Construction of Prostate‐Specific Expressed Recombinant Plasmids With High Transcriptional Activity of Prostate‐Specific Membrane Antigen (PSMA) Promoter/Enhancer

Zeng, Hao; Wu, Qi; Li, Hong; Wang, Qiang; Lu, Yiping; Li, Xiang; Wang, Fang; Zhao, Fu-Jun; Ding, Zhengyu; Yang, Yue

doi:10.1002/j.1939-4640.2005.tb01088.x

Cited by 14 publications

(18 citation statements)

References 21 publications

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“…Unlike other prostate‐specific proteins (PSA, PAP, probasin), PSMA expression is downregulated by androgen, 6 which makes it a more useful marker in the treatment of androgen‐independent prostate carcinoma. Our previous work confirmed that a single PSMA enhancer with a PSMA promoter has the strongest regulatory activity in PSMA‐positive cells when different controlling element combinations were evaluated 7 …”

Section: Introductionsupporting

confidence: 71%

“…Plasmid DNA was harvested from E. coli strain JM109 using a plasmid miniprep kit (Omega). For a reporter vector, the recombinant plasmids prostate‐specific membrane antigen enhancer/promoter ‐enhanced green fluorescent protein (pPSMA E/P ‐EGFP) and pPSMA E/P ‐UPRT were constructed and screened by our department; 7 while pEGFP‐N1 (Clontech, Mountain View, CA, USA) were used as a control plasmid.…”

Section: Methodsmentioning

confidence: 99%

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Improved effects of a double suicide gene system on prostate cancer cells by targeted regulation of prostate‐specific membrane antigen promoter and enhancer

et al. 2008

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Objective: To explore the specific killing effect on prostate cancer cells of a dual cytosine deaminase (CD) and uracil phosphoribosyltransferase (UPRT) expression plasmid system controlled by the prostate-specific membrane antigen (PSMA) promoter and enhancer. Methods: The CD gene was used to construct the recombinant plasmid prostate-specific membrane antigenpromoter/enhancer-CD (pPSMAE/P-CD). The specific regulatory function of the pPSMAE/P promoter was demonstrated by detection of enhanced green fluorescent protein (EGFP) expression in the LNCaP cell line. Survival of cells transfected with different plasmids and treated with 5-fluorocytosine (5-FC) was measured by microculture tetrazolium assay. Cell cycle changes were measured by flow cytometry. Results: Target-specific expression of PSMAE/P was observed in the prostate cancer cell line. Cytotoxicity of 5-FC was greater against LNCaP cells transfected with pPSMAE/P-CD and UPRT and pPSMAE/P-CD than control groups. Percentages of cells in S phase were 37.5% (LNCaP) and 30.6% (5-FC treatment) in the un-transfected groups, whereas they were 23.9% and 12.4% in the double and single suicide gene groups, respectively. Conclusions: Our findings confirm the cytotoxic efficacy of the pPSMAE/P-CD + 5-FC and pPSMAE/P-CD and UPRT + 5-FC suicide gene systems. The CD and UPRT gene system quickly and directly converted 5-FC into 5-FU, and then into toxic metabolites. The CD and UPRT double suicide gene system was more effective in inducing tumor cell apoptosis with 5-FC than the single suicide gene system. Thus, this construct can specifically target prostate cancer cells and might have a role in gene therapy against prostate cancer.

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Section: Introductionsupporting

confidence: 71%

Section: Methodsmentioning

confidence: 99%

Improved effects of a double suicide gene system on prostate cancer cells by targeted regulation of prostate‐specific membrane antigen promoter and enhancer

et al. 2008

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“…More important, our results serve as a proof of principle to deploy alternative PCa-specific promoters that can distinguish malignant and benign disease. Several genes are upregulated at the transcriptional level specifically in PCa, including Differential Display Code 3 (DD3/PCA3), prostate- specific membrane antigen, and a-methylacyl-CoA racemase (32–34). Given the extensive track record of the TSTA method, it should be feasible to use the promoters of these genes to develop a parallel transcription-amplified imaging strategy to specifically image intraprostatic PCa foci and better guide treatments.…”

Section: Discussionmentioning

confidence: 99%

In Vivo Imaging of Intraprostatic-Specific Gene Transcription by PET

Pouliot

Karanikolas²,

Johnson³

et al. 2011

J Nucl Med

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Better intraprostatic cancer imaging techniques are needed to guide clinicians in prostate cancer treatment decisions. Because many genes are specifically overexpressed in cancer cells, one strategy to improve prostate cancer detection is to image intraprostatic cancer-specific transcriptional activity. Because of the obstacles of weak cancer-or tissue-specific promoter activity and bladder clearance of many PET tracers, intraprostatic PET of gene transcriptional activity has not been previously reported. Methods: The two-step transcriptional amplification (TSTA) system that amplifies the prostate-specific antigen promoter activity was used for PET imaging of the reporter gene herpes simplex virus type-1 sr39 thymidine kinase (HSV1-sr39tk). The TSTA-sr39tk system was injected directly into prostates or prostatic tumors as a replication-incompetent adenovirus (AdTSTA-sr39tk) and imaged using PET. Results: AdTSTA-sr39tk was able to image prostate-specific antigen promoter transcriptional activity by 9-(4-18 F-fluoro-3-[hydroxymethyl]butyl)guanine PET, in both mouse and canine prostates in vivo. Ex vivo small-animal PET images, scintigraphic counts, and sr39tk expression analysis confirmed the specificity of the observed signal. Conclusion: Here, by combining the TSTA-amplified signal with a protocol for tracer administration, we show that in vivo PET detection of transcriptional activity is possible in both mouse and immunocompetent canine prostates. These results suggest that imaging applications using transcription-based tumor-specific promoters should be pursued to better visualize cancer foci that escape detection by conventional biopsies.

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“…The tandem structure of a single enhancer and a promoter exerts the strongest driving effect with a driving capability 53 times of that of the PSMA promoter alone. Importantly, EGFP and UPRT are only expressed in PSMA‐expressing LNCaP cells (Zeng et al, 2005). These results not only proved the cell‐specific driving capabilities of PSMAe/p but also laid a solid foundation for targeted therapy of prostate cancer.…”

Section: Introductionmentioning

confidence: 99%

Cell‐selective gene silencing in prostate cancer LNCap cells using prostate‐specific membrane antigen promoter and enhancer in vitro and in vivo

Liu

Sun

2012

Cell Biology International

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RNAi (RNA interference) has been widely used to silence specific genes. However, RNAi may also cause off‐target silencing and elicit non‐specific side effects. To achieve cell‐specific gene silencing, a cell‐selective promoter has to be used to drive RNAi expression. Furthermore, different terminators of cell‐selective promoters may cause different silencing efficacies. In order to explore the best promoter and terminator combination and prove the cell‐selective gene silencing effect of PSMAe/p (prostate‐specific membrane antigen enhancer/promoter), we first constructed three plasmids by using PSMAe/p and three different terminators [poly(A), minipoly(A) and poly(U)] to explore the cell‐selective driving ability of PSMAe/p by targeting EGFP (enhanced green fluorescent protein) in LNCaP, PC‐3, EJ and HEK‐293 (human embryonic kidney) cells. Then we chose NS (nucleostemin), an important endogenous gene of prostate cancer, and constructed the NS‐targeting shRNA (small‐hairpin RNA) expression plasmid by using PSMAe/p—poly(A) combination. Cell proliferation, cell cycle and early apoptosis in vitro and xenograft tumour growth in BALB/c nude mice in vivo were detected after NS knockdown. Results showed that PSMAe/p can drive EGFP silencing in LNCaP, not in PC‐3, EJ and HEK‐293 cells and PSMAe/p—poly(A) combination achieved the best silencing efficacy. Then PSMAe/p‐shNS‐poly(A) drives NS knockdown in LNCaP cells, not in PC‐3, EJ and HEK‐293 cells. Furthermore, RNAi‐mediated NS knockdown not only reduces cell proliferation rate, reduces the percentage of S‐stage cells and increases the percentage of G1‐stage cells and increases the early apoptosis ratio in LNCaP cells in vitro, but also inhibited the LNCaP xenograft tumour growth in BALB/c nude mice in vivo by intratumoural injection. In conclusion, we have demonstrated that PSMAe/p—poly(A) combination is a promising delivery system for targeted RNAi gene therapy of prostate cancer. We showed one effective antitumour strategy by targeting NS protein, an important target in prostate cancer, with PSMAe/p‐shNS‐poly(A). These results serve as an important step for developing novel strategies to treat prostate cancer.

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Construction of Prostate‐Specific Expressed Recombinant Plasmids With High Transcriptional Activity of Prostate‐Specific Membrane Antigen (PSMA) Promoter/Enhancer

Cited by 14 publications

References 21 publications

Improved effects of a double suicide gene system on prostate cancer cells by targeted regulation of prostate‐specific membrane antigen promoter and enhancer

Improved effects of a double suicide gene system on prostate cancer cells by targeted regulation of prostate‐specific membrane antigen promoter and enhancer

In Vivo Imaging of Intraprostatic-Specific Gene Transcription by PET

Cell‐selective gene silencing in prostate cancer LNCap cells using prostate‐specific membrane antigen promoter and enhancer in vitro and in vivo

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