Fig 1.
Alcohol induces DNA damage in MCF-7 cells.
A) MCF-7 cells were treated with alcohol (0, 0.1%, 0.2%, 0.4%, or 0.8% v/v) for 2 hours. Then DNA damage was quantified using an 8-OHdG ELISA assay and was performed in triplicate. The graph represents the relative levels of DNA damage (8-OHdG) as compared to the control. Values are presented as the mean ± standard error of the mean (S.E.) (**p<0.01). B) MCF-7 cells were cultured on glass coverslips and treated with various doses of alcohol (0, 0.2%, 0.4%, or 0.8% v/v) for 2 hours. After incubation in alcohol, cells were fixed and stained with p-H2AX primary and Alexa-488-conjugated secondary antibodies. Then, nuclei were counterstained with DAPI. Representative images are shown for each control and treatment group with p-H2AX foci indicating DSBs in the nuclei. C) The graph depicts the mean number of foci per cell (± S.E.) in each group as shown in B (**p<0.01).
Fig 2.
Alcohol activates the DNA damage/p53 pathway.
MCF-7 cells were treated with alcohol (0, 0.1%, 0.2%, 0.4%, or 0.8% v/v) for 2 hours. Then cells were harvested and protein was extracted for Western blot analysis of the indicated markers.
Fig 3.
Alcohol increases p53 transcriptional activity and induces cell cycle arrest.
A) Relative mRNA levels of TP53 are shown from MCF-7 cells treated with alcohol (0, 0.2%, or 0.4% v/v) for 6 hours. Values are presented as the mean ± S.E. B) MCF-7 cells transiently transfected with the p53 luciferase reporter plasmid (MCF-7/p53-luc cells) were treated with alcohol (0, 0.2%, or 0.4% v/v) for 2 hours. Then, the cell lysates were prepared for reporter assays. The luciferase activity relative to the control for each sample is shown. Values are presented as the mean ± S.E. (**p<0.01). C) MCF-7 cells were treated with various doses of alcohol (0, 0.1%, 0.2%, or 0.4% v/v) for 6 hours. mRNA levels of p21 and Bax were measured using qPCR. Relative fold changes for the alcohol-treated samples are displayed as compared to the respective control. Values are presented as the mean ± S.E. (**p<0.01). D) MCF-7 cells were treated with alcohol (0, 0.1%, 0.2%, 0.4%, or 0.8% v/v) for 6 hours and then harvested for protein expression. p21 and Bax protein levels were determined by Western blot analysis. E) MCF-7 cells were exposed to alcohol (0, 0.2%, 0.4%, or 0.8% v/v) for 24 hours before cells were fixed and prepared for FACS analysis as described in the Materials and Methods. F) The graph depicts the average percentage of cells (± S.E.) in G0/G1, S, and G2/M phase (*p<0.05; **p<0.01 as compared to the control) from three replicate experiments.
Fig 4.
p53 knockdown alleviates alcohol-induced cell cycle arrest.
A) p53 was knocked down in MCF-7 cells that were stably transfected with p53 siRNA as verified by Western blot analysis. B) MCF-7 control (MCF-7/Con) and MCF-7 p53-knockdown (MCF-7/sip53) cells were treated with various doses of alcohol (0, 0.2%, or 0.4% v/v) for 2 hours. Protein expression of p53 targets, p21 and Bax, were detected by Western blot analysis. C) MCF-7/Con and MCF-7/sip53 cells were treated with alcohol (0 or 0.8% v/v) for 24 hours. Then, the cells were prepared for FACS analysis. D) The graph depicts the average percentage of cells (± S.E.) in G0/G1, S, and G2/M phase (*p<0.05; **p<0.01) from three replicate experiments.
Fig 5.
p53 knockdown increases alcohol-induced DNA damage.
A) MCF-7/Con and MCF-7/sip53 cells were cultured on glass coverslips and exposed to various doses of alcohol (0 or 0.2% v/v) for 2 hours. Then, the cells were fixed and stained with p-H2AX primary and Alexa-488-conjugated secondary antibodies. The nuclei were counterstained with DAPI. Representative images are shown for each control and treatment group with p-H2AX foci formation in the nuclei. B) The graph depicts the mean number of foci per cell (± S.E.) in each group as shown in A (**p<0.01). C) MCF-7/Con and MCF-7/sip53 cells were exposed to various doses of alcohol (0, 0.1%, 0.2%, or 0.4% v/v) for 2 hours before cells were harvested and prepped for Western blot analysis. p-H2AX, H2AX, and β-actin protein levels are shown.
Fig 6.
Nutlin-3 (an Mdm2 inhibitor) increases G0/G1 arrest induced by alcohol exposure.
A) MCF-7 cells that were pretreated with 0 or 1 μM of Nutlin-3 for 1 hour were exposed to alcohol (0 or 0.8% v/v) for 24 hours. Cell cycle arrest was measured by FACS analysis. B) The graph depicts the average percentage of cells (± S.E.) in G0/G1, S, and G2/M phase (*p<0.05; **p<0.01) from three replicate experiments. C) p53, p21, Bax, p-H2AX, and H2AX protein expression were determined by Western blot analysis of cells that were pretreated with 0 or 1 μM of Nutlin-3 for 1 hour and then treated with alcohol (0, 0.2%, or 0.4% v/v) for 6 hours.