Szöke B, Horváth J, Halmos G, Rékási Z, Groot K, Nagy A, Schally A V
Endocrine, Polypeptide, and Cancer Institute, Veterans Administration Medical Center, New Orleans, LA 70146.
Peptides. 1994;15(2):359-66. doi: 10.1016/0196-9781(94)90024-8.
The binding and internalization of a cytotoxic analogue of luteinizing hormone-releasing hormone (LH-RH), T-98 (agonist [D-Lys6]LH-RH linked to glutaryl-2-(hydroxymethyl)anthraquinone), by rat anterior pituitary cells was investigated. Analogue T-98 was bound to pituitary membrane binding sites for LH-RH with a high affinity (Kd = 1.2 nM) and was 17 times more potent in releasing luteinizing hormone (LH) from superfused rat pituitary cells than LH-RH. The labeling of this cytotoxic LH-RH analogue was carried out both with radioactive (125I) and nonradioactive iodine. Monoiodination of the Tyr5 residue of T-98 did not significantly affect its binding affinity but greatly decreased its LH-releasing activity to about 3% of the original value. Di-iodination in the same position lowered binding affinity twenty-threefold and further diminished LH-releasing potency. [125I]T-98 was found to bind very strongly to polystyrene, which precluded the use of regular tissue culture plasticware in our experiments. In pituitary cells cultured in glass vials, binding and internalization of [125I]T-98 were observed, which were time and temperature dependent, and which could be inhibited by excess unlabeled analogue. No enzymatic degradation of labeled T-98 was detected in the culture medium during the incubation. Our results indicate that T-98 is internalized by pituitary gonadotropes through receptor-mediated endocytosis. Because this new class of compounds was designed as anticancer drugs, our findings also suggest that this cytotoxic LH-RH agonist may also be internalized by LH-RH receptors present in breast, prostate, ovarian, and other tumors.
研究了促黄体生成激素释放激素（LH-RH）的细胞毒性类似物T-98（激动剂[D-Lys6]LH-RH与戊二酰-2-（羟甲基）蒽醌相连）被大鼠垂体前叶细胞的结合和内化情况。类似物T-98以高亲和力（Kd = 1.2 nM）与LH-RH的垂体膜结合位点结合，并且在从灌流的大鼠垂体细胞中释放促黄体生成激素（LH）方面比LH-RH强17倍。这种细胞毒性LH-RH类似物的标记采用放射性（125I）和非放射性碘进行。T-98的Tyr5残基单碘化对其结合亲和力没有显著影响，但大大降低了其LH释放活性至约原始值的3%。同一位置的双碘化使结合亲和力降低了23倍，并进一步降低了LH释放效力。发现[125I]T-98与聚苯乙烯结合非常紧密，这使得我们的实验中无法使用常规的组织培养塑料制品。在玻璃小瓶中培养的垂体细胞中，观察到了[125I]T-98的结合和内化，它们具有时间和温度依赖性，并且可以被过量的未标记类似物抑制。在孵育过程中，未在培养基中检测到标记的T-98的酶促降解。我们的结果表明，T-98通过受体介导的内吞作用被垂体促性腺激素细胞内化。由于这类新化合物被设计为抗癌药物，我们的发现还表明，这种细胞毒性LH-RH激动剂也可能被存在于乳腺、前列腺、卵巢和其他肿瘤中的LH-RH受体内化。
