**3.5 C3H Chromosome 1 alleles increase spontaneous hepatocarcinogenesis**

To determine whether *Hcs7* influences spontaneous as well as DEN-induced hepatocarcinogenesis, homozygous male mice were aged to 15 months and tumors were counted (Table 1). B6.C3H-Ch1 males developed significantly more liver tumors than B6 males (onesided *P* < 0.01). The 4-fold higher multiplicity and the 2.5-fold higher incidence in this line closely resemble the higher multiplicity and incidence seen in inbred C3H males (*P* > 0.85). Similarly, line 1R33 males developed twice as many tumors and had 50% higher incidence than B6. The difference in tumor multiplicity was significant (one-sided *P* < 0.04). Males of the proximally congenic line 1R11, which developed approximately 3-fold more tumors than B6 when treated with DEN, had approximately 25% higher incidence and developed approximately 25% more spontaneous tumors than B6, but this difference was not significant (one-sided *P* > 0.18). This analysis of spontaneous tumor incidence indicates that the majority of the difference between C3H and B6 can be ascribed to alleles on Chromosome 1, including alleles in the *Hcs7* region.


Table 1. B6.C3H-Ch1 and 1R33 males are more susceptible to spontaneous liver tumors than B6.

Inbred and recombinant male mice were left untreated to 15 months of age and liver tumors were enumerated. N, the number of mice per group.

Many line 1R33 males developed severe skin disease. Twenty-eight males died or were euthanized prior to the 15-month time point (as compared to three B6, eight C3H, ten B6.C3H-Ch1, and eight line 1R11 males). These line 1R33 males developed skin problems with a frequency that differed from both B6 and the B6.C3H-Ch1 line, suggesting that multiple cooperating genes along Chromosome 1 must be derived from the same strain to prevent skin lesions. *Ifi202b* and neighboring genes have been implicated in autoimmune skin disease in distal Chromosome 1 congenic lines derived from B6 and the NZB (New Zealand Black) strain (Choubey et al., 2010; Panchanathan et al., 2011).

### **3.6** *Hcs7* **affects early lesion growth**

To determine when *Hcs7* influences tumorigenesis, we measured preneoplastic lesion size in B6, C3H, and congenic lines that had been treated with DEN at 12 days of age. Livers were collected at 16 and 24 weeks and frozen immediately on dry ice, followed by glucose-6-phosphatase staining to identify preneoplastic lesions. In the first experiment, lesions were measured in B6, line 1R11, and line 1R33 livers. By sixteen weeks, lesions in the moderately susceptible line 1R11 and the highly susceptible line 1R33 occupied 3.5 and 3.4 times, respectively, the volume occupied by B6 lesions (*P* < 10-3 for both lines; Table 2). At 24 weeks, 1R11 lesions occupied 1.5-fold more volume than B6 lesions (*P* > 0.15), while 1R33 lesions occupied 2.0-fold more volume than B6 lesions (*P* < 0.05). This result indicates that the growth advantage of lesions in the congenic lines begins prior to 16 weeks.

A second experiment yielded results for line 2R8 that were very similar to those obtained for the larger 1R33 congenic. Line 2R8 lesions occupied 3.4-fold more volume than B6 lesions at 16 weeks; the ratio was 3.3-fold at 24 weeks. This result confirms that *Hcs7* modifies preneoplastic lesion growth. This second experiment included C3H males, which developed lesions that occupied significantly more volume than the congenic at both time points (16 wks: *P* < 0.01; 24 wks: *P* < 10-4). C3H lesions occupied 17-fold more volume than B6 at 16 weeks, which increased to 33-fold more than B6 at 24 weeks. The magnitude of the growth effects in inbred C3H males suggests that *Hcs7* C3H alleles are not sufficient to recapitulate entirely the early lesion development seen in C3H inbred mice.

**Line N Incidence Multiplicity**  B6 46 0.21 0.28 ± 0.58 C3H 38 0.53 0.84 ± 1.10 B6.C3H-Ch1 17 0.47 1.35 ±2.21 1R33 31 0.35 0.65 ± 1.05 1R11 44 0.25 0.34 ± 0.71 Table 1. B6.C3H-Ch1 and 1R33 males are more susceptible to spontaneous liver tumors

Inbred and recombinant male mice were left untreated to 15 months of age and liver tumors

Many line 1R33 males developed severe skin disease. Twenty-eight males died or were euthanized prior to the 15-month time point (as compared to three B6, eight C3H, ten B6.C3H-Ch1, and eight line 1R11 males). These line 1R33 males developed skin problems with a frequency that differed from both B6 and the B6.C3H-Ch1 line, suggesting that multiple cooperating genes along Chromosome 1 must be derived from the same strain to prevent skin lesions. *Ifi202b* and neighboring genes have been implicated in autoimmune skin disease in distal Chromosome 1 congenic lines derived from B6 and the NZB (New

To determine when *Hcs7* influences tumorigenesis, we measured preneoplastic lesion size in B6, C3H, and congenic lines that had been treated with DEN at 12 days of age. Livers were collected at 16 and 24 weeks and frozen immediately on dry ice, followed by glucose-6-phosphatase staining to identify preneoplastic lesions. In the first experiment, lesions were measured in B6, line 1R11, and line 1R33 livers. By sixteen weeks, lesions in the moderately susceptible line 1R11 and the highly susceptible line 1R33 occupied 3.5 and 3.4 times, respectively, the volume occupied by B6 lesions (*P* < 10-3 for both lines; Table 2). At 24 weeks, 1R11 lesions occupied 1.5-fold more volume than B6 lesions (*P* > 0.15), while 1R33 lesions occupied 2.0-fold more volume than B6 lesions (*P* < 0.05). This result indicates that the growth advantage of lesions in the congenic lines begins

A second experiment yielded results for line 2R8 that were very similar to those obtained for the larger 1R33 congenic. Line 2R8 lesions occupied 3.4-fold more volume than B6 lesions at 16 weeks; the ratio was 3.3-fold at 24 weeks. This result confirms that *Hcs7* modifies preneoplastic lesion growth. This second experiment included C3H males, which developed lesions that occupied significantly more volume than the congenic at both time points (16 wks: *P* < 0.01; 24 wks: *P* < 10-4). C3H lesions occupied 17-fold more volume than B6 at 16 weeks, which increased to 33-fold more than B6 at 24 weeks. The magnitude of the growth effects in inbred C3H males suggests that *Hcs7* C3H alleles are not sufficient to

recapitulate entirely the early lesion development seen in C3H inbred mice.

were enumerated. N, the number of mice per group.

**3.6** *Hcs7* **affects early lesion growth** 

prior to 16 weeks.

Zealand Black) strain (Choubey et al., 2010; Panchanathan et al., 2011).

than B6.


Table 2. Distal Chromosome 1 alleles influence net lesion growth before 16 weeks.

Male mice were treated at 12 days of age with DEN and sacrificed at 16 or 24 weeks of age. Frozen liver sections were evaluated for the presence and size distribution of glucose-6 phosphatase-deficient foci. N, number of animals per group; VF ratio, volume fraction ratio relative to B6.
