ICANCERRESEARCH57.4164-4166. October1, 1997] Advances in Brief Genetic Mapping of Lung Cancer Modifier Loci Specifically Affecting Tumor Initiation and Progression' Giacomo Manenti, Manuela Gariboldi, Antonio Fiorino, Nicola Zanesi, Marco A. Pierotti, and Tommaso A. Dragani2 Division of Experimental Oncology A, istituto Nazionale per lo Studio e Ia Cura dei Tumori, 20133 Milan, italy affect lung tumor multiplicity. In the same cross, we have also mapped, on chromosome 4, a locus designated as Papgl specifically affecting the size of lung tumors. Materials and Methods Animals and Phenotype. Male and female SWR/J (W) and BALB/cJ (C) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were then bred in our laboratory to obtain CWF2 mice, which were given a single i.p. injection of 1000 mg of urethane dissolved in water (BDH Chem icals, Poole, United Kingdom) per kg body weight CWF2 male and female mice were killed at 40 weeks of age. The number of tumors on the surface of the lungs were counted after tissue fixation, and the diameter of each nodule was measured. Susceptibility of each mouse to lung tumor development was estimated using quantitative parameters, as described (7). Genetic Markers and Linkage Analysis. Primers for simple sequence length polymorphism markers were obtained from Research Genetics (Hunts ville, AL). Forty mice showing extreme phenotypes were genotyped with 231 markers covering all autosomes and chromosome X. Markers located on chromosomes showing putative linkage were typed in the whole CWF2 pop ulation (218 mice). Genetic linkage maps and linkage between parameters estimating lung cancer predisposition trait and genetic markers were deter mined as reported (3, 8, 9). Because the susceptible phenotype showed a nonnormal distribution, the linkage methods were applied to rank-transformed data (10), although the phenotypic values reported in Table 1 refer to untrans formed data. Results Loci Specifically Affecting Tumor Multiplicity. Whole-genome scanning of 40 extreme-phenotype mice with 23 1 markers showed significant linkage of a chromosome 18 region with lung tumor multiplicity (N). The lod score curve peaked at the Dl8Mit9 locus at 42 cM (Ref. 11; Fig. 1). The linkage between the Dl8Mit9 region and N was supported by a led score of 7.9. Mean lung tumor volume (V) showed only a marginal linkage at the same locus (led score of 2.1). ANOVA confirmed the linkage (P < 0.0001) between lung tumor multiplicity and Dl8Mit9, which explained —15% of the total phe notypic variance (Table 1). Values of lung tumor multiplicity by genotypic class at Dl8Mit9 indicate that BALB/c mice carry the resistance allele (Par2/+), and SWR/J mice carry the susceptibility allele (Par2/—). Resistance to multiplicity oflung cancer by Par2 was partially dominant, because heterozygous CWF2 animals showed an N value lower than the expected intermediate value for genetic effect of additive alleles but higher than that of the homozygous mice for the BALB/c allele (Table 1). The use of an intercross as compared to a backcross population had the advantage of allowing analysis of locus inheritance mode (i.e., partially dominant). The weak effect of Par2 on lung tumor size (V) behaved as a recessive trait, because only mice homozygous for the BALB/c allele showed a reduced V value, and heterozygous animals were not affected (Table 1). Therefore, Obata et a!. (6) and we have identified another locus (Par2) providing inherited resistance to lung tumorigenesis. However, in our CWF2 cross, Par2 accounts for about 15% of the phenotypic variance, whereas in the (A/i X BALB/c) X NJ backcross used by Abstract Mouse inbred strains with inherited predisposition and resistance to lung cancer provide a tool for the dissection of the complex genetics of this disease. In the present report, we have crossed the BALB/C with the SWR/J strain and performed whole-genome scanning for loci affecting lung tumor development in their F2 progeny. Both parental strains carry the pulmonary adenoma susceptibility 1 (Pasi) locus, a major locus af fecting predisposition to lung cancer in mice. On distal chromosome 18 and on centromere of chromosome 6, we have mapped two pulmonary adenoma resistance loci (Par2 and Par4, respectively), which reduce lung tumor multiplicity strongly, up to 15-fold. Par2 and Par4, however, do not affect lung tumor size, which is instead controlled by an additional locus that we have mapped on the central region of chromosome 4. We desig. nated this locus as â€oepulmonary adenoma progression 1― (Papgl), because it specifically modifies lung tumor size but not multiplicity. The present results, therefore, provide evidence for the existence of cancer modifier loci acting on specific stages of lung tumorigenesis Introduction In the last few years, progress in the genetics of an inherited predisposition to lung cancer in mice demonstrated that a major locus (Pasi) located on the distal region of chromosome 6 affects suscep tibility to lung tumorigenesis (1). Other minor loci may also contribute to the susceptibility trait (2). Recently, we have reported the mapping of a Mus spretus-derived locus that strongly inhibits the lung tumor igenesis in Pasl/+ mice (3). This locus, pulmonary adenoma resist ance 1 (Pan), maps on mouse chromosome 11, near the Rara locus (3). These findings provided the first evidence for the existence of a lung tumor resistance locus inhibiting the expression of an inherited predisposition to lung cancer. To further define the genetics of predisposition and resistance to lung cancer in mice, we have considered the BALB/c strain, which shows an intermediate susceptibility that is dominant over the high susceptibility of the A/J and SWR strains (4). Given that all three strains carry the Pasl/+ allele (5), the BALB/c mouse must carry lung tumor modifier loci that decrease expression of high susceptibility to lung tumorigenesis. Therefore, a cross between the BALB/c and one of the most susceptible strains would provide a clue to map lung cancer modifier loci in a common genetic background for Pasi. Accordingly, in a cross between the BALB/c and the SWRJJ mice, here we report the mapping of BALB/c-derived pulmonary adenoma resistance (Par2 and Par4) loci, on chromosomes 18 and 6. Our findings are in agreement with a recent report mapping the same Par2 locus on the distal region of chromosome 18 (6). Both loci specifically Received 7/14/97; accepted 8/8/97. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18U.S.C.Section1734solelyto indicatethisfact. I This work was supported in part by grants from Associazione and Fondazione Italiana Ricerca Cancro. 2 To whom requests for reprints should be addressed, at Istituto Nazionale Tumori, Via G. Venezian 1, 20133 Milan, Italy. Fax: 39-2-2390764; Phone: 39-2-2390642;, E-mail: dragani@istitutotumori.mi.it. 4164 on July 9, 2015. © 1997 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from