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. 2006 Oct 17;103(42):15404-9.
doi: 10.1073/pnas.0607031103. Epub 2006 Oct 9.

Acrolein Is a Major Cigarette-Related Lung Cancer Agent: Preferential Binding at p53 Mutational Hotspots and Inhibition of DNA Repair

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Acrolein Is a Major Cigarette-Related Lung Cancer Agent: Preferential Binding at p53 Mutational Hotspots and Inhibition of DNA Repair

Zhaohui Feng et al. Proc Natl Acad Sci U S A. .
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Abstract

The tumor suppressor gene p53 is frequently mutated in cigarette smoke (CS)-related lung cancer. The p53 binding pattern of carcinogenic polycyclic aromatic hydrocarbons (PAHs) found in CS coincides with the p53 mutational pattern found in lung cancer, and PAHs have thus been considered to be major culprits for lung cancer. However, compared with other carcinogenic compounds, such as aldehydes, the amount of PAHs in CS is minute. Acrolein (Acr) is abundant in CS, and it can directly adduct DNA. Acr-DNA adducts, similar to PAH-DNA adducts, induce predominantly G-to-T transversions in human cells. These findings raise the question of whether Acr-DNA adducts are responsible for p53 mutations in CS-related lung cancer. To determine the role of Acr-DNA adducts in p53 mutagenesis in CS-related lung cancer we mapped the distribution of Acr-DNA adducts at the sequence level in the p53 gene of lung cells using the UvrABC incision method in combination with ligation-mediated PCR. We found that the Acr-DNA binding pattern is similar to the p53 mutational pattern in human lung cancer. Acr preferentially binds at CpG sites, and this enhancement of binding is due to cytosine methylation at these sequences. Furthermore, we found that Acr can greatly reduce the DNA repair capacity for damage induced by benzo[a]pyrene diol epoxide. Together these results suggest that Acr is a major etiological agent for CS-related lung cancer and that it contributes to lung carcinogenesis through two detrimental effects: DNA damage and inhibition of DNA repair.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Chemical structures and detections of Acr–dG. (A) Chemical structure of Acr and Acr–dG adducts. (B) Identification of 32P-labeled Acr–DNA adducts by 2D TLC. Acr-modified DNA isolated from Acr-treated human cells and Acr-treated purified genomic DNA were digested with phosphodiesterase and nuclease P1, labeled with [γ-32P]ATP, and subjected to 2D TLC, as described in Materials and Methods. The standard Acr–dG adducts were obtained by reaction of Acr (0.2 M) with dGMP (2 mM) and then labeled with [γ-32P]ATP, and the major Acr–dG 3 adducts are indicated by circles. (a) DNA isolated from control cells. (b) Acr-modified genomic DNA. (c) DNA from Acr-treated cells. (d) Acr-modified dGMP. (e) Mixture of c and d.
Fig. 2.
Fig. 2.
Acr–dG and BPDE–dG distributions in the p53 gene. (A and B) Acr–dG and BPDE–dG adduct distribution in exons 5, 7, and 8 of the p53 gene of normal human lung cells treated with Acr (A) and BPDE (B). In A, NHBE cells and NHLF were treated with 20 μM Acr for 6 h, and in B, NHBE cells were treated with 1 μM BPDE for 30 min. Genomic DNA was then isolated, the DNA adduct distribution was mapped by the UvrABC/LMPCR method, and the DNA was separated by electrophoresis. A/G and T/C are Maxam and Gilbert reaction products (26). (C) Comparisons of the frequency of Acr–dG adduct distribution along the p53 gene in NHBE cells with the frequency of the p53 mutations in CS-related lung cancer (International Agency for Research on Cancer p53 Mutation database, http://www-p53.iarc.fr).
Fig. 3.
Fig. 3.
The effect of 5C cytosine methylation at CpG sites on Acr–dG adduct formation. Cytosines at CpG sites of 5′ 32P-labeled exon 5 (A) and 3′ 32P-labeled exon 7 (B) of p53 DNA fragments were methylated by SssI CpG methylase, and the DNA fragments with and without methylation treatment were modified with Acr (30 μM, 10-h incubation), treated with UvrABC nucleases, and separated by electrophoresis as previously described (25). A/G and T/C are Maxam and Gilbert reaction products. T/*C represents Maxam and Gilbert reaction products from methylated DNA fragments. *C represents the methylated cytosine, and the codon number of the bands corresponding to CpG sites is indicated by an asterisk.
Fig. 4.
Fig. 4.
Inhibition of the repair of BPDE–DNA adducts in human cells by Acr. (A) Repair inhibition determined by host cell reactivation assay. BPDE-modified luciferase reporter and unmodified β-galactosidase plasmids were cotransfected into NHLF treated with different concentrations of Acr for 1 h, and luciferase and β-galactosidase activities were measured 20 h after transfection. The relative repair capacity was calculated as the percentage of the relative luciferase activity of the plasmids in Acr-treated cells compared with untreated cells after normalization of the transfection frequency with β-galactosidase activity. (BD) Repair inhibition determined by in vitro DNA repair synthesis assay. BPDE-modified pUC18 and unmodified pBR322 plasmids were used as DNA substrates for in vitro DNA repair synthesis assay. (B) NHLF were treated with different concentrations of Acr for 1 h, and the cell extracts were used for repair assay. (C) Different concentrations of Acr were added directly into cell extracts prepared from untreated NHLF immediately before the start of repair assay. (Upper) Photograph of an ethidium bromide-stained gel. (Lower) Autoradiograph of the same gel. (D) The relative repair capacity was calculated as the percentage of the repair activity in Acr-treated samples to untreated samples. The data represent three independent experiments, and the error bars represent the standard deviation.

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