**3.3 C57BR/cdJ alleles between 175.35 and 178.64 Mb do not confer susceptibility**

We have shown previously that distal Chromosome 1 carries a modifier that causes 5- to 6-fold increased susceptibility in male C57BR/cdJ (BR) mice relative to B6 mice (Poole and Drinkwater, 1996; Bilger et al., 2004). To determine whether this *Hcif2* (formerly *Hcf2*) modifier might involve the same locus that confers susceptibility to C3H, we used B6.BR-Ch1 congenic mice to derive mice carrying smaller congenic regions and counted their tumors at 32 weeks (Figure 3). The line carrying the largest BR region, B6.BR.R4, developed fewer tumors than B6 (though not significantly; *P* > 0.37). Similarly, lines B6.BR.R5A and B6.BR.R5B, which carry part of the minimal region and extend distally beyond 191 Mb (R5A) or proximally beyond 165 Mb (R5B), also developed the same number of tumors as B6 or fewer (*P* > 0.86 and *P* > 0.68, respectively). All three recombinant lines developed significantly fewer tumors than the parental B6.BR-Ch1 line (not shown; *P* < 0.03 for all). These results indicate that *Hcif2* is unlikely to lie in the 175.35 to 178.64 *Hcs7* region.

confers a 7- to 10-fold increase in tumor multiplicity and lies between 172.9 and 178.64 Mb. This region carries 132 genes (www.ensembl.org; 8/2011). Line 2R7, which extends distally from 175.35 Mb, was also highly susceptible relative to B6, developing almost 9-fold more tumors (*P* < 10-5). In addition, line 2R7 is approximately 3-fold more susceptible than line 2R5, which carries C3H alleles distal of 178.9 Mb (*P* < 0.02). Together with the 2R16 results that place *Hcs7* between 172.9 and 178.64 Mb, these data suggest that *Hcs7* lies in the 3.3 Mb between 175.35 and 178.64 Mb. This region carries 44 genes (www.ensembl.org; 8/2011).

Again, the remaining lines suggest that the pattern of modifiers along the chromosome is complex. Lines 2R9 and 2R7 both carry C3H alleles to near the telomere and line 2R9 carries more C3H alleles than 2R7 proximally (their breakpoints differ by about 100 Kb), but line 2R9 is resistant (2R9 vs B6: *P* > 0.24). Similarly, the resistance of line 2R11 (2R11 vs. B6: *P* > 0.09), which, like line 2R9, was derived from sensitive line 2R3, together with the susceptibility of lines 2R2, 2R10, 2R15, 2R7, and 2R5, suggests there are additional modifiers both proximal to and distal of the 175.4 to 178.64 Mb minimal region. Alternatively, some

Recombination between B6 and C3H was suppressed in the minimal susceptibility region, between 175.4 and 178.4 Mb. No recombinants were observed among approximately 1350 segregating progeny, although 24 would be expected. This difference is highly significant (*P* < 10-6). Recombination is frequently suppressed by chromosomal rearrangements such as

**3.2 B6 alleles on distal Chr 1 are sufficient to suppress hepatocarcinogenesis on a** 

**3.3 C57BR/cdJ alleles between 175.35 and 178.64 Mb do not confer susceptibility** 

We have shown previously that distal Chromosome 1 carries a modifier that causes 5- to 6-fold increased susceptibility in male C57BR/cdJ (BR) mice relative to B6 mice (Poole and Drinkwater, 1996; Bilger et al., 2004). To determine whether this *Hcif2* (formerly *Hcf2*) modifier might involve the same locus that confers susceptibility to C3H, we used B6.BR-Ch1 congenic mice to derive mice carrying smaller congenic regions and counted their tumors at 32 weeks (Figure 3). The line carrying the largest BR region, B6.BR.R4, developed fewer tumors than B6 (though not significantly; *P* > 0.37). Similarly, lines B6.BR.R5A and B6.BR.R5B, which carry part of the minimal region and extend distally beyond 191 Mb (R5A) or proximally beyond 165 Mb (R5B), also developed the same number of tumors as B6 or fewer (*P* > 0.86 and *P* > 0.68, respectively). All three recombinant lines developed significantly fewer tumors than the parental B6.BR-Ch1 line (not shown; *P* < 0.03 for all). These results indicate that *Hcif2* is unlikely to lie in the 175.35 to 178.64 *Hcs7* region.

To determine whether B6 Chromosome 1 alleles can confer resistance to a sensitive C3H background (as C3H alleles confer sensitivity to a resistant B6 background), we generated C3H.B6-Ch1 congenics using ten generations of backcrossing B6 to C3H, selecting B6 alleles on distal Chromosome 1 at each generation. Heterozygous and homozygous congenic males and control inbred C3H males were injected with DEN at 12 days of age and their tumors were counted at 32 weeks (Figure 3). While heterozygosity for B6 alleles had no effect on tumorigenesis (*P*>0.84), homozygosity caused a 3.8-fold reduction in tumor multiplicity

lines may have undergone rearrangement in the susceptibility region.

inversions (Kirkpatrick, 2010).

**C3H background** 

(*P*<10-3).
