Cisplatin-DNA damage in p21WAF1/Cip1 deficient mouse keratinocytes exposed to cisplatin.
In response to DNA damage, cell cycle arrest, apoptosis, and DNA repair are mediated by a TP53 pathway that induces p21(WAF1/Cip1). The chemotherapeutic drug cis-diamminedichloroplatinum-II (cisplatin) damages cellular DNA by forming cis-diammineplatinum-N(7)-d[GpG] and cis-diammine-platinum-N(7)-d[ApG] adducts. To investigate the role of p21, skin keratinocytes from p21(WAF1/Cip1) wild-type (+/+), heterozygous (+/-), and null (-/-) mice, cultured in calcium levels designed to maintain a proliferating state, were exposed to 5 microM 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/mug DNA for p21(WAF1/Cip1) (+/+), (+/-) and (-/-) cells, respectively [P < 0.05 for (+/+) versus (+/-) and (-/-) versus (+/-)]. In order to understand underlying events, we examined p21(WAF1/Cip1) messenger RNA (mRNA), cell cycle arrest, and apoptosis in these cells. At 48 h of cisplatin exposure p21(WAF1/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 p21(WAF1/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 p21(WAF1/Cip1) controls activity resulting in either low Pt-DNA adduct formation or enhanced Pt-DNA adduct removal.
van Gijssel
Hilde E
HE
Laboratory of Cellular Carcinogenesis and Tumor Promotion, CCR, National Cancer Institute, National Institutes of Health, Building 37 Room 4032, Bethesda, MD 20892-4255, USA.
Leil
Tarek A
TA
Weinberg
Wendy C
WC
Divi
Rao L
RL
Olivero
Ofelia A
OA
Poirier
Miriam C
MC
D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000187:drug effects; D002453:Cell Cycle / Q000187:drug effects; D002460:Cell Line; D002945:Cisplatin / Q000633:toxicity*; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics*; D018736:DNA Adducts / Q000378:metabolism; D004249:DNA Damage / Q000187:drug effects*; D018628:Gene Dosage / Q000187:drug effects / Q000502:physiology; D015603:Keratinocytes / Q000187:drug effects*; D051379:Mice; D018345:Mice, Knockout; D013997:Time Factors
C493957:Cdkn1a protein, mouse; D050759:Cyclin-Dependent Kinase Inhibitor p21; D018736:DNA Adducts; D002945:Cisplatin