Confluent T24P and HT-1376 human bladder carcinoma cells were subcutaneously injected into the back of female CD1 nude mice, 6-8 weeks old.10 days after cell inoculation the developing tumors were measured. The ability to inhibit tumor growth by the single promoter DTA expression vectors (P4-DTA, H19-DTA) and by the double promoter DTA expression vector (H19-DTA-P4-DTA) was tested. Intratumoral injections of 25 μg of either DTA expressing constructs or Luc expressing constructs were given 10, 12 and 14 days after cells inoculation. In vivo Jet-PEI/DNA complexes with a ratio of PEI nitrogen to DNA phosphate of 6 were prepared in a solution of 5% w/v glucose. Tumor dimensions were measured, and the tumor volume was calculated according to the formula width2 × length × 0.5. The animals were sacrificed 3 days after the last treatment, the tumors were excised and their ex-vivo weight and volume were measured.

BACKGROUND: The human IGF2-P4 and H19 promoters are highly active in a variety of human cancers (including bladder cancer), while existing at a nearly undetectable level in the surrounding normal tissue.Single promoter vectors expressing diphtheria toxin A-fragment (DTA) under the control regulation of IGF2-P4 or H19 regulatory sequences (IGF2-P4-DTA and H19-DTA) were previously successfully used in cell lines, animal models and recently in human patients with superficial cell carcinoma of the bladder (treated with H19-DTA). However this targeted medicine approach could be limited, as not all cancer patients express high levels of H19. Hence, a double promoter DTA-expressing vector was created, carrying on a single construct two separate genes expressing the diphtheria toxin A-fragment (DTA), from two different regulatory sequences, selected from the cancer-specific promoters H19 and IGF2-P4. METHODS: H19 and IGF2-P4 gene expression was tested in samples of Transitional Cell Carcinoma (TCC) of the bladder by in-situ hybridization (ISH) and by quantitative Real-Time PCR (qRT-PCR). The therapeutic potential of the double promoter toxin vector H19-DTA-IGF2-P4-DTA was tested in TCC cell lines and in heterotopic and orthotopic animal models of bladder cancer. RESULTS: Nearly 100% of TCC patients highly expressed IGF2-P4 and H19, as determined by ISH and by qRT-PCR. The double promoter vector exhibited superior tumor growth inhibition activity compared to the single promoter expression vectors, in cell lines and in heterotopic and orthotopic bladder tumors. CONCLUSIONS: Our findings show that bladder tumors may be successfully treated by intravesical instillation of the double promoter vector H19-DTA-P4-DTA.Overall, the double promoter vector exhibited enhanced anti-cancer activity relative to single promoter expression vectors carrying either gene alone.