Establishment of novel gene therapy targeting gynecologic tumors

我们建立了一种新的方法来抑制肿瘤细胞中的端粒酶活性，方法是使用2-5-腺苷酸连接的反义DNA来对抗人端粒酶RNA组分(HTR)。这种反义DNA有效地阻断了宫颈癌ME180细胞的端粒酶活性。2-5A反义HTR对ME180细胞体外生长有明显抑制作用。令人惊讶的是，在治疗后24-48小时内观察到细胞生长抑制，比预期的要早得多。在这一较短的时间内，端粒长度并没有缩短。这些发现表明，端粒酶的阻断通过端粒不依赖的机制导致细胞生长抑制。我们证实了2-5A反义HTR对细胞生长的抑制是由于诱导了细胞凋亡。对2-5A反义hTR诱导细胞凋亡机制的进一步分析正在进行中，并建立了新的肿瘤基因治疗载体系统。我们先前克隆了人端粒酶逆转录酶(HTERT)的启动子，该启动子具有高度的癌细胞特异性。因此，我们利用该启动子在基因递送系统中表达肿瘤特异性基因。将caspase-8、FADD等多种凋亡诱导基因与hTERT启动子结合，作为肿瘤基因治疗的载体。这些载体的导入有效地诱导了癌细胞的凋亡，但也诱导了周围正常组织的凋亡。

We established novel methods that inhibit telomerase activity in cancer cells using 2-5Adenylate-linked antisense DNA against human telomerase RNA component (hTR). This antisense DNA effectively blocked telomerase activity in cervical cancer ME180 cells. Growth of ME180 cells in vitro was significantly inhibited by the treatment with 2-5A anti-hTR. Surprisingly, inhibition of cell growth was observed in 24-48 hr after treatment, quite earlier than expected. Telomere length was not shortened in this short period. These findings suggest that blockade of telomerase led to cell growth inhibition via telomere-independent mechanisms. We confirmed that growth inhibition of cells by the treatment with 2-5A anti-hTR was due to induction of apoptosis. The further anaylsis of mechanisms how 2-5A anti-hTR induces apoptosis is on going.We also established novel vector system for cancer gene therapy. We previously cloned promoter of human telomerase reverse transcriptase (hTERT), which is highly specific to cancer cells. We thus used this promoter for cancer-specific gene expression in gene delivery system. Various apoptosis-inducible genes, such as caspase-8 and FADD, were combined with hTERT promoter and used as vectors for cancer gene therapy. Introduction of these vectors effectively induced apoptosis of cancer cells but of surrounding normal tissues.