2 million T24P and HT-1376 human bladder carcinoma cells were subcutaneously injected into the back of CD1 nude mice.10, 12 and 14 days after cell inoculation, 25 µg plasmid DNA were complexed with 3 µl in vivo-jetPEI (N/P ratio of 6) and intratumorally injected into the developing tumors. The mice were sacrificed 3 days after the last injection. CD1 nude mice were also used to develop orthotopic superficial bladder tumors. Mice were anesthetized and the bladder was catheterized. Subsequently, a 0.1 ml suspension of PBS containing 10 million T24P human bladder carcinoma cells was instilled into the bladder. Seven days after cell instillation, mice received 20 μg of the toxin vector H19-DTA-P4-DTA complexed with 2.4 μl of the jetPEI (N/P ratio = 6) in 50 μl glucose 5% (w/v).The resulting mixture was vortex-mixed and left for 10-15 minutes at room temperature and subsequently instilled into the mice bladder transurethrally using the catheter. The same treatments were repeated after 3 days.The animals were sacrificed 4 days after the last plasmid instillation.

BACKGROUND: The human IGF2-P3 and IGF2-P4 promoters are highly active in bladder carcinoma, while existing at a nearly undetectable level in the surrounding normal tissue. A double promoter DTA-expressing vector was created, carrying on a single construct two separate genes expressing diphtheria toxin A-fragment (DTA), from two different regulatory sequences, selected from the cancer-specific promoters IGF2-P3 and IGF2-P4. METHODS: IGF2-P3 and IGF2-P4 expression was tested in samples of urothelial carcinoma (UC) of the bladder (n=67) by RT-PCR or by ISH. The therapeutic potential of single promoter expression vectors (P3-DTA and P4-DTA) was tested and compared to the double promoter toxin vector P4-DTA-P3-DTA in UC cell lines and in heterotopic and orthotopic animal models for bladder cancer. RESULTS: Nearly 86% of UC patients highly expressed IGF2-P4 and IGF2-P3, as determined by ISH. The double promoter vector (P4-DTA-P3-DTA) exhibited superior ability to inhibit tumor development by 68% (P=0.004) compared to the single promoter expression vectors, in heterotopic bladder tumors. The average size of the P4-DTA-P3-DTA bladder tumors (in orthotopically treated mice) was 83% smaller (P<0.001) than that of the control group. CONCLUSIONS: Overall, the double promoter vector exhibited enhanced anti-cancer activity relative to single promoter expression vectors carrying either gene alone. Our findings show that bladder tumors may be successfully treated by intravesical instillation of the double promoter vector P4-DTA-P3-DTA.