Depletion of WRN enhances DNA damage in HeLa cells exposed to the benzene metabolite, hydroquinone.
Werner syndrome is a progeroid disorder caused by mutations of the WRN gene. The encoded WRN protein belongs to the family of RecQ helicases that plays a role in the maintenance of genomic stability. Single nucleotide polymorphisms in WRN have been associated with an increased risk for some cancers and were recently linked to benzene hematotoxicity. To further address the role of WRN in benzene toxicity, we employed RNA interference (RNAi) to silence endogenous WRN in HeLa cells and examined the susceptibility of these WRN-depleted cells to the toxic effects of the benzene metabolite hydroquinone. HeLa cells were used as the experimental model because RNAi is highly effective in this system producing almost complete depletion of the target protein. Depletion of WRN led to a decrease in cell proliferation and an enhanced susceptibility to hydroquinone cytotoxicity as revealed by an increase in necrosis. WRN-depleted HeLa cells treated with hydroquinone also displayed an increase in the amount of DNA double-strand breaks as determined by the Comet assay, and an elevated DNA damage response as indicated by the sevenfold induction of gammaH2AX and acetyl-p53 (Lys373 and Lys382) over control levels. Together, these results show that WRN plays an important role in the protection of HeLa cells against the toxicity of the benzene metabolite hydroquinone, specifically in mounting a normal DNA damage response following the induction of DNA double-strand breaks. Further studies in bone marrow-derived stem or progenitor cells are required to confirm our findings in HeLa cells and expand our ability to extrapolate the results to benzene toxicity in humans.
Galván
Noé
N
Molecular Epidemiology and Toxicology Laboratory, School of Public Health, University of California at Berkeley, Berkeley, CA 94720, USA.
Lim
Sophia
S
Zmugg
Stephan
S
Smith
Martyn T
MT
Zhang
Luoping
L
D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D001554:Benzene / Q000378:metabolism; D015153:Blotting, Western; D049109:Cell Proliferation / Q000187:drug effects; D020552:Comet Assay; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects; D004249:DNA Damage; D004260:DNA Repair; D005090:Exodeoxyribonucleases / Q000235:genetics*; D005434:Flow Cytometry; D006367:HeLa Cells; D006657:Histones / Q000378:metabolism; D006801:Humans; D006873:Hydroquinones / Q000378:metabolism / Q000494:pharmacology*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D053484:RecQ Helicases / Q000235:genetics*; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D000071657:Werner Syndrome Helicase
C468783:H2AX protein, human; D006657:Histones; D006873:Hydroquinones; D034741:RNA, Small Interfering; D016159:Tumor Suppressor Protein p53; D005090:Exodeoxyribonucleases; D053484:RecQ Helicases; C098785:WRN protein, human; D000071657:Werner Syndrome Helicase; D001554:Benzene; C031927:hydroquinone