Tumor-specific adenovirus-mediated PUMA gene transfer using the survivin promoter enhances radiosensitivity of breast cancer cells in vitro and in vivo.
BACKGROUND
Survivin is a member of the inhibitors of apoptosis protein family, is expressed in most of human cancers. Thus, we hypothesized that using a survivin promoter-driven vector system, an apoptosis-inducible gene may be preferentially restricted to survivin-positive tumor cells. PUMA (p53 upregulated modulator of apoptosis) was recently identified a potent proapoptotic molecule. In the present study, our aim is to investigate whether adenovirus-mediated PUMA gene transfer using the survivin promoter could specifically enhance radiosensitivity of breast cancer cells in vitro and in vivo.


METHODS
Firstly, we performed RT-PCR assay to detect survivin mRNA expression in four human breast cancer cell lines and two normal cell lines. Then, we constructed plasmid vectors expressing luciferase or EGFP reporter gene driven by the survivin core promoter and evaluated its transcriptional activity in vitro. Next, a survivin promoter-driven adenoviral system expressing PUMA gene was constructed. Western blotting analysis of PUMA protein expression in MCF-7 and MCF210 cells treated with various adenovirus (empty, driven by CMV promoter or survivin promoter). Followingly, the effects of PUMA overexpression on the in vitro radiosensitivity of breast cancer cells were investigated by clonogenic formation assay. Cellular apoptosis were determined by FCM and TUNEL assays. The expression and activities of cellular caspase-3 were assayed by Western blot and colorimetry. Finally, we investigated the effect of PUMA overexpression on the radiosensitivity of breast carcinoma cells in vivo.


RESULTS
The survivin mRNA expression was obviously upregulated in breast cancer cell lines but in normal cell lines. Moreover, the survivin promoter could drive high-level expression of luciferase or EGFP in breast cells but not in normal cells. Using a survivin promoter-driven adenoviral system expressing PUMA gene, we found that PUMA expression was specifically and efficiently induced in MCF-7 cell but not in MCF210 cell. Furthermore, we observed that the novel adenovirus system could significantly enhance radiosensitivity of MCF-7 cell in vitro and in vivo, which might be associated with apoptosis induction by activating cellular caspase-3.


CONCLUSIONS
Our findings indicated that the survivin promoter-driven adenovirus system expression PUMA gene might be explored as a potential tool for radiosensitization of human breast cancer cells with tumor specificity and high efficacy.
Wang
Rui
R
Research Center, Tangdu Hospital, Fourth Military Medical University, Xi'an, Shaanxi Province, People's Republic of China.
Wang
Xi
X
Li
Bin
B
Lin
Fang
F
Dong
Ke
K
Gao
Ping
P
Zhang
Hui-Zhong
HZ
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D017209:Apoptosis / Q000235:genetics; D051017:Apoptosis Regulatory Proteins / Q000235:genetics*; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000235:genetics*; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D005260:Female; D015870:Gene Expression; D015972:Gene Expression Regulation, Neoplastic; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D020287:In Situ Nick-End Labeling; D051034:Inhibitor of Apoptosis Proteins; D051379:Mice; D008819:Mice, Nude; D008869:Microtubule-Associated Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D011836:Radiation Tolerance / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000077022:Survivin; D023041:Xenograft Model Antitumor Assays
D051017:Apoptosis Regulatory Proteins; C438378:BBC3 protein, human; C107489:BIRC5 protein, human; D051034:Inhibitor of Apoptosis Proteins; D008869:Microtubule-Associated Proteins; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D000077022:Survivin; D053148:Caspase 3