Mutagenesis Advance Access originally published online on December 8, 2006
Mutagenesis 2007 22(1):49-54; doi:10.1093/mutage/gel050
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Published by Oxford University Press 2006
Cisplatin–DNA damage in p21WAF1/Cip1 deficient mouse keratinocytes exposed to cisplatin
1 Laboratory of Cellular Carcinogenesis and Tumor Promotion, CCR, National Cancer Institute, Building 37 Room 4032, NIH Bethesda, MD 20892-4255 2 Laboratory of ImmunoBiology, Office of Biotechnology Products, CDER, US Food and Drug Administration Bethesda, MD 20892 3 Valley City State University, 101 College Street S.W. Valley City, ND 58072 4 Department of Oncology, Mayo Clinic 200 First Street SW., Rochester, MN 55905, USA
In response to DNA damage, cell cycle arrest, apoptosis, and DNA repair are mediated by a TP53 pathway that induces p21WAF1/Cip1. The chemotherapeutic drug cis-diamminedichloroplatinum-II (cisplatin) damages cellular DNA by forming cis-diammineplatinum-N7-d[GpG] and cis-diammine-platinum-N7-d[ApG] adducts. To investigate the role of p21, skin keratinocytes from p21WAF1/Cip1 wild-type (+/+), heterozygous (+/–), and null (–/–) mice, cultured in calcium levels designed to maintain a proliferating state, were exposed to 5 µM cisplatin continuously for 0, 8, 24, 48 and 72 h. At all time points the (+/–) cells had the fewest Pt-DNA adducts, and at 24 h mean Pt-DNA adduct levels were 541, 153 and 779 fmol adduct/µg DNA for p21WAF1/Cip1 (+/+), (+/–) and (–/–) cells, respectively [P < 0.05 for (+/+) versus (+/–) and (–/–) versus (+/–)]. In order to understand underlying events, we examined p21WAF1/Cip1 messenger RNA (mRNA), cell cycle arrest, and apoptosis in these cells. At 48 h of cisplatin exposure p21WAF1/Cip1 mRNA expression was 2-fold higher in the (+/+) cells, compared to the (+/–) cells. At 24 h, the % of cells in S-phase in cisplatin-exposed cultures, compared to unexposed cultures, was decreased by 51, 40 and 11% in p21WAF1/Cip1 (+/+), (+/–) and (–/–) cells, respectively (P = 0.04, ANOVA). At 24, 48 and 72 h the % of cisplatin-exposed (+/+) cells in apoptosis was 9.4–10.5%, while the cisplatin-exposed (+/–) and (–/–) cells had 1.2–3.7% of cells in apoptosis. The data support the interpretation that DNA replication arrest and apoptosis do not completely explain the low levels of Pt-DNA adducts in the (+/–) cells, and suggest that p21WAF1/Cip1 controls activity resulting in either low Pt-DNA adduct formation or enhanced Pt-DNA adduct removal.
*To whom correspondence should be addressed at: Carcinogen–DNA Interactions Section, National Cancer Institute, Building 37, Room 4032, NIH, 37 Convent Dr, MSC-4255, Bethesda, MD 20892-4255, USA. Email: poirierm{at}exchange.nih.gov