Matrix-induced fragmentation of P3'-N5' phosphoramidate-containing DNA: high-throughput MALDI-TOF analysis of genomic sequence polymorphisms.

Chemical and enzymatic approaches were used to produce polynucleotide fragments containing acid-labile internucleotide P3'-N5' phosphoramidate bonds, either in a surface-bound form or in solution. The primer extension reaction utilizing 5'-amino-5'-deoxynucleoside 5'-triphosphates generates polynucleotides that can be fragmented into short, easy-to-analyze pieces simply by being premixed with the acidic matrices typically used for MALDI-TOF mass spectrometry of nucleic acids. This leads to detection procedures that are simple, robust and easy to automate. Utilizing this approach, a polymorphic site in the human ADRB3 gene was interrogated. Primer extensions with phosphoramidate analogs of dNTPs allowed for unambiguous discrimination of all possible genotypes.

Targeting of lung cancer mutational hotspots by polycyclic aromatic hydrocarbons.

BACKGROUND: Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in combustion products of organic matter, including cigarette smoke. Metabolically activated diol epoxides of these compounds, including benzo[a]pyrene diol epoxide (B[a]PDE), have been suggested as causative agents in the development of lung cancer. We previously mapped the distribution of B[a]PDE adducts within the p53 tumor suppressor gene (also known as TP53), which is mutated in 60% of human lung cancers, and found that B[a]PDE adducts preferentially form at lung cancer mutational hotspots (codons 154, 157, 158, 245, 248, and 273). Other PAHs may be important in lung cancer as well. METHODS: Here we have mapped the distribution of adducts induced by diol epoxides of additional PAHs: chrysene (CDE), 5-methylchrysene (5-MCDE), 6-methylchrysene (6-MCDE), benzo[c]phenanthrene (B[c]PDE), and benzo[g]chrysene (B[g]CDE) within exons 5, 7, and 8 of the p53 gene in human bronchial epithelial cells. RESULTS: CDE exposure produced only low levels of adducts. Exposure of cells to the other activated PAHs resulted in DNA damage patterns similar to those previously observed with B[a]PDE but with some distinct differences. 5-MCDE, 6-MCDE, B[g]CDE, and B[c]PDE efficiently induced adducts at guanines within codons 154, 156, 157, 158, and 159 of exon 5, codons 237, 245 and 248 of exon 7, and codon 273 of exon 8, but the relative levels of adducts at each site varied for each compound. B[g]CDE, B[c]PDE, and 5-MCDE induced damage at codon 158 more selectively than 6-MCDE or B[a]PDE. The sites most strongly involved in PAH adduct formation were also the sites of highest mutation frequency (codons 157, 158, 245, 248, and 273). CONCLUSION: The data suggest that PAHs contribute to the mutational spectrum in human lung cancer.

Expression of base excision repair enzymes in rat and mouse liver is induced by peroxisome proliferators and is dependent upon carcinogenic potency.

Elevated and sustained cell replication, together with a decrease in apoptosis, is considered to be the main mechanism of hepatic tumor promotion due to peroxisome proliferators. In contrast, the role of oxidative stress and DNA damage in the carcinogenic mechanism is less well understood. In view of possible induction of DNA damage by peroxisome proliferators, DNA repair mechanisms may be an important factor to consider in the mechanism of action of these compounds. Here, the ability of peroxisome proliferators to induce expression of base excision repair enzymes was examined. WY-14,643, a potent carcinogen, increased expression of several base excision DNA repair enzymes in a dose- and time-dependent manner. Importantly, expression of enzymes that do not repair oxidative DNA damage was not changed. Moreover, less potent members of the peroxisome proliferator group had much weaker or no effects on expression of DNA repair enzymes when compared with WY-14,643. Collectively, these data suggest that DNA base excision repair may be an important factor in peroxisome proliferator-induced carcinogenesis and that induction of DNA repair might provide further evidence supporting a role of oxidative DNA damage by peroxisome proliferators.

Use of UvrABC nuclease to quantify benzo[a]pyrene diol epoxide-DNA adduct formation at methylated versus unmethylated CpG sites in the p53 gene.

We have used the UvrABC nuclease incision method in combination with ligation-mediated polymerase chain reaction (LMPCR) techniques to map and quantify (+/-)anti-7beta, 8alpha-dihydroxy-9alpha, 10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]-pyrene (BPDE) adduct formation in the p53 gene of human cells. We found that BPDE adduct formation, as revealed by UvrABC incision, preferentially occurred at methylated CpG sites that correspond to the mutational hotspots observed in human lung cancers. Our hypothesis is that it is this methylated CpG sequence-dependent preferential adduct formation, rather than selective growth advantage, that is the major determinant of the p53 mutation pattern in human cancers. Given the far reaching ramifications of such conclusions for cancer etiology, a legitimate question is raised regarding the reliability of using the UvrABC incision method for quantifying and determining the sequence-dependency of adduct formation. Is the higher frequency of UvrABC cutting at methylated versus unmethylated CpG sites due to the preference of the nuclease for cutting at those sites or due to the preferential formation of BPDE adducts at those sites? In order to distinguish between these two possibilities, we have analyzed the kinetics of UvrABC incision at BPDE adducts formed at either methylated CpG sites versus other sequences, or unmethylated CpG sites versus other sequences in exon 5 of the p53 gene. We have found that the UvrABC cutting kinetics are identical for both cases. On the basis of these results we conclude that under proper cutting conditions, UvrABC nuclease reacts with and incises with equal efficiency, BPDE adducts formed at methylated or unmethylated CpG sites as well as other sequences, and that the extent of UvrABC incision accurately reflects the extent of BPDE-DNA adduct formation. These conclusions were further supported by results obtained using a DNA synthesis blockage assay.

Quantitation and mapping of aflatoxin B1-induced DNA damage in genomic DNA using aflatoxin B1-8,9-epoxide and microsomal activation systems.

Aflatoxin B1 (AFB1) is a mutagenic and carcinogenic mycotoxin which may play a role in the etiology of human liver cancer. In vitro studies have shown that AFB1 adducts form primarily at the N7 position of guanine. Using quantitative PCR (QPCR) and ligation-mediated PCR (LMPCR), we have mapped total AFB1 adducts in genomic DNA treated with AFB1-8,9-epoxide and in hepatocytes exposed to AFB1 activated by rat liver microsomes or human liver and enterocyte microsomal preparations. The p53 gene-specific adduct frequencies in DNA, modified in cells with 40-400 microM AFB1, were 0.07-0.74 adducts per kilobase (kb). In vitro modification with 0. 1-4 ng AFB1-8,9-epoxide per microgram DNA produced 0.03-0.58 lesions per kb. The adduct patterns obtained with the epoxide and the different microsomal systems were virtually identical indicating that adducts form with a similar sequence-specificity in vitro and in vivo. The lesions were detected exclusively at guanines with a preference towards GpG and methylated CpG sequences. The methods utilizing QPCR and LMPCR thus provide means to assess gene-specific and sequence-specific AFB1 damage. The results also prove that microsomally-mediated damage is a suitable method for avoiding manipulations with very unstable DNA-reactive metabolites and that this damage can be detected by QPCR and LMPCR.

Formation and repair of DNA lesions in the p53 gene: relation to cancer mutations?

The number and diversity of mutations in the p53 mutation data base provides indirect evidence that implicates environmental mutagens in human carcinogenesis. The p53 gene has a large mutational target size; more than 280 out of 393 amino acids are found mutated in tumors. We argue that there is possibly a limited involvement of selection for specific mutations in the central domain of the protein, and that the distribution of DNA damage along the p53 gene caused by environmental carcinogens can be correlated with the mutational spectra, i.e., hotspots and types of mutations, of certain cancers. This concept has been validated by experiments with sunlight and the cigarette smoke component benzo[a]pyrene representing the polycyclic aromatic hydrocarbon class of carcinogens. The damage/repair data obtained for these mutagens can predict certain parameters of the mutational spectra including the distribution of hotspots in human nonmelanoma skin cancers and lung cancers from smokers. Future studies with suspected mutagens may help to implicate causative agents involved in other cancers, such as colon and breast cancer, where the exact carcinogen has not yet been identified but an environmental factor is suspected.

Slow repair of bulky DNA adducts along the nontranscribed strand of the human p53 gene may explain the strand bias of transversion mutations in cancers.

Using UvrABC incision in combination with ligation-mediated PCR (LMPCR) we have previously shown that benzo(a)pyrene diol epoxide (BPDE) adduct formation along the nontranscribed strand of the human p53 gene is highly selective; the preferential binding sites coincide with the major mutation hotspots found in human lung cancers. Both sequence-dependent adduct formation and repair may contribute to these mutation hotspots in tumor tissues. To test this possibility, we have extended our previous studies by mapping the BPDE adduct distribution in the transcribed strand of the p53 gene and quantifying the rates of repair for individual damaged bases in exons 5, 7, and 8 for both DNA strands of this gene in normal human fibroblasts. We found that: (i) on both strands, BPDE adducts preferentially form at CpG sequences, and (ii) repair of BPDE adducts in the transcribed DNA strand is consistently faster than repair of adducts in the nontranscribed strand, while repair at the major damage hotspots (guanines at codons 157, 248 and 273) in the nontranscribed strand is two to four times slower than repair at other damage sites. These results strongly suggest that both preferential adduct formation and slow repair lead to hotspots for mutations at codons 157, 248 and 273, and that the strand bias of bulky adduct repair is primarily responsible for the strand bias of G to T transversion mutations observed in the p53 gene in human cancers.

The p53 codon 249 mutational hotspot in hepatocellular carcinoma is not related to selective formation or persistence of aflatoxin B1 adducts.

Sequence-dependent formation and lack of repair of polycyclic aromatic hydrocarbon-induced DNA adducts correlates well with the positions of p53 mutational hotspots in smoking-related lung cancers (Denissenko et al, 1996, 1998). The mycotoxin aflatoxin B1 (AFB1) is considered to be a major causative agent in hepatocellular carcinoma (HCC) in regions with presumed high food contamination by AFB1. A unique mutational hotspot, a G to T transversion at the third base of codon 249 of the p53 gene is observed in these tumors. To test whether a selectivity of AFB1 adduct formation is related to this peculiar mutational spectrum, we have mapped AFB1-DNA adducts at nucleotide resolution using ligation-mediated PCR and terminal transferase-dependent PCR. Human HepG2 cells were exposed to AFB1 metabolically activated in the presence of rat liver microsomes. Significant adduct formation was seen at the third base of codon 249. However, this was not the major site of AFB1 adducts and strong adduction was also observed at codons 226, 243, 244, 245 and 248 in exon 7 of the p53 gene and at several codons in exon 8. The damage at codon 249 does not consist of a unique abasic site or ring-opened aflatoxin B1 adduct but rather is consistent with the principal N7-guanine adduct of AFB1. Time course experiments indicate that, under the conditions used, AFB1 adducts are not removed in a strand-selective manner and adduct removal from the third base of codon 249 proceeds at a relatively fast rate (50% in 7 h). The incomplete correspondence between sites of persistent AFB1 damage and the specific codon 249 mutation suggests that AFB1 may not be involved in mutation of this site or that additional mechanisms such as parallel infection with hepatitis B virus may be required for selection of codon 249 mutants in HCC.

PCR-based approaches to adduct analysis.

Ligation-mediated polymerase chain reaction (LMPCR) is a PCR-based method for the detection of DNA adducts at individual nucleotide positions in mammalian genes. Adduct-specific enzymes, such as T4 endonuclease V, various base excision repair enzymes, UvrABC nuclease, and chemical cleavage techniques can be used to convert the adducts into DNA strand breaks. The positions of these breaks are then detected by LMPCR. This method has been used primarily to map the distribution of UV-induced DNA lesions and adducts of polycyclic aromatic hydrocarbons. The number and diversity of mutations in the p53 mutation database provides indirect evidence that environmental mutagens may be involved in human carcinogenesis. We hypothesize that there is a limited involvement of selection for specific mutations in the central domain of the p53 protein, and that the distribution of DNA damage along the p53 gene caused by environmental carcinogens can be correlated with the mutational spectra, i.e. hotspots and types of mutations, of certain cancers. This concept has been validated by experiments with sunlight and the cigarette smoke component benzo[a]pyrene representing the polycyclic aromatic hydrocarbon class of carcinogens. The damage and repair data obtained for these mutagens can predict certain parameters of the mutational spectra of human non-melanoma skin cancers and lung cancers from smokers. Future studies with suspected mutagens may help to implicate causative agents involved in other cancers, where the exact carcinogen has not yet been identified but an environmental factor is suspected.

Sunlight induces pyrimidine dimers preferentially at 5-methylcytosine bases.

The most prevalent DNA lesion induced by UV irradiation is the cyclobutane pyrimidine dimer (CPD), which forms at positions of neighboring pyrimidines. Here we show that the rare DNA base 5-methylcytosine is the preferred target for CPD formation when cells are irradiated with natural sunlight. We have mapped the distribution of CPDs formed in normal human keratinocytes along exons of the p53 gene. Codons 196, 245, 248, and 282, which are mutational hot spots in skin cancers, are only weakly to moderately susceptible to formation of CPDs after irradiation with UVC (254 nm) or UVB (320 nm) light sources. However, when cells were exposed to natural sunlight, CPD formation was enhanced up to 15-fold at these codons due to the presence of 5-methylcytosine bases. These results suggest that CPDs containing 5-methylcytosine may play an important role in formation of sunlight-induced skin tumors and that methylation of CpG sequences, besides being involved in spontaneous mutagenesis processes, can also create preferential targets for environmental mutagens and carcinogens.

Cytosine methylation determines hot spots of DNA damage in the human P53 gene.

In the P53 tumor suppressor gene, a remarkably large number of somatic mutations are found at methylated CpG dinucleotides. We have previously mapped the distribution of (+/-) anti-7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy -7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) adducts along the human P53 gene [Denissenko, M. F., Pao, A., Tang, M.-s. & Pfeifer, G. P. (1996) Science 274, 430-432]. Strong and selective formation of adducts occurred at guanines in CpG sequences of codons 157, 248, and 273, which are the major mutational hot spots in lung cancer. Chromatin structure was not involved in preferential modification of these sites by BPDE. To investigate other possible mechanisms underlying the selectivity of BPDE binding, we have mapped the adducts in plasmid DNA containing genomic P53 sequences. The adduct profile obtained was different from that in genomic DNA. However, when cytosines at CpG sequences were converted to 5-methylcytosines by the CpG-specific methylase SssI and the DNA was subsequently treated with BPDE, adduct hot spots were created which were similar to those seen in genomic DNA where all CpGs are methylated. A strong positive effect of 5-methylcytosine on BPDE adduct formation at CpG sites was also documented with sequences of the PGK1 gene derived from an active or inactive human X chromosome and having differential methylation patterns. These results show that methylated CpG dinucleotides, in addition to being an endogenous promutagenic factor, may represent a preferential target for exogenous chemical carcinogens. The data open new avenues concerning the reasons that the majority of mutational hot spots in human genes are at CpGs.

Preferential formation of benzo[a]pyrene adducts at lung cancer mutational hotspots in P53.

Cigarette smoke carcinogens such as benzo[a]pyrene are implicated in the development of lung cancer. The distribution of benzo[a]pyrene diol epoxide (BPDE) adducts along exons of the P53 gene in BPDE-treated HeLa cells and bronchial epithelial cells was mapped at nucleotide resolution. Strong and selective adduct formation occurred at guanine positions in codons 157, 248, and 273. These same positions are the major mutational hotspots in human lung cancers. Thus, targeted adduct formation rather than phenotypic selection appears to shape the P53 mutational spectrum in lung cancer. These results provide a direct etiological link between a defined chemical carcinogen and human cancer.

Site-specific induction and repair of benzo[a]pyrene diol epoxide DNA damage in human H-ras protooncogene as revealed by restriction cleavage inhibition.

Most genotoxic DNA base modifications localized at key genomic sequences constitute the molecular alterations crucial or mutagenesis and tumorigenesis. We have utilized lesion-rendered inhibition of restriction endonuclease cleavage for the analysis of site-specific DNA damage induced by (+/-)-7,8-dihydroxy-anti-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (benzo[a]pyrene diol epoxide, anti-BPDE) in human genes. The H-ras protooncogene and insulin gene sequences were used as targets for modification in vitro and in vivo. Selective induction of individual facultative bands, resulting from covalent modification of the cognate recognition sites, was observed in modified plasmid DNA for a number of restriction nucleases. The ras gene-specific damage, at the PstI, BstYI, NotI and BstEII recognition sites, was visualized and quantitated in human genomic DNA adducted by anti-BPDE. Repair of lesions at hexanucleotide sequences and/or regions surrounding the restriction site, was assessed as a gradual disappearance of facultative bands in DNA from repair-proficient human fibroblasts exposed to the carcinogen in confluent culture. Efficiency of the PstI site-specific repair was compared at low and high levels of initial damage. Higher genotoxic dose caused a decrease in the extent of adduct removal from the bulk DNA, while the specific site of the ras gene was still subject to fast repair. No measurable PstI site-specific repair was detected in the insulin gene. These results show the region-selective induction of bulky anti-BPDE DNA damage in non-related genomic targets and suggest that repair of these lesions in human cells proceeds with the efficiency tightly controlled at different levels of initial genotoxic load.

Assessment of DNA damage and repair in specific genomic regions by quantitative immuno-coupled PCR.

Fine analysis of DNA damage and repair at the subgenomic level has indicated a microheterogeneity of DNA repair in mammalian cells, including human. In addition to the well established Southern hybridization-based approach to investigate gene-specific DNA damage and repair, alternative methods utilizing the sensitivity of PCR have been evaluated. The latter technique has relied on decreased PCR amplification due to damage in template DNA. We have developed a novel quantitative assay combining the selective recovery of DNA damage containing genomic fragments with the PCR amplification. DNA isolated from 7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) treated human skin fibroblasts was immunoprecipitated with polyclonal antibody BP-1. Recovered target sequences were amplified by PCR using primers encompassing a 149 bp target region around codon 12 of the H-ras proto-oncogene. Quantitative DNA damage specific response was observed with nanogram amounts of genomic DNA. This approach allowed analysis of the initial DNA damage at a level less than 1 anti-BPDE adduct per 6.4 kbp ras gene fragment. Repair proficient GM637 cells exposed to 2 microM anti-BPDE showed a faster removal of the adducts from the H-ras gene segment than from the genome overall. Gene-specific repair was not apparent in GM4429 xeroderma pigmentosum (complementation group A) cells. The established technique could be extended to the quantitative measurement of the repair of diverse DNA base lesions in any genomic region of known sequence.

Rapid activation of apoptosis in human promyelocytic leukemic cells by (+/-)-anti-benzo[a]pyrene diol epoxide induced DNA damage.

Genotoxic damage in responsive mammalian cells is implicated as a critical event in the induction of apoptosis. We have evaluated the time course of activation of apoptosis in HL-60 cells following treatment with (+/-)-anti-BPDE metabolite, a well established DNA damaging carcinogen. Programmed cell death, determined by typical cellular and molecular markers of apoptosis, was apparent with 1.5 h following treatment with varying concentrations of (+/-)-anti-BPDE. The fraction of apoptotic cell population and corresponding DNA fragmentation indicated a minimum threshold damage (approximately 1-2 x 10(-5) adduct/base or about 1 putative lesion/human gene) necessary to elicit apoptosis. Suppression of apoptosis (60-90% inhibition of DNA fragmentation) by 3-aminobenzamide and lack of an effect by aphidicolin indicated the role of poly(ADP-ribosylation) but not of blocked DNA replication. Our data suggest that DNA damage triggers an acute response that sets the responsive cells on path of an "immediate apoptotic" mode of cell death.