Differential gene expression profiles in colon epithelium of two rat strains with distinct susceptibility to colon carcinogenesis after exposure to PhIP in combination with dietary high fat

Colon cancers develop through accumulation of multiple genetic and epigenetic alterations in colon epithelial cells, and the environment of the genetically altered epithelial cells may also have a substantial impact on their further development to cancer. In the present study, groups of 6-week-old F344 and ACI male rats, the former strain being susceptible to colon carcinogenesis induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and the latter being relatively resistant, were subjected to a long-term carcinogenesis experiment using our intermittent feeding protocol of PhIP in combination with a high-fat diet, which serves as a relevant risk factor that promotes the development of colon cancers. Animals were sacrificed at 60 weeks, and global gene expression analyses of normal parts of colon epithelial tissues were conducted using a high-density oligonucleotide microarray to elucidate the differential gene expression profile (environment) in normal colonic regions between F344 and ACI strains. Of 8799 entries on the RatU34A array, 74 genes exhibited 3-fold or greater variation. A subset of genes encoding ribosomal RNAs and proteins were highly preferentially expressed in the F344 strain. In addition, genes encoding fatty acid binding proteins and the peroxisome membrane protein 70 appeared up-regulated in the susceptible F344 strain. In the ACI strain, a mismatch repair gene, Msh2 , was preferentially expressed, at approximately 20-fold the F344 level, along with a gene encoding a detoxification enzyme, catechol-O-methyltransferase. The combined effects of the repertoire of these differentially expressed genes in normal colon epithelial tissues may account for the distinct susceptibilities of F344 and ACI strains to colon carcinogenesis.     

Chromosomal mapping of genes controlling development, histological grade, depth of invasion, and size of rat stomach carcinomas

Rat stomach cancers induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) are widely used as a model of differentiated-type human stomach cancers. ACI/N (ACT) rats are susceptible and BUF/Nac (BUF) rats are resistant to MNNG-induced stomach carcinogenesis, and the presence of an autosomal gene with a dominant BUF allele has been suggested. In this study, we performed a carcinogenicity test by giving MNNG in drinking water to 117 male ACI x (ACIxBUF)F1 backcross rats. Each of 100 effective rats was diagnosed for its "carcinoma development" and when it was bearing stomach carcinoma(s), for histological grade, depth of invasion, and size and number of tumors. Carcinoma development was diagnosed based both on the age of the rat and on the presence of stomach carcinoma(s). Linkage analysis was performed with the genotypes of 161 loci, covering 1637 cM of the rat genome. Contrary to our original expectations, the most influential gene was the one on chromosome (chr.) 15, Gastric cancer susceptibility gene 1 (Gcs1), which confers susceptibility to stomach carcinogenesis (LOD, 3.8) with a dominant BUF allele by promoting conversion from adenomas to carcinomas. Two resistance genes on chr. 4 and chr. 3, Gastric cancer resistance gene 1 (Gcr1) and Gcr2, were shown to confer dominant resistance (LOD, 2.7 and 2.6, respectively). Gcs1, Gcr1, and Gcr2 exerted additive effects on the development of stomach carcinomas. A gene on chr. 16, Gcr3, was indicated to reduce the depth of invasion (LOD, 2.2) and sizes of tumors (LOD, 1.9). No linkage was obtained using the number of tumors. These findings show that the coordinate effect of a susceptibility gene, Gcs1, and two resistance genes, Gcr1 and Gcr2, is responsible for the development of MNNG-induced stomach carcinomas and that Gcr3 is responsible for the growth of a stomach carcinoma, reflected in the depth of invasion and in the tumor size.     

Expression of the zinc transporter ZnT4 is decreased in the progression from early prostate disease to invasive prostate cancer

We have utilized oligonucleotide microarrays to identify novel genes of potential clinical and biological importance in prostate cancer. RNA from 74 prostate cancers and 164 normal body samples representing 40 different tissues were analysed using a customized Affymetrix GeneChip oligonucleotide microarray representative of over 90% of the expressed human genome. The gene for the zinc transporter ZnT4 was one of several genes that displayed significantly higher expression in prostate cancer compared to normal tissues from other organs. A polyclonal antipeptide antibody was used to demonstrate ZnT4 expression in the epithelium of all 165 elements of benign and 326 elements of localized prostate cancers examined and in nine of 10 advanced prostate cancer specimens by immunohistochemistry. Interestingly, decreased intensity of ZnT4 immunoreactivity occurred in the progression from benign to invasive localized prostate cancer and to metastatic disease. Immunofluorescence analysis and surface biotinylation studies of cells expressing ZnT4 localised the protein to intracellular vesicles and to the plasma membrane. These findings are consistent with a role for ZnT4 in vesicular transport of zinc to the cell membrane and potentially in efflux of zinc in the prostate.     

Genetic analysis of the susceptibility in rats to aberrant crypt foci formation by 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine, PhIP.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most abundant heterocyclic amine produced while cooking fish and meat, induces aberrant crypt foci (ACF) and colon cancers in rats. We previously reported that F344 rats were sensitive and ACI rats resistant to ACF formation by PhIP, and that the genetic susceptibility in F344 rats to ACF formation by PhIP was autosomally dominant over ACI rats. To identify candidate susceptibility genes in F344 rats, a preliminary genome-wide linkage analysis was employed using a subset of 170 progeny of (F344 x ACI)F1 x ACI backcross rats with either high or low sensitivity to ACF formation by PhIP. Three chromosomes, 1, 6 and 16, demonstrated the presence of loci with a logarithm of the odds (lod) scores of more than 1.0, and a susceptible gene for ACF formation by PhIP was suggested to reside on chromosomes 16.     

Hormone therapy failure in human prostate cancer: analysis by complementary DNA and tissue microarrays.

BACKGROUND: The molecular mechanisms underlying the progression of prostate cancer during hormonal therapy have remained poorly understood. In this study, we developed a new strategy for the identification of differentially expressed genes in hormone-refractory human prostate cancer by use of a combination of complementary DNA (cDNA) and tissue microarray technologies. METHODS: Differences in gene expression between hormone-refractory CWR22R prostate cancer xenografts (human prostate cancer transplanted into nude mice) and a xenograft of the parental, hormone-sensitive CWR22 strain were analyzed by use of cDNA microarray technology. To validate the data from cDNA microarrays on clinical prostate cancer specimens, a tissue microarray of specimens from 26 prostates with benign prostatic hyperplasia, 208 primary prostate cancers, and 30 hormone-refractory local recurrences was constructed and used for immunohistochemical detection of protein expression. RESULTS: Among 5184 genes surveyed with cDNA microarray technology, expression of 37 (0.7%) was increased more than twofold in the hormone-refractory CWR22R xenografts compared with the CWR22 xenograft; expression of 135 (2.6%) genes was reduced by more than 50%. The genes encoding insulin-like growth factor-binding protein 2 (IGFBP2) and 27-kd heat-shock protein (HSP27) were among the most consistently overexpressed genes in the CWR22R tumors. Immunohistochemical analysis of tissue microarrays demonstrated high expression of IGFBP2 protein in 100% of the hormone-refractory clinical tumors, in 36% of the primary tumors, and in 0% of the benign prostatic specimens (two-sided P =.0001). Overexpression of HSP27 protein was demonstrated in 31% of the hormone-refractory tumors, in 5% of the primary tumors, and in 0% of the benign prostatic specimens (two-sided P =.0001). CONCLUSIONS: The combination of cDNA and tissue microarray technologies enables rapid identification of genes associated with progression of prostate cancer to the hormone-refractory state and may facilitate analysis of the role of the encoded gene products in the pathogenesis of human prostate cancer.     

Profiling and selection of genes differentially expressed in the pylorus of rat strains with different proliferative responses and stomach cancer susceptibility

Rat stomach cancers induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) are widely used as a model of differentiated-type human stomach cancers. ACI/NJcl (ACI) rats show persistent and strong cell proliferation in response to gastric mucosal damage by MNNG while BUF/NacJcl (BUF) rats show transient and limited cell proliferation. This difference is considered as one of the mechanisms for the high susceptibility of ACI rats to MNNG-induced stomach carcinogenesis. To identify genes involved in the differential induction of cell proliferation, cDNA subtraction was performed using RNA isolated from the pylorus of ACI and BUF rats treated with MNNG. By the temporal patterns of their expressions, the isolated 16 genes were overviewed and clustered into groups. Expression of the genes in group 1 (such as MHC class I and class II genes and interferon-inducible genes Iigp, Mx2 and Ubd) was induced by MNNG treatment, and the genes in group 2 (such as cellular retinoic acid-binding protein II (CrabpII)) were constantly expressed regardless of MNNG treatment. Then, expression profiles among multiple rat strains were compared with the extents of induction of cell proliferation. Iigp, CrabpII and EST222005 were found to show relatively good accordance, and these three genes were considered as candidates for genes that control differential induction of cell proliferation. Presence of polymorphisms at the genomic DNA level was indicated for CrabpII and EST222005, and these two genes were considered to be better candidates than IIGP: It was shown that the temporal profiles and profiles among strains, taking advantage of animal models, are useful to select candidate genes from a collection of genes isolated by various genome-wide scanning methods.     

A strong candidate gene for the Papg1 locus on mouse chromosome 4 affecting lung tumor progression

Lung cancer is the leading cause of cancer death among both men and women, accounting for more than 28% of all cancer deaths. In fact, more people die of lung cancer than of colon, breast, and prostate cancers combined. Although lung cancer is largely induced by smoking, there is strong evidence for genetic susceptibility and gene-environment interactions in the development of lung cancer. Inbred mouse models offer an effective means of identifying candidate lung cancer susceptibility loci since genetic heterogeneity and enormous variation in exposure levels to environmental agents make it difficult to identify lung cancer susceptibility loci in humans. Papg-1 (pulmonary adenoma progression 1) was previously mapped to a region on mouse chromosome 4. This locus contains a candidate gene, Cdkn2a also referred to as Ink4a/Arf, which dually encodes two established tumor suppressors p16(INK4a) and ARF. Cdkn2a became a primary candidate for Papg-1 for two reasons: (1) two haplotypes of mouse Cdkn2a were found to segregate with differential genetic susceptibility to lung tumor progression in mice; and (2) in vitro studies showed that the p16(INK4a) allele from the BALB/cJ mouse had a significantly decreased ability to bind and inhibit CDK6 and to suppress cell growth when compared with the p16(INK4a) allele from the A/J mouse. Here, we report that mice with a heterozygous deficiency for the A/J Cdkn2a allele were significantly more susceptible to lung tumor progression than mice with a heterozygous deficiency for a BALB/cJ Cdkn2a allele, when compared to their respective wild type mice. These results offer strong evidence that naturally occurring variation of p16(INK4a) influences susceptibility to enhance lung tumor progression making it a strong candidate for the lung tumor progression locus, Papg-1.     

Ectonucleoside triphosphate diphosphohydrolase 6 expression in testis and testicular cancer and its implication in cisplatin resistance

The development of resistance to cisplatin during treatment of testicular cancer constitutes a major obstacle to the cure of testicular cancer. To resolve its mechanism will provide useful information for making clinical decisions. We found 4 and 15 gene expressions were, respectively, up-regulated and down-regulated in cisplatin-resistant testicular cancer NEC-8/DDP cells compared with their parental NEC-8 cells (about 2.5-fold) using cDNA microarray analysis. After screening, we selected the ENTPD6 among these 19 genes. ENTPD6 was less expressed in cancerous region compared with that in non-cancerous lesion. In addition, ENTPD6 expression in seminoma was higher than that in other testicular tumors. ENTPD6 expression was involved in cellular sensitivity to cisplatin through an interaction with E-cadherin. ENTPD6 is a promising molecular biomarker of cisplatin resistance in testicular cancer, and also a novel therapeutical target modulating cisplatin resistance in testicular cancer.     

Clinical validation of candidate genes associated with prostate cancer progression in the CWR22 model system using tissue microarrays

To explore molecular mechanisms of prostate cancer progression, we applied tissue microarrays (TMAs) to analyze expression of candidate gene targets discovered by cDNA microarray analysis of the CWR22 xenograft model system. A TMA with 544 clinical specimens from different stages of disease progression was probed by mRNA in situ hybridization and protein immunohistochemistry. There was an excellent correlation (r = 0.96; n = 16) between the expression levels of the genes in the xenografts by cDNA microarray and mRNA in situ hybridization on a TMA. One of the most highly overexpressed genes in hormone-refractory CWR22R xenografts was the S100P gene. This gene, coding for a calcium signaling molecule implicated in the loss of senescence, was also significantly associated with progression in clinical tumors by TMA analysis (P < 0.001), suggesting dysregulation of this pathway in hormone-refractory and metastatic prostate cancers. Conversely, two genes that were down-regulated during tumor progression in the CWR22 model system were validated in vivo: crystallin mu (CRYM) and a LIM-domain protein LMO4 both showed significantly lower mRNA levels in hormone-refractory tumors as compared with primary tumors (P < 0.001). These results illustrate a strategy for rapid clinical validation at the mRNA and protein level of gene targets found to be differentially expressed in cDNA microarray experiments of model systems of cancer.     

Metallothionein is up-regulated under hypoxia and promotes the survival of human prostate cancer cells

Tumor hypoxia is a common feature of several cancers, including prostate cancer, and is associated with tumor progression, acquisition of anti-apoptotic potential and therapeutic resistance. We explored hypoxia-inducible genes and examined the effect of knockdown of a target molecule with small interference RNA (siRNA) on the proliferation of human prostate cancer cells. Human prostate cancer cell lines (LNCaP and PC-3) were cultured in normoxia (21% O2) or hypoxia (0.5% O2). Hypoxia-inducible genes were identified by cDNA microarray analysis. Metallothionein (MT) expression was assessed by real-time RT-PCR, Western blot analysis and immunohistochemical staining. siRNA was transfected to knock down MT expression, and the cell cycle and apoptosis were evaluated by flow cytometry analysis. In cDNA microarray analysis, 22 genes (including MT) were up-regulated under hypoxia. MT-1X and MT-2A were up-regulated in real-time RT-PCR. In particular, MT-2A was increased 3-fold in LNCaP and 8-fold in PC-3. The siRNA-MT-2A treatment resulted in a 20% inhibition of cell growth and induced apoptosis in both LNCaP and PC-3. In human prostate tissue, intense staining of MT was observed in cancer cells and residual cancer cells after androgen ablation therapy, while normal tissue was only stained in patches. In conclusion, MT was up-regulated under hypoxia in prostate cancer cells and overexpressed in prostate cancer tissue and residual cancer cells after androgen ablation therapy. As down-regulation of MT by siRNA inhibited cell growth and induced cell death, MT may be a new molecular target for the treatment of human prostate cancer.     

Identification and chromosome mapping of loci predisposing to colorectal cancer that control Wnt/beta-catenin pathway and progression of early lesions in the rat

Sporadic colorectal cancer (CRC) is a major health concern worldwide. Epidemiologic evidence suggests a polygenic predisposition to CRC, but the genes responsible remain unknown. Here, we performed genome-wide scanning of male (ACI/SegHsd x Wistar-Furth)F2 (AWF2) rats to map susceptibility genes influencing the evolution of early colorectal lesions to adenocarcinoma following 1,2-dimethylhydrazine administration. Phenotypic analysis revealed higher incidence/multiplicity and lower size of adenomas in ACI/SegHsd (ACI) and (ACI/SegHsd x Wistar-Furth)F1 (AWF1) than Wistar-Furth (WF) rats and higher incidence/multiplicity of poorly differentiated adenocarcinomas in WF than ACI rats, with intermediate values in AWF1 rats. Linkage analysis of 138 AWF2 rats identified three loci on chromosomes 4, 15 and 18 in significant linkage with lesion multiplicity that were identified as rat Colon cancer resistance (rCcr) 1, rCcr2 and rCcr3, respectively. Seven other loci on chromosomes 5, 6, 15, 17, 18 and 20 were in suggestive linkage with adenoma/adenocarcinoma multiplicity/surface area. Six of them were identified as rCcr4-9 and a locus on chromosome 5 was identified as a susceptibility locus, rCcs1. Significant interactions between rCcr3 and rCcr6, rCcr6 and rCcr8 and rCcr5 and rCcr9, and four novel epistatic loci controlling multiplicity/size of colorectal lesions were discovered. Apc, located at rCcr3, did not show functional promoter polymorphisms. However, influence of susceptibility/resistance genes on Wnt/beta-catenin pathway was shown by defective beta-catenin inactivation in WF but not in ACI and AWF1 rat adenocarcinomas. These data indicate that inheritance of predisposition to CRC depends on interplays of several genetic factors, and suggest a possible mechanism of polygenic control of CRC progression.     

基因芯片分析前列腺癌细胞系中转移相关基因

目的:通过基因芯片技术检测具有不同转移潜能的前列腺癌细胞系LNCaP和C4-2中表达差异的基因,寻找前列腺癌转移相关基因.方法:采用TRIzol一步法提取LNCaP和C4-2细胞的总RNA,并纯化mRNA,反转录合成荧光分子标记的cDNA探针,与基因芯片杂交.采用Genepix Pro 6.0图像分析软件进行芯片图像分析,把图像信号转化为数字信号,然后以差异大于等于2倍的标准来确定差异表达基因.结果:在LNCaP与C4-2细胞中,表达差异的基因共有417个,上调基因301个,下调基因116个.分析显示差异表达基因分别与细胞增殖、代谢、细胞因子、细胞转移等相关,其中发现7个基因与肿瘤细胞的转移相关,分别为EGFR、EGF、PIK3R1、Cyclin E、HYAL1、GNAI1、AZGP1.结论:筛选出LNCaP与C4-2细胞系中7个转移相关基因,为进一步研究前列腺癌转移机制奠定了基础.     

Expression microarray analysis reveals genes associated with in vitro resistance to cisplatin in a cell line model

We aimed to investigate the mechanisms of cisplatin resistance using an in vitro cancer model. A derivative breast cancer cell line (MCF-7CR) was established which demonstrated significant resistance to cisplatin at clinically relevant low concentrations compared to the MCF-7 parental cell line. Expression microarray analysis was used to identify targets from a 3k cancer-related oligonucleotide platform which were differentially expressed between the derivative and parental cell lines. Real-time quantitative PCR was used to confirm the difference in expression of a subset of genes which demonstrated significant up- or down-regulation. Using expression microarray analysis a total of 28 genes were identified to be differentially expressed (by at least 2-fold) between the MCF-7 and MCF-7CR cells. Real-time quantitative PCR expression analysis confirmed the differential expression of a selection of these genes (ACTG2, ARHD, CTSL, GSTM3, GSTM4 and EHF) between the two cell lines. An in vitro model of cisplatin resistance has been established and expression microarray analysis revealed 28 genes which may be associated with cisplatin resistance.