Feeding a High-Fat Diet for a Limited Duration Increases Cancer Incidence in a Breast Cancer Model.

High-fat intake by young Asian women impacts the risk of breast cancer. Understanding the underlying molecular mechanisms may be essential for disease prevention in Asia as well as globally. We aimed to examine the effects of corn oil- and animal fat-based high-fat diets (32.9 and 31.4%, respectively, of fat energy ratio as compared to 12.3% in the standard diet) on mammary carcinogenesis and alterations in gene expression and epigenetic statuses in the mammary gland during the growth stages in a rat model. An increased incidence of carcinomas was observed after the cessation of high-fat feeding. In addition, rapid tumor growth and elevations in Celsr2 expression, which may be a result of DNA hypomethylation patterns in the 3' untranslated region of the gene were noted in the animal fat group. In the human breast carcinoma cell line MCF7, a marginal decrease in cell viability was observed following the knockdown of Celsr2, suggesting that the animal fat-associated risk of cancer is partly due to the deregulation of mammary cell proliferation via non-metabolic gene functions. The present results will contribute to the development of strategies for controlling the food-associated risk of breast cancer, particularly in younger age groups.

o-Anisidine Dimer, 2-Methoxy-N4-(2-methoxyphenyl) Benzene-1,4-diamine, in Rat Urine Associated with Urinary bladder Carcinogenesis.

Monocyclic aromatic amines, o-toluidine (o-Tol) and its structural analog o-anisidine (o-Ans), are IARC Group 1 and Group 2A urinary bladder carcinogens, respectively, and are involved in metabolic activation and DNA damage. Our recent study revealed that 2-methyl-N4-(2-methylphenyl) benzene-1,4-diamine (MMBD), a p-semidine-type homodimer of o-Tol, was detected and identified in an in vitro reaction of o-Tol with S9 mix and in vivo urinary samples of o-Tol-exposed rats. Potent mutagenic, genotoxic, and cytotoxic activities were reported with MMBD, suggesting its involvement in urinary bladder carcinogenesis. However, it remains unknown whether o-Ans is converted to active metabolites to induce DNA damage in a similar manner as o-Tol. In this study, we report that a novel o-Ans metabolite, 2-methoxy-N4-(2-methoxyphenyl) benzene-1,4-diamine (MxMxBD), a dimer by head-to-tail binding (p-semidine form), was for the first time identified in o-Ans-exposed rat urine. MxMxBD induced a stronger mutagenicity in N-acetyltransferase overexpressed Salmonella typhimurium strains and potent genotoxicity and cytotoxicity in human bladder carcinoma T24 cells compared with o-Ans. These results suggest that MxMxBD may to some extent contribute toward urinary bladder carcinogenesis. In addition to homodimerization, such as MxMxBD, heterodimerizations were observed when o-Ans was coincubated with o-Tol or aniline (Ani) in in vitro reactions with S9 mix. This study highlights the important consideration of homodimerizations and heterodimerizations of monocyclic aromatic amines, including o-Ans, o-Tol, and Ani, in the evaluation of the combined exposure risk of bladder carcinogenesis.

An organoid-based carcinogenesis model induced by in vitro chemical treatment.

Animal carcinogenesis models induced by environmental chemicals have been widely used for basic and applied cancer research. However, establishment of in vitro or ex vivo models is essential for molecular mechanistic elucidation of early events in carcinogenesis, leading to clarification of the total mode of action. In the present study, to establish an organoid-based chemical carcinogenesis model, mouse organoids were treated in vitro with 4 genotoxic chemicals, e.g. ethyl methanesulfonate (EMS), acrylamide (AA), diethylnitrosamine (DEN) and 7,12-dimethylbenz[a]anthracene (DMBA) to examine their tumorigenicity after injection to nude mice. The four chemicals were reported to induce lung, liver or mammary carcinomas in mouse models. DMBA-treated mammary tissue-derived organoids with Trp53 heterozygous knockout exhibited tumorigenicity, but not those with wild-type Trp53, reflecting previous reports of corresponding animal models. Treatment of lung organoids with or without Trp53 knockout with EMS or AA resulted in carcinogenic histopathological characteristics, and the activation of oncogenic kinases was demonstrated in the nodules from the nude mouse subcutis. DEN-treated liver (biliary tract) organoids also had an increased number of similar changes. In conclusion, an ex vivo model for chemical carcinogenesis was established using normal mouse tissue-derived organoids. This model will be applied to detect early molecular events, leading to clarification of the mode of action of chemical carcinogenesis.

Organoid-based ex vivo reconstitution of Kras-driven pancreatic ductal carcinogenesis.

The organoid culture technique has been recently applied to modeling carcinogenesis in several organs. To further explore its potential and gain novel insights into tumorigenesis, we here investigated whether pancreatic ductal adenocarcinoma (PDA) could be generated as subcutaneous tumors in immunocompromised nude mice, by genetic engineering of normal organoids. As expected, acute induction of KrasG12Din vitro occasionally led to development of tiny nodules compatible with early lesions known as pancreatic intraepithelial neoplasia (PanIN). KrasG12D-expressing cells were enriched after inoculation in the subcutis, yet proved rather declined during culture, suggesting that its advantage might depend on surrounding environments. Depletion of growth factors or concurrent Trp53 deletion resulted in its robust enrichment, invariably leading to development of PanIN or large high-grade adenocarcinoma, respectively, consistent with in vivo mouse studies for the same genotype. Progression from PanIN was also recapitulated by subsequent knockdown of common tumor suppressors, whereas the impact of Tgfbr2 deletion was only partially recapitulated, illustrating genotype-dependent requirement of the pancreatic niche for tumorigenesis. Intriguingly, analysis of tumor-derived organoids revealed that KrasG12D-expressing cells with spontaneous deletion of wild-type Kras were positively selected and exhibited an aging-related mutation signature in nude mice, mirroring the pathogenesis of human PDA, and that the sphere-forming potential and orthotopic tumorigenicity in syngenic mice were significantly augmented. These observations highlighted the relevance of the subcutis of nude mice in promoting PDA development despite its ectopic nature. Taken together, pancreatic carcinogenesis could be considerably recapitulated with organoids, which would probably serve as a novel disease model.

Kras-driven heterotopic tumor development from hepatobiliary organoids.

Cancers arising from the biliary tract are refractory to conventional therapies, requiring the development of novel therapeutics. However, only a limited number of genetically engineered mouse models have been created, partly because of time-consuming work required. Besides, liver-specific gene manipulation mostly resulted in concurrent development of hepatocellular carcinoma, another type of liver cancer, and gallbladder-restricted gene targeting is still not feasible. Consequently, establishment of cancer type-specific disease modeling remains a technical challenge. To address this issue, we took an alternative cell-based approach to quickly induce tumorigenesis ex vivo. Specifically, murine primary organoids from liver and gallbladder were transduced with lentiviral vectors to reconstitute genetic alterations common in biliary tract cancers, followed by inoculation in immunodeficient mice. Although any single genetic alteration did not induce tumors, mutant Kras and repression of major tumor suppressors cooperated for tumor development within 2 months. Induced lesions varied among normal, dysplastic and papillary lesions to adenocarcinoma, recapitulating multistep tumorigenesis even in a heterotopic situation. We further demonstrated that two putative oncogenes in intrahepatic cholangiocellular carcinoma, mutant Pik3ca and FGFR2-AHCYL1 fusion, were rather modest drivers for liver-derived organoids, probably requiring additional mutations or hepatic niche to robustly induce full-blown tumors. Thus, we showed that cancer cells could be readily generated from primary cells in the biliary tract, at least in cases where genetic factors play dominant roles. Collectively, this study will likely contribute to gaining mechanistic insights into biliary carcinogenesis and providing valuable resources for drug discovery.

Shortcuts to intestinal carcinogenesis by genetic engineering in organoids.

Inactivation of the Adenomatous polyposis coli (APC) gene is an initiating and the most relevant event in most sporadic cases of colorectal cancer, providing a rationale for using Apc-mutant mice as the disease model. Whereas carcinogenesis has been observed only at the organism level, the recent development of the organoid culture technique has enabled long-term propagation of intestinal stem cells in a physiological setting, raising the possibility that organoids could serve as an alternative platform for modeling colon carcinogenesis. Indeed, it is demonstrated in the present study that lentivirus-based RNAi-mediated knockdown of Apc in intestinal organoids gave rise to subcutaneous tumors upon inoculation in immunodeficient mice. Reconstitution of common genetic aberrations in organoids resulted in development of various lesions, ranging from aberrant crypt foci to full-blown cancer, recapitulating multi-step colorectal tumorigenesis. Due to its simplicity and utility, similar organoid-based approaches have been applied to both murine and human cells in many investigations, to gain mechanistic insight into tumorigenesis, to validate putative tumor suppressor genes or oncogenes, and to establish preclinical models for drug discovery. In this review article, we provide a multifaceted overview of these types of approaches that will likely accelerate and advance research on colon cancer.

Genetic reconstitution of tumorigenesis in primary intestinal cells.

Animal models for human colorectal cancer recapitulate multistep carcinogenesis that is typically initiated by activation of the Wnt pathway. Although potential roles of both genetic and environmental modifiers have been extensively investigated in vivo, it remains elusive whether epithelial cells definitely require interaction with stromal cells or microflora for tumor development. Here we show that tumor development could be simply induced independently of intestinal microenvironment, even with WT murine primary intestinal cells alone. We developed an efficient method for lentiviral transduction of intestinal organoids in 3D culture. Despite seemingly antiproliferative effects by knockdown of adenomatous polyposis coli (APC), we managed to reproducibly induce APC-inactivated intestinal organoids. As predicted, these organoids were constitutively active in the Wnt signaling pathway and proved tumorigenic when injected into nude mice, yielding highly proliferative tubular epithelial glands accompanied by prominent stromal tissue. Consistent with cellular transformation, tumor-derived epithelial cells acquired sphere formation potential, gave rise to secondary tumors on retransplantation, and highly expressed cancer stem cell markers. Inactivation of p53 or phosphatase and tensin homolog deleted from chromosome 10, or activation of Kras, promoted tumor development only in the context of APC suppression, consistent with earlier genetic studies. These findings clearly indicated that genetic cooperation for intestinal tumorigenesis could be essentially recapitulated in intestinal organoids without generating gene-modified mice. Taken together, this in vitro model for colon cancer described herein could potentially provide unique opportunities for carcinogenesis studies by serving as a substitute or complement to the currently standard approaches.

Bcl11b SWI/SNF-complex subunit modulates intestinal adenoma and regeneration after γ-irradiation through Wnt/ß-catenin pathway.

SWI/SNF chromatin remodeling complexes constitute a highly related family of multi-subunit complexes to modulate transcription, and SWI/SNF subunit genes are collectively mutated in 20% of all human cancers. Bcl11b is a SWI/SNF subunit and acts as a haploinsufficient tumor suppressor in leukemia/lymphomas. Here, we show expression of Bcl11b in intestinal crypt cells and promotion of intestinal tumorigenesis by Bcl11b attenuation in Apc (min/+) mice. Of importance, mutations or allelic loss of BCL11B was detected in one-third of human colon cancers. We also show that attenuated Bcl11b activity in the crypt base columnar (CBC) cells expressing the Lgr5 stem cell marker enhanced regeneration of intestinal epithelial cells after the radiation-induced injury. Interestingly, BCL11B introduction in human cell lines downregulated transcription of ß-catenin target genes, whereas Bcl11b attenuation in Lgr5(+) CBCs increased expression of ß-catenin targets including c-Myc and cyclin D1. Together, our results argue that Bcl11b impairment promotes tumor development in mouse and human intestine at least in part through deregulation of ß-catenin pathway.

Newly defined aberrant crypt foci as a marker for dysplasia in the rat colon.

Dysplasia represents a preneoplastic status in multistep colon carcinogenesis. Whereas laborious preparation of thin sections is required for its diagnosis, we here show that newly defined aberrant crypt foci (ACF) simply mark the majority of the dysplasia on the whole colon. Specifically, decoloring of the azoxymethane-treated rat colon after scoring classical ACF (cACF) resulted in visualization of a subset of aberrant crypts that remained densely stained. They were morphologically classified into three subtypes, of which two with compressed luminal openings proved highly correlated with dysplasia. Accordingly, we designated those foci harboring either of the two crypt subtypes as dysplasia-associated ACF (dACF). By serially applying different detection methods for known preneoplastic lesions to the same colon, we showed that most dACF had already been identified as cACF, and a few newly identified dACF contained an entire population of more advanced lesions, such as flat ACF and mucin-depleted foci. Consequently, integrative scoring of cACF and dACF enabled capture of all early lesions of the colon. Furthermore, 94% of the dACF showed dysplasia and 90% of the dysplastic lesions proved to be dACF. Thus, dACF is a promising marker for dysplasia, likely facilitating precise identification of the early stages of colon carcinogenesis.

AKT is critically involved in cooperation between obesity and the dietary carcinogen amino-1-methyl-6-phenylimidazo [4,5-b] (PhIP) toward colon carcinogenesis in rats.

Obesity is highly associated with colon cancer development. Whereas it is generally attributed to pro-tumorigenic effects of high fat diet (HFD), we here show that a common genetic basis for predisposition to obesity and colon cancer might also underlie the close association. Comparison across multiple rat strains revealed that strains prone to colon tumorigenesis initiated by a dietary carcinogen amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP) tended to develop obesity. Through transcriptome and extensive immunoblotting analyses, we identified the basal level of activated AKT in colonic crypts as a biomarker for the common predisposition. Notably, PhIP induced activation of AKT, which could persist for several weeks under a low fat diet (LFD), but not under HFD. On the other hand, PhIP and HFD independently induced Wnt pathway activation and inhibited apoptosis, through distinct mechanisms involving GSK-3ß, caspase 3 and poly-ADP ribose polymerase (PARP). Taken together, these observations provide mechanistic insights into how PhIP-induced activation of AKT might cooperate with HFD at multiple levels toward development of colon cancer.

Different types of reactions to E7386 among colorectal cancer patient-derived organoids and corresponding CAFs.

Colorectal cancer (CRC) harbors genetic alterations in a component of the Wnt signaling pathway in ~90% of cases. In addition, the Wnt signaling pathway has been previously suggested to serve a notable role in the pathophysiology of CRC cells and cancer-associated fibroblasts (CAFs). In the present study, the possible effects of E7386, a selective inhibitor of the interaction between ß-catenin and the cAMP response element-binding protein-binding protein, were evaluated using organoids and the corresponding CAFs derived from patients with CRC. E7386 at 100 nM was revealed to decrease the viability of CRC organoids and CAFs. Analysis of the gene expression profiles revealed marked changes in the expression levels of different types of cancer-associated genes associated with E7386 concentrations in the organoids and/or CAFs, such as those regulating glucose and amino acid metabolism [phosphoenolpyruvate carboxykinase 2, asparagine synthetase (glutamine-hydrolyzing), phosphoserine aminotransferase 1 and phosphoglycerate dehydrogenase], stimulation of natural killer cell-mediated cytotoxicity (UL16-binding protein 1) and modification of the Wnt/ß-catenin signaling pathway (indicated by very low density lipoprotein receptor). Results of the hydrophilic metabolome analysis in the organoids were consistent with those of the transcriptomic analysis. In vivo experiments used corresponding xenograft models, although changes in volume of tumor tissues could not be observed at 50 mg/kg body weight twice a day for 14 days, results on the protein expression levels partially supported those in the in vitro experiments. In conclusion, different types of reactions, such as alterations in the glucose and amino acid metabolic pathways, stimulation of stress responses and NK-cell mediated cytotoxicity and components in the Wnt/ß-catenin signaling pathway, to E7386 in the CRC organoids and corresponding CAFs were observed under conditions with decreased cell viability. However, further mechanistic studies to clarify the relationships with Wnt/ß-catenin signaling pathway and these reactions using preclinical models and biomarker studies using clinical human samples are required for verification of the present preclinical biomarkers.

Establishment of Novel Genotoxicity Assay System Using Murine Normal Epithelial Tissue-Derived Organoids.

Short-/middle-term and simple prediction studies for carcinogenesis are needed for the safety assessment of chemical substances. To establish a novel genotoxicity assay with an in vivo mimicking system, we prepared murine colonic/pulmonary organoids from gpt delta mice according to the general procedure using collagenase/dispase and cultured them in a 3D environment. When the organoids were exposed to foodborne carcinogens-2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) and acrylamide (AA)-in the presence of metabolic activation systems, mutation frequencies (MFs) occurring in the gpt gene dose-dependently increased. Moreover, the mutation spectrum analysis indicated predominant G:C to T:A transversion with PhIP, and A:T to C:G and A:T to T:A transversion with AA. These data correspond to those of a previous study describing in vivo mutagenicity in gpt delta mice. However, organoids derived from the liver, a non-target tissue of PhIP-carcinogenesis, also demonstrated genotoxicity with a potency comparable to colonic organoids. Organoids and PhIP were directly incubated in the presence of metabolic activation systems; therefore, there was a lack of organ specificity, as observed in vivo. Additionally, PhIP-DNA adduct levels were comparable in hepatic and colonic organoids after PhIP exposure. Taken together, the organoids prepared in the present study may be helpful to predict chemical carcinogenesis.

Re-expression of REG family and DUOXs genes in CRC organoids by co-culturing with CAFs.

Organoids derived from epithelial tumors have recently been utilized as a preclinical model in basic and translational studies. This model is considered to represent the original tumor in terms of 3D structure, genetic and cellular heterogeneity, but not tumor microenvironment. In this study, we established organoids and paired cancer-associated fibroblasts (CAFs) from surgical specimens of colorectal carcinomas (CRCs), and evaluated gene expression profiles in organoids with and without co-culture with CAFs to assess interactions between tumor cells and CAFs in tumor tissues. We found that the expression levels of several genes, which are highly expressed in original CRC tissues, were downregulated in organoids but re-expressed in organoids by co-culturing with CAFs. They comprised immune response- and external stimulus-related genes, e.g., REG family and dual oxidases (DUOXs), which are known to have malignant functions, leading tumor cells to proliferative and/or anti-apoptotic states and drug resistant phenotypes. In addition, the degree of differential induction of REG1 and DUOX2 in the co-culture system varied depending on CAFs from each CRC case. In conclusion, the co-culture system of CRC organoids with paired CAFs was able to partially reproduce the tumor microenvironment.

Effects of the scid mutation on X-ray-induced deletions in the brain and spleen of gpt delta mice.

BACKGROUND: DNA-dependent protein kinase (DNA-PK), consisting of a Ku heterodimer (Ku70/80) and a large catalytic subunit (DNA-PKcs), plays an important role in the repair of DNA double-strand breaks via non-homologous end-joining (NHEJ) in mammalian cells. Severe combined immunodeficient (scid) mice carry a mutation in the gene encoding DNA-PKcs and are sensitive to ionizing radiation. To examine the roles of DNA-PKcs in the generation of deletion mutations in vivo, we crossed scid mice with gpt delta transgenic mice for detecting mutations. RESULTS: The scid and wild-type (WT) gpt delta transgenic mice were irradiated with a single X-ray dose of 10 Gy, and Spi- mutant frequencies (MFs) were determined in the brain and spleen 2 days after irradiation. Irradiation with X-rays significantly enhanced Spi- MF in both organs in the scid and WT mice. The MFs in the brain of irradiated scid mice were significantly lower than those in WT mice, i.e., 2.9 ± 1.0 × 10- 6 versus 5.0 ± 1.1 × 10- 6 (P < 0.001), respectively. In the spleen, however, both mouse strains exhibited similar MFs, i.e., 4.1 ± 1.8 × 10- 6 versus 4.8 ± 1.4 × 10- 6. Unirradiated scid and WT mice did not exhibit significant differences in MFs in either organ. CONCLUSIONS: DNA-PKcs is unessential for the induction of deletion mutations in the spleen, while it plays a role in this in the brain. Therefore, the contribution of DNA-PKcs to NHEJ may be organ-specific.

SND1, a component of RNA-induced silencing complex, is up-regulated in human colon cancers and implicated in early stage colon carcinogenesis.

Colon cancers have been shown to develop after accumulation of multiple genetic and epigenetic alterations with changes in global gene expression profiles, contributing to the establishment of widely diverse phenotypes. Transcriptional and posttranscriptional regulation of gene expression by small RNA species, such as the small interfering RNA and microRNA and the RNA-induced silencing complex (RISC), is currently drawing major interest with regard to cancer development. SND1, also called Tudor-SN and p100 and recently reported to be a component of RISC, is among the list of highly expressed genes in human colon cancers. In the present study, we showed remarkable up-regulation of SND1 mRNA in human colon cancer tissues, even in early-stage lesions, and also in colon cancer cell lines. When mouse Snd1 was stably overexpressed in IEC6 rat intestinal epithelial cells, contact inhibition was lost and cell growth was promoted, even after the cells became confluent. Intriguingly, IEC6 cells with high levels of Snd1 also showed an altered distribution of E-cadherin from the cell membrane to the cytoplasm, suggesting loss of cellular polarity. Furthermore, the adenomatous polyposis coli (Apc) protein was coincidentally down-regulated, with no significant changes in the Apc mRNA level. Immunohistochemical analysis using chemically induced colonic lesions developed in rats revealed overexpression of Snd1 not only in colon cancers but also in aberrant crypt foci, putative precancerous lesions of the colon. Up-regulation of SND1 may thus occur at a very early stage in colon carcinogenesis and contribute to the posttranscriptional regulation of key players in colon cancer development, including APC and beta-catenin.

Effect of running training on DMH-induced aberrant crypt foci in rat colon.

PURPOSE: We examined the effects of treadmill-running training on the induction of aberrant crypt foci (ACF), which is the first step of colon cancer induction, in the colonic mucosa of rats injected with 1,2-dimethylhydrazine (DMH). METHODS: Four-week-old F344 rats (N = 38) were randomly assigned to training (19 rats) and control (19 rats) groups. After a week, all rats were given DMH (20 mg.kg(-1) body weight) once a week for 2 wk. Running training was started at age 7 wk (speed: 10 m.min(-1), 0% grade, 120 min.d(-1), 5 d.wk(-1)). After 4 wk of training, the rats were sacrificed and the colon was removed, opened, and counted for ACF with 0.2% methylene blue staining. RESULTS: Running training resulted in lower body- (P < 0.01) and adipose fat weight (P < 0.05). The numbers of ACF and total AC were significantly lower in the running training group than in the control group (P < 0.05). The occurrences of one, three, and five aberrant crypts per focus were also significantly lower in the running training group than in the control group (P < 0.05). The ratios of total AC/ACF did not significantly differ between the running training and control groups. CONCLUSIONS: The results of the present investigation suggest that low-intensity running training inhibits the DMH-induced initiation of colon ACF development.

Cilostazol and enzymatically modified isoquercitrin attenuate experimental colitis and colon cancer in mice by inhibiting cell proliferation and inflammation.

We previously reported the anti-inflammatory effects of cilostazol, a selective inhibitor of phosphodiesterase 3, and two antioxidants, enzymatically modified isoquercitrin and α-lipoic acid in a dextran sodium sulphate-induced colitis mouse model. We further examined the chemopreventive effects of these substances in a murine azoxymethane/dextran sodium sulphate -induced colorectal carcinoma model and compared the effects with those of the well-known anticancer natural plant pigment, anthocyanin. In addition, the effects on cell proliferation activity were evaluated in colon cancer cell lines and mucosal epithelial cells in a model of acute dextran sodium sulphate-induced colitis. Cilostazol and enzymatically modified isoquercitrin improved the outcome of azoxymethane/dextran sodium sulphate-induced colorectal cancer along with anthocyanin though inhibiting inflammation and cell proliferation, but the effect of α-lipoic acid was minimal. Inhibition of cell proliferation by cilostazol was confirmed in vitro. In the acute dextran sodium sulphate-induced colitis model, cilostazol and enzymatically modified isoquercitrin prevented the decrease in epithelial proliferative cells. These results indicate that cilostazol and enzymatically modified isoquercitrin first exhibited an anti-dextran sodium sulphate effect at the initial stage of colitis and then showed antitumour effects throughout subsequent inflammation-related cancer developmental stages.

Genomic Landscape of Experimental Bladder Cancer in Rodents and Its Application to Human Bladder Cancer: Gene Amplification and Potential Overexpression of Cyp2a5/CYP2A6 Are Associated with the Invasive Phenotype.

Non-muscle invasive (superficial) bladder cancer is a low-grade malignancy with good prognosis, while muscle invasive (invasive) bladder cancer is a high-grade malignancy with poor prognosis. N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) induces superficial bladder cancers with papillary morphology in rats and invasive bladder cancers with infiltrating phenotype in mice. In this study, we analyzed genomic landscapes of rodent BBN-induced bladder cancers using array-based comparative genomic hybridization (array CGH). While no significant copy number alterations were detected in superficial bladder tumors in rats, copy number gains in chromosomal regions 2D-E1, 7qA3, 9F2, and 11C-D were detected in invasive bladder tumors in mice. Amplification of representative genes located on 2D-E1 and 7qA3 chromosomal regions was confirmed by quantitative PCR. Cyp2a22 and Cyp2a5 genes but not Cyp2g1, Cyp2a12, and Rab4b genes on mouse chromosome 7qA3 were amplified in invasive bladder cancers. Although the human ortholog gene of Cyp2a22 has not been confirmed, the mouse Cyp2a5 gene is the ortholog of the human CYP2A6 gene located in chromosomal region 19q13.2, and CYP2A6 was identified by database search as one of the closest human homolog to mouse Cyp2a22. Considering a possibility that this region may be related to mouse 7qA3, we analyzed CYP2A6 copy number and expression in human bladder cancer using cell lines and resected tumor specimens. Although only one of eight cell lines showed more than one copy increase of the CYP2A6 gene, CYP2A6 amplification was detected in six out of 18 primary bladder tumors where it was associated with the invasive phenotype. Immunohistochemical analyses of 118 primary bladder tumors revealed that CYP2A6 protein expression was also higher in invasive tumors, especially in those of the scattered type. Together, these findings indicate that the amplification and overexpression of the CYP2A6 gene are characteristic of human bladder cancers with increased malignancy and that CYP2A6 can be a candidate prognostic biomarker in this type of cancer.

Differential staining of dysplastic aberrant crypt foci in the colon facilitates prediction of carcinogenic potentials of chemicals in rats.

We developed a novel and simple method to identify dysplastic aberrant crypt foci (ACF) induced in rats by colon carcinogens more efficiently and selectively without conducting laborious histological examination, which usually requires enough time to get final diagnosis. By adding a simple decolorization process with 70% methanol after conventional 0.2% methylene blue staining, dysplastic ACF could be differentially contrasted. To examine the validity of this novel method, which we refer to as differential staining, we analyzed colonic lesions induced by three heterocyclic amines, including 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, and found that the number of dysplastic ACF detected more precisely reflected their carcinogenic potential than the total numbers of ACF.