Cloning and analysis of human UroplakinII promoter and its application for gene therapy in bladder cancer

Abstract

The differential expression of the desired gene product in the target tissue is central for gene therapy. One approach is to use a tissue-specific promoter to drive therapeutic gene expression. UroplakinII (UPII) is a urothelium-specific membrane protein. To investigate the feasibility of targeting gene therapy for bladder cancer, a DNA fragment of 2542-bp upstream of the UPII gene was amplified by PCR and linked to a promoterless firefly luciferase reporter gene. The transient transfection showed that the DNA fragment resulted in preferential expression in bladder carcinoma cells, with negligible expression in nonurothelium cells. Furthermore, the DNA segment located between À2545 and À1608 decided the tissue-specificity of the UPII promoter, the segment located between À328 and À4 being the core promoter of UPII. We generated two recombinant adenoviruses under the control of the UPII promoter: Ad-hUPII-GFP, carrying green fluorescence protein (GFP), and Ad-hUPII-TNF, carrying the tumor necrosis factor alpha (TNFa). ELISA revealed that the secretion of TNFa by Ad-hUPII-TNF-infected bladder cancer cells was significantly higher than Ad-hUPII-TNF-infected nonurothelium cells. The conditioned medium from Ad-hUPII-TNF-infected bladder cancer cells apparently inhibited the proliferation of L929 cells, a TNFa-sensitive cell line, comparing to Ad-hUPII-TNF-infected nonurothelium cells. Intravesical inoculation with Ad-hUPII-TNF inhibited tumor growth in the orthotopic human bladder cancer model. The sustained high level of TNFa in urine was identified with ELISA. Taken together, these data suggest that most of the cis elements that confer the bladder-specificity and differentiationdependent expression of the human UPII gene reside in the 2542-bp sequence, and TNFa driven by the human UPII (hUPII) promoter is effective in the specific inhibition of bladder cancer growth both in vivo and in vitro. These results may yield a new therapeutic approach for bladder cancer and provide information on the molecular regulation of urothelial growth, differentiation, and disease.

Electrostatic interaction of tumor-targeting adenoviruses with aminoclay acquires enhanced infectivity to tumor cells inside the bladder and has better cytotoxic activity

“…Development of gene therapy in bladder cancer has focused on modifying the mutated urothelial cells and restoring normal functions of tumor suppressor genes. In previous studies, adenovirus-mediated gene therapies for bladder cancer patients have been developed in the following strategies: (1) to induce apoptosis by delivering tumor suppressor genes, such as p53 (Werthman et al., 1996 ); (2) to overexpress toxic genes (El-Zawahry et al., 2006 ); (3) to enhance host anticancer immunity by overexpressing immune modulators (Adam et al., 2007 ); and (4) to lyse cancer cells by virus amplification (Zhu et al., 2004 ). In those strategies, adenovirus-based gene therapy has demonstrated significant tumor-suppressive efficacy in preclinical models but has shown no significant benefits in the clinic.…”

Antitumor effects of bladder cancer-specific adenovirus carrying E1A-androgen receptor in bladder cancer

“…33 There is a broad perspective to treating bladder cancer by targeting UPII promoter and a powerful antitumor effect in vitro and in vivo . 34–36 …”

The possibilities of practical application of transgenic mammalian species generated by somatic cell cloning in pharmacology, veterinary medicine and xenotransplantology

“…Although many reports have described the mouse UPII (mUPII) promoter as primarily urothelium selective so far, ectopic (most often negligible) expression of a transgene under the 3.6 kb mUPII promoter was also detected in the brain, kidneys and testis of some transgenic mouse lines and murine cell lines (Lin et al 1995, Kerr et al 1998, Zhu et al 2004, Kwon et al 2006. Kwon et al (2006) cloned an 8.8 kb pig UPII (pUPII) promoter region and investigated which cells within the bladder and urethra express a transgene consisting of the pUPII promoter fused to recombinant human erythropoietin (rhEPO) or a luciferase gene.…”

“…Furthermore, it has been shown that mammalian urothelial plaques contain four major integral membrane proteins: UP-Ia (27 kDa), UP-Ib (28 kDa), UPII (15 kDa), and UPIII (47 kDa) . Immunohistochemical survey of various tissues found UPs to be mammalian urothelium specific proteins that represent excellent markers for an advanced stage of urothelial differentiation (Lin et al 1995, Zhu et al 2004.…”