胆管癌p53-bax线粒体凋亡通路的甲基化研究

目的探讨胆管癌p53-bax线粒体凋亡通路中多个基因的甲基化状态及其在胆管癌发生过程中的意义。方法采用甲基化特异性聚合酶链反应（MSP），对胆管癌组织和癌旁组织中的p14^ARF、DAPK和TMS1／ASC基因启动子的甲基化状态进行检测，并对胆管癌组织中p53基因外显子5～8进行DNA序列分析。结果36例胆管癌组织标本中，有24例（66．7％）至少存在1个抑癌基因的甲基化，其中p14^ARF、DAPK和TMS1／ASC基因甲基化的比率分别为25．0％、30．6％和36．1％；癌旁组织中，有5例（13．9％）存在抑癌基因的甲基化，其中TMS1／ASC3例（8．3％），DAPK2例（5．6％）。36例胆管癌组织标本巾，有22例（61．1％）存在p53基因的突变。p53突变伴1个以上抑癌基因甲基化者共14例，占38．9％，其发生率与胆管癌的病理类型、分化程度和浸润深度有关（P〈0．05）。结论p53-bax线粒体凋亡通路中，DNA甲基化是胆管癌中常见的分子事件。癌旁组织中，DAPK和TMS1／ASC基因的甲基化率虽然较低，但可能有早期诊断意义。p53突变伴抑癌基因的甲基化与胆管癌的病理生物学行为有关，并趋向于较高的恶性程度。     

Epigenetic inactivation of paired box gene 5, a novel tumor suppressor gene, through direct upregulation of p53 is associated with prognosis in gastric cancer patients

Using genome-wide methylation screening, we identified that paired box gene 5 (PAX5) is involved in human cancer development. However, the function of PAX5 in gastric cancer (GC) development is largely unclear. We analyzed its epigenetic inactivation, biological functions and clinical application in GC. PAX5 was silenced in seven out of eight GC cell lines. A significant downregulation was also detected in paired gastric tumors compared with adjacent non-cancerous tissues. The downregulation of PAX5 was closely linked to the promoter hypermethylation status and could be restored with demethylation treatment. Ectopic expression of PAX5 in silenced GC cell lines (AGS and BGC823) inhibited colony formation and cell viability, arrested cell cycle, induced apoptosis, suppressed cell migration and invasion and repressed tumorigenicity in nude mice. Consistent with the induction of apoptosis by PAX5 in vitro, terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) staining showed significantly enhanced apoptotic cells in PAX5-expressed tumors compared with the vector control tumors. On the other hand, knockdown of PAX5 by PAX5-short hairpin RNA increased the cell viability and proliferation. The anti-tumorigenic function of PAX5 was revealed to be mediated by upregulating downstream targets of tumor protein 53 (p53), p21, BCL2-associated X protein, metastasis suppressor 1 and tissue inhibitors of metalloproteinase 1, and downregulating BCL2, cyclin D1, mesenchymal-epithelial transition factor (MET) and matrix metalloproteinase 1. Immunoprecipitation assay demonstrated that PAX5 directly bound to the promoters of p53 and MET. Moreover, PAX5 hypermethylation was detected in 77% (144 of 187) of primary GCs compared with 10.5% (2/19) of normal gastric tissues (P<0.0001). GC patients with PAX5 methylation had a significant poor survival compared with the unmethylated cases as demonstrated by Cox regression model and log-rank test. In conclusion, PAX5 is a novel functional tumor suppressor in gastric carcinogenesis. Detection of methylated PAX5 can be utilized as an independent prognostic factor in GC.     

Hypermethylation of the RASSF1A tumor suppressor gene in Japanese clear cell renal cell carcinoma

Hypermethylation associated inactivation of RASSF1A tumor suppressor gene at chromosome 3p21.3 has been observed in several human malignancies. Relatively high (91%) or low (23%) frequencies were reported in the methylation status of promoter region of the RASSF1A gene in clear cell renal carcinoma (RCC) depending on the country the report was from. To clarify exact contribution of the hypermethylation of RASSF1A gene in the development of RCC in Japan, we analyzed the methylation status of the RASSF1A promoter region in 50 Japanese clear cell RCC and RCC cell lines. Although relatively high frequency of hypermethylation in RASSF1A promoter (39 of 50 tumors, 78%) was observed, most of matched proximal normal tissue DNA also showed weak methylation. By comparison with methylation level of adapted normal kidney tissue DNA, tumor preferential hypermethylation in RASSF1A promoter was recognized as 40% (20/50 matched sets) of primary clear cell RCCs. Hypermethylation in RASSF1A promoter was observed in 36% (15/42) and 64% (5/8) of stage I-II or III-IV tumors, and also observed in 42% (11/26) and 38% (9/24) of our tumor samples with pathological grade I or II, respectively. In addition, 16 of 19 RCC cell lines (84%) showed complete or partial methylation of RASSF1A promoter region. There was no association between the frequency of RASSF1A methylation and inactivation of VHL tumor suppressor gene in either primary RCCs or RCC cell lines. Our results showed tumor specific RASSF1A promoter hypermethylation in up to 40% of low grade or low stage clear cell RCCs. It is essential to compare the methylation status of RASSF1A promoter in tumor with normal tissue to understand tumor specific hypermethylation. Since considerable cases of normal kidney are hypermethylated, contribution of the RASSF1A for the development and progression of kidney cancer may be more complex than expected.     

MicroRNA-145 is regulated by DNA methylation and p53 gene mutation in prostate cancer

MiR-145 is downregulated in various cancers including prostate cancer. However, the underlying mechanisms of miR-145 downregulation are not fully understood. Here, we reported that miR-145 was silenced through DNA hypermethylation and p53 mutation status in laser capture microdissected (LCM) prostate cancer and matched adjacent normal tissues. In 22 of 27 (81%) prostate tissues, miR-145 was significantly downregulated in the cancer compared with the normal tissues. Further studies on miR-145 downregulation mechanism showed that miR-145 is methylated at the promoter region in both prostate cancer tissues and 50 different types of cancer cell lines. In seven cancer cell lines with miR-145 hypermethylation, 5-aza-2'-deoxycytidine treatment dramatically induced miR-145 expression. Interestingly, we also found a significant correlation between miR-145 expression and the status of p53 gene in both LCM prostate tissues and 47 cancer cell lines. In 29 cell lines with mutant p53, miR-145 levels were downregulated in 28 lines (97%), whereas in 18 cell lines with wild-type p53 (WT p53), miR-145 levels were downregulated in only 6 lines (33%, P < 0.001). Electrophoretic mobility shift assay showed that p53 binds to the p53 response element upstream of miR-145, but the binding was inhibited by hypermethylation. To further confirm that p53 binding to miR-145 could regulate miR-145 expression, we transfected WT p53 and MUT p53 into PC-3 cells and found that miR-145 is upregulated by WT p53 but not with MUTp53. The apoptotic cells are increased after WT p53 transfection. In summary, this is the first report documenting that downregulation of miR-145 is through DNA methylation and p53 mutation pathways in prostate cancer.     

胆管癌p53-Bax线粒体凋亡通路DNA甲基化机制的研究

Objective: To study the methylation status of several genes on p53-Bax mitochondrial apoptosis pathway and clinical significance in cholangiocarcinoma. Methods: Promoter hypermethylation of DAPK, p14 and ASC genes were de-tected by methylation-specific PCR. p53 gene status (exon 5-8) were examined by automated sequencing, combined with the clinical documents of patients by statistics analysis. Results: (1) We found 66.7% of 36 cases cholangiocarcinoma had meth-ylation of at least one tumor suppressor gene. The frequency of tumor suppressor gene methylation in cholangiocarcinoma was: p14 (24%), DAPK (30.6%), TMSI/ASC (36.1%). The frequency of tumor suppressor gene methylation in tissues near cancer was: DAPK (5.6%), TMSI/ASC (8.3%). (2) p53 gene mutations were found in 22 of 36 patients (61.1%). (3) There were no statistically relationship among the methylation of DAPK, p14 and ASC genes. There were negative relationship differences between the methylation of p14 and p53 gene mutation (P<0.05). (4) p53 gene mutation combined with the methylation of tumor suppressor were 14 cases (38.9%). There were statistically differences on extent of pathologic biology, differentiation and invasion (P<0.05). Conclusion: Our study indicated that methylation of p53-Bax mitochondrial apoptosis pathway in cholangiocarcinoma was a common epigenetic event. Although the methylation of ASC, DAPK genes was low, it might be significance for early diagnosis, p53 gene mutation combined with the methylation of tumor suppressor might be relationship with pathologic biology, it trended to more malignancy.     

Methylation-associated silencing of TU3A in human cancers

TU3A, located on 3p21.1, was originally identified as a candidate tumor suppressor gene in renal cell carcinoma (RCC). Recently, down-regulation of TU3A expression has been reported not only in RCC but also in other types of cancers. However, no studies have evaluated the mechanism underlying TU3A inactivation. In the present study, we first examined the expression and promoter CpG island methylation of TU3A in RCC. TU3A mRNA was slightly or not expressed in 3 RCC cell lines (ACHN, Caki-1 and NC65). Bisulfite sequencing of the TU3A promoter and treatment of the RCC cell lines with 5-aza-2'-deoxycytidine and/or trichostatin A revealed an association between TU3A expression and promoter hypermethylation. Next, we analyzed TU3A methylation in primary RCC by using combined bisulfite restriction analysis. Mean methylated fraction was 19.2% (range: 0-57.3%) in 53 conventional RCCs and 2.3% (range: 0-12.7%) in 24 corresponding normal kidneys. We defined a methylation fraction of >20% as hypermethylation. TU3A hypermethylation was detected in 22 (41.5%) of 53 RCCs and significantly associated with advanced tumor stage (>T2 vs. T1 and T2: P=0.005, > or = N1 or M1 vs. N0M0: P=0.001) and poor disease-specific survival (P=0.0038). Furthermore, we observed promoter hyper-methylation of TU3A in several types of cancer cell lines and primary cancers of the bladder and testis. To our knowledge, the present study is the first to demonstrate the epigenetic inactivation of TU3A in human cancers. The findings of this study warrant further study to investigate the role of TU3A methylation in cancer development.     

Interferon regulatory factor 8 functions as a tumor suppressor in renal cell carcinoma and its promoter methylation is associated with patient poor prognosis

Interferon regulatory factor 8 (IRF8), as a central element of IFN-γ-signaling, plays a critical role in tumor suppression. However, its expression and underlying molecular mechanism remain elusive in renal cell carcinoma (RCC). Here, we examined IRF8 expression and methylation in RCC cell lines and primary tumors, and further assessed its tumor suppressive functions. We found that IRF8 was widely expressed in human normal tissues including kidney, but frequently downregulated by promoter methylation in RCC cell lines. IRF8 methylation was detected in 25% of primary tumors, but not in adjacent non-malignant renal tissues, and associated with higher tumor nuclear grade of RCC. Ectopic expression of IRF8 inhibited colony formation and migration abilities of RCC cells, through inducing cell cycle G2/M arrest and apoptosis. IFN-γ could induce IRF8 expression in RCC cells, together with increased cleaved-PARP. We further found that IRF8 inhibited expression of oncogenes YAP1 and Survivin, as well as upregulated expression of tumor suppressor genes CASP1, p21 and PTEN. Collectively, our data demonstrate that IRF8 as a functional tumor suppressor is frequently methylated in RCC, and IRF8-mediated interferon signaling is involved in RCC pathogenesis.     

Hypermethylation of the proapoptotic gene <Emphasis Type="Italic">TMS1/ASC</Emphasis>: prognostic importance in glioblastoma multiforme

The identification of clinical subsets of glioblastomas (GBM) associated with different molecular genetic profiles had opened the possibility to design tailored therapies to individual patients. One of the most intrigued subtypes is the long-term survival (LTS) GBM, which responds better to current therapies. The present investigation on GBM from 50 consecutive GBM displaying classic survival and seven LTS GBM is based on molecular epigenetic, clinical and histopathological analyses. Our aim was to recognize biomarkers useful to distinguish LTS from classic GBM. We analyzed the promoter methylation status of key regulator genes implicated in tumor invasion (TIMP2, TIMP3), apoptosis and inflammation (TMS1/ASC, DAPK) as well as overall survival, therapy status and tumor pathological features. For the first purpose a methylation-specific PCR approach was performed to analyze the CpG island promoter methylation status of each gene. The overall TMS1/ASC methylation rate in the 57 analyzed tumors was 21.05%. Hypermethylation of TMS1/ASC was significantly more frequent in LTS GBM (57.1% vs. 16%, P=0.029, Fisher's exact test). DAPK promoter hypermethylation was only observed in the LTS subset (14.3%) whereas TIMP2 and TIMP3 were unmethylated in both GBM collectives. Our results strongly suggest that, compared to classic GBM, LTS GBM display distinct epigenetic characteristics which might provide additional prognostic biomarkers for the assessment of this malignancy.     

Methylation of ASC/TMS1, a proapoptotic gene responsible for activating procaspase-1, in human colorectal cancer

ASC/TMS1, a proapoptotic activator of procaspase-1, was reported to be aberrantly methylated in human breast cancer. We found that ASC was methylated in three of five human colon cancer cell lines lacking ASC protein expression. Demethylation treatment of these cell lines lacking ASC with 5-aza-2'-deoxycytidine partially restored ASC expression. Methylated ASC was also detected in six of ten colorectal cancer tissues. Although clear down-regulation of ASC in the whole region of a tumor tissue was hardly observed by immunostaining with anti-ASC mAb, complete suppression of ASC was identified in a minor population of the colorectal tumor cells. The biological significance of ASC methylation inducible ASC suppression in colorectal cancer will be discussed.     

DNA Hypermethylation of Cell Cycle (p15 and p16) and Apoptotic (p14, p53, DAPK and TMS1) Genes in Peripheral Blood of Leukemia Patients

Aberrant DNA methylation of tumor suppressor genes has been reported in all major types of leukemiawith potential involvement in the inactivation of regulatory cell cycle and apoptosis genes. However, most ofthe previous reports did not show the extent of concurrent methylation of multiple genes in the four leukemiatypes. Here, we analyzed six key genes (p14, p15, p16, p53, DAPK and TMS1) for DNA methylation usingmethylation specific PCR to analyze peripheral blood of 78 leukemia patients (24 CML, 25 CLL, 12 AML, and17 ALL) and 24 healthy volunteers. In CML, methylation was detected for p15 (11%), p16 (9%), p53 (23%) andDAPK (23%), in CLL, p14 (25%), p15 (19%), p16 (12%), p53 (17%) and DAPK (36%), in AML, p14 (8%), p15(45%), p53 (9%) and DAPK (17%) and in ALL, p15 (14%), p16 (8%), and p53 (8%). This study highlightedan essential role of DAPK methylation in chronic leukemia in contrast to p15 methylation in the acute cases,whereas TMS1 hypermethylation was absent in all cases. Furthermore, hypermethylation of multiple genesper patient was observed, with obvious selectiveness in the 9p21 chromosomal region genes (p14, p15 and p16).Interestingly, methylation of p15 increased the risk of methylation in p53, and vice versa, by five folds (p=0.03)indicating possible synergistic epigenetic disruption of different phases of the cell cycle or between the cell cycleand apoptosis. The investigation of multiple relationships between methylated genes might shed light on tumorspecific inactivation of the cell cycle and apoptotic pathways.     

ASC/TMS1, a caspase-1 activating adaptor,is downregulated by aberrant methylation in human melanoma

ASC/TMS1 is an adaptor protein activating caspase-1 that stimulates processing of proIL-1beta and proIL-18. ASC was reported to be aberrantly methylated and silenced in human breast cancers. In our present study, ASC expression was examined in 12 melanoma cell lines by Western blot analysis and in 18 benign melanocytic nevi and 32 melanoma tissues by immunohistochemical staining. ASC expression was absent or reduced in 7 of 12 (58.3%) cell lines and in 20 of 32 (62.5%) melanoma tissues, whereas all 18 benign melanocytic nevi showed intensive ASC expression. To investigate whether ASC silencing in melanoma is involved in aberrant methylation, methylation specific PCR was carried out. Five of ten (50%) melanoma tissues exhibited methylation in CpG island of ASC companied with reduced ASC expression. Six of twelve (50%) melanoma cell lines showed aberrant methylation in the ASC gene, and 4 of the 6 (66.7%) methylation positive cell lines exhibited reduced ASC expression. We characterized methylation patterns in melanoma cell lines by using bisulfite genomic sequencing, and found that the degree of aberrant methylation correlated with the level of reductive ASC expression. Treatment with demethylating agent 5-aza-2'-deoxycytidine resulted in both demethylation of the ASC gene and the upregulation of ASC expression in the methylation positive melanoma cell lines. Our study shows that ASC is downregulated in melanoma, and that its suppression is partially mediated by aberrant methylation.     

Identification of novel target genes by an epigenetic reactivation screen of renal cancer

Aberrant promoter hypermethylation is a common mechanism for inactivation of tumor suppressor genes in cancer cells. To generate a global profile of genes silenced by hypermethylation in renal cell cancer (RCC), we did an expression microarray-based analysis of genes reactivated in the 786-0, ACHN, HRC51, and HRC59 RCC lines after treatment with the demethylating drug 5-aza-2 deoxycytidine and histone deacetylation inhibiting drug trichostatin A. Between 111 to 170 genes were found to have at least 3-fold up-regulation of expression after treatment in each cell line. To establish the specificity of the screen for identification of genes, epigenetically silenced in cancer cells, we validated a subset of 12 up-regulated genes. Three genes (IGFBP1, IGFBP3, and COL1A1) showed promoter methylation in tumor DNA but were unmethylated in normal cell DNA. One gene (GDF15) was methylated in normal cells but more densely methylated in tumor cells. One gene (PLAU) showed cancer cell-specific methylation that did not correlate well with expression status. The remaining seven genes had unmethylated promoters, although at least one of these genes (TGM2) may be regulated by RASSF1A, which was methylated in the RCC lines. Thus, we were able to show that up-regulation of at least 6 of the 12 genes examined was due to epigenetic reactivation. The IGFBP1, IGFBP3, and COL1A1 gene promoter regions were found to be frequently methylated in primary renal cell tumors, and further study will provide insight into the biology of the disease and facilitate translational studies in renal cancer.     

Paclitaxel/Taxol sensitivity in human renal cell carcinoma is not determined by the p53 status

In this study, we analyzed the role of the p53 status for paclitaxel/Taxol sensitivity in renal cell carcinomas (RCCs) of the clear cell type. Using immunohistochemistry, nuclear p53 accumulation could not be correlated to the paclitaxel/Taxol sensitivity. DNA sequencing detected a p53 gene mutation in two out of eight RCC cell lines, i.e. in exon 8 (cell line clearCa-6), and in exon 9 (cell line clearCa-5). No correlation, however, was found between the p53 status of our RCC cell lines and their paclitaxel/Taxol sensitivity as indicated by the IC50 values. However, paclitaxel-induced growth inhibition in paclitaxel-sensitive RCC cell lines was accompanied by an increase in apoptosis, irrespective of their p53 status. Although CD95 up-regulation was observed in renal cell carcinoma with wild-type p53 upon paclitaxel treatment, paclitaxel-induced apoptosis itself is triggered independently from the CD95 system. In conclusion, the p53 status cannot predict paclitaxel/Taxol sensitivity in RCC cell lines of the clear cell type.