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Mutagenesis Advance Access published online on December 8, 2006
Mutagenesis, doi:10.1093/mutage/gel050
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Published by Oxford University Press 2006
Received August 1, 2006
Revised September 13, 2006
Accepted October 2, 2006

Original article

Cisplatin-DNA damage in p21WAF1/Cip1 deficient mouse keratinocytes exposed to cisplatin

Hilde E. van Gijssel 1, Tarek A. Leil 2, Wendy C. Weinberg 3, Rao L. Divi 4, Ofelia A. Olivero 4, and Miriam C. Poirier 4 *

1 Laboratory of Cellular Carcinogenesis and Tumor Promotion, CCR, National Cancer Institute, Building 37 Room 4032, NIH, Bethesda, MD 20892-4255, USA; Valley City State University, 101 College Street S.W., Valley City, ND 58072, USA
2 Laboratory of Cellular Carcinogenesis and Tumor Promotion, CCR, National Cancer Institute, Building 37 Room 4032, NIH, Bethesda, MD 20892-4255, USA; Department of Oncology, Mayo Clinic, 200 First Street SW., Rochester, MN 55905, USA
3 Laboratory of ImmunoBiology, Office of Biotechnology Products, CDER, US Food and Drug Administration, Bethesda, MD 20892, USA
4 Laboratory of Cellular Carcinogenesis and Tumor Promotion, CCR, National Cancer Institute, Building 37 Room 4032, NIH, Bethesda, MD 20892-4255, USA

* To whom correspondence should be addressed.
Miriam C. Poirier, E-mail: poirierm{at}exchange.nih.gov


  Abstract

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.


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