ANALYSIS OF TR3 RECEPTOR IN APOPTOSIS OF PROSTATE CANCER AND APPLICATION FOR DIAGNOSIS OF PROSTATE CANCER RECCURENCE

(1) TR3孤儿受体在药物性前列腺凋亡中的作用在雄激素应答型LNCaP人前列腺癌细胞中，人TR3孤儿受体可通过雄激素或生长因子（即EGF或FGF）的刺激快速诱导。相比之下，阉割雄激素也能诱导TR3孤儿受体基因在大鼠腹侧前列腺中表达，并发生以DNA断裂阶梯为特征的程序性细胞死亡（凋亡）。这一现象促使我们进一步分析人类TR3孤儿受体在凋亡诱导的前列腺癌细胞中的作用。Northern blot分析表明，在LNCaP细胞中，10 mM钙离子载体或300 mM依托泊苷（VP 16）处理能快速诱导人TR3孤儿受体。反义TR3孤儿受体稳定转染LNCaP细胞表明，与对照（载体）稳定转染LNCaP细胞相比，需要更高浓度的etopo苷才能显示类似的凋亡。更多的数据表明，人类TR3孤儿受体可能在药物诱导的人前列腺癌细胞凋亡中发挥重要作用。(2) TR3孤儿受体a激素反应元件在人TR3孤儿受体CNTFR基因α成分的第5个内含子中发现了CNTER-NERB（5′-AAAGGTCA-3′）诱导人睫状神经营养因子受体（CNTFR）基因的内含子增强子。电泳迁移位移法证实体外表达的TR3与CNTFR-NBRE之间存在特异性结合。利用CAT进行的报告基因分析表明，CNTFR具有增强子活性，可被TR3受体以剂量依赖性方式诱导。在没有CNTFR-BNBRE的情况下，这种诱导作用显著降低。综上所述，这些结果表明CNTFR- nbre足以介导TR3的作用，诱导CNTFR增强子活性。因此，我们的发现可能表明CNTFR是TR3调控的靶基因，并扩大了TR3在神经系统中的作用。(3)前列腺癌组织中TR3受体的原位杂交。最近，Jenkins等人用荧光原位杂交技术检测了前列腺癌组织和转移性淋巴结中c-myc扩增的清晰数据。我们在人前列腺癌、良性前列腺肥大和正常前列腺组织中的原位杂交数据显示，TR3孤儿受体mRNA的表达模式与c-myc的表达模式非常相似。更有趣的是，TR3孤儿受体mrna在前列腺癌区域比在邻近的正常或良性肥厚组织中表达得更高。此外，这些表达在低分化腺癌区高度可见。这些数据表明，TR3孤儿受体可能与c-myc癌基因一样，在前列腺癌的发生或进展中发挥重要作用。(4)端粒酶在前列腺癌组织中的活性。我们分析了端粒酶活性与前列腺癌病理分化的关系。端粒酶活性检测采用基于pcr的端粒重复扩增法（TRAP）检测。结果显示，大部分前列腺癌组织（90%）显示端粒酶活性，原发和活检标本中端粒酶活性较高，低分化腺癌中端粒酶活性较高。RT-PCR检测催化亚基hTERT的表达。结果显示，与正常前列腺组织相比，70%的前列腺癌组织中表达量更高。少

(1) A role of TR3 orphan receptor in drug-induced prostate apoptosisIn androgen-responsive LNCaP human prostatic cancer cells, human TR3 orphan receptor can be rapidly induced by stimulation of androgen or growth factors (i.e., EGF or FGF). In contrast, ablation of androgen by castration can also induce the expression of TR3 orphan receptor gene in rat ventral prostate which underwent programmed cell death (apoptosis) characterized by DNA fragmentation ladder. This phenomenon prompted us to further analyze the roles of human TR3 orphan receptor in apoptosis-induced prostate cancer cells. Northern blot analysis shows that human TR3 orphan receptor can be induced rapidly after treatment of 10 mM calcium ionophore or 300 mM etoposide (VP 16) in LNCaP cells. Antisense TR3 orphan receptor stable transfectant LNCaP cells indicated a much higher concentration of etoposide was needed to show the similar apoptosis as compared to the control (vector) stable transfectant LNCaP cells. Together, ou … More r data suggest that human TR3 orphan receptor may play an important role of drug-induced apoptosis in human prostate cancer cells.(2) Induction of an intronic enhancer of the human ciliary neurotrophic factor receptor (CNTFR) gene by the TR3 orphan receptorA hormone response element, CNTER-NERB (5'-AAAGGTCA-3') has been identified in the fifth intron of the alpha component of CNTFR gene for the human TR3 orphan receptor. A specific binding between in vitro expressed TR3 and CNTFR-NBRE was demonstrated by using electrophoretic mobility shift assay. A reporter gene assay using CAT showed that CNTFR has an enhancer activity that could be induced by TR3 receptor in a dose-dependent manner. This induction was significantly reduced in the absence of CNTFR-BNBRE. Together, these results indicate CNTFR-NBRE is sufficient to mediate TR3 action in inducing the enhancer activity of CNTFR. Our finding may, therefore, suggest CNTFR is a target gene regulated by TR3 and expand the role of TR3 in the nervous system.(3) In situ hybridization of TR3 receptor in prostate cancer tissues.Recently, Jenkins et al. showed clear data in which c-myc amplification in prostate cancer tissues and metastatic lymphnodes was detected using fluorescence in situ hybridization. Our in situ hybridization data in human prostate cancer, benign prostate hypertrophy, and normal prostate tissues revealed that the expression patterns of TR3 orphan receptor mRNA were very similar to those of c-myc. More interestingly, TR3 orphan receptor mRNAs were more highly expressed in prostate cancer area than in adjacent normal or benign hypertrophic tissue. Furthermore, these expressions were highly seen in poorly differentiated adenocarcinoma area. These data suggest that the TR3 orphan receptor may play some important roles in development or progression of prostate cancer in the same manner as the c-myc oncogene.(4) Telomerase activities in prostate cancer tissues.We analyzed the telomerase activity in association with the pathological differentiation of prostate cancer. Telomerase activity was examined by PCR-based telomeric repeat amplification protocol (TRAP) assay. The results showed that most of prostate cancer tissues (90%) displayed telomerase activity and high activity in primary and biopsy specimens were more frequent detected in poorly differentiated adenocarcinoma. Also, the expression of catalytic subunit, hTERT, was detected by RT-PCR. It revealed that the expression in 70% of prostate cancer tissues was more highly seen in comparison with those in normal prostate tissues. Less