Identification of human uroplakin II promoter and its use in the construction of CG8840, a urothelium-specific adenovirus variant that eliminates established bladder tumors in combination with docetaxel.
Uroplakins (UPs) are a group of integral membrane proteins that are synthesized as the major differentiation products of mammalian urothelium. UPII gene expression is bladder specific and differentiation dependent, but very little is known about its transcription response elements. To identify the promoter elements, a DNA fragment of 2239 bp upstream of the UPII gene was amplified by PCR and linked to a promoterless firefly luciferase reporter gene. Transient transfection experiments showed that the DNA segment located between -1809 and +1 bp resulted in preferential expression in bladder carcinoma cells with negligible expression in nonurothelial cells. This promoter was engineered into adenovirus (Ad) type 5 to drive the expression of the E1A and E1B genes and to create an attenuated replication-competent Ad variant, termed CG8840. Viral replication and the cytopathic effect of CG8840 were evaluated by virus yield and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays in bladder transitional cell carcinoma (TCC) cell lines RT4 and SW780; nonbladder cancer cell lines G361 (melanoma), LNCaP (prostate cancer), PA-1 (ovarian cancer), and U118 (brain cancer); and human primary cells including lung fibroblasts, bladder smooth muscle cells, and mammary epithelial cells. CG8840 replicated in and eliminated bladder TCC efficiently with high specificity (10,000:1) in comparison with nonbladder cells. The antitumor activity of CG8840 was examined in BALB/c nu/nu mice carrying s.c. human TCC xenografts. Intratumoral and i.v. administration of CG8840 in RT4 human bladder cancer xenografts caused significant (P < 0.01) inhibition of tumor growth. Synergistic antitumor efficacy was observed when CG8840 was combined with docetaxel, resulting in significant regression of RT4 bladder cancer xenograft tumors within 6 weeks after i.v. administration of CG8840 (3.33 x 10(9) plaque-forming units/animal on day 1) and docetaxel (20 mg/kg on days 2, 6, and 9). These results demonstrate the utility of the UPII promoter in the generation of urothelium-specific adenoviral vectors and provide a potential foundation for the development of bladder tumor-specific oncolytic viral therapies.
Zhang
Jane
J
Cell Genesys, Inc., Foster City, California 94404, USA.
Ramesh
Nagarajan
N
Chen
Yu
Y
Li
Yuanhao
Y
Dilley
Jeanette
J
Working
Peter
P
Yu
De-Chao
DC
D017501:Adenovirus E1B Proteins / Q000235:genetics; D000260:Adenoviruses, Human / Q000235:genetics / Q000502:physiology*; D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D002295:Carcinoma, Transitional Cell / Q000188:drug therapy / Q000235:genetics / Q000628:therapy* / Q000821:virology; D003131:Combined Modality Therapy; D003588:Cytopathogenic Effect, Viral; D000077143:Docetaxel; D004357:Drug Synergism; D017353:Gene Deletion; D005809:Genes, Regulator; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics*; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D017239:Paclitaxel / Q000031:analogs & derivatives* / Q000494:pharmacology*; D011401:Promoter Regions, Genetic / Q000235:genetics; D043823:Taxoids; D001749:Urinary Bladder Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000628:therapy* / Q000821:virology; D060170:Uroplakin II; D014779:Virus Replication / Q000235:genetics / Q000502:physiology; D023041:Xenograft Model Antitumor Assays
D017501:Adenovirus E1B Proteins; D000972:Antineoplastic Agents, Phytogenic; D008565:Membrane Proteins; D043823:Taxoids; C554658:UPK2 protein, human; C554659:Upk2 protein, mouse; D060170:Uroplakin II; D000077143:Docetaxel; D017239:Paclitaxel