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.

Polycyclic aromatic hydrocarbon-DNA adducts determined by semiquantitative immunohistochemistry in human esophageal biopsies taken in 1985.

Esophageal endoscopic biopsy samples were obtained in 1985 in Linxian, China, a region with very high esophageal cancer incidence rates, and where ingested food is known to contain substantial amounts of polycyclic aromatic hydrocarbons (PAHs). In this study, the automated cellular imaging system (ACIS) was used for localization and semi-quantitation of PAH-DNA adducts. Fresh tissue sections were cut from archived paraffin blocks and incubated with an antiserum elicited against DNA modified with 7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene (BPDE). Nuclear PAH-DNA adduct staining was observed in four out of five human samples incubated with the anti-BPDE-DNA. By visual inspection, nuclei in the basal layer of the esophageal epithelium had higher levels of PAH-DNA adducts compared to those found in the adjacent superficial squamous layer. Nuclear PAH-DNA staining was absent in serial sections incubated with either normal rabbit serum or BPDE-DNA-antiserum previously absorbed with the immunogen BPDE-DNA. Semi-quantitative evaluation by ACIS revealed that per nucleus values for PAH-DNA adducts in the basal layer of the esophageal epithelium were 5- to 40-fold higher than those in the adjacent superficial squamous layer (P < 0.0001), using a random effects model). This pilot study demonstrates the presence of PAH-DNA adducts in archived paraffin-embedded endoscopic esophageal biopsy samples that are close to 20 years old, and suggests that an appropriate set of archived samples could be used to prospectively correlate PAH-DNA adduct formation with risk of esophageal cancer development.

Blockage of transcription as a trigger for p53 accumulation by 2-acetylaminofluorene DNA-adducts.

The hepatocarcinogen 2-acetylaminofluorene is one of the most studied experimental carcinogens. We have shown previously that normal rat hepatocytes accumulate the tumour suppressor p53 after exposure to this compound while preneoplastic rat hepatocytes do not. We suggested that the lack of p53 response may confer a growth advantage on preneoplastic hepatocytes and may be an important factor in hepatic tumor promotion by 2-acetylaminofluorene and other genotoxic compounds. Inhibition of RNA polymerase II driven transcription by DNA lesions may constitute one of the mechanisms leading to accumulation of the tumour suppressor p53. We have investigated the accumulation of p53 by structurally different DNA lesions of 2-acetylaminofluorene for which the rate of nucleotide excision repair (NER) and inhibition of transcription are known. Experiments were performed with NER proficient human fibroblasts as well as repair deficient xeroderma pigmentosum group A (XPA) cells, XPC cells [only transcription coupled repair (TCR)] and Cockayne syndrome (CS)B cells [only global genome repair (GGR)]. The cells were exposed to N-acetoxy-acetylaminofluorene (NAAAF) in the presence or absence of paraoxon inducing dG-C8-AAF or dG-C8-AF adducts respectively. Both treatments led to accumulation of p53 in all cells. However, dG-C8-AAF adducts produced greater p53 induction than dG-C8-AF adducts. The percentage p53-positive cells was highest and the threshold for p53 accumulation was lowest in XPA and CSB cells. Our results further demonstrate that both the potency of a lesion to inhibit transcription as well as the restoration of RNA synthesis determines the magnitude of p53 induction.

Semiquantitation of polycyclic aromatic hydrocarbon-DNA adducts in human esophagus by immunohistochemistry and the automated cellular imaging system.

It has been suggested that ingestion of polycyclic aromatic hydrocarbons (PAHs) may contribute to the high incidence and mortality of esophageal cancer in Linxian, China. To explore this relationship a semiquantitative immunohistochemical staining method was developed for localization of PAH-DNA adducts. Nuclear color intensity (bright field average pink intensity per nucleus for >1000 cells) was measured using the ChromaVision Automated Cellular Imaging System (ACIS). Paraffin-embedded sections of cultured human keratinocytes exposed to increasing concentrations of 7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene (BPDE) were incubated with BPDE-DNA antiserum and served as an internal positive control (standard curve). Values for nuclear staining intensity correlated directly with BPDE exposure concentration (r(2) = 0.99) and were reproducible. DNA adduct levels determined by BPDE-DNA chemiluminescence immunoassay in DNA from BPDE-exposed keratinocytes, correlated with BPDE exposure concentrations (r(2) = 0.99), showing that nuclear staining intensity determined by ACIS correlated directly with BPDE-DNA adduct levels determined by chemiluminescence immunoassay. The ACIS methodology was applied to 5 human samples from Linxian, and significantly positive nuclear PAH-DNA adduct staining was observed in this group when compared with esophageal tissue from 4 laboratory-housed monkey controls and 6 samples obtained at autopsy from smokers and nonsmokers in the United States. Nuclear PAH-DNA staining was absent from Linxian samples when serial sections were incubated with normal rabbit serum (negative control) and was significantly reduced on incubation with BPDE-DNA antiserum absorbed previously with the immunogen BPDE-DNA. These results appear to support the hypothesis that high PAH exposure levels may be etiologically associated with the development of esophageal cancer in Linxian.