GnRH-II receptor-like antigenicity in human placenta and in cancers of the human reproductive organs
GnRH-II receptor-like antigenicity in human placenta and in cancers of the human reproductive organs
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DOI：
10.1530/eje.1.02005
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发表时间：
2005-10-01
影响因子：
5.8
通讯作者：
Gründker, C
中科院分区：
文献类型：
--
作者：
Eicke, N;Günthert, AR;Gründker, C
我们最近已经证明GnRH-II对人子宫内膜和卵巢癌细胞株的抗增殖活性不是通过GnRH-I受体介导的。人促性腺激素释放激素-II的功能性受体尚未确定。在这项研究中，我们利用与锁孔帽状血蓝蛋白通过半胱氨酸残基偶联的第三胞外区的多肽(YSPTMLTEVPPC)制备了一种抗人GnRH-II受体的多克隆抗血清。数据库搜索显示，在任何其他人类基因中都没有相同的肽序列。为了避免与两个相似氨基酸序列的交叉反应，用这些多肽预吸收抗血清。用兔抗人GnRH-II受体抗血清对人胎盘、子宫内膜癌、卵巢癌和前列腺癌的免疫组织学切片显示GnRH-II受体样染色。人子宫内膜癌细胞系和卵巢癌细胞系细胞膜的Western印迹分析显示约43 kDa的条带，而绒猴卵巢细胞膜的Western印迹分析显示约54 kDa的条带。为了鉴定GnRH-II受体样抗原，我们使用了光亲和标记技术。I-125标记的(4-azidobenzoyl)-N-hydroxysuccinimide-[D-LyS(6)]-GnRH-II(10(-9)M)与人子宫内膜和卵巢癌细胞的细胞膜发生光化学反应，在约43 kDa处产生一条带。在竞争实验中，促性腺激素释放激素-I激动剂曲普瑞林(10(-7)M)对I-125标记的(4-azidobenzoyl)-N-hydroxysuccinimide-[D-LyS6]-GnRH-II与其结合部位的结合有微弱的减少。GnRH-I拮抗剂Cetrorelx(10(-7)M)的降幅明显大于GnRH-II激动剂[D-Lys(6)]-GnRH-II(10(-7)M)。用兔抗人GnRH-II受体抗血清对同一凝胶进行Western印迹分析，确定该条带为GnRH-II受体样抗原。
We have recently demonstrated that the antiproliferative activity of GnRH-II on human endometrial and ovarian cancer cell lines is not mediated through the GnRH-I receptor. A functional receptor for human GnRH-II has not yet been identified. In this study, we have generated a polyclonal antiserum to the putative human GnRH-II receptor using a peptide (YSPTMLTEVPPC) corresponding to the third extracellular domain coupled to keyhole limpet haemocyanin via the Cys residue. A database search showed no identical peptide sequences in any other human gene. To avoid cross-reactions against two similar amino acid sequences the antiserum was pre-absorbed using these peptides. Immune histological sections of human placenta and human endometrial, ovarian and prostate cancers using rabbit anti-human GnRH-II receptor antiserum showed GnRH-II receptor-like staining. Western blot analysis of cell membrane preparations of human endometrial and ovarian cancer cell lines yielded a band at approximately 43 kDa whereas Western blot analysis of cell membrane preparations of ovaries obtained from the marmoset monkey (Callithrix jacchus) yielded a band at approximately 54 kDa. To identify the GnRH-II receptor-like antigen we used the photo-affinity labelling technique. Photochemical reaction of I-125-labelled (4-azidobenzoyl)-N-hydroxysuccinimide-[D-LyS(6)]-GnRH-II (10(-9) M) with cell membrane preparations of human endometrial and ovarian cancer cells yielded a band at approximately 43kDa. In competition experiments, the GnRH-I agonist Triptorelin (10(-7) M) showed a weak decrease of I-125-labelled (4-azidobenzoyl)-N-hydroxysuccinimide-[D-LyS6]-GnRH-II binding to its binding site. The GnRH-I antagonist Cetrorelix (10(-7) M) showed a clearly stronger decrease, whereas GnRH-II agonist [D-Lys(6)]-GnRH-II (10(-7) M) was the most potent competitor. Western blot analysis of the same gel using rabbit anti-human GnRH-II receptor antiserum identified this band as GnRH-II receptor-like antigen.