Decreased expression of the adenomatous polyposis coli (Apc) and mutated in colorectal cancer (Mcc) genes in mouse lung neoplasia.

A decrease in the intracellular concentrations of the transcripts for some tumor suppressor genes has been found during murine lung tumorigenesis; for p15INK4b and p16INK4a, this was due to homozygous deletions. We report here a decrease in the mRNA levels of the mutated in colorectal cancer (Mcc) and adenomatous polyposis coli (Apc) genes in mouse lung tumors and some neoplastic cell lines. This was assessed both by northern blotting and reverse transcriptase-polymerase chain reaction of RNA isolated from lung tumors that had been induced by urethane, N-nitrosodiethylamine, or 3-methylcholanthrene in (A/J x C57BL/6) F1 or A/J mice. A reduced amount of both Mcc and Apc messages was also seen when two neoplastic cell lines, a spontaneous transformant (E9) and a line derived from a chemically induced solid tumor (82-132), were compared with two independently derived nontumorigenic cell lines (E10 and C10); E9 was derived from E10, and all of these lines are probably of alveolar type 2 cell origin. A cell line derived from a chemically induced papillary lung tumor probably of bronchiolar Clara cell origin (LM2) had Mcc mRNA levels similar to those of C10 and E10 but reduced Apc mRNA levels. A line (p53-823) derived from a papillary tumor that arose in a mouse with a mutated p53 transgene had a reduced amount of the Mcc gene product only. These differential changes in the relative amounts of Apc and Mcc messages in LM2 and p53-823) cells may serve as useful models for studying the regulation of their expression. Both messages had half-lives of 6-9 h in normal E10 and neoplastic E9 cells, so decreased message stability does not account for these reductions. This is the first report of estimated degradation rates of these mRNAs. Apc and Mcc message content did not vary as a function of growth status of the cell lines. Single-strand conformation polymorphism analysis did not reveal mutations in Apc coding regions known to have a high mutation frequency in human colon tumors. Loss of heterozygosity of Apc and Mcc was not found in tumors that developed in the F1 mice, implying a lack of allelic deletions. These changes in tumor suppressor gene expression may contribute to the development and maintenance of neoplasia in lung epithelium.

Tamoxifen causes gene mutations in the livers of lambda/lacI transgenic rats.

Tamoxifen, a rat liver carcinogen, was administered to female lambda/lacI transgenic rats at a dose of 20 mg/kg body weight by gavage for 6 weeks, and the animals were sacrificed 2 weeks later. Tamoxifen induced liver DNA adducts and caused a significant increase in mutation frequency (MF) of approximately 3-fold at the lacI gene in liver DNA. Liver DNA from animals dosed with tamoxifen at 10 mg/kg also showed a similar increase in MF. The mutations were characterized by a raised proportion of: (a) G:C to T:A transversions; (b) insertions of base pairs; and (c) deletions of pairs of G:C base pairs. These observations indicate that tamoxifen induces a distinct spectrum of mutations compared with that found in controls. Toremifene, a noncarcinogenic analogue of tamoxifen with similar estrogenic/antiestrogenic properties examined at 20 mg/kg body weight using the same dosing regime as tamoxifen was not mutagenic. A single oral dose of the rat liver carcinogen aflatoxin B1 (0.5 mg/kg) also significantly raised the MF. In conclusion, although tamoxifen is not mutagenic in regulatory short-term tests, it is a gene mutagen in the rat liver.

Mutational spectra of tamoxifen-induced mutations in the livers of lacI transgenic rats.

Tamoxifen, an important drug in breast cancer treatment, causes liver cancer in rats. The standard range of in vitro tests have failed to show that it causes DNA damage, but 32P-postlabelling and DNA-binding studies have shown that tamoxifen forms DNA adducts in rat liver. In 1995 a transgenic rat (Big Blue; Stratagene, La Jolla, CA) became available which harbours the bacterial lacI gene, thereby allowing the in vivo study of tamoxifen mutagenesis. Recently, we [Styles JA et al. (1996): Toxicologist 30; 161] showed that tamoxifen caused on increase in the mutation frequency at the lacI gene in these transgenic rats. In this study, we report on our preliminary analysis of the mutational spectra of 33 control and 38 tamoxifen-induced mutant lacI genes. Plasmid DNA containing the lacI gene was isolated from the mutant phages and its DNA sequence determined. In the control animal group, 81% of the mutant lacI genes were point mutations, whilst in the tamoxifen-treated group, 62% of the mutant lacI genes were point mutations. Of the tamoxifen-induced mutants, 43% were GC-->TA transversions and 70% of point mutations. In the control group, GC-->TA transversions were 19% of all mutations and 24% of point mutations. Thus, compared with control animals, tamoxifen treatment had significantly increased the proportion of GC-->TA transversions.

Histochemical characterization of non-neuroendocrine tumors and neuroendocrine cell hyperplasia induced in hamster lung by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone with or without hyperoxia.

Lung tumors induced by 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK) with or without hyperoxia have frequent K-ras mutations but only rare p53 mutations, suggesting that this may be a model for non-small cell lung cancers. The goals of the present study were (1) to characterize the histopathology of lung tumors induced in hamsters by NNK with or without O2 and (2) as a corollary, to quantitate the pulmonary neuroendocrine cell hyperplasia in the different treatment groups early and late in the treatment period. Lung tumors induced by NNK with or without O2 were 71% adenomas, 22% adenocarcinomas, approximately 4% bronchoalveolar carcinomas, and approximately 4% squamous/adenosquamous carcinomas. One-half of all tumors were positive for the Clara cell antigen CC10 and 21% of NNK-induced tumors were mucin positive, compared with 2% of NNK/O2-induced tumors (P = 0.003). Immunostaining for PGP9.5 was positive in 5% of tumors induced by NNK alone, but in none of NNK/O2-induced tumors (P = 0.024). Abundant proliferating cell nuclear antigen occurred in 55% of NNK-induced tumors, compared with 19% of NNK/O2-induced tumors (P = 0.009). These data indicate that NNK with or without O2 induces non-neuroendocrine lung tumors. Hyperoxia appears to inhibit cell proliferation and suppress mucinous and partial neuroendocrine differentiation in some of these tumors.

Six-month exposure of strain A/J mice to cigarette sidestream smoke: cell kinetics and lung tumor data.

Male strain A/J mice were exposed to sidestream smoke (SS) generated from burning Kentucky 1R4F reference cigarettes. Chamber concentrations were 4 mg/m3 of total suspended respirable particulate matter (TSP). Animals were exposed 6 hr a day, 5 days a week. One-week cumulative labeling indices were significantly increased in the large intrapulmonary airways during the 1st week and in the respiratory epithelium of the nasal and maxillar turbinates during the first 3 weeks of exposure and then returned to control values. Subsequently, signs of increased cell proliferation were again found in the nasal and maxillar turbinates during the 9th and 16th exposure weeks. The experiment was terminated after 6 months. The number of animals bearing lung tumors was the same in smoke-exposed as in filtered air-exposed animals as was the average number of tumors per lung. Analysis of the DNA of individual tumors obtained from exposed and control mice for K-ras mutations suggested that exon 2 might be a specific target for SS. It was concluded that (1) duration of exposure was too short or (2) concentration of TSP was too low to reveal a possible carcinogenic potential of SS in strain A/J mice or that (3) SS is not carcinogenic in strain A mice.

K-ras and p53 point mutations in 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced hamster lung tumors.

Lung tumors were induced in Syrian golden hamsters by s.c. injection of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). After 40 weeks lung tumor tissue was isolated. Administration of the NNK and exposure of the animals to an atmosphere of 65% oxygen resulted in a statistically significant reduction in tumor size but did not alter the histological tumor type or tumor incidence when compared with carcinogen treated animals maintained under ambient air. Histologically, lung tumors had the morphologic features of adenomas and adenocarcinomas with approximately 15% being squamous cell carcinomas. Lung tumors were examined for mutations in the Ki-ras oncogene and the p53 tumor suppressor gene by direct sequencing. The Ki-ras mutation frequency in RNA isolated from pooled tumors and in DNA isolated from individual tumors were found to be identical. Activated Ki-ras alleles were detected in 77-94% of tumors. All mutations observed (from a total of 65) except one were GC-AT. The Ki-ras mutations resulted in amino acid substitutions at either codons 12 or 13. No mutations were detected at the 61st codon. Examination of the same tumors for p53 mutations showed only one point mutation. We conclude that the NNK treatment in Syrian golden hamsters results in a distinctive mutation pattern in the Ki-ras gene whereas p53 gene mutations may not play a major role at this stage in hamster lung tumorigenesis.

K-ras activation in non-small cell lung cancer in the dog.

To investigate the role of K-ras mutations in canine non-small cell lung cancer, we first determined the nucleotide sequence of the normal canine K-ras gene and then examined 21 canine lung tumors for activating K-ras mutations. Canine K-ras was analyzed by direct sequencing of polymerase chain reaction products generated with oligonucleotide primers derived from the human K-ras sequence. Four nucleotide differences were found between the canine and human K-ras sequence from position 5 to 211. The deduced amino acid sequence of the canine gene was identical to that of the human. Activated K-ras alleles were detected in 5 of the 21 canine lung tumors examined. The activating lesions were point mutations, predominantly in codon 12. Of the 14 adenocarcinomas examined, 2 (14%) had K-ras mutations. Two of 5 (40%) adenosquamous carcinomas and the only large cell carcinoma also contained activated alleles. The overall frequency of K-ras point mutation in non-small cell lung cancer (25%) is similar to that reported in human non-small cell lung cancer. We conclude that K-ras activation by point mutation is associated with, but not necessary for, non-small cell lung cancer development in the dog.

Nucleotide sequence of canine c-N-ras: codons 1 to 71.

The c-N-ras gene has been implicated often in the genesis and/or progression of human leukemias. To our knowledge, the sequence of this gene in the dog has not been reported. Using a system of asymmetric reamplification of double-stranded polymerase chain reaction (PCR) products, we have sequenced normal canine c-N-ras mRNA from position -26 to +213, including codons 12, 13, and 61, which are the sites where oncogenic mutations are most commonly observed. The canine c-N-ras sequence has close homology with the human sequence in this area; there were only 6 observed base differences in nucleotide sequence and none resulted in a change of the encoded amino acid. The results of this study set the stage for directed searches for c-N-ras mutations in experimentally induced and naturally acquired neoplasms of the dog.

Mutational analysis of a dominant oncogene (c-Ki-ras-2) and a tumor suppressor gene (p53) in hamster lung tumorigenesis.

In human lung cancers, alterations of both a dominant oncogene (ras) and a tumor suppressor gene (p53) have been identified. Polymerase chain reaction (PCR) analysis of mRNA was used to amplify the c-Ki-ras-2 and p53 genes from Syrian golden hamsters. The PCR products were confirmed by predicted-size analysis, probing with nonradioactive (biotin-labeled) oligonucleotides, and direct sequencing. Lung tumors were produced in hamsters by repeated injections of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Of six tumors examined, three (50%) had mutations in codon 12 of Ki-ras. Examination of the conserved regions of p53 revealed no mutations. We conclude that NNK-induced carcinogenesis in the hamster results in characteristic alterations of Ki-ras but may not necessarily involve the p53 gene.

Increased c-Ki-ras expression in hamster lung exposed to N-nitrosodiethylamine and hyperoxia as detected by the polymerase chain reaction.

Neuroendocrine lung cancers can be induced in hamsters within 8-12 weeks by combined exposure to N-nitrosodiethylamine (DEN) and hyperoxia. The expression of the c-Ki-ras gene in this lung cancer model was studied using polymerase chain reaction analysis of mRNA (RNA/PCR). We used four different groups of hamsters, exposed for 6 weeks to DEN with hyperoxia (60% oxygen), DEN, hyperoxia, or ambient air, respectively. Total RNA was isolated from lung tissues and cDNA made prior to PCR amplification. A 234-bp product was amplified from c-Ki-ras cDNA and quantitated using scanning laser densitometry. The data obtained were normalized to the expression of the house keeping gene B-actin. The c-Ki-ras products were present after amplification of all hamster lung RNA samples. The hamster lungs exposed to DEN with hyperoxia displayed higher c-Ki-ras protooncogene expression than hamsters exposed to DEN, hyperoxia, or ambient air alone. Since the animals studied were sacrificed at 6 weeks, prior to the appearance of tumors, we conclude that this increased expression may indicate a role for c-Ki-ras in the initial steps in malignant transformation of neuroendocrine cells.

Diamine uptake by rat lung type II cells in vitro.

Lung epithelial type II cells are responsible for synthesising and secreting pulmonary surfactant which reduces surface tension and prevents lung collapse. Type II cells replace type I cells and can proliferate in response to alveolar injury. An important aspect of this proliferation may be the ability of type II cells to accumulate amines actively, particularly the endogenous diamine putrescine. Putrescine is accumulated into isolated alveolar type II cells by an energy-dependent process. The uptake obeys saturation kinetics for which an apparent Km of 14.7 microM and Vmax of 130 pmol/micrograms DNA/hr was derived. The inhibitory effects of structurally similar amines on putrescine accumulation are described. As the herbicide paraquat has been suggested to share the same uptake system as putrescine from lung slice studies, this phenomenon was investigated in type II cell cultures. The results demonstrated that paraquat is a partially competitive inhibitor of putrescine accumulation in the cells. The Ki for the inhibition of putrescine uptake by paraquat in type II cells was calculated to be 69 microM, a value which closely matches the Km for paraquat (70 microM) predicted from lung slice studies. In molecular terms, the partial nature of the competition indicates that paraquat and putrescine do not occupy identical sites. Saturation of its site by paraquat reduced the affinity of putrescine 3.6-fold, but did not abolish it.

Acute effects of the Bowman-Birk protease inhibitor in mice.

The soybean-derived Bowman-Birk inhibitor (BBI) has been shown to inhibit carcinogenesis in both in vitro and in vivo model systems. In the present study, protease enzyme activity in selected tissues of male strain A mice was measured by hydrolysis of the synthetic substrate Boc-Val-Pro-Arg-MCA (t-butoxycarbornylvalylprolylarginine 7-amido-4-methylcoumarin). When added to homogenates of lung, liver and kidney in vitro, purified BBI inhibited hydrolytic activity at concentrations ranging from 10-100 microM. In vivo, hydrolytic activity was found to be significantly and in a dose-dependent manner, decreased in the lung as early as 2 h after i.p. injection of purified BBI. Less inhibition was found in the liver and kidney after in vivo administration of purified BBI. A crude preparation of BBI, given at 100 mg/kg, had no influence on the overall disposition of radiolabeled benzo[alpha]pyrene in mice although it decreased the hepatic activities of cytochrome P-450, 7-ethoxycoumarin-O-deethylase and ethoxy resorufin-O-deethylase. It is concluded that the chemopreventive effects of BBI on mouse lung tumor development are most likely mediated through its protease-inhibitory properties in the target organ rather than by a non-specific effect on metabolism and disposition of carcinogens.

Clara cell cultures from the mouse and their reaction to bronchiolar toxins.

The major aim of this study was to determine if small numbers of freshly isolated mouse Clara cells could be used to rapidly screen the toxic effects of a number of diverse pulmonary toxins. A short-term (20 hr) culture of functionally competent (nitotetrazolium reductase positive) Clara cells was developed. In this culture the Clara cells were allowed to attach to an extracellular matrix in 96-well multiwell plates containing a culture medium of DCCM 1 and Ultroser G (0.4%). Pulmonary toxins (a total of 26 agents with concentrations ranging from 10(-7) M to 10(-3) M) were examined for their ability to reduce the attachment efficiency of functionally competent Clara cells and TD50 values (the amount of toxin required to reduce normal attachment efficiency by 50%) were calculated. With the possible exception of some halogenated hydrocarbons, the simple toxicity test in vitro correlated well with the known effects of the bronchiolar necrotic agents in vivo. For 13 compounds studied there was a direct correlation between TD50 values in vitro and LD50 values (mostly oral) in rodents in vivo, the correlation coefficient of the regression line being 0.783.

Isolation of Clara cells from the mouse lung.

A method is described for isolating Clara cells from the mouse lung that does not require the technique of elutriation. Mouse lungs totally perfused of blood are instilled with crystalline trypsin (0.25%) and incubated for the optimum time of 15 min. The lung tissue is chopped, mechanically agitated, and sequentially filtered to obtain a primary digest of 3 to 5 x 10(6) cells. Clara cells, identified routinely by histochemical localization of NADPH diaphorase, using the stain nitrotetrazolium blue (NBT), accounts for between 20 to 40% of the cells in the primary digest. Layering the cells of the primary digest on a discontinuous Percoll gradient followed by centrifugation gives rise to a major band of cells, 52% that are Clara cells (0.77 +/- 0.28 x 10(6)/mouse). A second method was devised to purify the Clara cells by simply centrifuging (32g, 6 min, 10 degrees C) the primary digest and discarding the supernatant that contained only a few NBT positive cells. When this process was repeated three times, the final pellet contained 68% Clara cells realizing 0.55 +/- 0.16 x 10(6) cells/mouse. The cells have typical Clara cell morphology as confirmed by electron microscopy and have a high level of P-450 enzymes (7-ethoxycoumarin deethylase and coumarin hydroxylase). Furthermore, the primary digests and the purified isolates contain less than 1% alveolar Type II cells, although such cells, identified by the histochemical localization of alkaline phosphatase, can be obtained by a second, more extensive digestion procedure. The simple procedure described for the isolation of mouse Clara cells could be further advanced if methods could be devised to prevent the loss of NADPH diaphorase activity during enzymatic digestion and cell centrifugation.

Isolation, biochemical characterization, and culture of lung type II cells of the rat.

A method is described for the isolation of rat lung epithelial Type II cells using trypsin digestion of tissue to release cells for subsequent separation by Percoll gradient centrifugation. Both the concentration of trypsin and the age (body weight) of the rat affect the yield from primary digestion and the final number of Type II cells obtained. A lung weighing 1 g from a 200 g rat yields approximately 30 X 10(6) washed Type II cells (approximately 25% of the total estimated lung population). These cells have a plating efficiency of 40-50% after 48 h of culture. The cells have a high alkaline to acid phosphatase ratio (usually greater than 4.0) compared with that of alveolar macrophages (0.1) and accumulate putrescine by an active transport mechanism with an apparent KM between 8 and 14 microM. Together with studies of [3H]thymidine uptake into DNA, which is maximal between 48 and 72 h of culture, these quantitative measurements form a good basis for investigating the interactions between a number of chemical agents and Type II cells in vitro.