Epistatic interactions govern chemically-induced lung tumor susceptibility and Kras mutation site in murine C57BL/6J-ChrA/J chromosome substitution strains

Abstract

Cancer susceptibility results from interactions between sensitivity and resistance alleles. We employed murine chromosome substitution strains to study how resistance alleles affected sensitive alleles during chemically-induced lung carcinogenesis. The C57BL/6J-Chr#(A/J) strains, constructed by selectively breeding sensitive A/J and resistant C57BL/6J (B6) mice, each contain one pair of A/J chromosomes within an otherwise B6 genome. Pas1, the major locus responsible for this differential strain response to urethane carcinogenesis, resides on Chr 6, but C57BL/6J-Chr6(A/J) mice (hereafter CSS-6) developed few tumors following a single urethane injection, which demonstrates epistatic interactions with other B6 alleles. CSS6 mice developed dozens of lung tumors after chronic urethane exposure, however, indicating that these epistatic interactions could be overcome by repeated carcinogen administration. Unlike A/J, but similar to B6 mice, CSS6 mice were resistant to lung carcinogenesis induced by 3-methylcholanthrene (MCA). Tumor multiplicity increased if BHT administration followed urethane exposure, showing that a Chr 6 gene(s) regulates sensitivity to chemically-induced tumor promotion. Unlike A/J tumors (predominantly codon 61 A-->T transversions), Kras mutations in tumors induced by urethane in CSS-6 mice were similar to B6 tumors (codon 61 A-->G transitions). DNA repair genes not located on Chr 6 may determine the nature of Kras mutations. CSS-6 mice are a valuable resource for testing the ability of candidate genes to modulate lung carcinogenesis.

Fig. 1. Lung tumor development following multiple injections of urethane

Average tumor multiplicity (A) and tumor diameter (B) in B6, CSS6, CSS11, and A/J mice treated with either a single 1mg/g dose of urethane (white bars) or 6 weekly 1 mg/g urethane doses (black bars). Mice were sacrificed 20 wks after the first urethane injection and tumors evaluated. Error bars represent SEM. (A) 6 urethane vs. 1 urethane: all strains have significant differences in multiplicity with chronic dosing (*p< 0.001). (B) The diameters of lung tumors from A/J mice treated with 6 urethanes are significantly larger than those from A/J mice treated with a single urethane injection as well as similarly treated B6, CSS6, and CSS11 mice (*p < 0.001). Tumors in CSS11 mice are larger than B6 (#p < 0.05. (C) (B6 × CSS6) F1, B6, and CSS6 mice were subjected to 6 wkly urethane injections, and tumors counted 20 wks after the first injection. **p < 0.01 vs. B6 and CSS6.

Fig. 2. Time course of lung tumor development

Time courses of tumor multiplicity (A) and diameter (B) after a single urethane exposure. A/J (▲), CSS6 (■), and B6 (●). * p < 0.01 vs. 16 wks. (C) H&E stain of A/J and CSS6 lung tumors 42 wks after urethane treatment. Bar is 100 μm.

Fig. 3. BHT-induced lung tumor promotion and pulmonary inflammation

(C) CSS6 mice were treated with MCA + vehicle (white bars) or MCA + BHT (black bars), and tumors counted 20 wks after MCA injection. *p < 0.03 vs. vehicle. (D) BAL macrophage (gray bars) and lymphocyte (black bars) contents in mice treated with 4 weekly BHT or corn oil vehicle (control) injections. *p < 0.05 vs. control. (E) B6 mice were given 6 wkly urethane injections followed by either 6 wkly BHT or vehicle injections. Tumors were harvested 20 wks after the initial urethane injection.

Fig. 4. Kras mutational analysis in A/J, B6, and CSS strains

(A) Strain survey of Kras codon 61 sequence in urethane-induced lung tumors from B6, CSS, and A/J mice. Kras codon 61 sequence: Wild type CAA, (white bars); CGA, (gray bars); CTA, (black bars). (B) Kras mutations as a function of time after mice received a single uethane injection in CSS6 and A/J mice. (C) Kras mutations as a function of number of urethane injections, one vs.6, in B6, CSS6, and A/J mice.