Development of Diagnosis and Treatment of Malignant Diseases Based on the Expression of DNaseγ

RT-PCR显示GnRH及其受体mRNA在人卵巢上皮癌细胞株（HTOA、OV-1063和OVCAR-3）和人子宫内膜癌细胞株（HEC-1和HHUA）中均有表达。RT-PCR检测细胞DNA酶γ和caspase 3激活的DNA酶（CAD）mRNA的表达。通过TUNEL染色测定，GnRH激动剂布舍瑞林和GnRH拮抗剂西曲瑞克增加了培养的HTOA细胞中的细胞凋亡发生率。用两种类似物的治疗以相同的程度抑制HTOA细胞系在裸鼠中的异种移植物的生长。在培养的HTOA细胞中，两种GnRH类似物均抑制EGF诱导的EGF受体酪氨酸磷酸化，并将细胞周期进程阻滞在G 0/G1期。这些发现使我们认为，这两种GnRH类似物的抑制作用可能是基于DNA酶激活诱导的细胞凋亡，抑制EGF诱导的信号通路或细胞周期停滞在G1期。另一方面，在上述5种细胞系中，使用我们先前制备的抗DNase γ多克隆抗体和单克隆抗体进行的Western印迹检测不能在这些细胞系中检测到DNaseγ蛋白的表达。并制备了特异性强的抗DNase γ单克隆抗体。该抗体已被证明可用于Western印迹和ELISA。

RT-PCR revealed the expression of mRNA for GnRH and its receptor in human epithelial ovarian cancer cell lines (HTOA, OV-1063 and OVCAR-3) and human endometrial cancer cell lines (HEC-1 and HHUA). Furthermore, the expression of mRNA of DNaseγ and CAD (caspase 3-activated DNase) was also detected by RT-PCR in these cell lines. The GnRH agonist, buserelin, and the GnRH antagonist, cetrorelix, increased the incidence of apoptosis in cultured HTOA cells as determined by TUNEL staining. The treatment with both analogs suppressed the growth of xenografts of HTOA cell line in nude mice to the same extent. In cultured HTOA cells, the both GnRH analogs suppressed EGF-induced tyrosine phosphorylation of EGF receptor and blocked cell cycle progression in G0/G1 phase. These findings lead us to suggest that the antineoplastic actions of both GnRH analogs might be based on apoptosis induced by the activation of DNase, suppression of EGF-induced signaling pathway or cell cycle arrest at G1. On the other hand, in the five cell lines described above, the expression of DNaseγ protein could not be detected in these cell lines by Western blotting using anti-DNaseγ polyclonal and monoclonal antibodies generated by us previously. Then a remarkably specific anti-DNaseγ monoclonal antibody was newly generated. This antibody has been shown to be useful for Western blotting and ELISA.