The investigation on the expression regulation and the role on chemosensitivity of a xenobiotic enzyme GSTP1 in lung cancer.

1.诱导肺腺癌细胞GSTP1启动子CpG岛甲基化。GSTP1启动子CpG岛未甲基化，免疫印迹法在A549肺腺癌细胞中检测到GSTP1。当我们将甲基化的寡核苷酸转导到这些细胞中时，GSTP1启动子的甲基化和GSTP1表达的降低被诱导。DNA甲基转移酶（DNA methyltransferases， DNMT）诱导CpG岛甲基化观察发现，DNMT1、3a、3b均在A549细胞中表达。我们克隆了DNMT3b的cDNA，并在A549细胞中建立了一个稳定的DNMT3b转染体。现在我们用亚硫酸酯测序来检测这些转染物中GSTP1 CpG岛的甲基化状态。我们在人肺源性成纤维细胞HFL-1和A549肺癌细胞中调控GSTP1的表达水平，研究GSTP1在肺源性成纤维细胞自发凋亡中的作用。GSTP1的缺失增加了两种细胞的自发凋亡和坏死。虽然我们研究了这一现象的机制，但我们没有发现这一现象与JNK途径或细胞内谷胱甘肽浓度之间的关系。GSTP1表达与化学敏感性的关系作为前期研究，我们转染GSTP1正义和反义载体，检测GSTP1对喜树碱诱导HeLa细胞凋亡的影响。转染GSTP1表达载体后，喜树碱诱导的HeLa细胞凋亡明显减弱。我们还观察到，用siRNA去除GSTP1会增加喜树碱诱导的A549肺癌细胞死亡。

1.Induction of CpG island methylation of GSTP1 promoter in lung adenocarcinoma cells.The CpG island of GSTP1 promoter was not methylated and GSTP1 was detected by immunoblotting in A549 lung adenocarcinoma cells. When we transduced the methylated oligonucleotides to these cells, methylation of the GSTP1 promoter and reduction of GSTP1 expression was induced.2.Induction of CpG island methylation with DNA methyltransferases (DNMT)It was observed that DNMT1, 3a, and 3b were all expressed in A549 cells. We cloned the cDNA of DNMT3b, and established a stable transfectant of DNMT3b from A549 cells. Now we check the methylation status of GSTP1 CpG island in these transfectants with bisulfite sequenting.3.Association between GSTP1 expression and viability of lung-derived cells (including lung cancer cells)We regulate the expression level of GSTP1 in human lung-derived fibroblast HFL-1 and A549 lung cancer cells, and investigated the role of GSTP1 on spontaneous apoptosis. Depletion of GSTP1 increased spontaneous apoptosis and necrosis in both kinds of cells. Although we investigated the mechanism of this phenomena, we found no relations between this phenomena and JNK pathway or intracellular concentration of glutathione.4.Association between GSTP1 expression and chemosensitivityAs a pilot study, we carried out transfection of GSTP1 sense and antisense vectors to examine effects of GSTP1 on apoptosis induced by camptothecin in HeLa cells. Camptothecin-induced apoptosis was attenuated by transfection of GSTP1 sense vector in HeLa cells.We also observed that GSTP1 depletion with siRNA increased camptothecin-induced cell death in A549 lung cancer cells.