Tobacco Documents Online

START OF DOCUMENT O0 0 0 I~o AQ12697

• Science On-Line: Mikhail F. Denissenko, et al... Page 1 of 5 LGo to SCIENCE On-Line wi~h fewer ~phicsl next MAGAZINE Preferential Formation of Benzo[a]pyrene Adducts at Lung Cancer Mutational Hotspots in P53 Mikhail F. Denissenko, Annie Pao, Moon-shong Tang," Gerd P. Pfeifer * 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 nucleofide 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. AQ12697 M. F. Denissenko and G. P. Pfeifer, Department of Biology, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA. A. Pao and M.-s. Tan& M. D. Anderson Cancer Center, University of Texas, Science Park, Smithville, TX 78957, USA. * To whom correspondence should be addressed. Lung cancer is currently the leading cause of cancer death in the United States and is also the most common type of tumor worldwide. Tobacco smoking is the single most important risk factor for lung cancer. Among the many components of cigarette smoke, polycyclic aromatic hydrocarbons are. strongly implicated as causative agents in the development of these cancers (~). Benzo[a]pyrene, which occurs in amounts of 20 to 40 ng per cigarette, is by far the best studied of these compounds and is one of the most potent mutagens and carcinogens known. The compound requires metabolic activati.on to become the ultimate carcinogenic metabolite, BPDE [(±)-anti-7[l,So; -dihydroxy-9~, 1 (k -epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene], which binds to DNA and forms predominantly covalent (+) tram adducts at the N2 position ofguanine 2(~). About 60% of human lung cancers contain mutations in the P53 tumor suppressor gene (3..). The P53 mutation database 4(~) includes more than 500 entries of sequenced P53 mutations for lung cancer. There is a large percentage of G to T transversion mutations in these tumors. Such mutations are hallmarks of mutagenesis involving certain types of p01ycyclic aromatic hydrocarbons, including BPDE 0.), but they can also be induced by other agents, including oxidative DNA damase (~. The distribution of mutations along the P53 gene in lung cancer is nonrandom but rather is characterized by several mutational hotspots, in particular, at codons 157, 248, and 273 (Fig. I), which correspond http:~www~sci~nc~mag~rg/science~scripts/disp~ay~fu~/274~5286~43~.htm1/&search-terms=benzd~/~a~ung+~ Oo 0 0 ~d 4~ O~

Science On-Line: MikhaJl F. Denissenko, et al... Page 2 of S to amino acids within the DNA binding domain of p53. Codon 157 is a mutational hotspot specific for lung cancer and does not occur as a hotspot in any other cancer, but the other two codons are affected in many different tumor types (~, 7). The majority oflung cancer mutations at these three co..~don positions are O to T transversions (~). Fig. 1. Frequency of P53 mutations in lung cancer by codon position. Numbers were obtained from the P53 database 4(~). Radon-associated lung cancers and cancers from nonsmokers were excluded. The sequences surrounding the mutational hotspot codons 157, 248, and 273 are indicated. The asterisks mark the mutated Gs within these codons. [Vi~0v Larger Version of this Image (22K GIF file)| To investigate the relation between BPDE adduct formation and PS,~ mutations, we mapped the distribution ofBPDE adducts along the P.f3 gene using a modification of the ligation-mediated polymerase chain reaction (LMPCR) C~). HeLa cells were treated with various concentrations of BPDE (_~), and DNA was isolated and cleaved at the sites of modified bases with the UvrABC nudease complex from Escher/c/da coil (LQ). UvrABC makes a dual incision 5~ and 3~ to the adducted base, and the 3~ incision occurs specifically at the fourth nudeotide position 3~ to a BPDE adduct (L].). These break positions can then be visualized by LMPCR in which PS.~-speC-tfic • oligonudeofide primers were used 1(~ J~. Figure 2A shows an analysis ofthe upper (nontranscribed) DNA strand of exon 5. One of the strongest BPDE-dedved signals along the exon is seen at codon 157, which is one of the major mutational hotspots in lung cancer. In ex0n 7, the two guanine positions within the frequently mutated codon 248 are the preferred uu'gets for BPDE adduct " formation (Fig.2~B). The same is true for exon 8, where the strongest signal corresponds to a BPDE adduct at the guan~e within the mutational hotspot codon 273 (Fig. 2C). l i:i:~,~.~. ~'~-.~:....,.~..~,,;':,.~.,.~:,:_,~. Fig. 2. Distribution ofBPDE adducts alongP53 exons in HeLa the distribution ofedducts in P53 was determ~ed after cleavage i~ .~'-~'~ ~ with UvrABC nuclease and LMPCR l(.!.L l_Z). Adduct-specilic bands ,, ! ~ i migrate four nuclcotide positions faster than the corresponding ~ • bands in the Maxam-C-ilbert sequencing ladders (left three lanes). Some bands in the sequencing lanes are absent because 5-methy|c~Ttosines arc not cleaved (_~). (A) Exon 5, nontranscribed strand. (B) Exon 7, nontranscribed strand. (C) Exon 8, nontranscribed strand. Brackets indicate the positions of selected P53 codons. Asterisks mark the strongly modified G positions within codons 157, 248, and 273~ [View I.,m-gex Version of this Image (65K G~ file)] To analyze a cell type that is more representative of the target cell population during lung tissue transformation~ we performed similar experiments with normal human bronchial epithelial cells (I__~). The BPDE adduct patterns were generally similar between HeLa cells and normal bronchial cells. Most important, the adduct hotspots were the same in the bronchial epithelial c.~ls (Fig. 3_). AQ12697 http://www, scienccmag.org/science/scripts/display/full/274/5286/43 O. html/&search_terms=benz~O~idg~+lung+l 0 0 I~0 -!~ O)

Science On-Line: Mikhail F. Denissenko, et al... Page 3 of 5 Fig. 3. Distribution ofBPDE adducts along P53 exons in bronchial epithelial cells. Cells were treated with 4 ~tM of BPDE for 30 rain, and the distribution of adducts in P5£ was determined after cleavage with UvrABC nuclease and LMPCR. (A) Exon 5, nontranscribed strand. (B) Exon, 7, nontranscribed strand. (C) Exon 8, nontranscribed strand. Asterisks mark the strongly modified O positions within codons 157, 248, and 273. [View Larger Version of this Image (90K G[F file)] To test whether the sequence specificity is related to ¢hromafin structure, we comparod the adduct pattern in BPDE-treated HeLa cells with the pattern in BPDE-treated isolated genomic DNA. The two patterns were almost identical (.L~, which excludes chromatin structure as a major modulating factor for the cell types analyzed. It should be noted that the histogenesis of the different types of lung cancer is incompletely understood. Therefore, it is important that a similar adduct pattern was seen in three different celt types: HeLa cells (Fig. 2_), bronchial cells (Fig. 3_), a~A normal human fibroblasts (.~). This pattern does not appear to be greatly modified by cell type-specific chromatin structure, which suggests that ~he same adduct pattern is likely to be present in the unidentified target cells for lung tissue transformation. Strong selectivity of BPDE binding at guanine positions in codons 157, 248, and 273 was not obscured in previous experiments in which a DNA polymerase fingerprint assay was used to detect adducts formed in carcinogen-treated plasmid DNA (~). The apparent discrepancies between our findings and those of this previous study could be due to different methylation patterns in/~ coil versus human DNAs; however, the discrepancies may also arise from differences in specificity and sensitivity of the detection method: The BPDE adduct hotspots are on the nonwanscribed DNA strand, which is expected to be repaired relatively inefficiently, according to the concept of transcription-coupled repair (~ 1_.~). A strand bias in repair is consistent with the majority (>9¢B4) of G to T mutations in lung cancer attributable to guanines on the nontranscribed strand (~, 4_). Codon 179, which is also fiequenfly mutated in lung tumors, is not a strong target for BPDE adduct formation (Fig. _2A). However, this oodon does not contain a guanine on the nontranscribed strand,, and the majority of mutations are A to O transitions at the second codon base 4f~). BPDE binds to guanine 20 times more etBdently than to adenine; thus, it is likely that these mutations are caused by another mutagenic component of cigarette smoke. Pronounced adduct formation was observed at codon 267 (Figs. 2C and 3_C), for which there is only one mutation entry in the P53 database. Here, the most strongly adducted base corresponds to the third codon position (CC~), and a mutation would not produce an amino acid change. It has been generally assumed that P$3 cancer mutations oc~.r frequently at specific codons because they are selected for in the cell transformation process. One possibility is that mutational hotspot codons are sites of preferred gain of function mutations or sites that are most important for tumor suppressor function of the protein. The presence of mutational hotspots has been correlated with crystal structure data obtained from a p53 protein-DNA complex (19). The most frequently mutated amino acids (residues 248 and 273) contact DNA directly, whereas some of'the other commonly mutated amino acids contribute to stabilization of the protein 1O~. In lung cancers, mutations in the http://www, sciencemag.~rg/s~ienee/scripts/disp~ay/fu~/274/52~6/43~.htm~/&search-terms=benzd~h/~+~ung+~ O0 0 0 O~ AQ12697

Science On-Line: Mikhail F. Denissenko, et al... Page 4 of 5 P$3 gene are found at more than 100 different sequence positions ffig. J.), and it is likely that all of these mutations can provide a growth advantage. These results, together with our current finding that P$3 mutation hotspots 157, 248, and 273 act as selective BPDE binding sites, suggest that P53 mutation hotspots are preferential targets for DNA-damaging agents and that selection may not necessarily play a major role in the occurrence of mutations at these sites. It is also of interest that two of the adduct hotspots (at codom 248 and 273) are at positiom that are common mutational sites not only in lung cancer but also in many other cancers. Almost all of the adduct hotspots were at CpO dinudeofides, although not all CpO sites were strong binding sites for BPDE. Because the CpG rites in the P$3 gene are methylated in every human tissue or cell type examined ~ g.Q.), the preferentially adducted sequence in vivo is 5-raethyl-CpG. Whether selective DNA damage also plays a role in the frequent occurrence oftmnsition mutations at specific CpO codons (codons 175, 245, 248, 273, and 252) remains to be determined. The coincidence of mutational hotspots and adduct hotspots suggests that benzo[a]pyrene metabolites or structurally related compounds are involved in tranfformation of human lung tissue. Our study thus provides a direct link between a defined cigarette smoke carcinogen and human cancer mutations. REFERENCES AND NOTES 1. S. S. Hecht, S. G. C, armella, S. E. Murphy, P. G. Foiles, F.-L. Chunlg J. Cell. Biochen~ SuppL 17F, 27 (1993) ~_~[]i~f~. 2. B. Singer and D. Gnmberger, Molecular Biology of Mutagens and Carcinogens (Plenum, Yodg 1983). 3. M. Hollstein, D. Sidransky, B. Vogelstein, C. C. Harrig ~.~cience 253, 49 (1991) [Medline]: M_ S. Greenblatt, W. P. Bennett, M HoIlstein, C. C. ~ Cancer Re& 54, 4855 (1994) 4. M. Hollstein eta/., Nucleic Acids Res. 24, 141 (1996) _[Medline]. 5. E. Eisenstadt, A. J. Warren, J. Porter, D. Atkins, J. H. l~Edlef0 Proc. NatL dcaal Sci. U.~A. 79, 1945 (1982) IMedline]; M. Mazur and B. G~ckma~ Somatic CelI MOl. Genet. 14, 393 0988) [M.~;[l~f.]; R.-H. Chert, V. M. Maher, J. J. McCormick, Proc. Natl. dead Sci. U.S.d. 87, 8680 (1990) IMedlinel: B. Ruggeri et al., ibid. 90, 1013 (1993) [Medline]. 6. T. Lindahl, Nature 362, 709 (1993) ['Medline]. 7. A. J. Levine et al.,An~ N.Y. dead Sci. 768, 111 (1995) IMedlin~. 8. G.P. Pfeifer, R. Drouin, A. D. Riggs, G. P. Holmquist, Pro~. Natl. dead SoL U.S.d. 88, 1374 (1991) [Medline). 9. HeLa $3 cells (American Type Culture Collection, Rookville, MD) were treated with medium containing 1, 2~ or 4 pM of freshly prepared BPDE (obtained from the National Cancer Institute repository, Midwest Research Institute, Kansas City, MO) for 30 rain at 37"C in the dark [ S. Venkataohalam, M. Denissenko, A. A. Wani, Carcinogenesis 16, 2029 (1995) [Medline]]. Controls were treated with solvent only (95% ethanol). 10.A. Sancar and M.-s. Tang. Photochem. Photobiol. 57, 905 (1993); B. Van Houten and A. Snowden, Bioessays 15, 51 (1993) [Me~.line]. 11.The purified DNA was treated with UvrABC (a 10-fold molar excess of protein over ! 04 nucleotides of DNA) under standard reaction conditions as described [M.-s. Tang, in Technologies for Detection of DNd Damage and Mutations, G. P. Pfeifer, Ed. (Plenum, New York, 1996), pp. 139-153]. After 90 min of incubation at 37"C, the proteins were removed by http~www.s~ien~emag.~rg~science/scripts~disp~ay~fu~274~5286/43~.htr~&search-terms=benz~+~ung+~ 0 4~ AQ 12697

• Science On-Line: Mikhail F. Denissenko, et al... Page 5 of 5 phenol extractions followed by diethyl ether extraction, and the DNA was purified further by repeated ethanol precipitations. UvrABC nuelease incises six to seven bases 5' and four bases 3' to a BPDE-modified purine, and under these reaction conditions, the cleavage at BPDE-DNA adducts by UvrABC nucleases is quantitative [M.-s. Tang, J. R. Pierce, R. P. Doisy, M. E. Nazimie~, M. C. MacLeod, Biochemistry 31, 8429 (1992)]. These results validate the UvrABC incision method for 0nalysis of the sequence selectivity of BPDE binding. Because the UvrABC incision at the 3' side ofBPDE-DNA addu~ts is very specific (four bases 3' to the adduct), LMPCR c4m be used to detenuine the BPDE addu~t distribution at nudeotide resolution. 12.UvrABC-induced strand breaks in the PS.~ 8ene we~, detected by use of LM~CR with P$3-specific primers [ S. Tornaletti, D. Rozek, G. P. Pfeifer, Oncogene 8, 2051 (1993) [Medli~¢]; S. Tornalet~i and G. P. Pfeifer, ~cience 263, 1436 (1994) I'Medlin~]. 13.S. Tornale~ti and G. P. Pfeifer, Onc.o~ene 10, 1493 (1995) _fMedline]. 14.Non~ml human bronchial epithelial ~ells (Clonetics, San Diego, CA) were ~ultured in growth medium recommended by the supplier. The c~gs were treated with 4 tam of BPDE as des~ibed 15.M F. Deni~enko, A. Pao, M_-s. Tan& G. P. Pfeifer, unpublished observations. 16./~ Puisieux, S. Lira, J. G-roopman, M. Ozturk, Can~ Res. 51, 6185 (1991) l~[edline~]. 17.I. Mellon, G. Spivak, P. C. I-Ianawalt, Cell $1, 241 0987) IMedline_]. 18.R.-K Chen, V. M. Maher, J. Brouwer, P. vande Putte, J. J. M~ormick, Proc. NatL Aca~ Sci. U.S.A. 8~, ~[3 ([992) [Medline]. 19.Y. Cho, S. Gorina, P. D. Jeffrey, N. P. Pavletich, Science 265, 346 (1994) lMedline]. 20.W.M. Rideout !~ G. A. Coetzee, A. F. Olumi, P. A. Jones, ~bi,~ 249, 1288 (1990) fMedline]. 21.We thank S. Bates for cell culture work. Supported by ~ gnmts CA65652 to G.P.P. and ES03124 to M.-s. T. 29 May 1996; accepted 19 August 1996 Volume 274, Number 5286, Issue ef 18 October 1996, pp. 430-432 01996 by The American Asmciatlen fer the Advancement of Science. AQ12697 http~//www~seiene~mag~rg~se~enee/s~ripts~disp~ay/fu~274/52$6~43~htm~&seaxch-terms=benzx~d1~a~-~ung+~ Oo 0 0 -I~