Enhancement of lung carcinogenesis initiated with 4-(N-hydroxymethylnitrosamino)-1-(3-pyridyl)-1-butanone by Ogg1 gene deficiency in female, but not male, mice.

The present study was conducted to assess involvement of oxidative stress in lung adeno-carcinogenesis, using mice deficient in the 8-hydroxyguanine DNA glycosylase 1 (Ogg1) gene encoding an enzyme that repairs an oxidative DNA injury 8-oxoguanine (8-oxoG). Furthermore, for comparison with the human case, mutations of mouse epidermal growth factor receptor (Egfr) and K-ras genes were examined. The homo- and heterozygously Ogg1 gene-deficient and wild-type mice (C57BL6/J origin), 6 weeks old, were administered 4-(N-hydroxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) by continuous subcutaneous infusion using an osmotic pump at a total dose of 6 mg/mouse for 1 week, then treated with one of 4 antioxidants (phenyl N-tert-butyl nitrone 0.13% in drinking water, resveratrol 20 ppm in diet, lactoferrin 2% in diet and bilberry powder 2% in diet) or no supplement for 33 weeks. Development of lung adenomas and preneoplastic atypical hypreplasias was significantly enhanced by the homo- and heterozygous Ogg1 gene deficiency only in female mice with intralesion accumulation of 8-oxoG. All antioxidants tended to inhibit enhanced adeno-carcinogenesis. The Egfr and K-ras gene mutations were detected at sites also found in human lung cancers with low incidences, while the Egfr gene mutation was detected for the first time in chemical lung carcinogenesis of animals. It is indicated that the Ogg1 gene deficiency enhances lung adeno-carcinogenesis in mice by virtue of accelerated oxidative stress. The presently utilized Ogg1 gene-deficient mice model may be useful to draw mechanism-based strategies to control human lung adenocarcinomas, especially in women.

Sodium phenylacetate inhibits the Ras/MAPK signaling pathway to induce reduction of the c-Raf-1 protein in human and canine breast cancer cells.

An aromatic fatty acid, phenylacetate (PA), has been shown to have cytostatic, antitumor and cell differentiation-inducing effects on various kinds of tumors. Previously, we have demonstrated cell growth inhibition, malignant phenotype reduction and cell differentiation effects of sodium phenylacetate (NaPA) treatment in a canine mammary tumor cell line. To clarify the molecular mechanism of these effects, we examined the expression of Ras/MAPK signaling pathway-related molecules in human and canine breast cancer cell lines, and found that the level of c-Raf-1 protein was reduced by 5, 10 and 20 mM of NaPA treatments, though Ras activation was maintained. Dephosphorylation of c-Raf-1 at Serine (Ser) 259, Ser 338, and Ser 621 were also seen in NaPA-treated cells. Downstream factors in the pathway, such as mitogen-activated protein kinase/ERK kinase (MEK)1/2 and ERK1/2, showed decreased activity, and accordingly, expressions of cyclinD1, c-myc, and inactivation of p90 ribosomal S6 kinase (RSK), which are MAPK targets, were reduced. We also observed the reduction of cell-cycle-promoted molecules, such as cdc1/cdk2, cdk4, PCNA cyclin A, and cyclin B, and the increased expression of p27kip1. Furthermore, expression of an epithelial marker, E-cadherin, was increased by NaPA treatment. These results suggest that one of the molecular targets of NaPA treatment was the reduction of c-Raf-1 protein, and that its reduction results in the decrease of malignant characteristics of tumor cells through blockage of the Ras/MAPK signaling pathway.

Expression and subcellular localization of E-cadherin, alpha-catenin, and beta-catenin in 8 feline mammary tumor cell lines.

Protein expression and subcellular localization of E-cadherin, alpha-catenin, and beta-catenin in 8 feline mammary tumor cell lines were examined by western blot analysis and fluorescence immunocytochemistry. A low E-cadherin expression was observed in FNN-m cells. Furthermore, compared to other cell lines, two E-cadherin bands existed in FMC-p1 cells and were localized in the perinuclear region; distinct radial lines were observed in the cytoplasm. A low alpha-catenin expression was observed in FON-m cells, but there were no apparent abnormalities in its localization. In contrast, similar levels of beta-catenin expression and cytoplasmic localization were observed in all cell lines.

Involvement of mutation-based inhibition of beta-catenin phosphorylation at Ser33 in the malignant progression of lung (pre)neoplastic lesions induced by N-nitrosobis(2-hydroxypropyl)amine in male Fischer 344 rats.

In this study we investigated the Ser33 phosphorylation status of beta-catenin protein in relation to genomic mutations in lung (pre)neoplastic lesions induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) in male Fischer 344 rats. Six-week-old animals received 2000 ppm of BHP in the drinking water for 8 weeks and were sacrificed 12 weeks thereafter. Histopathologically, 69 of 75 rats demonstrated multiple lung (pre)neoplastic lesions, classified into 27 slight and 33 advanced hyperplasias (preneoplasms) and 61 neoplasms, including adenomas, adenocarcinomas, and adenosquamous carcinomas. Nucleotide mutation analysis of the beta-catenin gene detected a total of 33 mutations in 12 assessed lung (pre)neoplastic lesions. The mutations tended to accumulate in positions near the phosphorylation region of the gene, between codons 33 and 45. Immunohistochemical analysis showed beta-catenin protein expression to be increased and its localization changed from the cell membrane to the cytoplasm and finally the nuclei with advancing malignancy of the lung lesions. In contrast, the expression of phosphorlyated beta-catenin protein at Ser33 was weakened in lung (pre)neoplastic lesions compared to normal lung tissues. These results suggest that BHP-induced mutation of the beta-catenin gene results in amino acid conversions in its product protein, which in turn lead to inhibition of phosphorylation of the protein and escape from protein degradation. These phenomena might contribute to the malignant progression of the lung (pre)neoplastic lesions, which start from the relatively early stage in lung carcinogenesis.

Retinoids induce growth inhibition and apoptosis in mast cell tumor cell lines.

Retinoids are well recognized as promising antitumor agents in humans. However, there have only been a few reports about the effect of retinoids in canine cancers. To investigate the antitumor effect of retinoids on mast cell tumors (MCT), inhibitory effect on cell growth and induction of apoptosis were examined in vitro. Although sensitivity of these cells differed among the cells, the growth of three MCT cell lines (CoMS, CM-MC and VI-MC) were inhibited dose dependently when they were treated with retinoids. FACS analysis of PI-stained nuclei revealed an apoptotic fraction in CM-MC cells about 30% when treated with retinoids, while those of control cells were less than 5%. Caspase-3 activation was observed after retinoid treatment in CM-MC cells. This was confirmed by inhibiting the retinoid-induced apoptosis using the pan-caspase inhibitor, ZVAD-FMK. Both retinoid receptors, RARs and RXRs, were detected by immunoprecipitation followed by western blot analysis in all the three MCT cells. These data suggests that retinoids inhibit the growth of MCTs partly through apoptosis, and this growth inhibition by retinoids may be mediated by RARs and RXRs. We conclude that retinoid may be a potential adjunctive chemotherapeutic agent for the treatment of canine MCT.

Relationship between retinoic acid receptor alpha gene expression and growth-inhibitory effect of all-trans retinoic acid on canine tumor cells.

Retinoids show antitumor effects on human acute promyelocytic leukemia and other tumors via retinoid receptors. In dogs, the role of retinoid receptors in inhibiting tumor development remains unclear. To evaluate the correlation between the degree of expression of retinoic acid receptor alpha (RARalpha) mRNA and the antiproliferative effects of all-trans retinoic acid (ATRA) treatments, expression analysis of RARalpha mRNA and cell growth inhibition assay were performed on 17 established canine tumor cell lines, including 6 mammary gland tumor (MGT) cell lines, 3 osteosarcoma cell lines, 5 melanoma cell lines, and 3 mast cell tumor (MCT) cell lines. Among the cell lines investigated, all 3 MCT cell lines showed high expression of RARalpha, and the most effective cell growth inhibition was observed in ATRA-treated MCT cell lines. However, remarkable antiproliferative effects of ATRA treatments were not observed on other tumor cell lines with moderate or low RARalpha mRNA expression. As a result of the relationship between RARalpha mRNA expression and ATRA treatment with regression analysis, statistically significant correlation was suggested. Furthermore, real-time quantitative polymerase chain reaction analysis of RARalpha was performed on MCT tissue samples of dogs with spontaneous disease, and 5 of 9 tissues showed high expression. These results suggest that ATRA may be an effective antitumor agent for MCT in dogs, and that prior measurement of expression of RARalpha mRNA may be a good indicator of the effectiveness of ATRA treatment.

Identification and characterization of a canine highly similar to retinoic acid receptor alpha.

A canine highly similar to retinoic acid receptor alpha (canine HS-RARa) cDNA was isolated from the spleen tissue. A database search and the alignment revealed that the canine cDNA was most similar to highly similar type of human RARa and was named canine HS-RARa. The expression of the genes encoding RARa in the dog was the highest in the testis and moderate in the blood, lymph node, mammary gland, pancreas, salivary gland, spleen, thyroid gland, tonsil and uterus. The nucleotide sequence encoded the 462-amino acid containing the conserved sequence motif of RARa. Though the amino acid sequences were well-conserved among species, some unique arrangements were observed within each class. In the phylogenetic analysis, each species separated according to their class. In the branch of mammals, the dog is in the cluster of humans, mice and western wild mice. However, hamsters and rats formed another branch.