate increased then decreased RAD51 whole cell protein to 1.09, 0.90, and 0.81 relative to control. RAD51 whole cell protein was decreased to 0.70, 0.28, and 0.11 relative to control after 72 h and further to 0.40, 0.20, and 0.06 relative to control after 120 h at 0.1, 0.2 and 0.3 μg/cm2 zinc chromate, respectively. All zinc chromate concentrations were significantly reduced compared with the control after prolonged 72 and 120 h exposure (p < .0001). However, the small reductions observed after 24 h zinc chromate exposure were not statistically significant.

Figure 1.

Figure 1.

Open in a new tab

Prolonged exposure to particulate Cr(VI) reduces RAD51 whole cell protein expression. This figure shows (A) representative Western blot images of whole cell RAD51 (all images have been cropped from original images). (B) This figure shows RAD51 whole cell protein (relative to control) significantly decreased with concentration and time after 72 and 120 h, but not 24 h exposure. GAPDH was used as a loading control. When comparing zinc chromate concentrations between time points, all concentrations were statistically significant (p < .01) between 24 and 72 h or 24 and 120 h exposure. Data represent the mean of 3 independent experiments. Error bars = standard error of the mean. Statistically different from the control: *p < .05; ***p < .0001.

Reduced RAD51 protein levels may be due to reduced RAD51 mRNA expression or increased RAD51 protein degradation resulting in lower RAD51 protein levels. We measured RAD51 protein half-life as 1 component of protein degradation to determine if particulate Cr(VI) reduces RAD51 protein levels by increasing degradation. To assess if particulate Cr(VI) affects RAD51 protein half-life, cells were treated with 0 or 0.2 μg/cm2 zinc chromate for 24 or 120 h and at the end of treatment a protein translation inhibitor, CHX, was added and a 12-h time course was performed to harvest protein. Figure 2A shows a representative Western blot for RAD51 whole cell protein after 24 or 120 h zinc chromate exposure and a 12-h CHX treatment time course. RAD51 whole cell protein levels decreased beginning at 4 h after the addition of CHX after 24 h 0 and 0.2 μg/cm2 zinc chromate exposure (Figure 2B). RAD51 protein was reduced 4 and 6 h after the addition of CHX in 120 h 0 and 0.2 μg/cm2 zinc chromate-treated cells, respectively (Figure 2C). There was no difference in RAD51 protein half-life comparing 0.2 μg/cm2 zinc chromate to the control or between time points. Table 1 shows the half-life of RAD51 calculated from the exponential lines of best fit of the data after 24 or 120 h zinc chromate exposure compared with the controls and confirms Cr(VI) has little effect on RAD51 protein half-life.

Figure 2.

Figure 2.

Open in a new tab

Particulate Cr(VI) has little effect on RAD51 protein half-life. This figure shows (A) representative Western blot images of RAD51 whole cell protein after 24 and 120 h zinc chromate exposure and 12 h CHX exposure (all images have been cropped from original images). Exposure to (B) 24 and (C) 120 h zinc chromate has little effect on RAD51 protein half-life relative to the 0 h CHX control for either the 0 or 0.2 μg/cm2 zinc chromate condition. GAPDH was used as a loading control. Dashed lines represent exponential trendlines for each set of data graphed on a log scale y-axis. Data represent the mean of 3 independent experiments. Error bars = standard error of the mean.

Table 1.

RAD51 Protein Half-Life in Hours After 24 or 120 h Exposure to 0 or 0.2 μg/cm2 Zinc Chromate

Zinc Chromate Conc. (μg/cm2) 24 h 120 h
0 5.12 ± 0.42 h 5.90 ± 1.05 h
0.2 4.27 ± 0.53 h 5.80 ± 1.25 h
Open in a new tab

Data represent the mean (± SEM) of 3 independent experiments.

The RAD51 protein half-life data indicates protein degradation is not significantly contributing to the reduction of RAD51 protein observed after prolonged particulate Cr(VI) exposure suggesting the effect is on protein production. To test this possibility, we measured RAD51 mRNA levels using qPCR. Particulate Cr(VI) inhibited RAD51 mRNA slightly after 24 h 0.1, 0.2, and 0.3 μg/cm2 zinc chromate exposure to 0.82, 0.73, and 0.62 relative to control although none were statistically significant (Figure 3). Prolonged exposure of 72 and 120 h to 0.1, 0.2, and 0.3 μg/cm2 zinc chromate reduced RAD51 mRNA to 0.49, 0.31, and 0.16 and 0.53, 0.21, and 0.25 relative to control, respectively. After prolonged exposure of both 72 and 120 h, RAD51 mRNA was significantly reduced at each concentration compared with control and compared with the respective concentration after 24 h exposure.

Figure 3.

Figure 3.

Open in a new tab

Particulate Cr(VI) inhibits RAD51 mRNA. This figure shows prolonged 72 and 120 h exposure to zinc chromate inhibits RAD51 mRNA levels. GAPDH was used to normalize RAD51 mRNA levels. RAD51 mRNA level was significantly reduced at all concentrations after 72 and 120 h exposure compared with control (*p < .05; ***p < .0001). RAD51 mRNA at all zinc chromate-treated concentrations was significantly decreased after 72 and 120 h compared with the respective concentration at 24 h exposure. Data represent the mean of 3 independent experiments. Error bars = standard error of the mean.

Particulate Cr(VI) Inhibits E2F1 Expression

The combination of protein half-life and qPCR data suggest the reduction of RAD51 protein following particulate Cr(VI) exposure is primarily a result of inhibited transcription. We sought to further investigate how Cr(VI) inhibits RAD51 transcription. We focused on E2F1, the predominant transcription factor for RAD51, and assessed if particulate Cr(VI) exposure affects E2F1 expression.

Figure 4A shows representative E2F1 whole cell Western blots. Similar to the effects on RAD51, particulate Cr(VI) did not affect E2F1 whole cell protein levels after 24 h exposure (0.84, 1.03, and 0.88 relative to control), but decreased levels to 0.57, 0.48, and 0.41 and 0.43, 0.47, and 0.55 relative to control after 72 and 120 h 0.1, 0.2, and 0.3 μg/cm2 zinc chromate exposure, respectively (Figure 4B). E2F1 whole cell protein was decreased significantly compared with the control after 72 h 0.3 μg/cm2 and 120 h 0.1 μg/cm2 zinc chromate, however, all zinc chromate-treated concentrations are clearly repressed compared with control at these time points.

Figure 4.

Figure 4.

Open in a new tab

Prolonged particulate Cr(VI) inhibits E2F1 whole cell protein. This figure shows E2F1 whole cell protein is reduced after prolonged 72 and 120 h exposure to zinc chromate. Data represent the mean of 3 independent experiments. Error bars = standard error of the mean. (A) Representative images of E2F1 whole cell protein Western blots (all images have been cropped from original images) Note: E2F1 and RAD51 were blotted on same membrane with GAPDH used as a shared loading control. (B) E2F1 whole cell protein (relative to control). Statistically significant compared with control: *p < .05.

E2F1 self-regulates its own transcription (Johnson et al., 1994) and we found prolonged particulate Cr(VI) exposure inhibits E2F1 protein expression. We tested if particulate Cr(VI) inhibits E2F1 mRNA levels using qPCR following particulate Cr(VI) exposure. Particulate Cr(VI) inhibited E2F1 mRNA levels after 24, 72, and 120 h exposure (Figure 5). After 24 h, E2F1 mRNA was reduced to 0.82, 0.55, and 0.56 relative to control following 0.1, 0.2, and 0.3 μg/cm2 zinc chromate, and further decreased to 0.45, 0.23, and 0.18 relative to control after 72 h. E2F1 mRNA remained low after 120 h decreasing to 0.55, 0.26, and 0.36 relative to control. At all time points, each concentration was significantly decreased compared with control except 24 h 0.1 μg/cm2 zinc chromate. All concentrations after prolonged exposure of 72 and 120 h were significantly decreased compared with the respective acute 24 h concentration.

Figure 5.

Figure 5.

Open in a new tab

Particulate Cr(VI) inhibits E2F1 mRNA levels. This figure shows 24, 72, and 120 h zinc chromate exposure inhibits E2F1 mRNA levels. GAPDH was used to normalize E2F1 mRNA levels. E2F1 mRNA level was significantly reduced at all concentrations compared with control except 24 h 0.1 μg/cm2 zinc chromate (***p < .0001). E2F1 mRNA at all zinc chromate-treated concentrations was significantly decreased after 72 and 120 h compared with the respective concentration at 24 h exposure. Data represent the mean of 3 independent experiments. Error bars = standard error of the mean.

Loss of E2F1 Inhibits RAD51 Nuclear Foci After Acute 24 h Particulate Cr(VI) Exposure

Prolonged Cr(VI) exposure induces loss of RAD51 foci formation, increased cytoplasmic accumulation of RAD51 protein and reduced RAD51 protein levels (Browning et al., 2016; Qin et al., 2014 and Figs. 1 and 2; Tamblyn et al., 2009). However, RAD51 exhibits the canonical response following 24 h particulate Cr(VI) exposure characterized by increased nuclear foci at double-strand breaks, low cytoplasmic accumulation, and unaffected or slightly increased protein levels (Browning et al., 2016; Qin et al., 2014; Tamblyn et al., 2009). Studies show loss of E2F1 can result in reduced RAD51 protein levels, nuclear foci formation and inhibited homologous recombination repair (Chen et al., 2011; Choi and Kim, 2019; Wu et al., 2014). Thus, given our discovery that Cr(VI) affects E2F1 in a similar manner as RAD51, we attempted to recapitulate the prolonged exposure outcomes after acute exposures by knocking down E2F1 and then treating for 24 h with particulate Cr(VI). We transfected cells with E2F1 siRNA for 48 h and treated with 0.2 μg/cm2 zinc chromate the last 24 h of the transfection. Cells were harvested for immunofluorescence staining, protein, and RNA.

Figure 6 shows E2F1 was successfully knocked down following transfection with 4 different E2F1 siRNAs after 24 h exposure to 0 and 0.2 μg/cm2 zinc chromate. All conditions were normalized to the untransfected 0 μg/cm2 zinc chromate control. A total of 0.2 μg/cm2 zinc chromate reduced E2F1 expression slightly in both the untransfected and non-targeting siRNA control compared with 0 μg/cm2 zinc chromate, respectively. This slight, but not significant reduction is the result we would expect after 24 h particulate Cr(VI) exposure. E2F1 protein was significantly decreased in all E2F1 siRNA conditions compared with the non-targeting siRNA control (p < .0001). E2F1 was knocked down to 0.30 or less compared with the non-targeting control.

Figure 6.

Figure 6.

Open in a new tab

Knockdown of E2F1 inhibits E2F1 protein levels. This figure shows E2F1 knockdown for 48 h leads to reduced E2F1 protein levels. Data represent the mean of at least 5 independent experiments. Error bars = standard error of the mean. E2F1 whole cell protein levels relative to the 0 μg/cm2 zinc chromate untransfected control. E2F1 levels were significantly decreased in all E2F1 siRNA conditions compared with the non-targeting siRNA control (†p < .0001).

We measured RAD51 nuclear foci after E2F1 knockdown and 24 h particulate Cr(VI) exposure to determine if E2F1 was necessary for RAD51 foci formation (Figure 7). Figure 7A shows representative images of WTHBF-6 cells exposed to 24 h 0 or 0.2 μg/cm2 zinc chromate with or without E2F1 siRNA transfection, which was used for RAD51 foci and cytoplasmic accumulation analysis. The untransfected and non-targeting siRNA control produced the expected significant increase in RAD51 foci (21% and 26% of cells, respectively p < .0001) previously shown to occur after acute exposure for 24 h to 0.2 μg/cm2 zinc chromate (Browning et al., 2016; Qin et al., 2014). In cells with E2F1 knocked down, there was no difference in RAD51 foci level in cells with no zinc chromate exposure across all conditions. This outcome is expected because untreated RAD51 foci levels are already below baseline level and without a stimulus to induce RAD51 foci formation no change would be observable. However, when comparing RAD51 foci levels after 0.2 μg/cm2 zinc chromate exposure, the percent of cells with RAD51 foci was decreased in all E2F1 knockdown conditions and was statistically significant in 3 of the 4 siRNAs considered when compared with the non-targeting siRNA control (p < .05). These data indicate loss of E2F1-inhibited RAD51 foci formation after acute 24 h particulate Cr(VI) exposure.

Figure 7.

Figure 7.

Open in a new tab

E2F1 knockdown decreases RAD51 nuclear foci after acute particulate Cr(VI) exposure. (A) Representative images of RAD51 subcellular localization using a 60× objective. DAPI stain (blue) designates the nuclei and green represents RAD51 (images have been cropped from original images). (B) This figure shows the percent of cells with more than 10 RAD51 foci per cell following 24 h particulate Cr(VI) exposure and E2F1 knockdown (untransfected 0 μg/cm2 zinc chromate controls subtracted). RAD51 foci were decreased in all E2F1 siRNA conditions after exposure to 0.2 μg/cm2 zinc chromate and 3 of the 4 conditions were statistically significant when compared with the non-targeting siRNA control (†p < 0.05). Data represent the mean of at least 2–3 independent experiments (E2F1 11 and 12; n = 2). Error bars = standard error of the mean.

RAD51 cytoplasmic accumulation is also a key phenotype observed after prolonged exposure to particulate Cr(VI) (Browning et al., 2016; Qin i, 2014). The mislocalization of RAD51 in the cytoplasm may inhibit RAD51 from participating in homologous recombination repair. We used immunofluorescence staining and confocal microscopy to analyze RAD51 cytoplasmic accumulation after E2F1 knockdown and 24 h particulate Cr(VI) exposure. Cells were considered positive for cytoplasmic accumulation if the cytoplasmic intensity was greater than 95% of control cells. After 24 h particulate Cr(VI) exposure, we would expect a normal RAD51 response indicated by no increase in RAD51 cytoplasmic accumulation in untransfected cells. This outcome is confirmed in the results which show 0.2 μg/cm2 zinc chromate control did not increase RAD51 cytoplasmic accumulation in the untransfected or the non-targeting siRNA control (Figure 8). We also observed no increase in RAD51 cytoplasmic accumulation in the E2F1 knockdown conditions after either 0 or 0.2 μg/cm2 zinc chromate exposure. The highest level of RAD51 cytoplasmic accumulation in any condition was 12% compared with 4% in the control (Figure 8).

Figure 8.

Figure 8.

Open in a new tab

E2F1 knockdown does not induce RAD51 cytoplasmic accumulation after 24 h particulate Cr(VI) exposure. This figure shows RAD51 cytoplasmic accumulation did not increase following E2F1 knockdown and 24 h particulate Cr(VI) exposure. Data represent the mean of 3 experiments for all conditions (except E2F1 9 and 10; n = 1). Error bars = standard error of the mean. No statistical significance was observed.

We are proposing E2F1’s transcription factor function would inhibit RAD51 expression. Less RAD51 protein may also explain the inhibited RAD51 nuclear foci response to acute 24 h particulate Cr(VI) exposure after E2F1 knockdown. To determine if RAD51 protein is suppressed following E2F1 knockdown and acute 24 h particulate Cr(VI) exposure, we measured RAD51 protein. Figure 9A shows representative Western blot images of E2F1 and RAD51 after E2F1 knockdown and 24 h exposure to particulate Cr(VI). We assessed the effect of the transfection on RAD51 protein expression and found although the transfection decreased RAD51 protein to 0.70 in the non-targeting siRNA condition compared with the untransfected condition, it was not significant (Figure 9B). In the untransfected and non-targeting control, we saw the expected result of no difference in RAD51 protein level between 0 and 0.2 μg/cm2 zinc chromate. Comparing the E2F1 knockdown conditions to the non-targeting siRNA control shows 3 of the 4 E2F1 siRNAs tested did not have a significant effect on RAD51 protein levels. These data indicate E2F1 knockdown does not affect RAD51 protein levels.

Figure 9.

Figure 9.

Open in a new tab

E2F1 knockdown does not inhibit RAD51 protein after 24 h particulate Cr(VI) exposure. This figure shows E2F1 knockdown for 48 h does not affect RAD51 protein levels. Data represent the mean of at least 5 independent experiments. Error bars = standard error of the mean. (A) Representative image of E2F1 and RAD51 Western blots (all images have been cropped from original images). (B) RAD51 whole cell protein levels relative to the 0 μg/cm2 zinc chromate untransfected control. There was no change in RAD51 protein level between 0 and 0.2 μg/cm2 zinc chromate in any of the conditions. RAD51 protein was decreased to 0.70 in the non-targeting siRNA control compared with the untransfected control, but this was not significant. RAD51 protein levels only significantly decreased in 1 E2F1 siRNA condition (E2F1 9; †††p < .001) compared with the non-targeting siRNA control, and only in the 0 μg/cm2 zinc chromate condition.

It is possible E2F1 knockdown inhibits RAD51 transcription, but does not inhibit protein levels. To test this possibility, we determined the effect of E2F1 knockdown on RAD51 mRNA levels using qPCR. We confirmed E2F1 knockdown inhibits E2F1 mRNA (Figure 10A). E2F1 mRNA was significantly inhibited in all E2F1 knockdown conditions compared with the non-targeting siRNA control except E2F1 10 after 0.2 μg/cm2 zinc chromate exposure. Exposure to 24 h 0.2 μg/cm2 zinc chromate also decreased E2F1 mRNA in all conditions, although only significantly in the untransfected control. The E2F1 mRNA reduction is consistent with our previous results (Figure 5). Next, we evaluated RAD51 mRNA levels following E2F1 knockdown focusing on 2 E2F1 siRNAs because of the similar results we observed across different siRNAs in previous experiments. Figure 10B shows RAD51 mRNA is decreased by 24 h exposure to 0.2 μg/cm2 zinc chromate however, none of these changes are significant. For example, 0.2 μg/cm2 zinc chromate reduced RAD51 mRNA to 0.64 compared with control similar to our previous experiments evaluating RAD51 mRNA after particulate Cr(VI) exposure (Figure 3). RAD51 mRNA was reduced by E2F1 siRNA 9 compared with the non-targeting control, but this reduction was not significant. E2F1 10 increased RAD51 mRNA slightly compared with the non-targeting control.

Figure 10.

Figure 10.

Open in a new tab

E2F1 knockdown does not consistently decrease RAD51 mRNA expression. This figure shows E2F1 knockdown does not inhibit RAD51 mRNA expression after acute 24 h particulate Cr(VI) exposure. Data represent the mean of 3 independent experiments. Error bars = standard error of the mean. (A) E2F1 mRNA were successfully reduced following E2F1 knockdown (†p < .05). Particulate Cr(VI) reduced E2F1 mRNA levels in all conditions, but was only significant in the untransfected control (***p < .0001). (B) RAD51 mRNA was unaffected by 0.2 μg/cm2 zinc chromate exposure or E2F1 knockdown. GAPDH was used as to normalize E2F1 and RAD51 mRNA levels.

E2F1 Nuclear Levels Are Unaffected by Particulate Cr(VI) Exposure

The literature shows loss of E2F1 results in RAD51 impairment after induction of DNA double-strand breaks (Chen et al., 2011; Choi and Kim, 2019; Wu et al., 2014). It is well-known exposure to particulate Cr(VI) induces DNA double-strand breaks and the repair of those breaks occurs through canonical homologous recombination (Bryant et al., 2006; Gastaldo et al., 2007; Helleday et al., 2000; Stackpole et al., 2007). However, the data indicate E2F1 does not modulate the RAD51 response to particulate Cr(VI). We began to develop alternative hypotheses that may explain this outcome. Although we showed E2F1 whole cell protein was significantly inhibited following prolonged exposure to particulate Cr(VI), it is possible in this case available protein is accumulated in the nucleus in order to preserve critical functions. Therefore, we measured E2F1 nuclear protein levels following particulate Cr(VI) exposure. Figure 11A shows representative Western blots of E2F1 nuclear protein. Particulate Cr(VI) had no effect on E2F1 nuclear protein levels after 24, 72, or 120 h exposure (Figure 11B). These data suggest although total levels of E2F1 are significantly suppressed following exposure to prolonged particulate Cr(VI), the levels of E2F1 protein remaining are sequestered in the nucleus.

Figure 11.

Figure 11.

Open in a new tab

Particulate Cr(VI) does not affect E2F1 nuclear protein levels. This figure shows E2F1 nuclear protein is unaffected by exposure to particulate Cr(VI). (A) Representative Western blots of E2F1 nuclear protein (all images have been cropped from original images). (B) E2F1 nuclear protein levels (relative to control) are unaffected following exposure to 24, 72, and 120 h particulate Cr(VI). H3 was used as loading control. Data represent the mean of 3 independent experiments. Error bars = standard error of the mean. No statistical significance was observed.

DISCUSSION

RAD51, the key protein in the effector step of homologous recombination, is impaired after prolonged exposure to particulate Cr(VI) while earlier steps in the pathway remain functional (Qin et al., 2014). Loss of RAD51 is characterized by inhibited nuclear foci and monofilament formation, increased cytoplasmic accumulation, and inhibited protein expression (Browning et al., 2016; Qin et al., 2014). Mechanisms of particulate Cr(VI)-inhibited RAD51 protein expression are unknown and was a major focus of this investigation.

We confirmed RAD51 protein expression decreased following prolonged, but not acute particulate Cr(VI) exposure consistent with previous studies (Browning et al., 2016; Qin et al., 2014). These data on prolonged exposures provide insight to key events in the timeline of particulate Cr(VI) carcinogenesis. Decreased levels of protein after prolonged exposure result from either increased protein degradation or decreased production of the protein. Bruno et al. (2016) characterized ubiquitin-mediated protein degradation as an important pathway of Cr(VI) exposure leading to deleterious cellular effects. However, our data show particulate Cr(VI) exposure had minimal effect on RAD51 protein half-life indicating protein degradation plays a minor role in the decrease of RAD51 protein levels. Thus, protein degradation may be an overall important pathway for Cr(VI), but it does not appear to have a major contribution to this specific protein.

In contrast, we did find a reduction in processes underlying protein production; specifically, RAD51 mRNA expression was decreased suggesting inhibited transcription is the primary mechanism responsible for the decrease of RAD51 protein. Although RAD51 mRNA began to decrease after acute 24 h exposure, RAD51 mRNA levels were significantly reduced after prolonged exposure consistent with the protein data. This result is supported by Manning et al. (1992), who demonstrated Cr(VI) inhibits transcription. Our data are the first to describe RAD51 mRNA expression following Cr(VI) exposure, for both particulate and soluble Cr(VI) compounds. These results are consistent with studies showing Cr(VI) downregulates expression of DNA repair genes and correlate with the trend in RAD51 protein levels previously reported after acute and prolonged Cr(VI) exposure (Browning et al., 2016; Hodges and Chipman, 2002; Hu et al., 2018; Pritchard et al., 2005; Qin et al., 2014; Takahashi et al., 2005).

E2F1 is the predominant transcription factor for RAD51. Studies show E2F1 is inhibited following exposure to other metals including arsenic and cadmium (Lam et al., 2014, 2015; Li et al., 2008; Wu et al., 2013). However, only 1 study investigated E2F1 after Cr(VI) exposure and found 24 h exposure increased E2F1 expression (Permenter et al., 2011). Prolonged exposures have not been investigated. Our data show E2F1 protein expression is unaffected after 24 exposure but decreases after 72 and 120 h exposure. We observe significant decrease of RAD51 mRNA after prolonged exposures correlating with prolonged Cr(VI)-inhibited E2F1 protein expression. E2F1 self-regulates its own transcription (Johnson et al., 1994). Therefore, the loss of E2F1 protein after prolonged exposure may explain reduced E2F1 mRNA observed after prolonged time points. Notably, RAD51 and E2F1 mRNA begin to decrease after 24 h suggesting transcription may be inhibited earlier than the decrease in protein is detected and there may be more than 1 mechanism for decreased mRNA levels at this time point.

Studies show loss of E2F1 inhibits RAD51 (Chen et al., 2011; Choi and Kim, 2019; Wu et al., 2014). We found knockdown of E2F1 did not inhibit RAD51 mRNA or protein expression or induce RAD51 cytoplasmic accumulation but did inhibit RAD51 nuclear foci formation. RAD51 protein levels were unaffected by E2F1 knockdown after exposure to acute 24 h particulate Cr(VI). These data indicate E2F1 does not modulate RAD51 protein levels in WTHBF-6 cells in response to particulate Cr(VI). Similarly, RAD51 mRNA were not affected by E2F1 knockdown. When we examined RAD51 localization, E2F1 knockdown did not induce RAD51 cytoplasmic accumulation observed after prolonged particulate Cr(VI) exposure indicating E2F1 may not be involved in mechanisms regulating RAD51 nuclear localization. The cytoplasmic accumulation result is contrary to our finding that E2F1 knockdown inhibited RAD51 nuclear foci formation after acute 24 h exposure to particulate Cr(VI). This result suggests E2F1 may affect RAD51 localization or stability at DNA double-strand breaks, but not general nuclear localization.

The results of our E2F1 knockdown experiments may be explained by the complex nature of the distinctive roles of E2F1 in RAD51 expression and function. We found E2F1 knockdown did not inhibit RAD51 mRNA expression after acute particulate Cr(VI) exposure. Although other studies have found E2F1 knockdown inhibits RAD51 mRNA expression, this is inconsistent with our results (Choi and Kim, 2019; Wu et al., 2014). It is possible the levels of E2F1 in our study after knockdown were not low enough to induce the RAD51 effect observed in other studies. An alternative explanation is although total E2F1 protein is significantly decreased after 72 and 120 h particulate Cr(VI) exposure, we found nuclear E2F1 protein levels only decreased after 120 h exposure. It appears what E2F1 protein is available is shuttled into the nucleus. In our E2F1 knockdown experiments what little E2F1 remained may also have been shuttled into the nucleus although it was not measured. Alternative transcription factors for RAD51 may also be compensating for the loss of E2F1 in the expression of RAD51 after particulate Cr(VI) exposure.

E2F1 knockdown did not induce RAD51 cytoplasmic accumulation after acute exposure to particulate Cr(VI). This result may be because E2F1 simply is not involved in the nuclear localization of RAD51 or the timing of the experiment did not allow the target effect to develop. Although RAD51 protein and mRNA expression and cytoplasmic accumulation were unaffected by E2F1 knockdown, RAD51 nuclear foci were inhibited. This result is consistent with studies showing loss of E2F1 reduces RAD51 nuclear foci at double-strand breaks and inhibits DNA repair (Chen et al., 2011; Choi and Kim, 2019; Wu et al., 2014). RAD51 nuclear foci normally increase in response to acute particulate Cr(VI) exposure (Browning et al., 2016; Qin et al., 2014). Our data show loss of E2F1 inhibited RAD51 nuclear foci formation after acute particulate Cr(VI) exposure. Studies show E2F1 can localize to DNA double-strand breaks and plays a role in the signaling and stability of DNA repair proteins (Chen et al., 2011; Choi and Kim, 2019; Liao et al., 2010; Lin et al., 2001). It is possible E2F1 facilitates RAD51 nuclear foci formation directly or through the regulation of mediator proteins. This direct involvement at DNA double-strand breaks may explain why E2F1 knockdown affected RAD51 nuclear foci but not the other endpoints we assessed. Future research will focus on the mechanisms of E2F1-inhibited RAD51 foci formation after particulate Cr(VI) exposure.

CONCLUSIONS

Particulate Cr(VI)-inhibited RAD51 protein levels after prolonged exposure are the result of inhibited expression not increased protein degradation processes. E2F1 protein and mRNA are also reduced by prolonged particulate Cr(VI) exposure. Loss of E2F1 inhibited RAD51 nuclear foci formation in response to acute particulate Cr(VI) exposure when they normally increase, but did not affect expression or cytoplasmic accumulation. These data suggest the predominant role of E2F1 after particulate Cr(VI) exposure may be in RAD51 nuclear foci formation at double-strand breaks and will be further investigated.

AUTHOR CONTRIBUTIONS

Rachel M. Speer: Conceptualization, data curation, formal analysis, investigation, methodology, validation, visualization, writing—review and editing. Jennifer H. Toyoda: Data curation, formal analysis, investigation, methodology, validation, visualization, writing—review and editing. Tayler J. Croom-Pérez: Data curation, formal analysis, investigation, methodology, validation, visualization, writing—review and editing. Ke Jian Liu: Conceptualization, writing—review and editing. John Pierce Wise, Sr.: Conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, project administration, resources, supervision, validation, visualization, writing—review and editing. All authors have approved these statements and the final article.

ACKNOWLEDGMENTS

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Environmental Health Sciences.

FUNDING

National Institute of Environmental Health Sciences (R01ES016893 to J.P.W., T32ES011564 to J.P.W., R.M.S., J.H.T., T.J.C., P30ES030283 to J.P.W.); the Jewish Heritage Foundation for Excellence [JPW]; the University of Louisville Graduate School Dissertation Completion Award [RMS]; and the University of Louisville.

ROLE OF THE FUNDING SOURCE

The funding sources were not involved in the study design, data collection, analysis and interpretation of the data, the writing of the article, or the decision to submit the article for publication.

DECLARATION OF CONFLICTING INTERESTS

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

REFERENCES

  1. Albertson D. G., Collins C., McCormick F., Gray J. W. (2003). Chromosome aberrations in solid tumors. Nat. Genet. 34, 369–376. [DOI] [PubMed] [Google Scholar]
  2. Baumann P., Benson F. E., West S. C. (1996). Human Rad51 protein promotes ATP-dependent homologous pairing and strand transfer reactions in vitro. Cell 87, 757–766. [DOI] [PubMed] [Google Scholar]
  3. Browning C. L., Qin Q., Kelly D. F., Prakash R., Vanoli F., Jasin M., Wise J. P. (2016). Prolonged particulate hexavalent chromium exposure suppresses homologous recombination repair in human lung cells. Toxicol. Sci. 153, 70–78. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bruno M., Ross J., Ge Y. (2016). Proteomic responses of BEAS-2B cells to nontoxic and toxic chromium: Protein indicators of cytotoxicity conversion. Toxicol. Lett. 264, 59–70. [DOI] [PubMed] [Google Scholar]
  5. Bryant H. E., , Ying S., and , Helleday T. (2006). Homologous recombination is involved in repair of chromium-induced DNA damage in mammalian cells. Mutat. Res. 599, 116–123. [DOI] [PubMed] [Google Scholar]
  6. Chen J., Zhu F., Weaks R. L., Biswas A. K., Guo R., Li Y., Johnson D. G. (2011). E2F1 promotes the recruitment of DNA repair factors to sites of DNA double-strand breaks. Cell Cycle 10, 1287–1294. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chen Q. Y., DesMarais T., Costa M. (2019). Metals and mechanisms of carcinogenesis. Annu. Rev. Pharmacol. Toxicol. 59, 537–554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Choi E.-H., Kim K. P. (2019). E2F1 facilitates DNA break repair by localizing to break sites and enhancing the expression of homologous recombination factors. Exp. Mol. Med. 51, 1–12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. D’Amours D., Jackson S. P. (2002). The MRE11 complex: At the crossroads of DNA repair and checkpoint signalling. Nat. Rev. Mol. Cell Biol. 3, 317–327. [DOI] [PubMed] [Google Scholar]
  10. Gastaldo J., Viau M., Bencokova Z., Joubert A., Charvet A.-M., Balosso J., Foray N. (2007). Lead contamination results in late and slowly repairable DNA double-strand breaks and impacts upon the ATM-dependent signaling pathways. Toxicol. Let. 173, 201–214. [DOI] [PubMed] [Google Scholar]
  11. Helleday T., Robert N., Dag J. (2000). Arsenic[III] and heavy metal ions induce intrachromosomal homologous recombination in the Hprt gene of V79 Chinese hamster cells. Environ. Mol. Mutagen. 35, 114–122. [DOI] [PubMed] [Google Scholar]
  12. Hodges N. J., Chipman J. K. (2002). Down-regulation of the DNA-repair endonuclease 8-Oxo-guanine DNA glycosylase 1 (HOGG1) by sodium dichromate in cultured human A549 lung carcinoma cells. Carcinogenesis 23, 55–60. [DOI] [PubMed] [Google Scholar]
  13. Holmes A., Wise S., John Wise J. P. (2008). Carcinogenicity of hexavalent chromium. Indian J. Med. Res.  128, 353–372. [PubMed] [Google Scholar]
  14. Holmes A. L., Wise S. S., Pelsue S. C., Aboueissa A.-M., Lingle W., Salisbury J., Gallagher J., Wise J. P. (2010). Chronic exposure to zinc chromate induces centrosome amplification and spindle assembly checkpoint bypass in human lung fibroblasts. Chem. Res. Toxicol. 23, 386–395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hu G., Li P., Cui X., Li Y., Zhang J., Zhai X., Yu S., Tang S., Zhao Z., Wang J., et al. (2018). Cr(VI)-induced methylation and down-regulation of DNA repair genes and its association with markers of genetic damage in workers and 16HBE cells. Environ. Pollut. 238, 833–843. [DOI] [PubMed] [Google Scholar]
  16. Janssen A., van der Burg M., Szuhai K., Kops G. J. P. L., Medema R. H. (2011). Chromosome segregation errors as a cause of DNA damage and structural chromosome aberrations. Science 333, 1895–1898. [DOI] [PubMed] [Google Scholar]
  17. Johnson D. G., Ohtani K., Nevins J. R. (1994). Autoregulatory control of E2F1 expression in response to positive and negative regulators of cell cycle progression. Genes Dev. 8, 1514–1525. [DOI] [PubMed] [Google Scholar]
  18. Kopec A. K., Thompson C. M., Kim S., Forgacs A. L., Zacharewski T. R. (2012). Comparative toxicogenomic analysis of oral Cr(VI) exposure effects in rat and mouse small intestinal epithelia. Toxicol. Appl. Pharmacol. 262, 124–138. [DOI] [PubMed] [Google Scholar]
  19. Lam S.-K., Li Y.-Y., Zheng C.-Y., Leung L. L., Ho J. C.-M. (2014). E2F1 Downregulation by arsenic trioxide in lung adenocarcinoma. Int. J. Oncol. 45, 2033–2043. [DOI] [PubMed] [Google Scholar]
  20. Li G.-Y., , Kim M., , Kim J.-H., , Lee M.-O., , Chung J.-H., and , Lee B.-H. (2008). Gene expression profiling in human lung fibroblast following cadmium exposure. Food Chem. Toxicol. 46, 1131–1137. [DOI] [PubMed] [Google Scholar]
  21. Liao C.-C., Tsai C. Y., Chang W.-C., Lee W.-H., Wang J.-M. (2010). RB·E2F1 complex mediates DNA damage responses through transcriptional regulation of ZBRK1. J. Biol. Chem. 285, 33134–33143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lin W.-C., Lin F.-T., Nevins J. R. (2001). Selective induction of E2F1 in response to DNA damage, mediated by ATM-dependent phosphorylation. Genes Dev. 15, 1833–1844. [PMC free article] [PubMed] [Google Scholar]
  23. Maeng S. H., Chung H. W., Kim K. J., Lee B. M., Shin Y. C., Kim S. J., Yu I. J. (2004). Chromosome aberration and lipid peroxidation in chromium-exposed workers. Biomarkers 9, 418–434. [DOI] [PubMed] [Google Scholar]
  24. Manning F. C. R., Xu J., Patierno S. R. (1992). Transcriptional inhibition by carcinogenic chromate: Relationship to DNA damage. Mol. Carcinog. 6, 270–279. [DOI] [PubMed] [Google Scholar]
  25. Permenter M. G., Lewis J. A., Jackson D. A. (2011). Exposure to Nickel, chromium, or cadmium causes distinct changes in the gene expression patterns of a rat liver derived cell line. PLoS One 6, e27730. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pritchard D. E., Ceryak S., Ramsey K. E., O'Brien T. J., Ha L., Fornsaglio J. L., Stephan D. A., Patierno S. R. (2005). Resistance to apoptosis, increased growth potential, and altered gene expression in cells that survived genotoxic hexavalent chromium [Cr(VI)] exposure. Mol. Cell. Biochem. 279, 169–181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Qin Q., Xie H., Wise S. S., Browning C. L., Thompson K. N., Holmes A. L., Wise J. P. (2014). Homologous recombination repair signaling in chemical carcinogenesis: Prolonged particulate hexavalent chromium exposure suppresses the Rad51 response in human lung cells. Toxicol. Sci. 142, 117–125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Rager J. E., Suh M., Chappell G. A., Thompson C. M., Proctor D. M. (2019). Review of transcriptomic responses to hexavalent chromium exposure in lung cells supports a role of epigenetic mediators in carcinogenesis. Toxicol. Lett. 305, 40–50. [DOI] [PubMed] [Google Scholar]
  29. San Filippo J., Sung P., Klein H. (2008). Mechanism of eukaryotic homologous recombination. Annu. Rev. Biochem. 77, 229–257. [DOI] [PubMed] [Google Scholar]
  30. Stackpole M. M., Wise S. S., Goodale B. C., Duzevik E. G., Munroe R. C., Thompson W. D., Thacker J., Thompson L. H., Hinz J. M., Wise J. P., et al. (2007). Homologous Recombination repair protects against particulate chromate-induced chromosome instability in Chinese hamster cells. Mutat. Res. Fundam. Mol. Mech. Mutagen. 625, 145–154. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sung P., Robberson D. L. (1995). DNA strand exchange mediated by a RAD51-SsDNA nucleoprotein filament with polarity opposite to that of RecA. Cell 82, 453–461. [DOI] [PubMed] [Google Scholar]
  32. Takahashi Y., Kondo K., Ishikawa S., Uchihara H., Fujino H., Sawada N., Miyoshi T., Sakiyama S., Izumi K., Monden Y. (2005). Microscopic analysis of the chromium content in the chromium-induced malignant and premalignant bronchial lesions of the rat. Environ. Res. 99, 267–272. [DOI] [PubMed] [Google Scholar]
  33. Tamblyn L., Li E., Sarras H., Srikanth P., Prakash Hande M., Peter McPherson J. (2009). A role for Mus81 in the repair of chromium-induced DNA damage. Mutat. Res. Fundam. Mol. Mech. Mutagen. 660, 57–65. [DOI] [PubMed] [Google Scholar]
  34. Williams R. S., Williams J. S., Tainer J. A. (2007). Mre11–Rad50–Nbs1 is a keystone complex connecting DNA repair machinery, double-strand break signaling, and the chromatin template. Biochem. Cell Biol. 85, 509–520. [DOI] [PubMed] [Google Scholar]
  35. Wise J. P., Wise S. S., Little J. E. (2002). The cytotoxicity and genotoxicity of particulate and soluble hexavalent chromium in human lung cells. Mutat. Res. Genet. Toxicol. Environ. Mutagen. 517, 221–229. [DOI] [PubMed] [Google Scholar]
  36. Wise S. S., Aboueissa A. E.-M., Martino J., Wise J. P. (2018). Hexavalent chromium–induced chromosome instability drives permanent and heritable numerical and structural changes and a DNA repair–deficient phenotype. Cancer Res. 78, 4203–4214. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Wise S. S., Elmore L. W., Holt S. E., Little J. E., Antonucci P. G., Bryant B. H., Pierce Wise Sr J. Sr. (2004). Telomerase-mediated lifespan extension of human bronchial cells does not affect hexavalent chromium-induced cytotoxicity or genotoxicity. Mol. Cell. Biochem. 255, 103–112. [DOI] [PubMed] [Google Scholar]
  38. Wise S. S., Holmes A. L., Qin Q., Xie H., Katsifis S. P., Thompson W. D., Wise J. P. (2010). Comparative genotoxicity and cytotoxicity of four hexavalent chromium compounds in human bronchial cells. Chem. Res. Toxicol. 23, 365–372. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Wise S. S., Schuler J. H. C., Katsifis S. P., Wise J. P. (2003). Barium chromate is cytotoxic and genotoxic to human lung cells. Environ. Mol. Mutagen. 42, 274–278. [DOI] [PubMed] [Google Scholar]
  40. Wu C.-H., , Tseng Y.-S., , Kao Y.-T., , Sheu H.-M., and , Liu H.-S. (2013). Low Concentration of Arsenic-Induced Aberrant Mitosis in Keratinocytes Through E2F1 Transcriptionally Regulated Aurora-A. Toxicol. Sci. 132, 43–52. [DOI] [PubMed] [Google Scholar]
  41. Wu M., Wang X., Mcgregor N., Pienta K. J., Zhang J. (2014). Dynamic regulation of Rad51 by E2F1 and P53 in prostate cancer cells upon drug-induced DNA damage under hypoxia. Mol. Pharmacol. 85, 866–876. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Xie H., Holmes A. L., Young J. L., Qin Q., Joyce K., Pelsue S. C., Peng C., Wise S. S., Jeevarajan A. S., Wallace W. T., et al. (2009). Zinc chromate induces chromosome instability and DNA double strand breaks in human lung cells. Toxicol. Appl. Pharmacol. 234, 293–299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Xie H., Wise S. S., Holmes A. L., Xu B., Wakeman T. P., Pelsue S. C., Singh N. P., Wise J. P. (2005). Carcinogenic lead chromate induces DNA double-strand breaks in human lung cells. Mut. Res. Genet. Toxicol. Environ. Mutagen. 586, 160–172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Xie H., Wise S. S., Wise J. P. (2008). Deficient repair of particulate hexavalent chromium-induced DNA double strand breaks leads to neoplastic transformation. Mutat. Res. Genet. Toxicol. Environ. Mutagen. 649, 230–238. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Toxicological Sciences are provided here courtesy of Oxford University Press

RESOURCES