BACKGROUND: TMS1/ASC is a bipartite protein comprising two protein-protein interactive domains: pyrin (PYD) and caspase recruitment domain (CARD). Proteins containing these domains play pivotal roles in regulating apo...BACKGROUND: TMS1/ASC is a bipartite protein comprising two protein-protein interactive domains: pyrin (PYD) and caspase recruitment domain (CARD). Proteins containing these domains play pivotal roles in regulating apoptosis and immune response pathways. The absence of TMS1/ ASC expression in some tumors is because methylation of the TMS1/ASC gene contributes to carcinogenesis and cancer development. We studied the methylation status of the TMS1/ASC gene and its clinical significance in cholangiocarcinoma. METHODS: Target DNA was modified by sodium bisulfite, coverting all unmethylated, but not methylated, cytosines to uracil, and subsequently by a nested amplification with primers specific for methylated versus unmethylated DNA. The PCR product was detected by gel electrophoresis and combined with the clinical records of patients. RESULTS: Aberrant methylation of the TMS1/ASC gene was detected in specimens of colorectal cancer tissues from 13 (36.1%) of 36 patients, and specimens of adjacent normal tissues from 3 patients (8.3%). No statistical differences were seen in the extent of differentiation and invasion, lymph node metastasis, and pathologic type between the methylated and unmethylated tissues (P】0.05). CONCLUSIONS: The frequency of TMS1/ASC gene methylation in cholangiocarcinoma is high, but it is not related to pathologic changes. The TMS1/ASC gene is probably suppressed by methylation, and is resistant to apoptosis and immunological surveillance. The gene epigenetically affected in methylated tissues could be associated with carcinogenesis of cholangiocarcinoma.展开更多
文摘BACKGROUND: TMS1/ASC is a bipartite protein comprising two protein-protein interactive domains: pyrin (PYD) and caspase recruitment domain (CARD). Proteins containing these domains play pivotal roles in regulating apoptosis and immune response pathways. The absence of TMS1/ ASC expression in some tumors is because methylation of the TMS1/ASC gene contributes to carcinogenesis and cancer development. We studied the methylation status of the TMS1/ASC gene and its clinical significance in cholangiocarcinoma. METHODS: Target DNA was modified by sodium bisulfite, coverting all unmethylated, but not methylated, cytosines to uracil, and subsequently by a nested amplification with primers specific for methylated versus unmethylated DNA. The PCR product was detected by gel electrophoresis and combined with the clinical records of patients. RESULTS: Aberrant methylation of the TMS1/ASC gene was detected in specimens of colorectal cancer tissues from 13 (36.1%) of 36 patients, and specimens of adjacent normal tissues from 3 patients (8.3%). No statistical differences were seen in the extent of differentiation and invasion, lymph node metastasis, and pathologic type between the methylated and unmethylated tissues (P】0.05). CONCLUSIONS: The frequency of TMS1/ASC gene methylation in cholangiocarcinoma is high, but it is not related to pathologic changes. The TMS1/ASC gene is probably suppressed by methylation, and is resistant to apoptosis and immunological surveillance. The gene epigenetically affected in methylated tissues could be associated with carcinogenesis of cholangiocarcinoma.
文摘目的检测吉西他滨（gemeitabine，GEM）诱导胰腺癌细胞株PANC-1中甲基化诱导静止基因（TMSl／ASC）的表达，并探讨半胱氨酸天冬氨酸酶-1（cysteine aspartase，caspase-1）、核转录因子（nuclear factor—κB，NF—κB）的活性与TMSl／ASC之间的关系。方法用噻唑蓝（methyl thiazolyl tetrazolium，MTr）比色法检测GEM在不同浓度（1、2、4、8、16μg／m1）及不同时间（24、48h）下对胰腺癌细胞PANC-1存活率的影响；RT—PCR技术检测PANC—1细胞在GEM（4．27μg／m1）刺激组（实验组）和空白组（对照组）培养24h和48h后TMS1／ASCmRNA的表达情况。用抗KB抑制蛋白（inhibitory protein of NF—κB，IKBR）多克隆抗体、抗caspase-1多克隆抗体和内参β—actin单克隆抗体通过Western blot技术检测并比较IKBa和caspase-1在实验组和对照组中的蛋白表达水平，从而分析NF．KB和caspase-1的活化状态。结果GEM对PANC-1细胞生长的抑制有浓度和时间依赖性，其半数抑制率（IC50）为24h4．27μg／ml；24h时实验组和对照组TMSl／ASC的表达量分别为（0．3±0．004）和（0．1±0．001），48h实验组和对照组分别为（0．63±0．007）和（0．21±0．006），2组相比较，差异有统计学意义（P〈0．01）。Westernblot结果显示：随着培养时间延长，caspase-1在对照组中未发生活化，GEM可促进其活化；GEM实验组与对照组相比It（Bet的表达量无明显变化，GEM不能促使NF—KB活化。结论GEM能抑制胰腺癌PANC-1细胞增殖并促进凋亡，随着作用时间的延长其药物敏感性减低。GEM作用-定时间可诱导TMS1／ASC表达增加，caspase-1可能参与GEM诱导胰腺癌细胞株PANC-1凋亡的信号转导途径，NF-κB不参与GEM诱导胰腺癌细胞株PANC—1凋亡的信号转导途径。
