Although the ras genes have long been established as proto-oncogenes, the dominant role of activated ras in cell transformation has been questioned. Previous studies have shown frequent loss of the wildtype Kras2 allele in both mouse and human lung adenocarcinomas. To address the possible tumor suppressor role of wildtype Kras2 in lung tumorigenesis, we have carried out a lung tumor bioassay in heterozygous Kras2-deficient mice. Mice with a heterozygous Kras2 deficiency were highly susceptible to the chemical induction of lung tumors when compared to wildtype mice. Activating Kras2 mutations were detected in all chemically induced lung tumors obtained from both wildtype and heterozygous Kras2-deficient mice. Furthermore, wildtype Kras2 inhibited colony formation and tumor development by transformed NIH/3T3 cells and a mouse lung tumor cell line containing an activated Kras2 allele. Allelic loss of wildtype Kras2 was found in 67% to 100% of chemically induced mouse lung adenocarcinomas that harbor a mutant Kras2 allele. Finally, an inverse correlation between the level of wildtype Kras2 expression and extracellular signal-regulated kinase (ERK) activity was observed in these cells. These data strongly suggest that wildtype Kras2 has tumor suppressor activity and is frequently lost during lung tumor progression.

Lung cancer, primarily associated with tobacco use, is the leading cause of cancer morbidity and mortality in the United States. Squamous cell carcinoma (SCC) is one of the four major histological types of lung cancer. Although there are several established models for lung adenoma and adenocarcinomas, there is no well-established mouse model for lung SCC. We treated eight different inbred strains of mice with N-nitroso-trischloroethylurea by skin painting and found that this regimen induced lung SCCs in five strains of mouse (SWR/J, NIH Swiss, A/J, BALB/cJ, and FVB/J) but not in the others (AKR/J, 129/svJ, and C57BL/6J). Mouse lung SCCs have similar histopathological features and keratin staining to human SCC. Moreover, a wide spectrum of abnormal lung squamous phenotypes including hyperplasia, metaplasia, carcinoma in situ, and invasive carcinoma, were observed. There are strain-specific differences in susceptibility to Lscc induction by N-nitroso-tris-chloroethylurea with NIH Swiss, A/J, and SWR/J mice developing scores of SCCs whereas the resistant strains AKR/J, 129/svJ, and C57BL/6J failed to develop any SCCs. FVB/J and BALB/cJ mice had an intermediate response. We conducted whole-genome linkage disequilibrium analysis in seven strains of mice, divided into three phenotype categories of susceptibility, using Fisher's exact test applied to 6,128 markers in publically available databases. Three markers were found significantly associated with susceptibility to SCC with the P < 0.05. They were D1Mit169, D3Mit178, and D18Mit91. Interestingly, none of these sites overlap with the major susceptibility loci associated with lung adenoma/adenocarcinoma development in mice. The mouse SCC described here is highly significant for preclinical studies of lung cancer chemopreventive agents because most human trials have been conducted against precancerous lesions for SCC. Furthermore, this model can be used in determining genetic modifiers that contribute to susceptibility or resistance to lung SCC development.

This report describes recent efforts to develop and apply small animal magnetic resonance imaging methods to the study of lung tumors in mice. Magnetic resonance (MR) images obtained with respiratory gating, with data collection synchronized with the respiration of the animal, allow visualization of submillimeter tumors in animals treated with a lung carcinogen. Comparison of the MR images with gross pathology of these lungs demonstrates the utility of the imaging methods for measuring tumor burden. As a noninvasive imaging modality that uses nonionizing radiation, MR is well suited to longitudinal studies aimed at understanding the factors that control the onset and development of lung tumors and their response to therapy in a wide variety of animal models.

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Streptococcus suis serotype 2 is an important zoonotic pathogen. Antimicrobial resistance phenotypes and genotypic characterizations of S. suis 2 from carrier sows and diseased pigs remain largely unknown. In this study, 96 swine S. suis type 2, 62 from healthy sows and 34 from diseased pigs, were analyzed. High frequency of tetracycline resistance was observed, followed by sulfonamides. The lowest resistance of S. suis 2 for β-lactams supports their use as the primary antibiotics to treat the infection of serotype 2. In contrast, 35 of 37 S. suis 2 with MLSB phenotypes were isolated from healthy sows, mostly encoded by the ermB and/or the mefA genes. Significantly lower frequency of mrp+/epf+/sly+ was observed among serotype 2 from healthy sows compared to those from diseased pigs. Furthermore, isolates from diseased pigs showed more homogeneously genetic patterns, with most of them clustered in pulsotypes A and E. The data indicate the genetic complexity of S. suis 2 between herds and a close linkage among isolates from healthy sows and diseased pigs. Moreover, many factors, such as extensive use of tetracycline or diffusion of Tn916 with tetM, might have favored for the pathogenicity and widespread dissemination of S. suis serotype 2.

Pulmonary adenoma susceptibility 1 (Pas1), located on chromosome 6, is the major locus affecting inherited predisposition to lung tumor development in mice. We have fine mapped the Pas1 locus to a region of Ϸ0.5 megabases by using congenic strains of mice, constructed by placing the Pas1 region of chromosome 6 from A͞J mice onto the genetic background of C57BL͞6J mice. Systematic characterization of Pas1 candidates establishes the Las1 (lung adenoma susceptibility 1) and Kras2 (Kirsten rat sarcoma oncogene 2) genes as primary candidates for the Pas1 locus. Clearly, Kras2 affects lung tumor progression only, and Las1 is likely to affect lung tumor multiplicity. L ung cancer is the leading cause of cancer death in men and women in the United States (1). Although lung cancer is largely induced by smoking, there is strong evidence for genetic susceptibility and gene-environment interactions in its development (2-5). However, genetic heterogeneity and enormous variation in exposure levels to environmental agents make it difficult to identify lung cancer susceptibility loci in humans. Thus, inbred mouse models offer an effective means of identifying candidate lung cancer susceptibility loci (6)(7)(8)(9)(10)(11)(12)(13)(14). Inbred strains of mice have different susceptibilities to spontaneous and carcinogen-induced lung tumor formation (6, 7). The A͞J strain is the most susceptible to lung tumorigenesis, whereas the C3H and C57BL͞6J strains are among the most resistant (6, 7). Linkage study using (A͞J ϫ C3H͞HeJ)F 2 and (A͞J ϫ C57BL͞6J)F 2 mice has demonstrated that pulmonary adenoma susceptibility 1 (Pas1) is the major lung tumor susceptibility locus in mice, which has been mapped to the distal region of chromosome 6 and accounts for Ϸ50% of the phenotypic variance (9, 10). Here we provide definitive evidence to support the candidacy of both Las1 (lung adenoma susceptibility 1) and Kras2 (Kirsten rat sarcoma oncogene 2) as the Pas1 genes on mouse chromosome 6. We are not aware of the identification of any other candidates for this quantitative trait locus (QTL) in either mouse models or humans since the initial mapping of the Pas1 QTL in 1993 (9). Materials and MethodsConstruction of Congenic Strains. Inbred A͞J and C57BL͞6J mice were purchased from The Jackson Laboratory. The basic breeding scheme in the study was to put an Ϸ26.1-centimorgan fragment of chromosome 6, encompassed by D6MIT54 and D6MIT373 markers from the lung tumor-susceptible A͞J strain, onto the genetic background of lung tumor-resistant C57BL͞6J mice. A͞J mice were crossed initially to C57BL͞6J mice. F 1 progeny were backcrossed to C57BL͞6J mice to produce the first backcross generation (N 2 ). The N 2 generation heterozygous for the chromosome region of interest was then backcrossed again to C57BL͞6J mice to produce the N 3 generation. This process was repeated for a total of seven backcrosses. At the N 5 generation, additional microsatellite markers on chromosomes 9, 10, 17, and 19 (D9MIT75, D9MIT355, D9MIT35, D10MIT106, D10MIT2, D10MIT126, D17MIT...

Previous observation has shown that the wild-type Kras2 allele is a suppressor of lung cancer in mice. Here we report that loss of heterozygosity (LOH) of chromosome 12p was detected in B50% of human lung adenocarcinomas and large cell carcinomas, and Kras2 mutations were detected at codon 12 in B40% of adenocarcinomas and large cell carcinomas. Interestingly, all of the lung adenocarcinomas and large cell carcinomas containing a Kras2 mutation exhibited allelic loss of the wild-type Kras2 allele when a correlation between LOH of the region on chromosome 12p and Kras2 mutation was made. These results from human lung cancer tissues provide a strong evidence in support of our previous observation in mouse models that the wild-type Kras2 is a tumor suppressor of lung cancer.