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

1.肺腺癌细胞GST 1启动子CpG岛甲基化的诱导A549肺腺癌细胞GST 1启动子CpG岛未甲基化，免疫印迹法检测到GST 1。当我们将甲基化的寡核苷酸转导到这些细胞中时，GSTP 1启动子的甲基化和GSTP 1表达的降低被诱导。2.用DNA甲基转移酶（DNMT）诱导CpG岛甲基化观察到DNMT 1、3a和3b在A549细胞中均表达。我们克隆了DNMT 3b的cDNA，并从A549细胞中建立了DNMT 3b的稳定转染子。3. GST 1表达与肺源性细胞（包括肺癌细胞）存活率的关系我们通过调节GST 1在人肺源性成纤维细胞HFL-1和A549肺癌细胞中的表达水平，研究GST 1在肺源性成纤维细胞HFL-1和A549肺癌细胞自发凋亡中的作用。GSTP 1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发生机制，但没有发现这一现象与JNK通路或细胞内谷胱甘肽浓度的关系。4. GST P1表达与化疗敏感性的关系作为初步研究，我们进行了GST P1正义和反义载体转染HeLa细胞，以检测GST P1对喜树碱诱导的细胞凋亡的影响。转染GST P1正义表达载体可减弱喜树碱诱导的HeLa细胞凋亡，并可增加喜树碱诱导的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.