Levy Shauna, Zhou Beilan, Ballian Nikiforos, Li Zhijun, Liu Shi-He, Feanny Mark, Wang Xiao-Ping, Blanchard D Kay, Brunicardi F Charles
The Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, Texas 77030, USA.
J Surg Res. 2006 Nov;136(1):154-60. doi: 10.1016/j.jss.2006.05.021. Epub 2006 Sep 27.
Transcription factor PDX-1 is expressed by human pancreatic and breast cancers. Although cytotoxicity of PDX-1-directed RIP-TK/GCV gene therapy to pancreatic cancer cells has been demonstrated, the efficacy of this treatment in breast cancer cells is unknown. The purpose of this study was to determine the expression of PDX-1 and its effect on RIP activation in two human breast cancer cell lines, AU565 and T47D. We also investigated the efficacy of RIP-TK/GCV gene therapy and examined whether exogenous PDX-1 to would enhance its cytotoxic effect.
RT-PCR was used to determine PDX-1 expression. Gene constructs RSVLacZ and RIPLacZ were used for transient transfection and LacZ expression was determined using reporter assays. T47D cells were also transfected with adenoviral vectors. Cells were transfected with RIP-TK and the suboptimal level of GCV was determined for each cell line. Following GCV treatment, cytotoxicity was measured using MTS assays. The effect of exogenous PDX-1 on LacZ expression and RIP-TK cytotoxicity was determined.
PDX-1 mRNA was expressed in human breast cancer cells and activated the RIP. Exogenous PDX-1 enhanced LacZ expression in AU565 cells but not in T47D cells. Adenoviral transfection was more efficient in T47D cells than non-viral transfection. RIP-TK treatment was cytotoxic to AU565 and T47D cells and this effect was enhanced by exogenous PDX-1 with both transfection methods.
RIP-TK/GCV therapy is cytotoxic to human breast cancer cells and exogenous PDX-1 enhances cytotoxicity. In vivo studies are necessary to determine the tumor specificity and efficacy of this treatment.
转录因子PDX-1在人类胰腺癌和乳腺癌中表达。尽管已证实PDX-1导向的RIP-TK/GCV基因疗法对胰腺癌细胞具有细胞毒性，但该疗法在乳腺癌细胞中的疗效尚不清楚。本研究的目的是确定PDX-1在两种人乳腺癌细胞系AU565和T47D中的表达及其对RIP激活的影响。我们还研究了RIP-TK/GCV基因疗法的疗效，并检测外源性PDX-1是否会增强其细胞毒性作用。
采用RT-PCR检测PDX-1表达。基因构建体RSVLacZ和RIPLacZ用于瞬时转染，并使用报告基因检测法测定LacZ表达。T47D细胞也用腺病毒载体转染。用RIP-TK转染细胞，并确定每种细胞系的次优GCV水平。GCV处理后，使用MTS检测法测量细胞毒性。测定外源性PDX-1对LacZ表达和RIP-TK细胞毒性的影响。
PDX-1 mRNA在人乳腺癌细胞中表达并激活RIP。外源性PDX-1增强了AU565细胞中的LacZ表达，但在T47D细胞中未增强。腺病毒转染在T47D细胞中比非病毒转染更有效。RIP-TK处理对AU565和T47D细胞具有细胞毒性，并且两种转染方法中外源性PDX-1均增强了这种作用。
RIP-TK/GCV疗法对人乳腺癌细胞具有细胞毒性，外源性PDX-1增强细胞毒性。需要进行体内研究以确定该治疗的肿瘤特异性和疗效。
