Theodora R. Devereux

Wildtype Kras2 can inhibit lung carcinogenesis in mice

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.

p53 mutation hotspot in radon-associated lung cancer

Mutations in gene p53 are the most common defects in lung cancer and may be a pathway through which environmental carcinogens initiate cancer. We investigated p53 mutations in lung cancers from uranium miners with high radon exposure. 16 (31%) of 52 large-cell and squamous-cell cancers from miners contained the same AGG to ATG transversion at codon 249, including cancers from 3 or 5 miners who had never smoked. This specific mutation has been reported in only 1 of 241 published p53 mutations from lung cancers. The codon 249 mutation may be a marker for radon-induced lung cancer.

Mutation of β-catenin is an early event in chemically induced mouse hepatocellular carcinogenesis

β-catenin activation, and subsequent upregulation of Wnt-signaling, is an important event in the development of certain human and rodent cancers. Recently, mutations in the β-catenin gene in the region of the serine-threonine glycogen kinase (GSK)-3β phosphorylation target sites have been identified in hepatocellular neoplasms from humans and transgenic mice. In this study we examined 152 hepatocellular neoplasms from B6C3F1 mice included in five chemical treatment groups and controls for mutations in the β-catenin gene. Twenty of 29 hepatocellular neoplasms from mice treated with methyleugenol had point mutations at codons 32, 33, 34 or 41, sites which are mutated in colon and other cancers. Likewise, nine of 24 methylene chloride-induced hepatocellular neoplasms and 18 of 42 oxazepam-induced neoplasms exhibited similar mutations. In contrast, only three of 18 vinyl carbamate-induced liver tumors, one of 18 TCDD-induced liver tumors, and two of 22 spontaneous liver neoplasms had mutations in β-catenin. Thus, there appears to be a chemical specific involvement of β-catenin activation in mouse hepatocellular carcinogenesis. Expression analyses using Western blot and immunohistochemistry indicate that β-catenin protein accumulates along cell membranes following mutation. The finding of mutations in both adenomas and carcinomas from diverse chemical treatment groups and the immunostaining of β-catenin protein in an altered hepatocellular focus suggest that these alterations are early events in mouse hepatocellular carcinogenesis.

Aberrant Promoter Hypermethylation of the Death-Associated Protein Kinase Gene Is Early and Frequent in Murine Lung Tumors Induced by Cigarette Smoke and Tobacco Carcinogens

Loss of expression of the death-associated protein (DAP)-kinase gene by aberrant promoter methylation may play an important role in cancer development and progression. The purpose of this investigation was to determine the commonality for inactivation of the DAP-kinase gene in adenocarcinomas induced in mice by chronic exposure to mainstream cigarette smoke, the tobacco carcinogens 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and vinyl carbamate, and the occupational carcinogen methylene chloride. The timing for inactivation was also determined in alveolar hyperplasias that arise in lung cancer induced in the A/J mouse by NNK. The DAP-kinase gene was not expressed in three of five NNK-induced lung tumor-derived cell lines or in a spontaneously arising lung tumor-derived cell line. Treatment with 5-aza-2′-deoxycytidine restored expression; dense methylation throughout the DAP-kinase CpG island detected by bisulfite sequencing supported methylation as the inactivating event in these cell lines. Methylation-specific PCR detected inactivation of the DAP-kinase gene in 43% of tumors associated with cigarette smoke, a frequency similar to those reported in human non-small cell lung cancer. In addition, DAP-kinase methylation was detected in 52%, 60%, and 50% of tumors associated with NNK, vinyl carbamate, and methylene chloride, respectively. Methylation was observed at similar prevalence in both NNK-induced hyperplasias and adenocarcinomas (46% versus 52%), suggesting that inactivation of this gene is one pathway for tumor development in the mouse lung. Bisulfite sequencing of both premalignant and malignant lesions revealed dense methylation, substantiating that this gene is functionally inactivated at the earliest histological stages of adenocarcinoma development. This study is the first to use a murine model of cigarette smoke-induced lung cancer and demonstrate commonality for inactivation by promoter hypermethylation of a gene implicated in the development of this disease in humans.

Identification of Cytochrome P-450 Isozymes in Nonciliated Bronchiolar Epithelial (Clara) and Alveolar Type II Cells Isolated from Rabbit Lung

Two forms of cytochrome P-450 (P-450I and P-450II) have been shown by several techniques to be present in both nonciliated bronchiolar cells (Clara) and alveolar type II cells isolated from rabbit lung. In contrast, the alveolar macrophage contains little or none of these cytochromes. Cross-reactivity between antibodies to cytochrome P-450I or P-450II and detergent-digested microsomes prepared from 80% type II or 70% Clara cell fractions was shown by Ouchterlony double immunodiffusion. The presence of both cytochromes was also demonstrated by histochemical immunofluorescence in smears of type II cells stained by a modified Papanicolaou procedure and Clara cells stained with nitroblue tetrazolium. However, this same fluorescent antibody technique used for localization of rabbit pulmonary cytochromes P-450I and P-450II in tissue sections showed most of the immunofluorescence in the Clara cells of the bronchiolar epithelium. SDS-polyacrylamide gel electrophoresis of microsomes from either the type II or Clara cell fractions produced bands which corresponded to cytochrome P-450I (52,000 daltons) and cytochrome P-450II (58,000 daltons).

CTNNB1 mutations and β‐catenin protein accumulation in human hepatocellular carcinomas associated with high exposure to aflatoxin B1†

β‐Catenin plays a key role in the Wnt signaling pathway, and mutations of CTNNB1, the gene that encodes β‐catenin, have been identified in about one‐fourth of human hepatocellular carcinomas from regions of low aflatoxin B1 exposure. In this study 62 hepatocellular carcinomas (HCCs) from people highly exposed to aflatoxin B1 in Guangxi, Peoples Republic of China, were laser‐capture microdissected and examined for CTNNB1 mutations. In addition, 41 of the HCCs were evaluated for the presence of the β‐catenin protein by immunohistochemical methods. Twenty of the HCCs showed positive results for β‐catenin, with strong membrane staining, while adjacent non‐neoplastic liver tissue lacked or showed only weak membrane staining. One HCC, in which a CTNNB1 mutation was not detected, showed nuclear staining for the β‐catenin protein. Mutations of CTNNB1 were identified in five HCCs. These consisted of four point mutations in the glycogen serine kinase‐3β phosphorylation region of codons 32–45 and one deletion of codons 32–38. These mutations were similar to those previously reported for human HCC, although at a lower frequency. A signature mutation profile associated with aflatoxin B1 exposure could not be identified. The immunohistochemical findings indicate a role for accumulation of β‐catenin and possibly increased Wnt signaling in aflatoxin B1–associated HCC. The low frequency of CTNNB1 mutations, however, suggests that mutation of another Wnt signaling component, such as the Wnt scaffolding protein axin or the adenomatous polyposis coli protein, both of which modulate β‐catenin stability, also may be involved in aflatoxin‐associated HCC. Published 2001 Wiley‐Liss, Inc.

Isolation and identification of clara cells from rabbit lung

SummaryA procedure has been developed for the isolation of nonciliated bronchiolar epithelial cells (Clara cells) from rabbit lung. Following pulmonary lavage to eliminate macrosphages, cells (5% Clara cells) were released by digestion with 0.1% Protease I in HEPES-buffered balanced salt solution containing 0.5 mM ethylene glycol-bis-(β-aminoethyl ether)-N,N′-tetraacetic acid instilled through the trachea. These cells were then separated on the basis of size using the Beckman JE-6 elutriator rotor. The fourth fraction collected from the elutriator contained about 30% Clara cells. This fraction was then layered on a two-polymer aqueous phase system consisting of 5% dextran T500 (DT) and 3.8% polyethylene glycol 6000 (PEG) in sodium phosphate buffer. A cell fraction was obtained from the PEG phase, which included approximately 70% Clara cells. These cells were found to be greater than 90% viable by trypan blue dye exclusion.Identification of isolated Clara cells was confirmed by light microscopic observation of nitro blue tetrazolium staining and by ultrastructural characteristics as observed by electron microscopy.

Molecular profiling of mouse lung tumors: association with tumor progression, lung development, and human lung adenocarcinomas

We have performed oligonucleotide array analysis on various murine lung tissues [normal lungs, lung adenomas, and lung adenocarcinomas (ACs)] using Affymetrix U74Av2 GeneChips to examine the complex genetic changes occurring during lung carcinogenesis. Analysis yielded 20 novel genes differentially expressed in both lung adenomas and ACs versus normal lungs, including the tumor suppressor APC2 and the oncogene Ros 1. In addition, 50 genes were found to be differentially expressed in lung adenomas versus lung ACs, including the differentiation factor Hox C6, the oncogene Ets 2, and the Ras nuclear transport factor, nuclear transport factor 2. To understand the potential relationship between genes expressed in murine lung tumors and its relationship to altered gene expression observed during embryogenesis and postnatal development, tissues from embryonic lungs and from lungs of mice up to 4 weeks following birth were examined using Affymetrix U74Av2 GeneChips. From this analysis, approximately 1300 genes were determined to exhibit differential expression in fetal lung versus postnatal lung. When we compared lung adenomas, lung ACs, and normal lung parenchyma, 24 developmentally regulated genes were found aberrantly expressed in lung tumors; these included the cell cycle control factor CDC5, the cellular differentiation factor TEA domain 4, and the proapoptotic factor BNIP 2. Finally, we compared the murine lung tumor gene expression data to the expression of genes in human lung cancer, in order to assess the relevance of murine lung cancer models in the study of human AC formation. When the 17 human lung ACs and six human lung large cell carcinomas were examined, it was found that 13 of the 17 human lung ACs clustered tightly together in a pattern that was different from the remaining four human lung ACs and six large cell carcinomas, which exhibited a different pattern. Interestingly, the mouse lung adenomas appeared similar to 13 clustered ACs, while mouse lung ACs appeared more similar in pattern to the group consisting of four ACs and six large-cell carcinomas (LCCs). Nevertheless, when compared with the combined human ACs, 39 genes with similar expression changes in murine lung tumors and human ACs/LCCs were identified, such as the oncogene-related BCL7B, the cell cycle regulator CDK4, and the proapoptotic Endophilin B1. Overall, we have determined, for the first time, the expression profiles during murine lung tumor progression and have established, at the molecular level, an association between murine lung tumorigenesis and lung development. We have also attempted to compare the expression profiles found in mouse lung cancers and those in human lung ACs.

Analysis of genetic alterations in uterine leiomyomas and leiomyosarcomas by comparative genomic hybridization

Uterine leiomyomas are the most prevalent tumor type in women of reproductive age and are the most common reason for hysterectomies. Although uterine leiomyomas are considered to be benign, they are a major public health concern for women. In contrast, leiomyosarcomas are rare but highly malignant uterine tumors. They may arise in uteri with preexisting leiomyomas and histologically sometimes resemble leiomyomas, thus causing controversy about whether leiomyosarcomas arise within leiomyomas. In this study, we used comparative genomic hybridization (CGH) to identify genetic alterations unique to each tumor type and alterations that are common between the two tumors. We analyzed 14 cases of uterine leiomyomas and eight cases of uterine leiomyosarcomas. Only two of the 14 leiomyomas exhibited genetic alterations, and those were restricted to gains on chromosomes 14 and 19 and losses on chromosomes 1 and 4. In addition, 68 leiomyomas were examined for loss of heterozygosity on chromosomes 1 and 4, and only three tumors exhibited any losses. In contrast, all eight leiomyosarcomas showed gains and losses of DNA by CGH, and in many cases multiple changes were observed. The most commonly observed genetic aberration, occurring in five tumors, was gains on both arms of chromosome 1, suggesting that this chromosome contains loci involved in the development of leiomyosarcoma. Our results do not provide evidence for the progression from benign leiomyoma to malignant leiomyosarcoma. Moreover, the large number of random chromosomal alterations in the leiomyosarcomas suggests that increased genetic instability plays a role in the formation of these tumors. Mol. Carcinog. 19:273–279, 1997.

Xenobiotic metabolism by alveolar type II cells isolated from rabbit lung

Abstract A procedure for the isolation of alveolar type II cells from rabbit lung was developed. Following pulmonary lavage to minimize macrophage contamination, viable cells (30% type II cells) were released by digestion with 0.1% Protease type I (Sigma) in 4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid (HEPES)-buffered balanced salt solution containing 0.5 mM ethyleneglycolbis (amino-ethylether) tetra-acetate (EGTA) instilled via the trachea. Type II cells were enriched to 50–60 per cent purity by centrifugal elutriation. Density gradient centrifugation in metrizamide was used to increase the purity of the type II cells from the elutriator fraction to 80 per cent. Whole, freshly isolated alveolar type II cells metabolized 7-ethoxycoumarin at a rate of 30 pmoles umbelliferone formed per mg protein/min. However, only traces of coumarin hydroxylase activity were detected which could be accounted for by 1–2% Clara cell contamination in the type II cell fraction. NADPH-cytochrome c (cyt c) reductase activity in the sonicated type II cell fraction was 44 nmoles cyt c reduced per mg protein/min compared to 25 for lung homogenate. Benzo[a]pyrene hydroxylase and N,N-dimethylanilineN-oxidase activities were also demonstrated in the type II cell fraction to be 13 pmoles 3-OH benzo[a]pyrene · (mg protein)−1 · min−1 and 0.8 nmole DMA N-oxide · (mg protein)−1 · min−1 respectively. Microsomes prepared from the isolated type II cell fraction contained 74 pmoles cytochrome P-450/mg protein and 90 pmoles cytochrome b5/mg protein.