Cytotoxic gene therapy for human breast cancer in vitro.
BACKGROUND
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Levy
Shauna
S
The Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, Texas 77030, USA. cbrunica@bcm.tmc.edu
Zhou
Beilan
B
Ballian
Nikiforos
N
Li
Zhijun
Z
Liu
Shi-He
SH
Feanny
Mark
M
Wang
Xiao-Ping
XP
Blanchard
D Kay
DK
Brunicardi
F Charles
FC
D000256:Adenoviridae / Q000235:genetics; D000998:Antiviral Agents / Q000494:pharmacology; D001943:Breast Neoplasms / Q000235:genetics* / Q000628:therapy*; D045744:Cell Line, Tumor; D015774:Ganciclovir / Q000494:pharmacology; D015316:Genetic Therapy / Q000379:methods*; D018398:Homeodomain Proteins / Q000235:genetics*; D006801:Humans; D066298:In Vitro Techniques; D007763:Lac Operon; D012333:RNA, Messenger / Q000378:metabolism; D013937:Thymidine Kinase / Q000235:genetics; D015534:Trans-Activators / Q000235:genetics*; D014162:Transfection
D000998:Antiviral Agents; D018398:Homeodomain Proteins; D012333:RNA, Messenger; D015534:Trans-Activators; C084010:pancreatic and duodenal homeobox 1 protein; D013937:Thymidine Kinase; D015774:Ganciclovir