Cloning of the hamster p16 gene 5' upstream region and its aberrant methylation patterns in pancreatic cancer

The hamster model of pancreatic carcinogenesis is useful for understanding the development of human pancreatic cancer. However, there is only a small amount of hamster genetic information available for analyzing the gene alterations in hamster pancreatic cancers. Here, we determined the nucleotide sequence of the 5' upstream region of the hamster p16 gene using a suppression polymerase chain reaction method combined with gene-specific primers. Based on this sequence, we analyzed the methylation status of the 5' region by bisulfite sequencing in three normal pancreatic tissues and five pancreatic duct adenocarcinomas (PDAs). All five PDAs were highly methylated in the 5' upstream region and showed reduced expressions of the p16 gene, while the three normal samples were demethylated. The method described in this study is a highly effective and rapid technique for determining the 5' upstream region, and is applicable to epigenetic studies of the methylation status of this region.     

MPTP诱导的帕金森病小鼠模型黑质脑组织DNA甲基化研究

为研究DNA甲基化在帕金森病发病机制中的作用,本研究用环境毒素1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)连续腹腔给药诱导小鼠帕金森病(Parkison's disease,PD)模型,应用ELISA检测小鼠黑质脑组织总体甲基化水平,应用实时荧光定量PCR方法检测DNA甲基转移酶表达水平,探讨MPTP诱导的小鼠PD模型黑质部位是否存在DNA甲基化异常.进一步应用甲基化DNA免疫共沉淀结合DNA甲基化芯片方法,构建MPTP诱导的小鼠PD模型黑质脑组织DNA甲基化谱,并寻找DNA甲基化修饰异常的PD相关基因对其进行验证.结果表明,模型组小鼠黑质脑组织DNA总体甲基化水平较对照组显著降低,Dnmt1的表达水平显著增高.利用DNA甲基化芯片在全基因组内筛选出甲基化差异修饰位点共48个,涉及44个基因,这些甲基化差异基因参与信号转导、分子转运、转录调控、发育、细胞分化、凋亡调控、氧化应激、蛋白质降解等生物学过程.在甲基化差异修饰基因中,对Uchl1基因及Arih2基因进行了甲基化水平以及表达水平的验证.结果表明,模型组小鼠黑质脑组织Uchl1启动子区域甲基化水平较对照组增高,m RNA及蛋白质表达水平降低,Arih2启动子区域甲基化水平较对照组降低,m RNA及蛋白质表达水平增高.实验结果进一步证实,DNA甲基化修饰异常在帕金森病发病机制中有重要作用,环境因素(如MPTP)可以通过改变DNA甲基化修饰参与帕金森病的发生发展.     

Lack of involvement of metallothionein expression in pancreatic carcinogenesis by N-nitrosobis (2-oxopropyl) amine in Syrian hamsters.

The possible involvement of metallothionein (MT) in pancreatic ductal carcinogenesis by N-nitrosobis(2-oxopropyl) amine (BOP) in hamsters was investigated. Hamsters received subcutaneous (s.c.) injections with dissolved BOP to 70 mg/kg body weight (BW) followed 7 days later by 20 mg/kg BW BOP and they were sacrificed at 4, 11, 16 and 27 weeks after the beginning of the experiment. MT expression was studied by immunohistochemistry and MT contents were assayed biochemically. Pancreatic ductal hyperplasias were developed from 11 weeks on and carcinomas from 16 weeks on, the incidence of the latter reaching 73% at the end of experiment. However, while normal appearing proliferating duct cells were sometimes positive, MT expression was not evident in hyperplasia (H), atypical hyperplasia (AH) or carcinoma (C), and MT contents did not significantly differ in pancreas of hamsters receiving saline or BOP at any time point. The results suggest that MT is not involved in pancreatic duct carcinogenesis. However, the presence of MT in proliferating ducts not related to carcinogenesis may suggest some unknown role for MT in cellular homeostasis.     

Differences between pancreatropic nitrosamine carcinogens and N-nitrosodimethylamine in methylating DNA in various tissues of hamsters and rats

N-Nitrosobis(2-oxopropyl)amine (BOP) and N-nitroso(2-hydroxypropyl)(2-oxypropyl)amine (HPOP) induce pancreatic tumors in the Syrian hamster. BOP and HPOP target the kidneys, esophagus and upper respiratory system in rats, but the pancreas of this species is resistant to the above carcinogens. On the other hand, N-nitrosodimethylamine (DMN) induces hepatic and kidney tumors in the rat, and tumors of the liver and upper respiratory system in the hamster, but it is not known to affect the pancreas of either species. At equimolar doses, ratios of DMN versus BOP or HPOP mediated methylation in hamster liver DNA are 1.6 and 8.1, respectively. Respective ratios in the rat liver are 1.1 and 6.5. However, in both species equitoxic doses of BOP, HPOP and DMN induce similar levels of N7-methylguanine (N7-MeG) in hepatic DNA. At such doses methylation of kidney DNA is 24 and 14 times more extensive in BOP and HPOP than in DMN-treated hamsters. Similarly, ratios of N7-MeG in the pancreas of BOP and HPOP vs. DMN-treated hamsters are 10 and 5, respectively, while in the lung this ratio is 2.2 for both carcinogens. Levels of O6-methylguanine (O6-MeG) in the DNA of extrahepatic tissues are substantially greater in hamsters treated with BOP or HPOP than in those treated with an equitoxic dose of DMN. In rats, equitoxic doses of BOP and DMN induce similar levels of N7-MeG and O6-MeG in hepatic, kidney and lung DNA. However, levels of these adducts in pancreatic DNA are 2 times greater following BOP than DMN administration. Ratios of N7-MeG in pancreas, lung and kidney in HPOP vs. DMN-treated rats are 2.1, 2.7 and 2.1, respectively. Repair of O6-MeG is more effective in rat than in hamster liver, however in other tissues this is not always the case. Levels of O6-MeG in the pancreas of rats are reduced to half of their initial value between 40 and 50 h following the administration of 10, 50 or 20 mg/kg DMN, HPOP or BOP, respectively. However, half-lives for the repair of O6-MeG in hamster pancreas are 28, 62 and greater than 120 h at the respective doses of the above carcinogens. Since the above doses of DMN, HPOP and BOP induce 7, 19 and 41 nmol O6-MeG/mmol of guanine respectively in the hamster pancreas, it is suggested that the rate of repair could be a function of the initial concentration of this adduct. Differences between DMN and BOP or HPOP in methylating pancreatic DNA are sufficient to distinguish the latter two nitrosamines as pancreatic carcinogens for the hamster.     

Albumin and alpha-fetoprotein gene transcription in rat hepatoma cell lines is correlated with specific DNA hypomethylation and altered chromatin structure in the 5'' region.

We examined DNA methylation and DNase I hypersensitivity of the alpha-fetoprotein (AFP) and albumin gene region in hepatoma cell lines which showed drastic differences in the level of expression of these genes. We assayed for methylation of the CCGG sequences by using the restriction enzyme isoschizomers HpaII and MspI. We found two methylation sites located in the 5' region of the AFP gene and one in exon 1 of the albumin gene for which hypomethylation is correlated with gene expression. Another such site, located about 4,000 base pairs upstream from the AFP gene, seems to be correlated with the tissue specificity of the cells. DNase I-hypersensitive sites were mapped by using the indirect end-labeling technique with cloned genomic DNA probes. Three tissue-specific DNase I-hypersensitive sites were mapped in the 5' flanking region of the AFP gene when this gene was transcribed. Similarly, three tissue-specific DNase I-hypersensitive sites were detected upstream from the albumin gene in producing cell lines. In both cases, the most distal sites were maintained after cessation of gene activity and appear to be correlated with the potential expression of the gene. Interestingly, specific methylation sites are localized in the same DNA region as DNase I hypersensitive sites. This suggests that specific alterations of chromatin structure and changes in methylation pattern occur in specific critical regulatory regions upstream from the albumin and AFP genes in rat hepatoma cell lines.     

Involvement of aberrant DNA methylation on reduced expression of lysophosphatidic acid receptor-1 gene in rat tumor cell lines

Lysophosphatidic acid (LPA) is a bioactive phospholipid that stimulates cell proliferation, migration, and protects cells from apoptosis. It interacts with specific G protein-coupled transmembrane receptors. Recently, it has been reported that alterations of LPA receptor expression might be important in the malignant transformation of tumor cells. Therefore, to assess an involvement of DNA methylation in reduced expression of the LPA receptor-1 (lpa1) gene, we investigated the expression of the lpa1 gene and its DNA methylation patterns in rat tumor cell lines. Both rat brain-derived neuroblastoma B103 and liver-derived hepatoma RH7777 cells used in this study indicated no expression of lpa1. For the analysis of methylation status, bisulfite sequencing was performed with B103 and RH7777 cells, comparing with other lpa1 expressed cells and normal tissues of brain and liver. The lpa1 expressed cells and tissues were all unmethylated in this region of lpa1. In contrast, both B103 and RH7777 cells were highly methylated, correlating with reduced expression of the lpa1. Treatment with 5-aza 2'-deoxycytidine induced expression of lpa1 gene in B103 and RH7777 cells after 24h. In RH7777 cells treated with 5-aza 2'-deoxycytidine, stress fiber formation was also observed in response to LPA in RH7777 cells, but not in untreated RH7777 cells. These results suggest that aberrant DNA methylation of the lpa1 gene may be involved in its reduced expression in rat tumor cells.     

Methylation and downregulated expression of mac25/insulin-like growth factor binding protein-7 is associated with liver tumorigenesis in SV40T/t antigen transgenic mice, screened by restriction landmark genomic scanning for methylation (RLGS-M)

Restriction landmark genomic scanning for methylation (RLGS-M) was used to detect alterations in DNA methylation associated with murine SV40 T/t antigen-induced hepatocarcinogenesis. An altered locus/spot (S130) was cloned and found to correspond to sequences in the 5' flanking region and 5' portion of the cDNA for the murine mac25/insulin-like growth factor binding protein-7 (Igfbp-7) gene. IGFBPs are believed to be capable of binding insulin, Igf1, and Igf2 and modulating mitogenic effects. Previous studies have shown that Igf2 has an important role in promoting liver tumorigenesis. Quantitative PCR was used to access the methylation status of the NotI site just 5' to the coding region and the expression level of the mac25/igfbp-7 gene. The results indicated that the degree of methylation was inversely related to the expression level and is consistent with a role for DNA methylation in silencing mac25/Igfbp-7 gene expression and function for mac25/Igfbp-7 as a tumor suppressor gene.     

Differential methylation of the ornithine carbamoyl transferase gene on active and inactive mouse X chromosomes.

Ornithine carbamoyl transferase (Oct) is an X-linked gene which exhibits tissue-specific expression. To determine whether methylation of specific CpG sequences plays a role in dosage compensation or tissue-specific expression of the gene, 13 potentially methylatable sites were identified over a 30-kilobase (kb) region spanning from approximately 15 kb upstream to beyond exon II. Fragments of the Mus hortulanus Oct gene were used as probes to establish the degree of methylation at each site. By considering the methylation status in liver (expressing tissue) versus kidney (nonexpressing tissue) from male and female mice, the active and inactive genes could be investigated on active and inactive X-chromosome backgrounds. One MspI site, 12 kb 5' of the Oct-coding region, was cleaved by HpaII in liver DNA from males but not in kidney DNA from males and thus exhibited complete correlation with tissue-specific expression of the gene. Six other sites showed partial methylation, reflecting incomplete correlation with tissue-specific expression.