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Proc. Nadl. Acad. Sci. USAVol. 86, pp. 8328-8332, November 1989Biochemistry

Isolation of cDNAs encoding a human protein that binds selectivelyto DNA modified by the anticancer drug cis-diammine-dichloroplatinum(II)

(DNA binding protein/DNA damage/platinum complexes/cisplatin)

JEFFREY H. TONEY*, BRIAN A. DONAHUEt, PATTI J. KELLETT*, SUZANNE L. BRUHN*,JOHN M. ESSIGMANN*t, AND STEPHEN J. LIPPARD**Department of Chemistry and tWhitaker College of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA 02139

Contributed by Stephen J. Lippard, August 3, 1989

ABSTRACT DNA modified by the antitumor drug cis-diamminedichloroplatinum(II) (cis-DDP or cisplatin) was usedto identify a factor in mammalian cells that binds to cis-DDP-damaged DNA and hence may play a role in repair. Thisfactor selectively recognizes double-stranded DNA fragmentsmodified by cis-DDP or [Pt(en)CI2J (en, ethylenediamine).Little or no binding occurs to unmodified double-strandedDNA or to DNA modified with the clinically ineffective com-pounds trans-DDP and [Pt(dien)Cl]Cl (dien, diethylenetri-amine). Low levels of binding to single-stranded DNA modifiedby cis-DDP are observed. The apparent molecular mass of thefactor in a variety of mammalian cells is -100 kDa, asdetermined by modified Western blotting. Two recombinantphage have been isolated from a human B-cell Agtll library byusing a cis-DDP-modified DNA restriction fragment as a probe.The two clones have insert sizes of 1.88 and 1.44 kilobases andare aligned at their 5' ends. The polypeptides encoded by therecombinant phage exhibit DNA binding properties similar tothose of the cellular factor identified in crude extracts preparedfrom mammalian cells. Northern analysis with one ofthe clonesrevealed an mRNA of 2.8 kilobases that is conserved in humansand rodents. The methods used here should be applicable instudies of other damage-specific DNA binding proteins.

cis-Diamminedichloroplatinum(II) (cis-DDP or cisplatin) is aclinically important antitumor drug used for the treatment ofseveral human cancers, especially those of genitourinaryorigin (1). The biological target for cis-DDP is generallyaccepted to be DNA (2), and considerable information is nowavailable on binding of the drug to DNA and the structures ofits major adducts (3). Despite this progress, much remains tobe learned about the specificity of cis-DDP for certain tumorsas well as its activity relative to other platinum compounds.Both cis-DDP and its biologically inactive trans isomer

bind to DNA in vitro and in vivo, and the resultant adductscan serve as termination sites for DNA polymerase (4). DNAreplication is inhibited to similar extents by both cis andtrans-DDP in vivo at the same bound drug/nucleotide ratioItew vaWts71 * _sIr. %V% rT /--. "I'It / ss1 s- * X s

process or repair the DNA adducts of these various platinumcomplexes.To address this issue, we set out to identify cellular factors

that could play a role in processing DNA modified byplatinum(II) complexes. Previously identified prokaryoticrepair systems such as the UvrABC excision nuclease areknown to excise adducts from DNA modified by cis- but nottrans-DDP (6). In addition, one component of the repaircomplex, UvrA, possesses specific binding properties forDNA modified by damaging agents such as ultraviolet radi-ation (7, 8). It therefore seemed reasonable that, in mamma-lian cells, a similar damaged DNA binding factor would existhaving sufficient generality to recognize cis-DDP modifiedDNA as an initial step leading to repair.Our approach has involved the use of DNA modified by

cis-DDP as a probe for DNA binding factors present in crudemammalian cellular extracts. By exploiting recently devel-oped methodologies used to study sequence-specific DNAbinding factors (9), we have identified a cellular factor thatselectively binds to DNA modified by cis-DDP or [Pt(en)C121.Related cellular factors that bind specifically to DNA mod-ified by ultraviolet radiation or cis-DDP were reported whilethis work was in progress (10).As will be described separately (B.A.D., J.H.T., J.M.E.,

and S.J.L., unpublished results), cis-DDP- or [Pt(en)CI2]-modified DNA restriction fragments incubated with crudehuman cellular extracts exhibit pronounced mobility shiftswhen studied by gel electrophoresis. This behavior wasascribed to the presence of a human cellular factor that bindsDNA modified by these compounds and suggested that itmight be possible to isolate cDNA clones by using cis-DDP-modified DNA as a probe. As reported here, thisapproach proved to be successful, and clones encoding thecis-DDP-modified DNA binding domain of the factor are nowin hand.

MATERIALS AND METHODS(5). Why cis-IJJJJ, [Pt(en)CI2J (en, ethylenedliamine), and Preparation of Platinum Compounds and Platinated DNAcertain other complexes that form 1,2-intrastrand crosslinks Restriction Fragments. The compounds cis-DDP, trans-DDP,on DNA are biologically active, whereas trans-DDP and [Pt(dien)Cl]Cl, and [Pt(en)CI2] were prepared as described[Pt(dien)Cl]l (dien, diethylenetriamine) are not, is of partic- (11-14). The DNA probe used in these studies was a 422-ular interest. One possible reason is that cells differentially base-pair (bp) Ava I restriction fragment from M13mp18,

purified from low-melting-point agarose gels by phenol ex-H3N Cl H2N +HN Ns traction, followed by butanol extraction and ethanol precip-c \ H3N\ Hpt (NH itation. A portion of the DNA was allowed to react in 1 mMH3N' \Cl H2N' \ C NH' H C sodium phosphate/3 mM NaCl, pH 7.4 (buffer B) or 10 mMH3N ClNH3 2N Tris HCl, pH 7.5/1 mM Na2EDTA (TE) at a DNA phosphatecis-DDP Pt(en)C2] trans-DDP [Pt(dien)Cl]+ concentration of -10 nM, with the appropriate platinum

complex at a variety of formal drug/nucleotide (D/N) ratios,

Abbreviations: DDP, diamminedichloroplatinum(II); D/N, drug/nucleotide.

8328

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Proc. Nati. Acad. Sci. USA 86 (1989) 8329

at 370C for 12-16 hr in the presence of excess carrierM13mpl8 DNA. Unbound platinum complex was removedby ethanol precipitation of the DNA samples, followed byseveral washes of the pellet with 70% ethanol. Controlunplatinated DNA was processed the same way. Platinumand DNA concentrations were measured by flameless atomicabsorption spectroscopy and optical absorption spectropho-tometry, respectively.

Platinum-modified and control DNA fragments were la-beled with the Klenow fragment of DNA polymerase I and[a-32P]deoxycytidine triphosphate (>5000 Ci/mmol; 1 Ci =37 GBq; New England Nuclear) and purified by electropho-resis on native polyacrylamide gels (ref. 15, pp. 113, 178).Purity of the labeled DNA fragments was checked by nativepolyacrylamide gel electrophoresis.

Single-stranded DNA was prepared by boiling the unplat-inated, radiolabeled 422-bp restriction fragment and thenallowing the DNA to reanneal in the presence of a 10-foldmolar excess of M13mpl8 circular single-stranded DNA plusstrand. The 422-nucleotide plus strand was then resolved on,and isolated from, a native polyacrylamide gel and platinatedas described for the double-stranded DNA fragments.

Preparation of Crude Extracts. Mammalian nuclear andcytosolic extracts of HeLa cells (16) and Escherichia colilysogens (9) were prepared according to methods describedin the literature. Extracts used in these studies were derivedfrom either HeLa cells or E. coli strain SG 1161 (Ion-)lysogens, the latter being used to reduce proteolytic degra-dation of the B-galactosidase fusion protein/platinated DNAbinding factor.

Modified Western Blots. Extracts were prepared fromeither isopropyl f3-D-thiogalactopyranoside-induced lysogensor HeLa cells. Typically, 50 Ag of total protein per lane (17)was separated by SDS/PAGE (18) on an 8% separating geland transferred onto nitrocellulose (BA85; 0.45 um; Schlei-cher & Schuell) as described (19). After transfer, filters wereprocessed as described in the literature (9). To assay for DNAbinding, nitrocellulose filter-bound proteins were incubatedin binding buffer (30 mM Hepes-NaOH, pH 7.5/10 mMMgCl2/2 mM MnCl2/0.25% nonfat dry milk), using 20 ml per20 x 20 cm filter, with 32P-labeled DNA fragment (0.25-2.0x 10' cpm/ml; 10-100 pM). Poly(dI-dC)poly(dI-dC) wasadded as competitor for nonspecific DNA binding proteins at10 ,tg/ml or 40 ,uM. The incubations were run for 60 min atroom temperature with gentle agitation. In experiments inwhich single-stranded DNA was used as a probe, a mixtureof S ,ug/ml each of poly(dI-dC)-poly(dI-dC) and M13mpl8single-stranded (plus strand) DNA was used as competitor.Unbound DNA was then removed by washing the filterstwice at 4°C with binding buffer lacking MgCl2 and MnCl2.Protein-DNA complexes were detected by autoradiographywith the use of an intensifying screen at -80°C.

Screening a Human B-Cell Agt11 cDNA Library with cis-DDP-Modified DNA. Protein replica filters were prepared froman unamplified human B-cell cDNA library (Clontech) con-structed in the expression vector Agtll (20). The cDNA librarywas originally prepared by oligo(dT) priming ofpoly(A)+ RNA(21). Screening ofthe Agtll recombinants plated on E. coli hoststrain Y1090 was accomplished as described (9) with cis-DDP-modified 32P-labeled DNA. To screen clones for plati-nated DNA binding, each filter was incubated for 60 min atroom temperature in 10 or 25 ml ofTNE (10 mM TrisHCI, pH7.5/50 mM NaCI/1 mM Na2EDTA/1 mM dithiothreitol) in100- and 150-mm plates, respectively. The buffer contained32P-labeled DNA at a final concentration of -3 x 104 cpm/mlor 10 pM as well as both sonicated native and denatured calfthymus DNA with an average length of -1 kb at 1.0 and 5.0,ug/ml, respectively. The filters were then washed at roomtemperature three times for 10 min each with TNE, air dried,and autoradiographed at -800C with the use of an intensifying

screen for 24-48 hr. Putatively positive clones were re-screened for binding to cis-DDP-modified DNA. Secondaryscreens were carried out on 100-mm plates with plating mix-tures of -5 x 103 plaque-forming units of A phage, whiletertiary screens used plating mixtures of -100 plaque-formingunits. Two recombinant phage, APtl and APt2, were purifiedto homogeneity by this protocol.

Restriction Enzyme Mapping of Isolated cDNA Clones.Amplified phage stocks prepared from APtl and APt2 wereused to isolate A recombinant DNA (ref. 15, pp. 76-85). Eachclone was digested with a variety of restriction enzymes(International Biotechnologies; Bethesda Research Labora-tories). After electrophoretic separation, DNA fragmentswere transferred to a nitrocellulose filter (ref. 15, pp. 383-386). To determine homologies between the two cDNAclones, the filter was probed with a APt2 cDNA insert labeledwith [a-32P]deoxycytidine triphosphate by the Klenow frag-ment of DNA polymerase I (15). Hybridization was carriedout with 10% dextran sulfate in 50% (vol/vol) formamide for3 hr at 450C, and the filters were washed twice with 1xSSC/0.1% SDS (1x SSC = 0.15 M NaCI/15 mM trisodiumcitrate, pH 7.0) at room temperature followed by two addi-tional washes with 0.1x SSC/0.1% SDS at room tempera-ture. Autoradiography was carried out at -80'C with anintensifying screen.

Northern Analysis. Cytoplasmic RNA from human HeLa,hamster V79, and murine L1210 cells was isolated by apublished procedure (22). Twelve micrograms of RNA wasloaded in each lane and resolved on a 1% agarose gel contain-ing 6% formaldehyde, 20 mM Mops, 5 mM sodium acetate,and 1 mM Na2EDTA. RNA was transferred in 20x SSC bycapillary action to GeneScreenPlus (New England Nuclear).The APt2 DNA insert was labeled with [a-32P]deoxycytidinetriphosphate as described (23). The filter was probed with 106cpm of hybridization mixture per ml [45% formamide/10%odextran sulfate/0.1% sodium phosphate/50mM Tris HCl, pH7.5/5 x Denhardt's solution (1x Denhardt's solution = 0.02%bovine serum albumin/0.02% Ficoll/0.02% polyvinylpyrroli-done)/100 ,tg of sheared denatured salmon sperm DNA perml/0.5% SDS] at 42°C. Filters were washed twice with 2xSSC at 65°C followed by two additional washings with 1xSSC/0.1% SDS at 65°C. Autoradiography was carried out at-80°C with an intensifying screen.

RESULTSModified Western blotting was used to identify a factorpresent in HeLa cells that selectively binds to DNA modifiedby cis-DDP or [Pt(en)C12]. The size of the binding factor isestimated to be -100 kDa, although species of 28 kDa alsobound significantly to the cis-DDP- and [Pt(en)Clj2-modifiedprobes (Fig. 1B, lane 1). Only double-stranded DNA restric-tion fragments modified by cis-DDP or [Pt(en)C12] boundselectively to the human cellular factor. A low level ofbinding of this factor to single-stranded DNA modified bycis-DDP was seen, and little or no binding was observedwhen unmodified single- (data not shown) or double-strandedDNA restriction fragments were used as probes (Fig. 1). Noappreciable binding to the factor, using DNA modified withthe clinically ineffective trans-DDP or [Pt(dien)Cl]CI com-pounds, was observed compared with results for unplatinatedcontrol DNA (data not shown). The extent of binding of thefactor to DNA depended on the level of modification bycis-DDP (data not shown), with a detection limit of =2platinum adducts per 1000 nucleotides (bound D/N ratio of0.002).The selective binding of the HeLa cellular factor to DNA

modified by cis-DDP suggested that it might be possible toisolate cDNA clones by using cis-DDP-modified DNA as aprobe. From a primary screen of 360,000 phage plaques, two

Biochemistry: Toney et al.

Proc. Natl. Acad. Sci. USA 86 (1989)

A

1 2 3 4

200 -

92.5 -

69 -

B

1 23 4

p-

FIG. 1. DNA binding of crude extracts as assessed by modifiedWestern blotting. Lanes: 1, HeLa cytosolic extract; 2, bacteriallysogen crude extract from Agtll vector with no insert; 3, bacteriallysogen crude extract from APt2; 4, bacterial lysogen crude extractfrom APtl. Protein filters were incubated with 422-bp restrictionfragment unmodified (A) or modified (B) by cis-DDP (bound D/Nratio = 0.043) and washed; autoradiography was carried out over-night at -80TC with an intensifying screen. Relative molecularmasses (kDa) were determined by using prestained Rainbow proteinmarkers (Amersham).

recombinant phage, APt1 and APt2, were isolated from ahuman B-cell expression library probed with a 422-bp DNAfragment modified by cis-DDP at a bound D/N ratio of 0.04(Fig. 2). To demonstrate that these clones encode proteinsthat specifically bind to DNA modified by cis-DDP, E. colilysogens were prepared for each clone, as well as for thecloning vector lacking the insert. Crude extracts obtainedfrom induced lysogens were subjected to SDS/PAGE and theresolved proteins were transferred to nitrocellulose filters.After denaturation and renaturation (24), filters were probedwith either 32P-labeled unmodified 422-bp restriction frag-ment or the same DNA probe modified with cis-DDP (Fig. 1),[Pt(en)CI2], trans-DDP, or [Pt(dien)Cl]+. Parallel filters weretreated with a monoclonal antibody raised against /3-galactosidase, or stained with india ink to detect total protein(25). Only DNA modified by cis-DDP or [Pt(en)CI2] bound tothe nitrocellulose-immobilized proteins, in accord with re-sults obtained with the HeLa cellular factor (Fig. 1, lanes2-4). The detection limit of binding of the phage-encodedprotein to cis-DDP-modified DNA was found to be =2platinum adducts per 100 nucleotides (bound D/N ratio of0.02) (data not shown).

Restriction analysis of the two recombinant phage APtl andAPt2 indicated that they contain nucleotide sequences alignedat their 5' ends (Fig. 3) with insert sizes of 1.88 and 1.44kilobases (kb), respectively. Homology between the twoclones was confirmed by Southern analysis (data not shown).The apparent molecular mass of the fusion protein encodedby APt2 is estimated to be =168 kDa by SDS/PAGE, or =50kDa for the portion containing the cloned human B-cell

FIG. 2. Binding of DNA unmodified (A) or modified (B) bycis-DDP (bound D/N ratio = 0.040) to phage APt2 protein replicafilters. Phage APt2 was plated at -=100 plaque-forming units andprotein replica filters were incubated with the appropriate 32P-labeled

422-bp restriction fragment. Data shown are from the tertiary screen.

polypeptide (Fig. 1, lanes 3). Two predominant polypeptidesthat selectively bind to DNA modified by cis-DDP, separatedby --4 kDa (lanes 4), are observed in APtl lysogens. Theslower-migrating band corresponds to a molecular mass of-72 kDa. The faster-migrating band may arise from prote-olysis of the phage-encoded protein.

Expression of the APt2 gene was assessed by Northernanalysis of cytoplasmic RNA prepared from HeLa, murineleukemia L1210, and Chinese hamster V79 cells. The full-length message, conserved in these species, is 2.8 kb (Fig. 4).The predicted molecular mass of the full-length protein is 100kDa. This value is similar to the size of the binding factorobserved by modified Western analysis of HeLa cytosolicextracts (Fig. 1), suggesting that the clone encodes a portionof this same factor.

DISCUSSIONThe studies described here demonstrate that HeLa cellscontain a factor of apparent molecular mass 100 kDa that

A

46 -

30 -B

8330 Biochemistry: Toney et al.

'L""'t. ...- :AI., 0 Ak"1.

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uoW4

Proc. Natl. Acad. Sci. USA 86 (1989) 8331

cn

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9 z4

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C)

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IU ej

" = Lr00'.- :

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c

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FIG. 3. Restriction maps of recombinant phagesAPtl andAPt2showing alignment.

selectively binds to DNA modified by cis-DDP or [Pt(en)CI2.Two cDNA clones, designated APtl and APt2, were isolatedfrom a human B-cell library based on their ability to bindselectively to DNA modified by cis-DDP. Although wepresently do not know what other kinds of DNA damagewould be recognized by theAPtl- andAPt2-encoded proteins,it is highly unlikely that human cells would encode a bindingprotein specific for cis-DDP-damaged DNA. Some insightinto this question can be gained by considering the knownstructures of cis-DDP-DNA adducts.The DNA restriction fragment used in this study contained

a variety of favored cis-DDP binding sites, including 39 (dG)"sequences, where n - 2, as well as 40 d(AG) sequences. Inthe case of cis-DDP binding to DNA, -90% of platinumcoordination involves intrastrand crosslink formation at such(dG),, and d(AG) sequences (3). Thus, at a bound D/N ratioof0.04, or 17 Pt atoms per strand, -40%o of the most favorablebinding sites on the restriction fragment would be platinated.The structure of the d(GpG) adduct, an intrastrand crosslink

1 2 3

9.49 -

7.46

4.40 -

2.37 -

1.35 -

FIG. 4. Expression of APt2 in mammalian cell lines. Lanes: 1,HeLa; 2, Chinese hamster V79; 3, murine leukemia L1210. Twelvemicrograms of cytoplasmic RNA was loaded per lane.

in which platinum is bound to the N7 positions of adjacentguanine bases, has been elucidated by single crystal x-raydiffraction for cis-[Pt(NH3)2{d(pGpG)}] (26) and cis-[Pt(NH3)2{d(CpGpG)}I (27). In both cases, binding to plati-num unstacks the adjacent purine bases by 70O-90°. Recentgel electrophoresis studies revealed that double-strandedDNA containing either d(GpG) or d(ApG) intrastrand cis-{Pt(NH3)2}2+ crosslinks is also bent by 32°-35° toward themajor groove (ref. 28;S. F. Bellon and S.J.L., unpublishedresults). Adducts formed by trans-DDP or [Pt(dien)Cl]+ donot bend the duplex in a similar manner. We therefore suggestthat the structural motif recognized by the cis-DDP-damagedDNA binding factor may derive from geometric distortions inthe double helix that arise from the directed bending phe-nomenon. Moreover, covalent binding of the factor to theplatinum atom is unlikely, since DNA modified by trans-DDPand [Pt(dien)Cl]+, both of which induce quite different struc-tural changes in DNA (3), was not recognized by the factor.

It is interesting that DNA modified by the two activeantitumor compounds was recognized by the cellular factor,and it is conceivable that their anticancer activity might arisein some manner from the very occurrence of such a damagedDNA binding factor. One attractive possibility is that tumorcells are deficient in their ability to repair platinated DNA.Accordingly, active platinum compounds would selectivelykill tumor versus normal cells because platinum-DNA ad-ducts are more efficiently removed from the latter. Platinumcomplexes that bind DNA without signaling recognition andrepair by such cellular factors would, according to thismechanism, be ineffective as anticancer agents. We caution,however, that the situation is more complex, as revealed bythe relative insensitivity of cells to trans-DDP at doses atwhich cis-DDP is highly toxic (for discussion, see ref. 5). Analternative hypothesis is that the cellular factor is a regulatoryprotein, the activation or deactivation of which somehowtriggers toxic events in tumor cells. Further study of thesematters is clearly warranted.Northern blot analysis with the APt2 clone revealed an

encoded protein of 100 kDa in mammalian cells. The size ofa related factor identified in gel mobility-shift assays of crudeHeLa cellular extracts (B.A.D., J.H.T., J.M.E., and S.J.L.,unpublished results) has been computed from the results ofsucrose sedimentation analysis. From the S value of 5.6, themolecular mass was estimated to be -90 kDa. This value,which is sensitive to protein conformation, compares wellwith that measured by modified Western blots prepared fromthe same cellular extracts (Fig. 1). These results, takentogether, suggest that the cDNA clones described in thisreport encode the same or similar proteins present in mam-malian cells that selectively bind to DNA modified by cis-DDP or [Pt(en)CI2].The cis-DDP damage-specific recognition properties of the

present clones are reminiscent of proteins believed to play arole in DNA repair. Cellular factors that selectively bind toDNA containing ultraviolet photoproducts (10, 29-32), apu-rinic/apyrimidinic sites (33, 34), G-T mismatches (35), as wellas N-acetyl-2-aminofluorene adducts (36) have been reportedin human cells. None of these factors possesses endonucle-olytic activity against damaged DNA. The biological func-tions of these proteins are currently unknown, although inone case a factor that recognizes photoproducts on DNA hasrecently been reported to be missing in one complementationgroup ofxeroderma pigmentosum cells (10). Since xerodermapigmentosum cells are deficient in excision repair of damagedDNA, this observation lends credence to the notion that suchdamage-specific cellular factors may play a key role ineukaryotic DNA repair.The isolation of genes and proteins involved in mammalian

DNA repair has been a difficult task. One approach to cloninga family of human DNA repair genes has been to correct the

Biochemistry: Toney et al.

8332 Biochemistry: Toney et al.

sensitivity of excision repair-defective Chinese hamsterovary cells to DNA damaging agents by transfection of DNAfrom normal cells (for a review, see ref. 37). A secondmethod, which to date has been less successful, is thepurification of the relevant protein such that a portion of theamino acid sequence can be determined to aid in the designof oligonucleotide probes. To accomplish this goal, however,substantial amounts of purified protein are required. Thecloning technique used in the present study offers a powerfuladdition to these approaches, and it is likely to be useful forisolating cDNAs that encode other DNA damage-specificbinding proteins.

We thank A. Baldwin, W. Heiger-Bernays, P. Pil, P. Sharp, H.Singh, and W. I. Sundquist for helpful discussions. We also acknowl-edge Englehard Corporation for a loan of K2PtCI4 from whichplatinum complexes were synthesized. This research was supportedby National Institutes of Health Grants CA34486 (to S.J.L.) andCA40817 (to J.M.E.) and by a grant from Bristol-Myers Co. J.H.T.acknowledges support from a National Research Service Award(CA07951) and National Cancer Institute Training Grant CA09112,B.A.D. was supported by National Institute of EnvironmentalHealth Sciences Training Grant ES07020, and P.J.K. was supportedby National Cancer Institute Training Grant CA09112.

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