Identification of an X-linked Locus ModifyingMouse Skin Tumor Susceptibility

Kenneth George, Audrey Iacobucci, Jessica Uitto, and Thomas G. O’Brien*

Lankenau Institute for Medical Research, Wynnewood, Pennsylvania

The enhancing effect of overexpression of an ornithine decarboxylase (Odc) transgene on skin tumor susceptibilitycan be modified by genetic loci present in several inbred mouse strains. The BALB/cJ strain is among the most resistantstrains so far examined; tumor multiplicity following 7,12-dimethylbenz(a)anthracene (DMBA) treatment is reduced by

90% when the K6/ODC transgene is expressed on a BALB/cJ background versus the susceptible C57BL/6J background.Further, transgenic BALB/cJ males developed more tumors than females, indicating the presence of sex-dependentmodifier pathway. Analysis of 263 F2 intercross mice revealed significant linkage of markers on the X chromosome to

tumor multiplicity. This analyses as well as a similar genome-wide scan of 136 backcross mice found evidence for othermodifier loci on chromosomes 4, 6, and 17. Identification of these modifier genes should reveal the effector pathwaysresponsive to Odc overexpression that mediate susceptibility to skin tumorigenesis. � 2005 Wiley-Liss, Inc.

Key words: ornithine decarboxylase; transgenic mouse; tumor modifier loci; X-linked modifier

INTRODUCTION

Elevated tissue polyamine levels greatly increasesusceptibility of mouse skin to tumor development.Sustained high levels of putrescine and, to a lesserextent, spermidine can be achieved by skin-targetedtransgenic overexpression of ornithine decarboxy-lase (ODC), a regulatory enzyme for polyaminebiosynthesis [1], or spermine/spermidine acetyl-transferase (SSAT), the initial enzyme in the poly-amine catabolic pathway [2]. In each of thesetransgenic mouse models, a substantially greaterskin tumor response of transgenic mice is observedafter initiation/promotion protocols compared tonon-transgenic littermate controls. The cellularpathways/functions modulated by polyamines thatunderlie increased susceptibility to tumor develop-ment are not known. Despite the strong effect ofelevated polyamine levels on skin tumor suscept-ibility, this phenotype is nevertheless subject togenetic modification. In the K6/ODC transgenicmodel on a C57BL/6J (B6) background, introductionof alleles from several other inbred strains alteredtumor susceptibility both qualitatively and quanti-tatively [3]. Modifier loci affecting both polyamine-mediated tumor multiplicity and predisposition tocarcinoma development have been mapped in theB6, FVB, andC3H/HeJ strains. These loci appear to bedistinct from previously mapped skin tumor modi-fiers in crosses involving various standard inbredstrains [4–6]. Presumably, these Moo (‘‘modifier ofOdc’’) loci encode genes whose expression or func-tion is differentially affected in a strain-specificmanner by high intracellular polyamine levels. Theresults of tumorigenesis experiments in the K6/ODCmodel on a BALB/cJ (C) strain background indicated

a pronounced effect of gender on skin tumor multi-plicity, implicating for the first time a sex-dependentmodification pathway for skin tumorigenesis. Inthe present study, we utilized an interval mappingapproach-involving crosses of K6/ODC transgenicmice on the B6 and C strain backgrounds to detectgenetic modifier loci. In an intercross population,we detected strong linkage of a region on the Xchromosome to the tumor multiplicity phenotype.We have also obtained evidence for additionalmodifier loci on chromosomes 4, 6, and 17.

MATERIALS AND METHODS

Animals and Generation of Phenotypic Data

All mice used for tumorigenesis assays and geneticmapping experiments were hemizygous for the K6/ODC transgene, but on a variety of genetic back-grounds. BALB/cJ (C) andC57BL/6J (B6) femalemicewere obtained from The Jackson Laboratory (BarHarbor, ME). K6/ODC mice on the B6 and C strainbackgrounds are indistinguishable grossly, and theaberrant skin phenotype (alopecia, thickened skinwith intradermal epithelial cysts) is similar histo-logically in the two strains. To produce mice fortumorigenesis experiments and genetic mapping,

MOLECULAR CARCINOGENESIS 44:212–218 (2005)

� 2005 WILEY-LISS, INC.

Abbreviations: Odc, ornithine decarboxylase; DMBA, 7,12-dimethylbenz(a)anthracene; QTL, quantitative trait locus; Ar, androgenreceptor.

*Correspondence to: Lankenau Institute for Medical Research,100 Lancaster Avenue, Wynnewood, PA 19096.

Received 6 April 2005; Accepted 24 May 2005

DOI 10.1002/mc.20130

male K6/ODC mice on either a congenic B6 orpredominantly C background (fifth backcross gen-eration from B6CF1 (98.44% C genome)) were bredto female mice of the other strain to generate F1hybrid mice. Male K6/ODC F1 hybrid mice werethen bred to non-transgenic F1 females to produceK6/ODC.CB6F2 intercross mice or to B6 females toproduce K6/ODC.CB6F1xB6 backcross mice.To induce tumors, newborn mice, 1 d after birth,

were treated with 200 nmol 7, 12-dimethylbenz(a)-anthracene (DMBA) dissolved in 50 mL acetoneapplied with micropipettor to the mid-dorsal skin.Mice were not further treated, as transgene expres-sion in this model is an optimal tumor-promotingstimulus [1]. At weaning, transgenic mice wereidentified by their hair loss phenotype [7] andobserved for tumor development for up to 22 wk.The total number of skin tumors�2mmwere count-ed weekly, beginning at 6 wk after DMBA treatment.All tumors scored were squamous papillomas.

Biochemical Assays

For measurement of ODC enzymatic activity andtissue polyamine levels, 10 wk old male K6/ODC.B6and K6/ODC.C mice (three per group) were killedand4cm�6cmsectionsof dorsal skin excised, cut inhalf, and the pieces processed separately for ODCactivity measurements and polyamine determina-tions exactly as described previously (3). Results forODC activity are expressed as units/mg protein,where 1 unit¼1 nmol CO2 liberated per hour.Results for polyamines are expressed as nmol/mgDNA.

Genotyping

DNA was purified from spleens by standardmethods and subjected to amplification by PCRusing primers flanking microsatellite markers poly-morphic for the B6 andC strains. In general,markerswere chosen based on the marker sizes describedin the STS website (http://www-genome.wi.mi.mit.edu/cgi-bin/mouse/sts_info?database¼mouserelease)and were evenly spaced along each chromosome.Products of the PCR genotyping reactions wereseparated on 3% agarose gels containing ethidiumbromide or by capillary electrophoresis on an AB1Prism 310 Genetic Analyzer equipped with theGenescan (version 2.0) software analysis package.In the latter case, one of the PCR primers used wasfluorescently labeled with FAM, HEX, or NED.

Statistical Analyses

In both the K6/ODC.B6F1 x B6 backcross and theK6/ODC.CB6F2 intercross experiments, mice withextreme phenotypes (lowest and highest tumormultiplicities) were first genotyped for �100 poly-morphic microsatellites on all chromosomes. Theremaining mice were then genotyped for only thosemarkers with LOD values (see below) greater than

1.0. Once regions of interest were identified, addi-tional markers were used for finer mapping. The Q-link (from Dr. Norman Drinkwater, University ofWisconsin–Madison) and MapManager QTXb16(from Dr. Kenneth Manly, Roswell Park CancerInstitute) software programs were used for linkageanalyses. The Q-Link program uses nonparametricstatistics to calculate a Zw value for linkage, whichcan be converted to a LOD equivalent term, LODw,as described by Kruglyak and Lander [8]. TheMapManager QTX program uses interval-mappingtechniques to calculate a likelihood ratio statistic,which can be converted to a LOD score as describedby Balding et al. [9]. Both methods gave very similarresults for quantitative trait locus (QTL) detection.Empirical P-values for linkage data were derived bypermutation testing. Differences between tumormultiplicities of male and female K6/ODC.CBF1and K6/ODC.B6CF1mice were analyzed by the non-parametric Wilcoxon Rank Sum test in the Statviewpackage (SAS Institute, Cary, NC).

RESULTS

Effect of BALB/cJ Genes on K6/ODC-Mediated

Susceptibility to Skin Tumorigenesis

In the process of placing the K6/ODC transgeneon a variety of genetic backgrounds, we observed apronounced strain-dependent effect on the en-hanced skin tumor susceptibility phenotype con-ferred by transgene expression. Compared to thevery sensitive B6 strain, each of three additionalstrains tested (DBA/2, FVB, C3H/HeJ) exhibitedreduced susceptibility to challenge the carcinogenDMBA [3]. A similar result was observed when K6/ODCmice on a BALB/cJ backgroundwere exposed to200 nmol DMBA (Figure 1): a 90% reduction in

Figure 1. Comparison of the tumor response of K6/ODC mice ontwo different genetic backgrounds. Newborn mice (1 d old) on eithera C57BL/6J (&) or BALB/cJ (~) background were treated with DMBAas described in Materials and Methods. Beginning 6 wk later, tumors�2 mmwere counted weekly until tumor multiplicity plateaued (20–22 wk after DMBA). Both male and female mice on each geneticbackground were used.

SKIN TUMOR MODIFIER LOCI IN BALB/cJ MICE 213

tumor multiplicity was observed compared to trans-genic mice on the sensitive B6 background. Inaddition to the strong resistance of K6/ODC.C miceto tumor development, there was a very pronouncedsex difference in tumor response: of the 76 totaltumors present at 22wk after DMBA, 68were presentin males and only 8 in females. The mean tumormultiplicity was 2.4 tumors per male mouse versus0.3 tumors per femalemouse (P¼0.02, paired t-test).We have not previously observed a sex differencein tumor multiplicity in the K6/ODC model on 4other inbred strain backgrounds (3; andunpublisheddata).To investigate a genetic basis for this phenom-

enon, reciprocal F1 hybrid mice (K6/ODC.CB6F1,K6/ODC.B6CF1) involving Cmice and B6mice weregenerated and compared for tumor susceptibility.In these crosses, all females are genetically identical(XB6XC) but males differ in the parental origin oftheir sex chromosomes (XB6YC or XCYB6). Therefore,the presence of modifier genes on the X and/or Y-chromosomes could be inferred by comparing thetumor response of K6/ODC.CB6F1 and K6/ODC.B6CF1males.As shown inTable1, our results supportthis hypothesis: male K6/ODC.B6CF1 (XB6YC) micedeveloped 4.5 tumors/mouse after DMBA challengecompared to 1.2 tumors/mouse in K6/ODC.CB6F1(XCYB6) mice (P<0.0001, Wilcoxon Rank sum test).When the data from each F1 hybrid experimentwereanalyzed by gender, there was no difference in thetumor response of K6/ODC.CB6F1 males (XCYB6)and females (XCXB6), but there was for K6/ODC.B6CF1; males (XB6YC) developed 4.5 tumors/mouseversus 2.7 for females (XB6XC). This difference ishighly significant (P<0.0001, Wilcoxon Rank Sumtest). The simplest interpretation of these data is thatBALB/cJ mice harbor a dominant tumor resistanceallele on the X chromosome.

Effects of Genetic Background on Transgene Expression

Compared to K6/ODC mice on a B6 strain back-ground, K6/ODC.C have a similar phenotype,including hair loss beginning in the first few weeksafter birth leading to total alopecia, enhanced nailgrowth, andprogressive thickeningof the skindue toformation of large hair follicle-derived intradermalepithelial cysts [7]. All phenotypes in the K6/ODCmodel, including enhanced susceptibility to tumordevelopment, are absolutely dependent on trans-gene expression. Nevertheless, it is possible thatquantitative differences in a phenotype such astumor multiplicity after DMBA treatment might bedue to a difference in transgene expression. Specifi-cally, K6/ODC.C mice might have reduced ODCactivity and/or lower polyamine levels compared toK6/ODC.B6 mice. To investigate this possibility, wemeasured ODC activity and polyamine levels in theskin of K6/ODC.C and K6/ODC.B6 mice (Table 2).There was no significant difference in ODC specificactivity due to strain background (P¼0.41; 2-sidedt-test). Polyamine levels were actually twofold tothreefold higher in K6/ODC.C mice versus K6/ODC.B6 mice. From these results, it seems clear thatreduced ODC expression or lower polyamine levelscannot account for the resistance to tumor devel-opment of K6/ODC mice on the BALB/cJ strainbackground.

Linkage Analysis in K6/ODC.CB6F1� B6 Mice

To map the autosomal genetic loci responsible formodifying the effect of ODC overexpression onskin cancer susceptibility, a backcross population of136micewas generated bymating K6/ODCmales ona CB6F1 background to B6 female mice. Transgenicbackcross mice developed more tumors on averagethan transgenic CB6F1mice [5.73�0.4 (SE) tumors/mouse vs. 1.2�0.2 (SE) tumors/mouse)]. This differ-ence was not statistically different (P¼ 0.07) due tothe large variation in tumor multiplicity in thesegregating backcross population (range¼0–26).As expected, male K6/ODC backcross mice devel-oped more tumors than female mice (6.8 tumors/mouse vs. 3.8). When a genome-wide scan wasperformed utilizing �100 microsatellite markers,linkages to two chromosomal regions were found(Table 3). A region on chromosome 4 aroundD4Mit52 at 113.1 Mb showed significant linkage(peak LOD¼ 3.52) and a broad region of chromo-some 17 (24.0–50.9 Mb) showed suggestive linkage(LOD scores 2.34–3.17). In both cases, the BALB/cJloci encoded resistance alleles. It is probably thechromosome 17 locus that harbors the previouslycharacterized modifier locus (Moo1) identified incrosses between the B6 and C3H/HeJ strains [3], aswell as possible additional QTLs. Because of theresults from the reciprocal F1 hybrids describedabove (Table 1), there is presumably also a modifier

Table 1. Tumorigenesis Data in CB6F1 and B6CF1 Mice

Mouse strain Nb

Tumor multiplicity

All mice Males Females

K6/ODC.CB6F1 44 1.2 1.2 1.2K6/ODC.B6CF1 91 3.7 4.5a 2.7

Newborn mice (1 d after birth) were treated with 200 nmolDMBA. Beginning at 6 wk of age, tumors �2 mm were countedon each mouse weekly, until 20 wk after DMBA. The resultsshown are data from week 20, which was the maximal tumorresponse for each strain.aP< 0.0001 versus K6/ODC.B6CF1 female tumor multiplicity andK6/ODC.CB6F1 male tumor multiplicity, (Wilcoxon Rank Sumtest).bN, total number of mice in each experiment. Each experimentconsisted of approximately equal numbers of males and females.An outlier was removed from the female B6CF1 group, as itstumor number was >5� the mean tumor multiplicity.

214 GEORGE ET AL.

locus or loci on the X chromosome affecting tumormultiplicity, but due to the nature of the breedingscheme used (only transgenic male B6CF1 werebackcrossed to B6 females), recombination eventsinvolving the X chromosome could not be scored.

Linkage Anaylsis in K6/ODC.CB6F2 Intercross Mice

To map a presumptive X-chromosome-linkedmodifier, a large intercross population of mice(n¼263) was produced by breeding K6/ODC.CB6F1males to nontransgenic CB6F1 females. In thispopulation, themean tumormultiplicity was higherin males (4.35) than in females (2.27), as expected.A genome wide scan with �100 microsatellitemarkers revealed strong linkage of several closelyspaced chromosome X markers to the tumor multi-plicity phenotype (Table 4). Highly significantlinkages (LOD scores of 5.20–6.61) to markers inthe 89.6–92.5 Mb mid-chromosome region were

observed. We term this new QTL, Moo2. We alsodetected suggestive linkage of markers on chromo-some 17 (in the same region suggestive linkage wasfound in the CB6F1�B6 backcross) and of markerson distal chromosome 6. In this intercross we couldnot confirm the suggestive linkage of chromosome4 markers to tumor multiplicity observed in theCB6F1�B6 backcross (all LOD scores of chromo-some 4 markers were <1.0).

DISCUSSION

As a result of screening several inbred mousestrains for evidence of modifier loci affecting poly-amine-dependent skin tumor susceptibility, theBALB/cJ strain was found to be one of the mostresistant strains to tumordevelopment. In thewidelyused initiation/promotion model of skin carcino-genesis, the BALB/c mouse is considered a relativelyresistant strain to tumor development by this

Table 2. Effect of Genetic Background on K6/ODC Transgene Expression

StrainODC Sp. Act.units/mg prot

Polyamines nmol/mg DNA

Pua Spdb Spc

C57BL/6J 3.44� 0.75 163� 56 369� 63 81.7� 13.4BALB/cJ 2.79� 0.15 397� 51 1098� 81 265� 20

ODC activity and polyamine levels were measured in the dermis of 10 wk-old male K6/ODC mice on eachstrain background, as described in Materials and Methods. Results are the mean� SD of three mice perstrain.aPu, putrescine.bSpd, spermidine.cSp, spermine.

Table 3. Association of Markers Which Modify Tumor Multiplicity in CB6F1� B6 Mice

Phenotypic data Linkage data

Het* Homo{ Position, Mb LOD P-value

Chromosome 4D4Mit178 4.46 7.02 65.5 1.00D4Mit58 4.11 7.20 97.7 2.89D4Mit31 3.95 7.30 105.5 3.41 0.032D4Mit52 3.97 7.29 113.1 3.52 0.024D4Mit12 4.37 6.75 122.7 1.63

Chromosome 17D17Mit46 4.32 6.96 24.0 2.34D17Mit62 4.51 7.71 32.2 3.17 0.05D17Mit50 4.21 7.19 43.5 3.02 0.068D17Mit106 4.34 7.01 48.0 2.41D17Mit70 4.19 7.04 50.9 2.74D17Mit129 4.48 6.78 85.3 1.76

Mice with either low tumor multiplicity (two or less) or high multiplicity (12–24 tumors/mouse) weregenotyped for multiple markers on all chromosomes and then the remainder were genotyped only in regionsof LOD scores �1.0. Shown are the only chromosomal regions with LOD scores >2.0. Empirical P-valueswere obtained by permutation testing (10 000 permutations) as described by Doerge & Churchill [28].Phenotypic data are given as mean tumors/mouse for mice *heterozygous (Het) or {homozygous (Homo) foreach DNA marker. Chromosome positions in Mb are from the NCBI Mouse Build m33 sequence assembly.

SKIN TUMOR MODIFIER LOCI IN BALB/cJ MICE 215

treatment protocol [10]. However, the genetic deter-minants of skin tumor susceptibility in the K6/ODC transgenic model, which does not involve anexogenous promotion protocol, are likely to bedifferent than those governing susceptibility to theinitiation/promotion protocol. Indeed, theC57Bl/6Jstrain is themost susceptible genetic background forthe K6/ODC transgene of five strains so far exam-ined, yet it is considered one of the more resistantstrains to initiation/promotion [11]. As adirect resultof enforced overexpression of ODC in keratin 6-expressing keratinocytes, polyamine levels, espe-cially putrescine, are dramatically elevated in thismodel. Genetic modifiers identified in this modelmust be sensitive in somemanner to this constitutiveupregulation of intracellular polyamine levels. Onepossible mechanism could be transcription-based:strain-dependent sequence variation in the promo-ter regionsofpolyamine-regulated genes could resultin different steady-state mRNA levels. Alternatively,because of amino acid sequence variation, the func-tions of the gene products themselves may be af-fected todifferent extents bypolyamines. Since thesemechanisms are not mutually exclusive, in somecases both possibilities could be operative.

A novel finding of this study was the effect ofgender on DMBA-induced skin tumor development.This finding was surprising since susceptibility tochemically induced mouse skin tumorigenesis hasnot previously been shown to differ between sexes.Nagase et al. [4] reported an interaction of sex with amodifier locus on chromosome 7 in crosses betweenMus spretus andNIH/ola, but therewasno effect of sexby itself. However, genetic determinants of suscept-ibility to UV-induced skin carcinogenesis have beenreported: Noonan and colleagues have reportedstrain differences in both UV-induced immunosup-pression [12], a necessary condition for UV-inducedskin tumors. Interestingly, the two strains used byNoonan et al., were BALB/cAnNCr andC57BL/6NCr,strains very similar to those used in our studies.Further, using a reciprocal hybrid approach similarto that used in this study, these authors demon-strated sex dependence for UV-induced carcinogen-esis [13]. It is not likely, however, that the X-linkedmodifier of UV carcinogenesis is identical to Moo2detected in our model, since the susceptibility allelefor UV carcinogenesis is the BALB/c allele while theBALB/c Moo2 allele confers resistance to DMBA-induced tumorigenesis. Nevertheless, the results

Table 4. Association of Markers Which Modify Tumor Multiplicity in CB6F2 Mice

DNA marker

Phenotypic data Linkage data

BB* BC{ CC{ Position, Mb LOD P-value

Chromosome XDxMit166 4.60 2.65 3.13 44.0 2.17DxMit25 5.18 2.47 3.00 63.0 4.26DxMit119 5.14 2.51 3.06 64.0 4.02DxMit45 4.81 2.87 3.04 68.6 2.70DxMit114 5.57 2.38 2.74 89.6 6.61 0.0001DxMit16 5.76 2.51 2.92 90.8 5.98 0.0002DxMit41 5.46 1.96 3.12 92.5 6.26 0.0003DxMit64 4.98 1.98 3.12 97.6 5.20 0.0014DxMit79 5.21 2.80 2.95 120.9 3.72DxMit153 4.60 2.64 3.40 140.9 2.10

Chromosome 17D17Mit46 3.88 3.37 3.41 24.0 0.24D17Mit50 4.60 3.21 2.53 43.5 2.02 0.46D17Mit139 4.77 3.93 2.80 50.8 2.61 0.16D17Mit164 4.26 3.42 2.47 53.1 1.61D17Mit123 3.84 3.37 2.83 91.9 0.46

Chromosome 6D6Mit93 3.38 2.96 4.22 52.1 0.87D6Mit149 3.48 3.72 2.85 106.4 0.46D6Mit59 4.67 3.58 2.47 139.1 2.65 0.14D6Mit294 4.57 3.39 2.57 146.4 1.93D6Mit304 3.60 3.94 2.21 147.6 1.74

263 CB6F2 mice were genotyped for 98 microsatellite markers on all chromosomes, including X. Shown arethe only chromosomal regions with LOD scores >2.0. Empirical P-values were obtained by permutationtesting (10000 permutations). Phenotypic data are given as mean tumors/mouse for mice *heterozygous(BC) or {homozygous B6 (BB) or {C (CC) for each DNAmarker. For chromosome X markers, phenotypic datafor BB and CC genotypes include both female and male mice, while the BC genotype includes female miceonly. Chromosome positions in Mb are from the NCBI Mouse Build m33 sequence assembly.

216 GEORGE ET AL.

of Noonan et al. support the concept of X-linkedmodifiers of skin cancer.With respect to cutaneous squamous cell carcino-

mas (SCCs) in humans, several reports indicate amale bias for development of this cancer. In recentepidemiologic studies the male/female ratio forcutaneous SCC incidence was reported to be >2.0[14,15]. While environmental factors, especially sunexposure, probably account for some of this sexdifferential in cancer incidence, the influence ofgenetic (inherited) risk factors cannot be ruled out.Indeed, in a large cohort study, individuals witha personal history of nonmelanoma skin cancer(SCC and basal cell carcinoma) were at increased riskfor development of several other forms of cancerunrelated to sun exposure, suggestive of a geneticinfluence affecting susceptibility to multiple formsof cancer [16].Our initial tumor multiplicity data in male versus

female K6/ODC mice on a BALB/cJ backgroundimplicated a hormone-dependent modifier path-way, (not a genetic modifier effect, because only Calleles are present) while the results in K6/ODC.CB6F1 versus K6/ODC.B6CF1 reciprocal hybridsstrongly suggested a locus or loci on the BALB/c Xchromosome that confers resistance to polyamine-dependent tumor development. The detection of aQTL on the X chromosome affecting tumor sus-ceptibility in a large intercross population confirmedthe latter hypothesis. Highly significant LOD scoresfor linkage (5.20–6.61) were found for severalmidX-chromosome markers (Table 4). An intriguing can-didate gene in this regionof chromosomeXboundedby DXMit114 and DXMit41 (89.6–92.5 Mb) thatfulfills the criteria for both sex hormone dependenceand a genetic modifier is the androgen receptor (Ar)gene at 89.6 Mb. The androgen receptor protein is aligand (testosterone, dihydrotestosterone)-activatedtranscription factor known to be expressed in skin.Physiologically, Ar regulates hair follicle functionbut also influences such diverse processes as inflam-mation [17] and cutaneous wound healing [18].There is evidence fromhumanepidemiologic studiesthat variation in the AR gene modifies prostatecancer risk [19,20]. In humans, AR is highly poly-morphic with multiple alleles containing variableCAG and GGN repeats in the N-terminal codingregion of the gene. In mice, there are no reportedcoding region sequence variations amongst dif-ferent standard inbred strains, but inspection ofavailable databases reveals multiple single nucleo-tide polymorphisms in introns and the 30 and 50

flanking regions that may influence Ar expression[21,22]. In both rodents and humans, Odc is atranscriptional target gene of Ar [23,24]. We haverecently implicated variation in the human ODCgene in risk of colon adenoma recurrence [25] anddemonstrated an interaction between high riskvariants of ODC and AR influencing prostate cancer

risk [26]. These epidemiologic results support thepossibility that allelic variation in the Ar genebetween the B6 and C strains may explain the sexdifference in tumor susceptibility in the K6/ODCmodel.While the X-linked modifier apparently contri-

butes most to the tumor resistance phenotypeassociated with the BALB/cJ strain, other genetic lociwith possible effects on phenotype were detected inour analyses. Significant linkage to chromosome 4markers and suggestive linkages to chromosome 17and chromosome 6 markers were identified. TheC alleles of each of these loci confer resistance topolyamine-dependent tumor development. Thechromosome 17 region mapped in this study con-tains the Moo1 locus previously identified by us incrosses involving the B6 and C3H/Hej strains (3).In comparing the results from both the backcrossand intercross experiments, we failed to confirm theassociation of the chromosome 4 and 6 regions withthe tumor multiplicity phenotype. The reasonsfor this are not clear, but emphasize the need forcaution in interpreting suggestive or barely signifi-cant linkage results. The regions of chromosome 4and 17 loci identified in this study also containpotential modifier loci identified in other tumor-igenesis models involving BALB/c mice. Manentiet al. [27] mapped Papg1 (‘‘pulmonary adenomaprogression gene 1’’) to chromosome 4 at 105.5 Mb,close to D4Mit31-D4Mit52 (106–113.7 Mb). How-ever, this locus modified lung tumor volume (noeffect on tumor multiplicity) and the C allele con-ferred susceptibility, whereas in our model the Callele modifies tumor multiplicity and confersresistance. In the afore-mentioned UV skin carcino-genesis model, Clemens et al. [12] mapped a BALB/cmodifier locus for UV-induced immunosuppression(known to be associated with UV-induced skincarcinogenesis) to D17Mit49 (43.5 Mb), within thebroad region of chromosome 17 whereMoo1 resides.Finer mapping studies and eventual modifier geneidentification will be required before a rigorousconclusion concerning identity of these QTLs withthose reported in this study can be made.Because of the complexity of phenotypes such as

susceptibility to cancer, mouse models such as theK6/ODC model may be the only realistic way toquickly identify candidate tumor modifier loci inhumans. It is hoped that once theMoo loci are morefinely mapped and the genes identified, the homo-logous genes inhumans can be tested for their role inhuman skin cancer susceptibility.

ACKNOWLEDGMENTS

The authors thank Dr. Janet Sawicki for helpfuladvice and Loretta Rossino for editorial assistance.This work was supported by grant ES01664 (awardedto TGO) from the National Institute of Environ-mental Health Sciences, NIH, PHS, DHHS.

SKIN TUMOR MODIFIER LOCI IN BALB/cJ MICE 217

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