Lung Carcinogenesis In Mice Research Articles

Transforming Growth Factor-Beta Expression in Mouse Lung Carcinogenesis

Transforming growth factor-beta (TGF-β) is a multifunctional growth modulator that inhibits the proliferation of many epithelial cells while stimulating the proliferation of most fibroblasts. To examine the role of TGF-β in mouse lung chemically induced tumorigenesis, expression of the TGF-β1, -β2, and -β3 proteins was examined in A/J mice treated with the carcinogen urethane to induce lung adenomas using immunohistochemical staining analysis. Immunostaining for the TGF-β ligands was detected in the epithelium of the bronchioles of untreated A/J mice with immunostaining being more intense for TGF-β1 than for TGF-β2 and TGF-β3; immunostaining for each TGF-β ligand was also detected in the bronchiolar epithelium of urethane-treated A/J mice at levels similar to untreated mice. Immunostaining for the TGF-β ligands was also detected in adenomas by 2 months; staining for TGF-β1, -β2, and -β3 in adenomas was detected at levels comparable with bronchioles. Following treatment with urethane for 8 months, immunostaining for TGF-βs 1, 2, and 3 in bronchioles persisted at levels comparable to that in normal bronchioles and also persisted in adenomas, with staining for the TGF-β ligands being very prominent on the edge of the tumor. Expression of TGF-β1 mRNA was examined in urethane-treated mouse lung tissue using Northern blot hybridization; here, expression of TGF-β1 mRNA increased 2-fold in 3-month urethane-treated lung tissue and an additional 2.5-fold by 8 months following urethane administration. Expression of TGF-β1 mRNA was also examined in nontumorigenic and tumorigenic mouse lung cells; in these cells, expression of TGF-β1 mRNA was higher in the tumorigenic cells than in the nontumorigenic cell line. These data show that there is an increase in expression of TGF-β1 during tumorigenesis and suggest that TGF-β may play an important role in mouse lung carcinogenesis induced by urethane.

Smad4 (homolog of human DPC4) and Smad2 (homolog of human JV18-1): candidates for murine lung tumor resistance and suppressor genes.

In this study we investigated the mouse mad-related genes, Smad4/Dpc4 and Smad2 (homolog of JV18-1), as candidates for involvement in lung tumor resistance and suppression. These genes are located in a region of mouse chromosome 18 that is syntenic with human 18q21.1, where several genes that are mutated in various cancers have been mapped. A newly identified murine lung tumor resistance locus, Par2 has also been mapped to this region of chromosome 18. We found no mutations in the coding regions of either gene in 11 lung tumors from B6CF1 (C57BL/6 x BALB/c) mice by RT-PCR and SSCP/RFLP, suggesting that these genes are not mutated in lung carcinogenesis in this strain. Moreover, loss of heterozygosity in this region of chromosome 18 was not detected in 28 lung adenocarcinomas from B6CF1 mice, 17 lung adenocarcinomas from B6C3F1 mice or 18 lung adenocarcinomas from AB6F1 mice. These data provide evidence that a 'classical' tumor suppressor gene for mouse lung carcinogenesis in these strains does not reside in this region. In order to investigate Smad4/Dpc4 and Smad2 as candidates for the Par2 resistance locus mapped to this region, we also sequenced the coding regions of both genes in cDNA from normal lungs of A/J, BALB/c and C57BL/6 inbred strains of mice. No polymorphisms were detected in the coding region of Smad4. In Smad2, two sequence polymorphisms were identified that are not in the conserved regions of the gene. Northern blot analysis revealed no differential expression in normal lung tissue among the three strains for either gene. Thus, in this study we found no evidence that either Smad4 or Smad2 represents the Par2 lung tumor resistance locus or is a lung tumor suppressor gene in the B6CF1 mice.

Similar incidence of K-ras mutations in lung carcinomas of FVB/N mice and FVB/N mice carrying a mutant p53 transgene.

Mutated p53 genes are capable of complementing activated ras genes in the transformation of primary rat embryo fibroblasts in vitro. Mutations in both genes have also been found in several human cancers, including lung carcinomas. We generated transgenic mice containing a p53 construct with a missense mutation in exon 5 (ala135val) to study the role of p53 mutations in lung tumorigenesis, and to facilitate identification of other genetic events that might complement p53 mutations in mouse lung carcinogenesis. The p53 transgenic lines exhibited a higher frequency of lethal lung tumors than the parental FVB/N strain. We examined the spontaneously-arising lung carcinomas from mice expressing the mutated p53 transgene for K-ras mutations using single-stranded conformation polymorphism (SSCP) and/or direct sequencing approaches. Fifteen of 29 (52%) carcinomas contained mutations in the K-ras oncogene. Six of 15 of the K-ras mutations were in codon 61 and 9/15 were in codon 12. Subsequent analysis of spontaneous lung carcinomas from mice of the FVB/N parental strain showed that 9/12 (75%) carcinomas examined contained K-ras mutations. Two of these were in codon 12, one in codon 13, and 6 were in codon 61. These results demonstrate that the frequency of ras mutations does not differ between the p53 FVB/N transgenic mice and their parental FVB/N strain but suggest that a high frequency of mutations K-ras can be correlated with lung tumorigenesis in both groups of mice.

The inhibitory effect of vitamin E on pulmonary polyamine biosynthesis, cell proliferation and carcinogenesis in mice

We investigated the modulating effect of vitamin E on pulmonary polyamine biosynthesis, cell proliferation and carcinogenesis in mice treated with urethane. Pulmonary ornithine decarboxylase induction and subsequent polyamine accumulation were observed during the initiation and promotion phases of the urethane-induced lung carcinogenesis in mice. The increases of ODC activity and polyamine level during both phases were almost inhibited when a high vitamin E diet was provided. The urethane-increased level of pulmonary proliferating cell nuclear antigen as a marker of cell proliferation during the carcinogenesis was inhibited by vitamin E treatment. Also, vitamin E suppressed the urethane-induced elevation of pulmonary cyclooxygenase activity as a marker of tumor promotion. In conjugation with these events, vitamin E reduced the development of lung tumors in mice treated with urethane. These results indicated that vitamin E could act as a useful chemopreventive agent against lung carcinogenesis in mice due to the regulation of cell proliferation.

The inhibitory effect of vitamin E on K- ras mutation at an early stage of lung carcinogenesis in mice

In this study, we investigated the effect of vitamin E on the activation of the K- ras oncogene with a 61st codon A→T mutation at an early stage of urethane-induced lung carcinogenesis in mice. Thirty days after urethane injection, the K- ras mutation was detected in 64% of lung samples tested by mutant-allele-specific amplification. The consumption of a supplemented diet with about 20-times more vitamin E than the control diet, only during the promotion phase or during both the initiation and promotion phases of lung carcinogenesis, reduced the frequency of the mutation to 36 and 18%, respectively. Also, vitamin E suppressed the level of proliferating cell nuclear antigen as a marker of cell proliferation in the lungs of mice treated with urethane. These results support the notion that vitamin E is a useful chemopreventive agent against lung cancer. © 1997 Elsevier Science B.V.

The Detection of Chemically Initiated Cells Having the Mutation of K-rasGene at an Early Stage of Lung Carcinogenesis in Mice

The Detection of Chemically Initiated Cells Having the Mutation of K-rasGene at an Early Stage of Lung Carcinogenesis in Mice

Chemopreventive effect of perillyl alcohol on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone induced tumorigenesis in (C3H/HeJ X A/J)F1 mouse lung.

This study was designed to test the chemopreventive potential of perillyl alcohol, an inhibitor of farnesyltransferase, in a mouse lung tumor bioassay. Perillyl alcohol is a naturally occurring monoterpene found in lavender, cherries, and mint. We have shown previously that the majority of lung tumors in this bioassay have an activating mutation in the K-ras gene, which occurs early in the development of mouse lung carcinogenesis. The Ras protein undergoes a series of post-translational modifications, the first of which is farnesylation at the cysteine of the C-terminal CAAX motif. These modifications lead to the anchoring of Ras p21 to the plasma membrane in its biologically active state. Activated Ras p21 couples growth regulatory signals from receptor tyrosine kinases to cytoplasmic second messengers. In a preliminary study, we determined the maximum tolerated dose of perillyl alcohol to be 75 mg/kg body weight. For the bioassay, 5-week-old male (C3H/HeJ X A/J) F1 hybrid mice were randomized into trial groups, and treated with perillyl alcohol three times per week i.p., starting 1 week prior to initiation with the carcinogen NNK, and continuing for 22 weeks after initiation. Our results show a 22% reduction in tumor incidence, and a 58% reduction in tumor multiplicity. Our study demonstrates that perillyl alcohol is an effective chemopreventive compound in the mouse lung tumor bioassay.

Effect of video display terminal irradiation on urethane-induced lung carcinogenesis in mice

Female Swiss-derived SHR mice were exposed intraperitoneally to 4 injections of 1% urethane at the Ist, 8th, 54th and 61st days of the experiment (a single dose was 20 mg per mouse). Groups of mice starting from the Ist day were also exposed 5 days per week to 1 hour irradiation of video display terminal (VDT) or VDT+ screen filter (SF). The distance from VDT screen and bottom of animal cage was 38 cm. The experiment was finalized in 1 year. Longterm exposure of urethane-treated mice to both VDT or VDT+SF was followed by slight decrease in survival time of lung tumor-bearing animals. The tendency to increase of the incidence of lung tumors and the number of lung tumor per mouse was observed in mice exposed to urethane + VDT as compared to those exposed to urethane alone. The incidence of lung tumors and lung tumor multiplicity were significantly decreased in mice treated with urethan and VDT+SF in comparison to mice exposed to urethane + VDT. Thus, a slight promoting effect of VDT-induced irradiation on lung tumorigenesis was observed induced by urethane in mice, and SF alleviated this effect. The dose of urethane in these experiments was rather high to show more clearly the effect of VDT irradiation on lung carcinogenesis.

Perinatal lung carcinogenesis in mice: in vivo and in vitro correlation.

Perinatal lung carcinogenesis in susceptible (A) and resistant (C57B1/6J) to lung carcinogenesis mice was studied in (1) experiment in vivo and (2) in organ cultures of embryonic lung exposed to carcinogens directly (in vitro) or transplacentally (in utero): females were treated with carcinogens at 17-19 days of pregnancy, killed 1-2 days after the treatment and embryonic lungs were explanted in organ cultures. Transplacental and direct (in vitro) treatment with urethane and benz(a)pyrene (BP) induced in the explants: (1) stimulation of epithelial and mesenchymal cell proliferation; (2) increase of viability and survival as compared to the non-treated explants (growth stimulating effect); (3) morphological lesions (hyperplasia and squamous-cell metaplasia of epithelium, adenomatous growth and adenoma-like structures). All the alterations developed in treated lung explants depended both on host factors and carcinogenic treatment (type of carcinogen, dose, method of exposure, and duration of cultivation).

Cancer chemoprevention by natural carotenoids and their related compounds

As one of the most promising cancer chemopreventive agents, beta-carotene has been studied extensively. However, other natural carotenoids have also suppressed tumorigenesis, and some are more potent than beta-carotene. For example, alpha-carotene shows higher potency than beta-carotene in suppressing tumorigenesis in mouse skin and lung models. In the two-stage mouse skin carcinogenesis model (initiator, 7,12-dimethylbenz[a]anthracene; promoter, 12-O-tetradecanoylphorbol-13-acetate), topical application of alpha-carotene at a 200 nmol dose per painting twice a week significantly decreased the mean number of skin tumors per mouse. The greater potency of alpha-carotene over beta-carotene in suppression of tumor promotion was confirmed in the two-stage mouse lung carcinogenesis model (initiator, 4-nitro-quinoline-1-oxide; promoter, glycerol). Oral administration of alpha-carotene (0.05% in drinking water) significantly decreased the mean number of lung tumors per mouse. In contrast, beta-carotene showed no suppression of lung tumor formation under the same experimental conditions. Fucoxanthin, a carotenoid as abundant in nature as beta-carotene, was also found to have antitumorigenic activity in mouse skin and duodenum models. Thus, further studies on various natural carotenoids, other than beta-carotene, should be carried out in the field of cancer chemoprevention.

Lack of modifying potential of 8-methoxypsoralen in the promotion or progression stages of lung carcinogenesis in A/J female mice

Pretreatment with 8-methoxypsoralen (8-MOP), a potent human CYP2A6 inhibitor, strongly suppresses lung tumorigenesis by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in female A/J mice. Since it has been reported that CYP2A protein is highly expressed in NNK-induced lung adenomas and adenocarcinomas, potential anticancer properties of 8-MOP in female A/J mice were examined subsequent to initiation. The agent was administered at 100 ppm levels in the diet during the promotion phase (experimental weeks 1-16) and progression phase (experimental weeks 16-32) and mice were sacrificed for histopathological examination of lung tumor development at 16 and 32 weeks after the initiation of NNK treatment (2 mg/0.1 ml saline/mouse, i.p.), respectively. Chemopreventive effects of 8-MOP were not observed in either experiment. In addition, no modifying effects on hepatic mRNA levels for CYP2A5, considered to be the mouse ortholog of human CYP2A6, were evident. On immunohistochemical analysis, the CYP2A protein was found to be overexpressed in all lung adenomas and adenocarcinomas, with or without 8-MOP. It can be concluded from the present data that 8-MOP at 100 ppm in the diet does not prevent mouse lung carcinogenesis when administered in the post-initiation phase.

Effect of combined deficiency of fat and vitamin A on N-nitrosodiethylamine-induced lung carcinogenesis in mice

The present investigation shows the effect of combined dietary deprivation of fat and vitamin A on N-nitrosodiethylamine (NDEA) induced lung carcinogenesis in male Swiss NMRI mice. Lung tumors were induced by three intragastric administrations of NDEA (80, 80, 40, mg/kg body wt.) in normal saline with a gap of 4 weeks between each administration. The tumor incidence and tumor burden in control animals were found to be 39.1 and 3.9%, respectively. Fat deficiency decreased the tumor incidence to 30.4% and tumor burden to 3.7%. On the other hand, vitamin A deficiency increased them to 95.6 and 6.8%, respectively. Fat deprivation in the diet of animals fed with vitamin A deficient diet decreased the tumor burden and tumor incidence to 59.0 and 4.0%, respectively which were still more than in the control animals. This study suggests a protective role for low intake of fat in vitamin A deficiency.

Skin initiating action and lung carcinogenesis by derivatives of urethane (ethyl carbamate) and related compounds

The possibility that a metabolite might be implicated in the carcinogenic action of urethane on the lungs, and in its limited “initiating” action in the case of skin carcinogenesis, prompted the testing of a number of derivatives and analogues of urethane for both lung carcinogenesis and skin initiation in mice. The modifications in molecular structure of urethane involved (i) changes in the carbamyl portion, (ii) changes in its carboethoxy portion, (iii) phosphorylated derivatives, and (iv) addition compounds with keto and amino acids. Most of the compounds tested proved inactive, and among those that did give positive results, the effects were weak ; thus indicating that none of them represented the sought-for metabolite. Some tentative implications of the results are discussed as pointers for further enquiry.