白细胞介素-11类似物c(CGRRAGGSC)与人前列腺癌PC-3细胞的特异性结合研究

目的 探讨白细胞介素-11(IL-11)类似物环九肽c(CGRRAGGSC)与人前列腺癌PC-3细胞的体外结合和在前列腺癌骨转移模型的体内分布.方法 印迹蛋白分析PC-3细胞和正常前列腺细胞的IL-11受体(IL-11R)的表达.荧光检测c(CGRRAGGSC)与PC-3细胞的结合位点及内化.将28只荷瘤裸鼠随机分为7组,用99Tcm标记c(CGRRAGGSC)制备99Tcm-DTPA-c(CGRRAGGSC),经尾静脉注射0.74 MBq/0.2 ml,观察标记物在荷瘤鼠体内的生物分布.结果　PC-3细胞IL-11R是正常前列腺细胞的2.12倍.荧光示踪复染合成物结合在PC-3细胞膜及胞质.标记物在模型体内迅速持续聚集在瘤体,4 h瘤体、骨骼%ID/g达到峰值(15.12±1.19、7.03±1.83),其他脏器分布少,差异有统计学意义(F值依序为71.58、9.58,P＜0.05).结论　环九肽与PC-3细胞的结合具有特异性,标记物能与前列腺癌骨转移灶靶向结合.

Abstract：

Objective To assess the specific binding between interleukin-11 (IL-11) analogue c (CGRRAGGSC) and PC-3 cells in vitro and biostribution in bone metastases of prostate cancer in mice in vivo. Methods The expression changes of IL-11R in PC-3 tumors and normal prostate cells were examined by Western blotting. The binding site of the molecular target and internalization with PC-3 cells was observed under a fluorescence microscope. Twenty-eight tumor-bearing nude mice were divided into 7groups, and radioactive molecular probe 99Tcm-DT-PA- c (CGRRAG-GSC) was radiolabeled with 99Tcm.Biodistribution in nude mice bearing PC-3 tumors was observed and analyzed after intravenous injection of 99Tcm-DTPA-c (CGRRAGGSC) (0.74 MBq/0.2 mi). Results The expression of IL-11R in PC-3tumors group was 2. 2 times as the normal group. The counter stain molecular probe was combined with the PC-3 cell membrane and cytoplasm through fluorescence tracing. Uptake of the imaging probe in the tumor was increased. Radioactivity was abnormally concentrated in the tumor lesions and skeleton. Most apparent after 4 h ( 15. 12 ± 1.19, 7.03 ± 1.83), radiation was not obviously observed in other viscera ( F = 71.58,9. 58 ,P ＜ 0. 05). Conclusion c (CGRRAGGSC) peptide has the specific binding ability in targeting to PC-3 cells in vitro. Radioactive molecular probe 99Tcm-DTPA-c (CGRRAGGSC) has potential as a specific molecular target imaging agent for bone metastasis, such as prostate cancer.     

孕酮和米非司酮对前列腺癌PC-3细胞作用机理的研究

目的探讨孕酮和米非司酮促进前列腺癌PC-3细胞增殖作用的机理。方法细胞培养观察孕酮和米非司酮对前列腺癌PC-3细胞增殖的影响。RT-PCR检测PC-3细胞孕激素受体mRNA的表达。Westernblot检测孕酮对PC-3细胞细胞外信号调节激酶ERK1/2活性的影响,并观察米非司酮对孕酮的阻断效果。结果细胞培养7d,孕酮(100nmol/L)组细胞数量为(3.2±0.3)×105,明显高于正常培养组[(1.9±0.2)×105](P<0.05);米非司酮(10μmol/L)组为(1.3±0.2)×105,明显低于正常培养组(P<0.05)。PC-3细胞有孕激素受体mRNA的表达。孕酮可以激活ERK1/2,而米非司酮阻断孕酮对ERK1/2的激活,孕酮组与孕酮+米非司酮组p-ERK1/2条带强度差异有统计学意义(吸光度A值分别为70752±16377和12493±3478,P<0.05)。结论孕酮对PC-3细胞的促进增殖作用与激活细胞ERK1/2有关;米非司酮阻断孕酮对PC-3细胞ERK1/2的活化,从而抑制细胞增殖。     

槲皮素对前列腺癌PC-3细胞凋亡作用的研究

目的:研究槲皮素对人前列腺癌PC-3细胞的凋亡作用。方法:体外培养PC-3细胞,给予不同浓度(50、100、150、200、250μmol/L)的槲皮素处理,MTT法检测槲皮素对细胞抑制率;流式细胞仪检测细胞凋亡率;透射电镜观察细胞超微结构的变化。结果:槲皮素能抑制人前列腺癌PC-3细胞的体外生长,且呈时间与剂量依赖性,浓度由低到高,其24 h的抑制率(%)分别为3.01±1.32、4.84±1.73、20.35±1.30、16.78±1.89、27.25±4.01,48 h的抑制率(%)分别为10.18±1.16、6.22±0.04、24.29±4.19、22.4±4.26、41.42±5.43,当药物浓度>150μmol/L时P<0.05,具有统计学意义;流式细胞术显示PC-3细胞凋亡率随槲皮素浓度升高和作用时间延长而增加(P<0.05),其中浓度150μmol/L和200μmol/L组,24 h的凋亡率(%)分别19.10±0.28、26.55±0.78,48 h的凋亡率(%)分别为27.65±1.06、38.30±5.96;电镜观察到细胞的典型凋亡形态学变化。结论:在适当的条件下,槲皮素对人前列腺癌PC-3细胞增殖有明显的抑制作用,同时可诱导PC-3细胞凋亡,其具体作用机制有待进一步深入研究。     

A14125I-胰岛素与H22肝癌细胞胰岛素受体结合的研究

目的体外分析H22肝癌细胞与正常肝细胞膜的胰岛素受体表达,探讨胰岛素作为受体介导靶向治疗载体的可行性及临床应用前景.方法用氯氨T法制备A14125I-胰岛素,电泳分离纯化标记物并用自身置换比放射性测定和过量受体结合实验法鉴定标记物质量,再制备H22肝癌细胞及正常小鼠肝细胞,进行体外受体结合实验,分别测定125I-胰岛素与小鼠H22肝癌及正常肝细胞膜胰岛素受体结合的Kd值及Bmax值以及其竞争结合曲线.125I-胰岛素受体结合的Kd值及Bmax值用t检验分析,饱和结合曲线用Scatchard分析.结果小鼠H22肝癌细胞及正常小鼠肝细胞胰岛素受体Bmax值分别为(5.6±1.1) pmol/106细胞和(3.2±0.8) pmol/106细胞,高亲和力Kd值为(1.8±0.6) nmol/L及(2.1±0.9) nmol/L,低亲和力Kd值为(32.0±10.7) nmol/L及(37.0±12.3) nmol/L.癌细胞胰岛素受体Bmax值明显高于正常肝细胞(P＜0.05).受体结合实验表明,125I-胰岛素与H22肝癌及正常细胞的结合具有特异性.结论 125I-胰岛素与H22肝癌及正常小鼠肝细胞膜受体的结合具有高度特异性,H22肝癌细胞较正常小鼠肝细胞表达胰岛素受体增加,提示胰岛素可作为抗癌载体通过与肝癌细胞表面受体结合而介导肝癌的靶向治疗.     

促黄体激素释放激素-绿脓杆菌外毒素A与胃癌细胞受体结合竞争的研究

目的探讨重组人促黄体激素释放激素-绿脓杆菌外毒素A(LHRH-PE40)在胃癌细胞及正常细胞是否存在相应受体表达及存在竞争性抑制。方法将胃癌细胞系BGC-823及正常人肾细胞系293制备成细胞膜,利用125I标记的LHRH-PE40与两种细胞膜进行放射性配基分析, 以及与LHRH竞争结合分析。结果人肾细胞系293未见配基-受体特异性结合;而人胃癌细胞系 BGC-823的结合竞争符合特异性配基-受体结合、竞争。BGC-823细胞亲和力:Kd=(37．82±1．42) nmol／L,容量Bmax=475．00±17．86 pmol／mg。结论 LHRH是胃癌免疫治疗的有效靶点,LHRH- PE40对过度表达LHRH受体的胃癌均具有特异有效的抗肿瘤活性。     

双氢青蒿素对前列腺癌细胞PC-3M生长的影响及其机制探讨

目的:观察双氢青蒿素对雄激素非依赖性前列腺癌细胞株PC-3M细胞凋亡和血管内皮生长因子(VEGF)表达的影响。方法:不同浓度(0、25、50、100μmol/L)的双氢青蒿素分别作用于PC-3M细胞48 h,MTT法检测细胞生长活性;流式细胞仪测定细胞凋亡率;分光光度法检测细胞凋亡过程中caspase-3、caspase-8活性变化;半定量RT-PCR法检测PC-3M细胞内VEGF mRNA的表达;Western印迹法检测细胞VEGF蛋白表达。结果:双氢青蒿素能显著抑制PC-3M细胞的增殖,与对照组(0μmol/L)的细胞凋亡率(2.92±0.45)%相比,各剂量组(25、50、100μmol/L)的细胞凋亡率[(8.85±0.74)%,(12.83±0.84)%,(18.65±1.24)%]显著增加,caspase-8[(0.47±0.05)U/μg vs(1.22±0.15)U/μg,(1.76±0.07)U/μg,(2.91±0.24)U/μg]、caspase-3[(0.44±0.07)U/μg vs(0.95±0.08)U/μg,(1.48±0.14)U/μg,(2.92±0.45)U/μg]活性显著增加,呈剂量依赖性(P<0.01)。PC-3M细胞内VEGF mRNA的表达和蛋白表达呈剂量依赖性降低。结论:双氢青蒿素能显著抑制体外PC-3M细胞的生长,并促进其凋亡,机制可能与增加凋亡蛋白酶和抑制VEGF表达有关。     

多肽K237对PC-3M细胞增殖及bax、bcl-2 mRNA表达的影响

目的:研究多肽K237对体外培养的雄激素非依赖性前列腺癌PC-3M细胞的抑制作用,及其可能的作用机制。方法:将培养的PC-3M细胞分为4组:实验组(分别以50、100、200μmol/L的多肽K237处理48h)和对照组(K237浓度为0μmol/L),采用MTT法观察多肽K237对前列腺癌PC-3M细胞增殖的影响,用RT-PCR法检测bax、bcl-2mRNA表达的变化。结果:不同浓度的多肽K237处理48h后,PC-3M细胞形状变圆,体积变小,胞质透亮度下降,部分细胞脱落悬浮于培养液中。在50、100、200μmol/L的多肽K237作用48h后,MTT法检测的细胞生长抑制率分别为(12.6±0.95)%、(17.8±0.99)%、(27.2±1.12)%。RT-PCR结果显示:50、100、200μmol/L实验组和对照组的bax/β-actin值分别为0.919±0.071、0.971±0.083、0.992±0.102,(0.889±0.06),bcl-2/β-actin值分别为0.896±0.085、0.791±0.084、0.764±0.702,0.922±0.097,3组中上述两项指标均较对照组有明显变化(P均<0.01),其中,baxmRNA表达水平上调,而bcl-2mRNA表达水平下调,上述作用呈现剂量效应关系。结论:多肽K237可能通过影响bax、bcl-2mRNA的表达来诱导PC-3M细胞凋亡,从而抑制前列腺癌细胞的增殖。     

靶向ADAM17基因siRNA对前列腺癌PC-3细胞增殖能力的影响

目的:探讨靶向抑制ADAM17基因对雄激素非信赖性前列腺癌PC-3细胞增殖的影响。方法:应用ADAM17 siRNA转染PC-3细胞后,通过RT-PCR、Western印迹方法分别检测ADAM17 mRNA和蛋白表达变化;MTT、BrdU掺入法检测下调ADAM17对PC-3细胞的增殖和DNA合成能力的影响;流式细胞术检测ADAM17 siR-NA对PC-3细胞细胞周期的影响;Western印迹检测下调ADAM17对PC-3细胞增殖相关基因表达的影响。结果:两对ADAM17 siRNAs均可有效地降低PC-3细胞ADAM17 mRNA和蛋白的表达;MTT结果显示与对照组(0.80±0.51)相比,两对ADAM17 siRNAs均可显著抑制细胞的生长(0.43±0.57、0.44±0.64,P均<0.05);Br-dU掺入实验显示与对照组(0.79±0.72)相比,ADAM17 siRNAs均能显著下调DNA的合成能力(0.48±0.43、0.54±0.59,P<0.05);流式细胞术结果显示,与对照组(41.38±1.53)%相比,ADAM17 siRNAs可显著增加G1期细胞数量[(61.83±2.41)%、(59.78±1.92)%,P均<0.05]、降低S期细胞数量[从(33.51±1.47)%减少到(23.64±2.56)%、(25.24±1.86)%,P均<0.05],同时伴随着cyclin D1蛋白的表达下降而p21蛋白的表达升高。结论:ADAM17 siRNA可以通过下调cyclin D1、上调p21的表达而抑制前列腺癌PC-3细胞增殖,ADAM17可能成为前列腺癌基因治疗的靶点。     

选择性环氧化酶-2抑制剂NS398诱导前列腺癌PC-3细胞系凋亡的研究

目的观察选择性环氧化酶2(COX-2)抑制剂氮-2,环己氧-4,硝基苯-甲基磺胺 (NS398)在诱导COX-2表达阴性的前列腺癌PC-3细胞凋亡中的作用. 方法应用RT-PCR和Western blot的方法对PC-3细胞mRNA和蛋白水平的COX-2表达情况进行检测,四甲基偶氮唑蓝快速比色法观察不同浓度和时间NS398对PC-3细胞生存率的影响,流式细胞术检测100 μmol/L NS398作用24 h PC-3细胞的凋亡情况. 结果 mRNA和蛋白水平PC-3细胞COX-2均呈阴性表达;NS398可以抑制PC-3细胞的存活,并随浓度增加而增强,但无显著的时间依赖性;与对照组(10.563±2.582)%相比,100 μmol/L NS398诱导PC-3细胞早期凋亡比率(19.307±3.773)%显著增加,差异有统计学意义(P=0.01). 结论选择性COX-2抑制剂NS398可以诱导COX-2表达阴性的PC-3细胞凋亡,提示COX-2非依赖性途径的存在.     

羟基喜树碱对前列腺癌PC-3细胞凋亡作用的研究

目的:观察羟基喜树碱(HCPT)对前列腺癌PC-3细胞凋亡的影响,初步探讨其作用机制。方法:四甲基偶氮唑盐(MTT)检测不同浓度(1×10-1、1×10-2、1×10-3、1×10-4mg/ml)、不同作用时间(12、24、48h)下HCPT对PC-3细胞增殖的影响;吖啶橙/溴化乙锭(AO/EB)染色观察细胞凋亡情况;琼脂糖凝胶电泳观察凋亡细胞特征性DNA条带;流式细胞术测定HCPT诱导PC-3细胞凋亡率。结果:HCPT明显抑制PC-3细胞生长,呈时间、剂量依赖性,12、24、48h的IC50值分别为:6.50×10-2、2.35×10-2、5.31×10-3mg/ml;荧光显微镜下观察到经AO/EB染色的典型凋亡细胞;琼脂糖凝胶电泳可见凋亡细胞DNA呈规律的梯状条带;流式细胞术显示PC-3细胞凋亡率随HCPT浓度的增加而升高,浓度为1×10-3mg/ml时凋亡率达到最高峰(35.76%)。结论:HCPT可通过诱导凋亡较为明显地抑制PC-3细胞生长,但其作用机制目前尚需进一步研究。     

Cell-Surface labeling and internalization by a fluorescent inhibitor of prostate-specific membrane antigen

BACKGROUND: [corrected] Prostate-specific membrane antigen (PSMA) remains an attractive target for imaging and therapeutic applications for prostate cancer. Recent efforts have been made to conjugate inhibitors of PSMA with imaging agents. Compared to antibodies, small-molecule inhibitors of PSMA possess apparent advantages for in vivo applications. To date, there are no reports on the cellular fate of such constructs once bound the extracellular domain of PSMA. The present study was focused on precisely defining the binding specificity, time-dependent internalization, cellular localization, and retention of inhibitor conjugates targeted to PSMA on LNCaP cells. A novel fluorescent inhibitor was prepared as a model to examine these processes. METHODS: Fluorescence microscopy of LNCaP and PC-3 cell lines was used to monitor the specificity, time-dependent internalization, cellular localization, and retention of a fluorescent PSMA inhibitor. RESULTS: Fluorescent inhibitor 2 was found to be a potent inhibitor (IC50 = 0.35 nM) of purified PSMA. Its high affinity for PSMA on living cells was confirmed by antibody blocking and competitive binding experiments. Specificity for LNCaP cells was demonstrated as no labeling by 2 was observed for negative control PC-3 cells. Internalization of 2 by viable LNCaP cells was detected after 30 min incubation at 37 degrees C, followed by accumulation in the perinuclear endosomes. It was noted that internalized fluorescent inhibitor can be retained within endosomes for up to 150 min without loss of signal. CONCLUSIONS: Our results suggest that potent, small-molecule inhibitors of PSMA can be utilized as carriers for targeted delivery for prostate cancer for future imaging and therapeutic applications.     

Anoikis is regulated by BCL-2-independent pathways in human prostate carcinoma cells

BACKGROUND: Loss of contact with the extracellular matrix (ECM) triggers a specialized form of apoptosis known as "anoikis" in normal epithelial cells. Dependence on adhesion to ECM is often lost in transformed cells, and the degree of anchorage independence may vary in non-metastatic and metastatic cancer cells. BCL-2 oncoprotein overexpression correlates with the progression and metastases of prostate cancer. Materials and Methods We studied anoikis in suspension cultures of PC-3 and LNCaP prostate carcinoma cells selected for enhanced metastatic potential in vivo and in PC-3 and LNCaP cells stably transfected with BCL-2. Apoptosis-associated DNA fragmentation was measured by agarose gel electrophoresis and propidium iodide staining and flow cytometry. Expression of BCL-2 family polypeptides was determined by immunoblotting. RESULTS: Non-metastatic PC-3P cells were significantly more sensitive to anoikis than the metastatic PC-3 variants (PC-3M, PC-3M-PRO-4, and PC-3M-LN-4), but anoikis resistance did not correlate with metastatic potential in LNCaP-derived cell lines. Expression of BCL-2 was higher in metastatic PC-3 and LNCaP subclones compared to isogenic non-metastatic cells, but these levels were not affected by anoikis. Enforced overexpression of BCL-2 did not protect either PC-3P or LNCaP-PRO-5 cells from anoikis, even though it rendered them resistant to thapsigargin and inhibited cytochrome c release. Strikingly, cells that died of anoikis maintained their pretreatment levels of BCL-2, whereas the cells that survived anoikis expressed much lower levels of the protein. CONCLUSIONS: Sensitivity to anoikis is regulated by BCL-2 independent mechanisms in LNCaP and PC-3 prostate cancer cells.     

AsRed2示踪前列腺癌肺转移模型的建立

目的:使用携带红色荧光蛋白(AsRed2)的前列腺癌PC-3细胞(PR7细胞)建立可用活体荧光成像技术监测的前列腺癌肺转移模型。方法:分别采用MTT及Transwell实验比较前列腺癌PC-3与PR7细胞体外增殖、迁移及侵袭能力;将20只BALB/c裸鼠随机平均分为4组,分别尾静脉注射不同浓度PR7细胞悬液200μl,A组:1×107/ml,B组:2.5×107/ml,C组:5×107/ml,D组:2.5×107/ml,并于1周后重复注射2.5×107/ml细胞悬液200μl。第2、4、6、8周分别用活体荧光成像技术检测各组肺转移灶形成情况。结果:前列腺癌PC-3细胞与PR7细胞体外增殖、迁移、侵袭能力均无明显差异(P>0.05);第4周D组40%裸鼠用活体荧光成像检测到肺转移灶;第8周,A组20%、B组60%、C组100%、D组100%裸鼠可检测到肺转移灶;通过病理检查证实肺转移灶形成。结论:通过尾静脉注射携带红色荧光蛋白的前列腺癌PR7细胞可成功建立用活体荧光成像技术监测的前列腺癌肺转移模型。     

Androgen-independent growth is induced by neuropeptides in human prostate cancer cell lines

BACKGROUND: Androgen-independent growth leads to progressive prostate cancer after androgen-ablation therapy. This may be caused by altered specificity of the androgen receptor (AR), by ligand-independent stimulation of the AR, or by paracrine growth modulation by neuropeptides secreted by neuroendocrine (NE) cells. METHODS: We established and characterized the androgen-independent FGC-DCC from the androgen-dependent LNCaP fast growing colony (FGC) cell line. The androgen-independent DU-145, FGC-DCC, and PC-3, and the androgen-dependent LNCaP and PC-346C cell lines were used to study growth modulation of gastrin-releasing peptide (GRP), calcitonin (CT), serotonin (5-HT), and vasoactive intestinal peptide (VIP) by (3)H-thymidine incorporation. Specificity of the growth-modulating effects was tested with the anti-GRP monoclonal antibody 2A11 and induction of cAMP by neuropeptides. RESULTS: Androgen-independent growth stimulation by neuropeptides was shown in DU-145 and PC-346C. 2A11 inhibited GRP-induced (3)H-thymidine incorporation in DU-145 and PC-346C and inhibited proliferation of the FGC-DCC and PC-3 cell lines. With some exceptions, cAMP induction paralleled growth stimulation. Dideoxyadenosine (DDA) inhibited the GRP-induced growth effect in DU-145 and PC-346C, whereas oxadiazoloquinoxaline-1-one (ODQ) had no effect on (3)H-thymidine incorporation. None of the neuropeptides stimulated growth of LNCaP, FGC-DCC, or PC-3. CONCLUSIONS: GRP-induced growth of DU-145 and PC-346C was specific and cAMP-mediated. Androgen-independent growth of FGC-DCC cells was mainly due to an induction of Bcl-2 expression and possibly through the activation of an autocrine and NE-like pathway, as has been shown also for the PC-3 cell line. Growth induction of non-NE cells by neuropeptides could be a possible role for NE cells in clinical prostate cancer.     

抗前列腺特异抗原/抗人CD3双特异性单链抗体基因在HeLa细胞中的表达及亲和活性测定

目的　在HeLa细胞中表达抗前列腺特异抗原 (PSA ) /抗人CD3双特异性单链抗体(scFv)融合基因 ,并进行亲和活性测定。方法　在已经构建抗PSA/CD3双特异性scFv融合基因基础上 ,将融合基因克隆入真核表达载体pSecTag2 B中 ,并转染HeLa细胞进行表达。表达产物纯化后 ,用流式细胞仪进行亲和活性测定。结果　经SDS PAGE和Western印迹实验证实 ,表达产物的Mr约为 65 0 0 0。纯化后经流式细胞仪检测 ,可特异性地结合PC 3细胞和人外周血单个核细胞(PBMC)。结论　获得了可与PC 3细胞和PBMC特异结合的抗PSA/CD3双特异性scFv ,为进一步临床应用奠定了基础。     

1alpha,25-dihydroxyvitamin D3 induced growth inhibition of PC-3 prostate cancer cells requires an active transforming growth factor beta signaling pathway

BACKGROUND: Prostate cancer growth inhibition by 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) is best characterized in the androgen dependent LNCaP cell line, where treatment with this hormone causes cell cycle arrest and apoptosis. 1,25(OH)2D3 also inhibits the growth of PC-3 prostate cancer cells, but not through the induction of G1 arrest or apoptosis. In this study, we have sought to elucidate the mechanism/s involved in PC-3 cell growth inhibition by 1,25(OH)2D3. EXPERIMENTAL METHODS: We determined the effect of transforming growth factor beta (TGFbeta) blocking antibodies on 1,25(OH)2D3 mediated growth inhibition of PC-3 cells. In addition, we also studied the effects of 1,25(OH)2D3 on TGFbeta signaling and receptor expression. Finally, we assessed the role of TGFbeta signaling in the induction of the growth inhibitory protein, insulin like growth factor binding protein 3 (IGFBP-3), by 1,25(OH)2D3. RESULTS: We find that 1,25(OH)2D3 action in PC-3 cells is mediated through at least two distinct pathways, the TGFbeta pathway and the IGFBP-3 pathway. We show that 1,25(OH)2D3 treatment elevates TGFbeta production and signaling, as well as receptor levels, in PC-3 cells. Further, using a blocking antibody against TGFbeta substantially reduces 1,25(OH)2D3 mediated growth inhibition without affecting IGFBP-3 induction, suggesting that IGFBP-3, alone, is insufficient to inhibit the growth of PC-3 cells.     

Triiodothyronine modulates cell proliferation of human prostatic carcinoma cells by downregulation of the B-cell translocation gene 2

BACKGROUND: Studies suggest that triiodothyronine (T3) and cognate nuclear receptors (hTR) are involved in regulation of prostatic cell growth and differentiation. To probe mechanisms for T3 effects, we studied prostate carcinoma cells, investigating the effect of T3 on expression of the B-cell translocation gene 2 (BTG2), which regulates the G1/S transition of the cell cycle. METHODS: Effects of T3 on cell proliferation were determined by (3)H-thymidine incorporation. T3 modulation of BTG2 expression was investigated using immunoblots, Northern blots, and transient gene expression assays. The putative T3 response element was determined by electrophoretic mobility shift assay. RESULTS: T3 (0.1-1,000 nM) enhanced threefold the proliferation of prostate carcinoma cells and human androgen-dependent prostate carcinoma cells (LNCaP), but not PC-3 cells. T3 also inhibited BTG2 gene expression in LNCaP cells. Reporter assays showed that T3 downregulates by 50% promoter activity of the BTG2 gene in LNCaP cells but not PC-3 cells or thyroid-hormone receptor (TRbeta1)-overexpression PC-3 cells. Deleting the putative thyroid hormone response element (TRE; AGCGATGACCTCAGCG) blocked the inhibitory effect of T3 on BTG2 promoter activity. Electrophoretic mobility shift assays with purified TRbeta1 from in vitro translation, or with nuclear extracts from LNCaP cells and PC-3 cells, demonstrated the presence of T3 receptor binding sites in the TRE region. CONCLUSIONS: These results suggested that the T3 upregulates proliferation of LNCaP cells by downregulating BTG2 gene expression through the consensus TRE pathway.     

Prostate-tumor targeting of gene expression by lentiviral vectors containing elements of the probasin promoter

BACKGROUND: Lentiviruses are retroviruses that can infect and stably integrate into the chromatin of non-dividing cells. The purpose of this study was to determine whether lentiviral vectors containing the probasin (PB) promoter displayed prostate-specific, androgen-regulated, and persistent gene expression. METHODS: Three lentiviral-PB promoter/enhanced green fluorescent protein (EGFP)-reporter vectors together with a control lentiviral-CMV-EGFP, were tested by microscopy and flowcytometry for expression of EGFP after infection of human prostate cancer cells (LNCaP, PC-3, PC-3(hAR), and Du145 cells) and non-prostate cells (COS-1, HeLa, HeLa(hAR), and MCF-7 cells). RESULTS: All cells infected in vitro with lentiviral-CMV vectors expressed EGFP, whereas with lentiviral-PB vectors (the most potent being Lv-ARR(2)PB), reporter expression was only observed in LNCaP cells with a small amount seen in androgen-independent PC-3 cells. Stable or transient transfection of androgen receptor only raised EGFP expression in prostate-derived cell lines, but did not change tumor specificity. With Lv-ARR(2)PB infected LNCaP cells, androgens regulated EGFP both in vitro and in vivo. After intra-tumor injection of this vector, EGFP expression was observed in LNCaP tumors, but not in A-549 lung or CaKi-2 kidney tumors. CONCLUSIONS: Lv-ARR(2)PB may be an ideal vector for prostate-tumor targeting and for persistent, hormone-enhanced expression of a therapeutic gene to treat slow growing prostate tumors.     

Characterization of high-affinity receptors for bombesin/gastrin releasing peptide on the human prostate cancer cell lines PC-3 and DU-145: Internalization of receptor bound 125I-(Tyr4) bombesin by tumor cells

Specific receptors for bombesin/gastrin releasing peptide (GRP) on the androgen-independent human prostate cancer cell lines PC-3 and DU-145 were characterized. No specific binding of ¹²⁵I-[Tyr⁴]-bombesin to the androgen-dependent human prostate cancer cell line LNCaP was detectable. The binding of ¹²⁵I-[Tyr⁴]-bombesin to PC-3 and DU-145 cells was found to be time- and temperature-dependent, saturable, and reversible. Scatchard analysis revealed a single class of binding sites with high affinity (K_d 9.8 × 10^?11 M for PC-3, and 9.1 × 10-¹¹ M for DU-145 cells at 25°C) and with a binding capacity of 44,000 binding sites/cell and 19,000 binding sites/cell, respectively. Bound ¹²⁵I-[Tyr⁴]-bombesin was rapidly internalized by PC-3 cells. The nonhydrolyzable GTP analog GTP-gamma-S caused a dose-dependent inhibition of ¹²⁵I-[Tyr⁴]-bombesin binding to PC-3 and DU-145 cells, indicating that a G-protein (guanine nucleotide-binding protein) couples the bombesin receptor to intracellular effector systems. Bombesin and GRP(14-27) inhibited the binding of ¹²⁵I-[Tyr⁴]-bombesin to both cell lines in a dose-dependent manner with inhibition constants (K_i of 0.5 nM and 0.4 nM, respectively. Both cell lines express the bombesin/GRP preferring bombesin receptor subtype, since, in displacement studies, neuromedin B was more than 200 times less potent than bombesin and GRP(14-27) in inhibiting the binding of ¹²⁵I-[Tyr⁴]-bombesin. Two synthetic bombesin/GRP antagonists, RC-3095 and RC-3110, powerfully inhibited the specific binding of ¹²⁵I-[Tyr⁴]-bombesin with K_i 0.92 nM and 0.26 nM on PC-3 cells, and 3.3 nM and 0.89 nM on DU-145 cells, respectively. These findings indicate that the PC-3 and DU-145 human prostate cancer cell lines possess specific high-affinity receptors for bombesin/GRP, and are suitable models for the evaluation of the antineoplastic activity of new bombesin/GRP antagonists in the treatment of androgen-independent prostate cancer. © 1994 Wiley-Liss, Inc.