Gao Lie, Yang Quan-Hui, Xu Rong-Kun
Department of Physiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, China.
Sheng Li Xue Bao. 2005 Jun 25;57(3):319-27.
In order to investigate the molecular mechanisms of the inhibition of the proliferation of 17-beta-estradiol (E(2))-induced pituitary prolactin-secreting tumor (prolactinoma) by melatonin (MLT) in the rat, we examined the inhibitory effects of MLT on the proliferation of E(2)-induced prolactinoma of the rat and the suppressing effects of MLT on the enhancer elements mutation of PRL gene in vivo and in vitro. The results showed that the weights of prolactinomas in MLT groups, in which 0.25 mg or 0.50 mg per day per rat of MLT was administered subcutaneously at 18:00, were decreased significantly. Out of the dosage of MLT, such as 0.05, 1.00 mg and 2.00 mg per day per rat, the antitumor action of MLT is less or disappointing. Polymerase chain reaction (PCR) and DNA sequencing showed five mutations in the enhancer elements of PRL gene in prolactinoma, such as -1885 point mutation (C --> G), -1857 - -1855 substitution (ACA --> G), -1792 - -1791 insertion G, -1383 - -1382 insertion (GGTGTGTG), -1265 - -1250 deletion (GTGTGTGTGTGTGTGT). Excluding of -1885 point mutation (C --> G), the mutation in the prolactinoma treated with 0.25 mg per day per rat MLT was decreased, such as -1792 - -1791 without insertion of G, -1856 - -1855 deletion AC, -1385 - -1384 deletion TG, -1250 - -1253 deletion GTGT. Firefly luciferase reporter gene systems showed that the luminosity of enhancer elements-luciferase reporter fusion gene in normal pituitary, prolactinoma treated without or with 0.25 mg per day per rat MLT were (13448.17+/-3012.74), (161831.67+/-60996.01), and (10212.17+/-634.71) OD units. Compared with the normal pituitary, the activity of PRL gene enhancer elements in prolactinoma was increased by 11 times (P<0.001). Compared with the prolactinoma, the activity of PRL gene enhancer elements in prolactinoma treated with MLT was decreased by 93.69% (P<0.001). Analysis of the space structure of PRL gene enhancer elements showed that the bending index in prolactinoma was higher than that in prolactinoma treated with MLT, which was higher than that in the normal pituitary. These results demonstrate that one of the important molecular mechanisms of MLT inhibiting the proliferation of prolactinoma is related to the reduction of enhancer elements mutation of PRL gene. These data also suggest that MLT-induced attenuation of enhancer elements mutation of PRL gene is involved in decreasing the bending index and attenuating the higher expression of PRL gene.
为了研究褪黑素（MLT）抑制大鼠中17-β-雌二醇（E₂）诱导的垂体催乳素分泌瘤（催乳素瘤）增殖的分子机制，我们检测了MLT对大鼠E₂诱导的催乳素瘤增殖的抑制作用以及MLT在体内和体外对PRL基因增强子元件突变的抑制作用。结果显示，在18:00皮下注射MLT（每只大鼠每天0.25 mg或0.50 mg）的MLT组中，催乳素瘤的重量显著降低。在MLT的剂量中，如每只大鼠每天0.05 mg、1.00 mg和2.00 mg，MLT的抗肿瘤作用较小或令人失望。聚合酶链反应（PCR）和DNA测序显示催乳素瘤中PRL基因增强子元件有五个突变，如-1885位点突变（C→G）、-1857 - -1855替换（ACA→G）、-1792 - -1791插入G、-1383 - -1382插入（GGTGTGTG）、-1265 - -1250缺失（GTGTGTGTGTGTGTGT）。除-1885位点突变（C→G）外，每只大鼠每天用0.25 mg MLT治疗的催乳素瘤中的突变减少，如-1792 - -1791无G插入、-1856 - -1855缺失AC、-1385 - -1384缺失TG、-1250 - -1253缺失GTGT。萤火虫荧光素酶报告基因系统显示，正常垂体、未用MLT或每只大鼠每天用0.25 mg MLT治疗的催乳素瘤中增强子元件-荧光素酶报告融合基因的发光度分别为（13448.17±3012.74）、（161831.67±60996.01）和（10212.17±634.71）OD单位。与正常垂体相比，催乳素瘤中PRL基因增强子元件的活性增加了11倍（P<0.001）。与催乳素瘤相比，用MLT治疗的催乳素瘤中PRL基因增强子元件的活性降低了93.69%（P<0.001）。对PRL基因增强子元件空间结构的分析表明，催乳素瘤中的弯曲指数高于用MLT治疗的催乳素瘤，后者又高于正常垂体。这些结果表明，MLT抑制催乳素瘤增殖的重要分子机制之一与PRL基因增强子元件突变的减少有关。这些数据还表明，MLT诱导的PRL基因增强子元件突变的减弱参与了降低弯曲指数和减弱PRL基因的高表达。
