Abstract  In recent years several new mouse models for lung cancer have been described. These include models for both non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC). Tumorigenesis in these conditional mouse tumor models can be initiated in adult mice through Cre-recombinase-induced activation of oncogenic mutations in a subset of the cells. They present a marked improvement over mouse models that depend on carcinogen induction of tumors. These models permit us to study the consecutive steps involved in initiation and progression and allow us to address questions like the cell of origin, and the role of cancer stem cells in the maintenance of these tumors. They now need to be validated as suitable preclinical models for intervention studies in which questions with respect to therapy response and resistance can be addressed.

Abstract  Identifying the cells of origin of lung cancer may lead to new therapeutic strategies. Previous work has focused upon the putative bronchoalveolar stem cell at the bronchioalveolar duct junction as a cancer cell of origin when a codon 12 K-Ras mutant is induced via adenoviral Cre inhalation. In the present study, we use two "knock-in" Cre-estrogen receptor alleles to inducibly express K-RasG12D in CC10(+) epithelial cells and Sftpc(+) type II alveolar cells of the adult mouse lung. Analysis of these mice identifies type II cells, Clara cells in the terminal bronchioles, and putative bronchoalveolar stem cells as cells of origin for K-Ras-induced lung hyperplasia. However, only type II cells appear to progress to adenocarcinoma.

Abstract  Human lung cancer is responsible for approximately 30% of all cancer deaths worldwide with >160,000 deaths in the United States alone annually. Recent advances in the identification of novel mutations relevant to lung cancer from a myriad of genomic studies might translate into meaningful diagnostic and therapeutic progress. Towards this end, a genetic model animal system that can validate the oncogenic roles of these mutations in vivo would facilitate the understanding of the pathogenesis of lung cancer as well as provide ideal preclinical models for targeted therapy testing. The mouse is a promising model system, as complex human genetic traits causal to lung cancer, from inherited polymorphisms to somatic mutations, can be recapitulated in its genome via genetic manipulation. We present here a brief overview of the existing mouse models of lung cancers and the challenges and opportunities for building the next generation of lung cancer mouse models.

Abstract  Human adenocarcinoma (AC) is the most frequently diagnosed human lung cancer, and its absolute incidence is increasing dramatically. Compared to human lung AC, the A/J mouse-urethane model exhibits similar histological appearance and molecular changes. We examined the gene expression profiles of human and murine lung tissues (normal or AC) and compared the two species' datasets after aligning approximately 7500 orthologous genes. A list of 409 gene classifiers (P value <0.0001), common to both species (joint classifiers), showed significant, positive correlation in expression levels between the two species. A number of previously reported expression changes were recapitulated in both species, such as changes in glycolytic enzymes and cell-cycle proteins. Unexpectedly, joint classifiers in angiogenesis were uniformly down-regulated in tumor tissues. The eicosanoid pathway enzymes prostacyclin synthase (PGIS) and inducible prostaglandin E(2) synthase (PGES) were joint classifiers that showed opposite effects in lung AC (PGIS down-regulated; PGES up-regulated). Finally, tissue microarrays identified the same protein expression pattern for PGIS and PGES in 108 different non-small cell lung cancer biopsies, and the detection of PGIS had statistically significant prognostic value in patient survival. Thus, the A/J mouse-urethane model reflects significant molecular details of human lung AC, and comparison of changes in orthologous gene expression may provide novel insights into lung carcinogenesis.

Abstract  Alveolar type-II cells were isolated from the lungs of fetuses (day 18 of gestation) of the A/WySnAf (A/Sn) mouse strain, which were treated in utero at day 15 with the directly-acting carcinogen N-ethyl-N-nitrosourea (ENU). The isolated type-II cells were again treated with ENU during their initial growth in vitro. After a prolonged culture period, 5 cell lines were obtained, which were identified as type-II cell lines. Differences between cell lines were found with respect to contact-inhibited growth, cell doubling time and ability to grow in a serum-free medium. Two out of the 5 cell lines produced highly invasive type-II cell carcinomas after s.c. injection of 5 x 10(6) cells into nude mice. Thus, both tumorigenic and non-tumorigenic mouse alveolar type-II cell lines were derived after this combined in vivo and in vitro carcinogen treatment of fetal mouse alveolar type-II cells. This offers the possibility of studying in vitro the factors thought to influence lung tumorigenesis in vivo. In addition, our findings strongly suggest that alveolar type-II cells are the progenitor cells of malignant mouse lung tumors.

Abstract  Twenty-five mouse lung tumors induced by a single urethan treatment in female A/J, BALB/c, and (A/J x C3H/He)F1 (AC3) mice were analyzed for the presence of mutations at codon 61 of the Ki-ras gene and for the expression of the surfactant protein A (SP-A), retinoblastoma (Rb), growth arrest-specific-3 (gas-3), p53, c-myc, and thymidylate synthase (TS) genes. Ki-ras codon 61 mutations were detected in 22 of 25 tumor samples without differences among strains. In comparison with normal lungs, all the tumors showed increased SP-A mRNA levels, indicating their derivation from alveolar type II pneumocytes or Clara cells. Rb and gas-3 transcripts were instead found in all tumors at about tenfold and about 20-fold reduced levels, respectively. No apparent structural alterations or loss of heterozygosity at the Rb locus was detected in any tumors. The p53 mRNA was observed without variation in quantity or size in lung tumors and normal tissue. A threefold to fivefold c-myc overexpression was observed, without amplification of the gene. TS expression was only slightly increased, indicating no great differences in cell proliferation between lung tumors and normal tissue. Our data suggest that the pathogenesis of urethan-induced lung tumors in mice involves specific and recurrent molecular alterations (Ki-ras mutations, decrease of Rb and gas-3 expression, and increase of c-myc expression) that could represent different steps in lung carcinogenesis.

Abstract  Alveolar type II cell tumors were induced transplacentally by intraperitoneal injection of pregnant C3H/HeNCr MTV- or Swiss Webster mice with N-nitrosoethylurea at a dose of 0.5 mmol/kg and 0.74 mmol/kg. At different time points after birth (1-32 weeks), the entire lungs from 40 of the male offspring were inflated with Bouin's fixative, separated into lobes, and sectioned at 5 microns serially to detect every microscopic lesion. Results were compared with those obtained from examining only every 10th, 20th, or a single midlevel section from the same material. On average, 150 serial sections were prepared per mouse lung. Initially, only purely solid/alveolar or purely tubulopapillary types were observed but with tumor progression, papillary structures developed within solid tumors resulting in mixed neoplasms. Analyzing mouse lungs in step sections of every 10th section (50-60 microns), 5/238 (2%) of the tumors were missed, in step sections of every 20th section (100-120 microns), 16/238 (7%) of the tumors were not detected and usually less than half of the tumors were seen in the single mid-level section. The approximate size of the neoplasms is indicated by the total number of sections per tumor. The dimensions of tumors evaluated with step sections of 10 or 20 were comparable to the size observed with serial sections. It is concluded that the evaluation of mouse lung tumors in steps of approximately 50 microns is basically equivalent to the study of serial sections and appears to be a feasible method to assess the complete incidence, histological type, and size of all proliferative processes throughout the entire lung.

Abstract  The localization of surfactant apoprotein (SAP) and the Clara cell antigen(s) (CCA) was studied in naturally occurring and experimentally induced pulmonary hyperplasias and neoplasms by avidin-biotin peroxidase complex (ABC) immunocytochemistry. Lungs of B6C3F1 and A strain mice with naturally occurring lesions, B6C3F1 mice given injections of N-nitrosodiethylamine (DEN), BALB/c nu/nu or nu/+ mice exposed transplacentally on Day 16 of gestation to ethylnitrosourea (ENU), or BALB/c nu/+ mice exposed to ENU at 8-12 weeks of age were preserved in formalin or Bouin's fixative. After ABC immunocytochemistry, SAP was found in the cytoplasm of normal alveolar Type II cells; in the majority of cells in focal alveolar and solid hyperplasias originating in peribronchiolar or peripheral locations; and in solid, tubular, papillary, and mixed adenomas and carcinomas. The larger mixed-pattern neoplasms and small or large tubular neoplasms usually had the least number of cells with SAP. The majority of large papillary adenomas and carcinomas in BALB/c mice exposed to ENU and in untreated A strain mice contained SAP in the nuclei of many neoplastic cells but only in the cytoplasm of a few neoplastic cells. CCA was found in normal Clara cells of bronchi and bronchioles but not in any hyperplastic or neoplastic lesion of any mouse studied. This study provided immunocytochemical evidence that the vast majority of naturally occurring and experimentally induced pulmonary neoplasms of mice are alveolar Type II cell adenomas and carcinomas.

Abstract  There is now a great deal of data available that show that BHT enhances the development of lung tumors in mice. In many ways BHT behaves like a promoting agent. Interestingly, it also has tumor enhancing or promoting properties in other organs than mouse lung, such as rat liver, rat bladder, possibly rat GI tract, and in in vitro systems. The development of lung tumors by BHT may be influenced by comparatively low exposure regimens; the minimum dose found so far to be effective is 6 intraperitoneal injections of 50 mg/kg of BHT of feeding a diet containing 500 ppm of BHT for 2 weeks. While these findings seem to require that the continued use of BHT as a food additive needs to be reevaluated, it should be mentioned that other considerations have led to the conclusion that the use of BHT probably has a large margin of safety. This makes it important to establish the mechanism of action of BHT which at this time remains unknown.

Abstract  This chapter focuses on the genetics of murine lung tumors. Some studies indicate that susceptibility to lung tumorigenesis is determined by a single gene, while other studies suggest the involvement of multiple genes. Most studies on the genetics of lung tumorigenesis in mice have considered tumor incidence and the number of tumors per animal as the phenotype, without considering the size of neoplastic lesions. There is no relationship between the susceptibility of any given mouse strain to lung tumors and its susceptibility to tumors of other organs. Susceptibility to spontaneous lung tumor development is paralleled by susceptibility to the induction of the same tumor type by chemical carcinogens. The study and identification of genetic factors affecting inherited predisposition to lung tumorigenesis in mice are of great interest as a model system for understanding pathogenetic mechanisms. Inbred mice represent good model systems for the identification of the number and chromosomal localization of genetic loci predisposing lung tumor development. The knowledge of the genetics of lung tumor susceptibility in mice is growing very quickly. Lung tumor is a relatively common type of cancer in humans, and the familial clustering of cases is rare compared to colon and breast cancer, where both nonhereditary and familial cases are recognized. The murine strains predisposed to lung tumor development may provide a unique experimental system for the analysis of the genetics of these tumors.

Abstract  About 10 years have elapsed since the first whole-genome scanning studies in the mouse to identify loci that affect susceptibility or resistance to tumorigenesis. In that time, >100 cancer modifiers have been mapped, and four strong candidate genes have been identified. Cancer modifier loci affect almost all types of mouse tumorigenesis, with some loci acting on the entire tumorigenic process, whereas others act on specific stages, e.g., tumor initiation or tumor growth/progression. Present evidence indicates that the effects of cancer modifier loci are tissue-specific and restricted to tumor cells. However, a subset of such loci may be involved in different types of tumors, and several chromosomal regions show significant clustering of cancer modifier loci. Human homologues of mouse cancer modifier loci most likely exist and play a role in the risk of sporadic cancer, although present experimental evidence for this possibility is sparse. Mouse cancer modifier loci might serve as the basis for understanding the genetic and biochemical mechanisms of polygenic inheritance of cancer predisposition/resistance. Identification of homologous cancer modifier loci in humans might, in turn, provide a step toward the development of diagnostic, preventive, and therapeutic strategies that target these loci.

Abstract  The cyclin-dependent kinase inhibitor 2a (Cdkn2a) locus encodes two distinct tumor suppressors, p16INK4a and p19ARF, whose functions interrelate in the regulation of cell proliferation as key components of the retinoblastoma and p53 pathways, respectively. In many types of cancer, alterations of Cdkn2a abrogate the functions of both suppressors, implying that both are integral to the genesis of certain cancer types. While this has been observed in mouse lung adenocarcinogenesis, recent observations also suggested that naturally occurring variation at the Cdkn2a locus is probably operative in the development of these tumors. Firstly, two common haplotypes of mouse Cdkn2a have been identified, each of which encodes cosegregating variants of p16INK4a and p19ARF. The p16INK4a variants differ at amino acids 18 (histidine or proline) and 51 (valine or isoleucine), whereas the p19ARF variants differ only at amino acid 72 (histidine or arginine). Secondly, genetic resistance to lung tumor formation appears to segregate with one particular haplotype, which also is deleted preferentially in lung adenocarcinomas of Cdkn2a heterozygous mice. Here we attempt to explain these observations and to characterize further the roles of p16INK4 and p19ARF in mouse lung tumorigenesis by examining the function and expression of each of the variants of Cdkn2a. Functional analysis showed that the proline 18/isoleucine 51 p16INK4a variant was diminished in cdk6 binding, cdk6 inhibition and NIH/3T3 fibroblast growth suppression compared with the histidine 18/valine 51 variant, whereas both of the p19ARF variants suppressed growth with similar potencies. Also, the different alleles for p16INK4a and p19ARF were transcribed equally in the normal lungs of Cdkn2a heterozygotes, as determined by comparative reverse transcription-polymerase chain reaction-single-stranded conformation polymorphism analysis. These results indicate that strain-specific variation in p16INK4a function is exploited in mouse lung tumorigenesis and strongly implicate a role for p16INK4a in lung cancer predisposition and development.

Abstract  National Toxicology Program (NTP) inhalation studies demonstrated that cumene significantly increased the incidence of alveolar/bronchiolar adenomas and carcinomas in B6C3F1 mice. Cumene or isopropylbenzene is a component of crude oil used primarily in the production of phenol and acetone. The authors performed global gene expression analysis to distinguish patterns of gene regulation between cumene-induced tumors and normal lung tissue and to look for patterns based on the presence or absence of K-ras and p53 mutations in the tumors. Principal component analysis segregated the carcinomas into groups with and without K-ras mutations, but failed to separate the tumors based on p53 mutation status. Expression of genes associated with the Erk MAP kinase signaling pathway was significantly altered in carcinomas with K-ras mutations compared to tumors without K-ras mutations or normal lung. Gene expression analysis also suggested that cumene-induced carcinomas with K-ras mutations have greater malignant potential than those without mutations. In addition, significance analysis of function and expression (SAFE) demonstrated expression changes of genes regulated by histone modification in carcinomas with K-ras mutations. The gene expression analysis suggested the formation of alveolar/bronchiolar carcinomas in cumene-exposed mice typically involves mutation of K-ras, which results in increased Erk MAP kinase signaling and modification of histones.

Abstract  Divinylbenzene-HP is used for producing vinyl polymers. Divinylbenzene-HP was nominated for study by the National Cancer Institute because of the potential for worker exposure and the structural similarity of divinylbenzene to styrene, a potential human carcinogen. Male and female F344/N rats and B6C3F1 mice were exposed to divinylbenzene-HP (80%) by inhalation for 2 weeks, 3 months, or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, Escherichia coli, and mouse peripheral blood erythrocytes. 2-WEEK STUDY IN RATS Groups of five male and five female rats were exposed by whole body inhalation to divinylbenzene-HP at target concentrations of 0, 25, 50, 100, 200, or 400 ppm 6 hours plus T90 (12 minutes) per day, 5 days per week for 16 days. All rats survived to the end of the study. Significant decreases in mean body weights occurred in both male and female rats in the 400 ppm groups. Relative kidney weights of 50 ppm or greater males and relative liver weights of 200 and 400 ppm males were significantly greater than those of the chamber controls. A clear serous nasal/eye discharge was observed in groups of males exposed to 100 ppm or greater and females exposed to 50 ppm or greater. Minimal or mild rhinitis occurred in 400 ppm rats of both sexes. 2-WEEK STUDY IN MICE Groups of five male and five female mice were exposed by whole body inhalation to divinylbenzene-HP at target concentrations of 0, 25, 50, 100, 200, or 400 ppm for 6 hours plus T90 (12 minutes) per day, 5 days per week for 17 days. All 400 ppm males and females died on or before the second day of the study, and two male and two female 200 ppm mice died early. Mean body weights of 100 and 200 ppm males were significantly less than those of the chamber controls. Thymus weights of exposed groups of males were significantly less than those of the chamber controls, and relative liver weights of 100 and 200 ppm males were significantly increased. Kidney and liver weights of exposed groups of females were significantly greater than those of the chamber controls. Mice exposed to 200 and 400 ppm had liver lesions including degeneration, necrosis, hemorrhage or cytomegaly. Renal tubule necrosis and regeneration occurred at 200 ppm. Necrosis or metaplasia of nasal epithelium and glands occurred in the nose in all exposure groups. 3-MONTH STUDY IN RATS Groups of 10 male and 10 female rats were exposed to divinylbenzene-HP at concentrations of 0, 25, 50, 100, 200, or 400 ppm for 6 hours plus T90 (12 minutes) per day, 5 days per week for 14 weeks. All rats survived to the end of the study. There were no biologically significant changes in body weight in either sex. Nasal/eye discharge was noted in 400 ppm males and 100 ppm females. Kidney and liver weights of exposed groups of males and of 400 ppm females were generally greater than those of the chamber controls. In addition, the relative weights of the heart and testis were significantly increased in 200 and 400 ppm males. Incidences of degeneration of the olfactory epithelium in 200 and 400 ppm rats and basal cell hyperplasia of the olfactory epithelium in rats exposed to 100 ppm or greater were significantly increased. 3-MONTH STUDY IN MICE Groups of 10 male and 10 female mice were exposed to divinylbenzene-HP at concentrations of 0, 12.5, 25, 50, 100, or 200 ppm for 6 hours plus T90 (12 minutes) per day, 5 days per week for 14 weeks. All 200 ppm males and nine 200 ppm females died early. Final mean body weights were significantly lower in males and females exposed to 25, 50, or 100 ppm when compared with chamber controls. Lethargy or hypoactivity was observed in the higher exposure concentration groups. Exposure to divinylbenzene was associated with necrosis of the liver and kidney in 200 ppm males and females dying early. In all exposed groups of male and female mice, there was necrosis of nasal cavity lateral walls, olfactory epithelium, and glands with resultant atrophy of olfactory epithelium and glands in females. A lower number of animals had necrotic or degenerative changes of the upper respiratory tract. 2-YEAR STUDY IN RATS Groups of 50 male and 50 female rats were exposed to divinylbenzene-HP at concentrations of 0, 100, 200, or 400 ppm for 6 hours plus T90 (12 minutes) per day, 5 days per week for up to 105 weeks. Survival of 400 ppm females was significantly less than that of the chamber control group. Survival of all exposed groups of males was similar to that of the chamber control group. Mean body weights of 400 ppm males and females were significantly less than those of the controls during the second half of the study. Renal tubule carcinomas occurred in two of 50 males exposed to 400 ppm in the original kidney sections, an incidence that exceeded the historical control range. In 400 ppm males, the incidence of renal tubule hyperplasia was increased, and the incidence of nephropathy was significantly increased. Following combined analysis of single and step-section data, the incidences of renal tubule adenoma and adenoma or carcinoma (combined) were marginally higher in 200 and 400 ppm males, and the incidence of renal tubule hyperplasia was significantly increased in 400 ppm males. The incidences of malignant glial cell tumors (malignant astrocytoma and oligodendroglioma) in the brain were slightly increased in 100 and 200 ppm males, and the incidence in the 200 ppm group exceeded the historical range for chamber controls. There were increased incidences of degenerative and regenerative changes in the olfactory epithelium in the nose of all exposed groups of rats. The incidence of focal chronic inflammation in the lung of 400 ppm males was significantly greater than in the chamber control group. 2-YEAR STUDY IN MICE Groups of 50 male and 50 female mice were exposed to divinylbenzene-HP at concentrations of 0, 10, 30, or 100 ppm for 6 hours plus T90 (12 minutes) per day, 5 days per week for up to 105 weeks. Survival of all exposed groups of male and female mice was similar to that of the chamber controls. Mean body weights were lower relative to chamber controls in 100 ppm males and in 30 and 100 ppm females. The incidences of alveolar/bronchiolar adenoma and alveolar/bronchiolar adenoma or carcinoma (combined) in 100 ppm males were greater than chamber control incidences, but the incidences of adenoma or carcinoma (combined) were within the historical control range. The incidences of alveolar/bronchiolar adenoma and alveolar/bronchiolar adenoma or carcinoma (combined) in all exposed groups of females were generally greater than those of the chamber controls; the incidences were at the upper end or exceeded the historical control ranges. There was a greater incidence and severity of alveolar epithelial hyperplasia in 100 ppm females and a greater severity of this lesion in 30 ppm females, when compared to chamber controls. The incidences and/or severities of atypical bronchiole hyperplasia were significantly increased in all exposed groups of mice. Nonneoplastic nasal lesions occurred in most exposed mice. GENETIC TOXICOLOGY Divinylbenzene-HP was not mutagenic in any of three independent gene mutation assays using Salmonella typhimurium strains TA97, TA98, TA100, TA1535, or TA1537 or Escherichia coli tester strain WP2 uvrA with or without induced hamster or rat liver enzymes. No increases in the frequencies of micronucleated normochromatic erythrocytes or alterations in the percentages of polychromatic erythrocytes were seen in peripheral blood of male or female B6C3F1 mice exposed to divinylbenzene-HP by inhalation for 3 months. CONCLUSIONS Under the conditions of this 2-year inhalation study, there was equivocal evidence of carcinogenic activity of divinylbenzene-HP in male F344/N rats based upon the occurrence of carcinomas in the kidney and glial tumors in the brain. There was no evidence of carcinogenic activity in female F344/N rats exposed to 100, 200, or 400 ppm divinylbenzene-HP. There was no evidence of carcinogenic activity in male B6C3F1 mice exposed to 10, 30, or 100 ppm divinylbenzene-HP. There was equivocal evidence of carcinogenic activity of divinylbenzene-HP in female B6C3F1 mice based on the incidences of alveolar/bronchiolar adenoma or carcinoma (combined) in the lung. Exposure to divinylbenzene-HP caused nonneoplastic lesions of the nasal cavity in male and female rats and of the lung and nasal cavity in male and female mice.

Abstract  Pregnant C3H/HeNCr MTV- mice were given a single intraperitoneal injection of 0.5 mmol N-nitrosoethylurea/kg on days 14, 16, or 18 of gestation. Six of the male offspring were sacrificed for study at the ages of 2, 4, 8, 16, 32, and 52 weeks. Grossly visible lung tumors were counted and all lungs were sectioned completely, saving every tenth section for histologic evaluation. All N-nitrosoethylurea-induced mouse lung tumors have previously been shown to originate from alveolar type II cells. Lung tumors were diagnosed as solid, papillary, or mixed solid/papillary types, and at the largest area of each tumor, the perimeter was measured and compared with the number of sections per tumor. The fraction of tumors detected grossly depended on size and, on average, only 51% of neoplasms present were detected macroscopically. A significant correlation was seen between the mean number of histological sections and perimeter length per tumor, in particular for small and medium sized papillary neoplasms. The growth of solid tumors was limited to a maximum size, after which they progressed towards papillary types. The numbers of transplacentally induced mouse lung tumors were distributed in direct proportion to the weight of the individual lung lobes, unrelated to day of treatment of type or tumor. Tumor biology depended on the day of treatment reflecting numbers of degree of differentiation of fetal alveolar type II cells, i.e., the target cell: most tumors developed in offspring treated on day 16, tumor size was greater and progression from solid to papillary neoplasms faster at earlier treatments, increase in tumor multiplicity postnatally was only seen in mice treated late in gestation, and mice treated on day 14 or day 16 showed a consistent ratio of solid to papillary tumors.

Abstract  The continued progress of modelling lung cancer in mice has led not only to new means of understanding the molecular pathways governing human lung cancer, but it has also created a vast reservoir of alternative tools to test treatments against this malignancy. More sophisticated somatic mouse models for nonsmall cell lung cancer, small cell lung cancer and pulmonary squamous cell carcinoma have been generated that closely mimic human lung cancer. These models enable us to identify the cells of origin and the role of stem cells in the maintenance of the various types of lung cancer. Moreover, results of lung cancer intervention studies are now starting to reveal the full potential of these somatic mouse models as powerful pre-clinical models.

Abstract  Identifying the cells of origin of lung cancer may lead to new therapeutic strategies. Previous work has focused upon the putative bronchoalveolar stem cell at the bronchioalveolar duct junction as a cancer cell of origin when a codon 12 K-Ras mutant is induced via adenoviral Cre inhalation. In the present study, we use two "knock-in" Cre-estrogen receptor alleles to inducibly express K-RasG12D in CC10(+) epithelial cells and Sftpc(+) type II alveolar cells of the adult mouse lung. Analysis of these mice identifies type II cells, Clara cells in the terminal bronchioles, and putative bronchoalveolar stem cells as cells of origin for K-Ras-induced lung hyperplasia. However, only type II cells appear to progress to adenocarcinoma.

Abstract  To investigate the role of an activated K-Ras gene in the initiation and maintenance of lung adenocarcinomas, we developed transgenic mice that express murine K-Ras4b(G12D) under the control of doxycycline in type II pneumocytes. Focal proliferative lesions of alveolar type II pneumocytes were observed as early as seven days after induction with doxycycline; after two months of induction, the lungs contained adenomas and adenocarcinomas, with focal invasion of the pleura at later stages. Removal of doxycycline caused a rapid fall in levels of mutant K-Ras RNA and concomitant apoptotic regression of both the early proliferative lesions and the tumors. Tumor burden was dramatically decreased by three days after withdrawal, and tumors were undetectable after one month. When similar experiments were performed with animals deficient in either the p53 gene or the Ink4A/Arf locus, tumors arose more quickly (within one month of exposure to doxycycline) and displayed more obvious histological features of malignancy; nevertheless, these tumors also regressed rapidly when the inducer was removed, implying that continued production of mutant K-Ras is necessary to maintain the viability of tumor cells in the absence as well as the presence of tumor suppressor genes. We also show that the appearance and regression of these pulmonary tumors can be readily monitored in anesthetized transgenic animals by magnetic resonance imaging.

Abstract  In the A/J strain of mice, urethane (ethyl carbamate) induces lung hyperplasia, adenoma, and adenocarcinoma in a time-dependent manner. These distinct morphological stages may correlate with sequential molecular genetic changes in this mouse model. To test this hypothesis, we investigated the presence of mutations involving Ki-ras and p53 in urethane-induced lung lesions in A/J mice at early and late stages of tumorigenesis. We precisely microdissected 40 lung lesions from paraffin-embedded sections. Ki-ras mutations around codon 61 and p53 mutations in exons 5-8 were identified by polymerase chain reaction-single-strand conformation polymorphism and DNA sequencing techniques. In 29 early-stage lung lesions classified as hyperplasias (seven) or adenomas (22), we observed 19 Ki-ras mutations (66%), including three silent mutations and one double mutation at different codons, and one silent p53 mutation (3.5%). In 11 late-stage adenomas, we identified nine activating Ki-ras mutations (82%) and four missense p53 mutations (36%). These results indicate that Ki-ras mutations arise early, whereas p53 mutations occur relatively late during the benign stages of urethane-induced lung carcinogenesis in A/J mice.

Abstract  About 30% of human tumours carry ras gene mutations. Of the three genes in this family (composed of K-ras, N-ras and H-ras), K-ras is the most frequently mutated member in human tumours, including adenocarcinomas of the pancreas ( approximately 70-90% incidence), colon ( approximately 50%) and lung ( approximately 25-50%). To construct mouse tumour models involving K-ras, we used a new gene targeting procedure to create mouse strains carrying oncogenic alleles of K-ras that can be activated only on a spontaneous recombination event in the whole animal. Here we show that mice carrying these mutations were highly predisposed to a range of tumour types, predominantly early onset lung cancer. This model was further characterized by examining the effects of germline mutations in the tumour suppressor gene p53, which is known to be mutated along with K-ras in human tumours. This approach has several advantages over traditional transgenic strategies, including that it more closely recapitulates spontaneous oncogene activation as seen in human cancers.

Abstract  Lung cancer kills more Americans yearly than any other neoplastic process. Mortality rates have changed little over the past several decades, despite improvements in surgical techniques, radiation therapy and chemotherapy. The identification of mutations in oncogenes and tumor suppressor genes in human lung tumor specimens, including K-ras, p53, p16INK4a and Rb, offers molecular explanations for tumor development and resistance to therapy. Mouse models of human lung cancer may advance our understanding of this disease. The examination of mice which develop lung cancer either spontaneously or due to carcinogen exposure, and the creation of mouse strains harboring the specific genetic mutations found in human lung cancer are among strategies being pursued.