Imai A, Takagi H, Furui T, Horibe S, Fuseya T, Tamaya T
Department of Obstetrics and Gynecology, Gifu University School of Medicine, Japan.
Cancer. 1996 Jan 1;77(1):132-7. doi: 10.1002/(SICI)1097-0142(19960101)77:1<132::AID-CNCR22>3.0.CO;2-5.
Gonadotropin-releasing hormone (Gn-RH) receptor (Gn-RHR) has been demonstrated in epithelial ovarian carcinoma (Imai et al., Cancer 1994; 74:2555-61). To examine whether Gn-RHR mediates direct antiproliferative effects, we attempted to determine stimulatory regulation by Gn-RH of phosphotyrosine phosphatase (PTP) activity in plasma membranes isolated from ovarian carcinoma samples.
Surgically removed ovarian carcinomas were screened for Gn-RHR expression prior to plasma membrane isolation. The phosphotyrosine level was observed by: (1) immunoblotting of membrane extracts with antiphosphotyrosine antibodies, and (2) dephosphorylation from 32P-labeled membrane protein. Membrane PTP activity was determined using the synthetic substrate p-nitrophenyl in a spectrophotometric assay.
A Gn-RH analog alone, or guanosine thiotriphosphate (GTP-gamma-S) alone, caused a remarkable loss of phosphotyrosine from a 35-kD protein of the membranes; incubation with a Gn-RH analog and GTP-gamma-S produced a further dephosphorylation of this endogenous protein. The Gn-RH analog buserelin stimulated the PTP activity of the membranes in a dose-dependent manner (P < 0.01). GTP-gamma-S enhanced the stimulatory action of Gn-RH on PTP; GDP-gamma-S reversed the Gn-RH action. A similar stimulation of PTP was observed (P < 0.01) when carcinoma tissue slices were exposed to Gn-RH analog in vivo prior to assay in vitro.
Activation of PTP by Gn-RH stimulated the loss of phosphotyrosine from endogenous proteins through GTP-binding protein within plasma membrane isolated from Gn-RHR-expressing ovarian carcinoma. The antimitogenic action of the hormone may occur by counteracting tyrosine phosphorylation to promote cell growth.
促性腺激素释放激素（Gn-RH）受体（Gn-RHR）已在卵巢上皮癌中得到证实（今井等人，《癌症》1994年；74:2555 - 61）。为了研究Gn-RHR是否介导直接的抗增殖作用，我们试图确定Gn-RH对从卵巢癌样本中分离的质膜上磷酸酪氨酸磷酸酶（PTP）活性的刺激调节作用。
在分离质膜之前，对手术切除的卵巢癌进行Gn-RHR表达筛查。通过以下方法观察磷酸酪氨酸水平：（1）用抗磷酸酪氨酸抗体对膜提取物进行免疫印迹，以及（2）从32P标记的膜蛋白上进行去磷酸化。使用合成底物对硝基苯在分光光度法测定中测定膜PTP活性。
单独的Gn-RH类似物或单独的鸟苷三磷酸硫代（GTP-γ-S）会导致膜上一种35-kD蛋白的磷酸酪氨酸显著减少；与Gn-RH类似物和GTP-γ-S一起孵育会使这种内源性蛋白进一步去磷酸化。Gn-RH类似物布舍瑞林以剂量依赖方式刺激膜的PTP活性（P < 0.01）。GTP-γ-S增强了Gn-RH对PTP的刺激作用；GDP-γ-S则逆转了Gn-RH的作用。当癌组织切片在体外测定前在体内暴露于Gn-RH类似物时，观察到了类似的PTP刺激（P < 0.01）。
Gn-RH对PTP的激活通过从表达Gn-RHR的卵巢癌中分离的质膜内的GTP结合蛋白刺激内源性蛋白磷酸酪氨酸的丢失。该激素的抗有丝分裂作用可能通过抵消酪氨酸磷酸化来促进细胞生长而发生。
