Andriani F, Nan B, Yu J, Li X, Weigel N L, McPhaul M J, Kasper S, Kagawa S, Fang B, Matusik R J, Denner L, Marcelli M
Department of Medicine, Baylor College of Medicine, and VA Medical Center, Houston, TX 77030, USA.
J Natl Cancer Inst. 2001 Sep 5;93(17):1314-24. doi: 10.1093/jnci/93.17.1314.
Adenovirus-mediated overexpression of the apoptosis-inducing protein Bax can induce apoptosis in prostate cancer cell lines. Constitutive overexpression of Bax could result in unwanted apoptosis in every site of accidental Bax accumulation in vivo. Therefore, we developed an adenoviral construct (Av-ARR2PB-Bax) in which the probasin promoter, modified to contain two androgen response elements, drives Bax expression. This promoter would be expected to limit expression of Bax to cells expressing the androgen receptor.
A variety of androgen receptor (AR)-positive and -negative cell lines of prostatic or nonprostatic origin were infected with Av-ARR2PB-Bax or a control virus, Av-ARR2PB-CAT, in which the same promoter drives expression of the chloramphenicol acetyl transferase-reporter gene. Bax expression and apoptosis in vitro were assessed by western blot analysis. Tumor size and apoptosis in vivo were assessed after four weekly injections of Av-ARR2PB-Bax or Av-ARR2PB-CAT into subcutaneous LNCaP xenografts growing in uncastrated male mice. All statistical tests were two-sided.
Bax was overexpressed in an androgen-dependent way in AR-positive cell lines of prostatic origin but not in AR-positive cells of nonprostatic origin or in AR-negative cell lines of either prostatic or nonprostatic origin. The androgen dihydrotestosterone activated apoptosis in LNCaP cells infected with Av-ARR2PB-Bax but not in those infected with Av-ARR2PB-CAT. Av-ARR2PB-Bax-injected LNCaP xenograft tumors decreased in tumor size from 34.1 mm3 (95% confidence interval [CI] = 25.1 mm3 to 43.1 mm3) to 24.6 mm3 (95% CI = -2.5 mm3 to 51.7 mm3), but the difference was not statistically significant (P =.5). Tumors injected with Av-ARR2PB-CAT increased in size, from 28.9 mm3 (95% CI = 12.7 mm3 to 45.1 mm3) to 206 mm3 (95% CI = 122 mm3 to 290 mm3) (P =.002) and contained statistically significant more apoptotic cells (23.3% [95% CI = 21.1% to 25.6%] versus 9.5% [95% CI = 8.0% to 11.1]) (P<.001).
Av-ARR2PB-Bax induces androgen-dependent therapeutic apoptosis in vitro and in vivo by activating apoptosis in AR-positive cells derived specifically from prostatic epithelium and does not affect nonprostatic cells.
腺病毒介导的凋亡诱导蛋白Bax的过表达可诱导前列腺癌细胞系凋亡。Bax的组成型过表达可能会在体内Bax意外积累的每个部位导致不必要的凋亡。因此，我们构建了一种腺病毒载体（Av-ARR2PB-Bax），其中经修饰含有两个雄激素反应元件的前列腺素启动子驱动Bax表达。预期该启动子可将Bax的表达限制在表达雄激素受体的细胞中。
用Av-ARR2PB-Bax或对照病毒Av-ARR2PB-CAT感染多种前列腺或非前列腺来源的雄激素受体（AR）阳性和阴性细胞系，在对照病毒中相同的启动子驱动氯霉素乙酰转移酶报告基因的表达。通过蛋白质印迹分析评估体外Bax表达和凋亡情况。在未阉割雄性小鼠皮下种植的LNCaP异种移植物中每周注射一次Av-ARR2PB-Bax或Av-ARR2PB-CAT，共四周后，评估体内肿瘤大小和凋亡情况。所有统计检验均为双侧检验。
在前列腺来源的AR阳性细胞系中，Bax以雄激素依赖的方式过表达，但在非前列腺来源的AR阳性细胞或前列腺及非前列腺来源的AR阴性细胞系中未过表达。雄激素双氢睾酮可激活感染Av-ARR2PB-Bax的LNCaP细胞中的凋亡，但不能激活感染Av-ARR2PB-CAT的细胞中的凋亡。注射Av-ARR2PB-Bax的LNCaP异种移植瘤体积从34.1立方毫米（95%置信区间[CI]=25.1立方毫米至43.1立方毫米）减小至24.6立方毫米（95%CI=-2.5立方毫米至51.7立方毫米），但差异无统计学意义（P=0.5）。注射Av-ARR2PB-CAT的肿瘤体积增大，从28.9立方毫米（95%CI=12.7立方毫米至45.1立方毫米）增大至206立方毫米（95%CI=122立方毫米至290立方毫米）（P=0.002），且凋亡细胞在统计学上显著增多（23.3%[95%CI=21.1%至25.6%]对9.5%[95%CI=8.0%至11.1%]）（P<0.001）。
Av-ARR2PB-Bax通过激活源自前列腺上皮的AR阳性细胞中的凋亡，在体外和体内诱导雄激素依赖的治疗性凋亡，且不影响非前列腺细胞。
