Apoptosis of bladder transitional cell carcinoma T24 cells induced by adenovirus-mediated inducible nitric oxide synthase gene transfection

Abstract

Objectives: To investigate the effects of adenovirus-mediated inducible nitric oxide synthase gene transfection on bladder transitional cell carcinoma T24 cells, and to provide novel insights and approaches to clinical therapies against bladder transitional cell carcinoma.

Methods: Firstly, construct recombinant adenovirus vector pAd-iNOS of iNOS, followed by transfection of pAd-iNOS into HECK293 packaging cells. Thirdly, harvest recombinant adenovirus rAd-iNOS after amplification and purification procedures. Finally, transfect the recombinant adenovirus rAd-iNOS into human bladder carcinoma T24 cells and examine the effect of rAd-iNOS transfection on apoptosis of T24 and possible mechanism.

Results: As shown by this study, the recombinant adenovirus rAd-iNOS was constructed successfully. The virus titer was 5.8×10(8) PFU/mL and recombinant was verified by PCR analysis. Transfection of adenovirus rAd-iNOS into T24 cells could induce secretion of high NO concentration, P53 protein expression up-regulation, as well as promotion of T24 cell apoptosis.

Conclusions: The transfection of human bladder carcinoma T24 cells from recombinant adenovirus rAd-iNOS was confirmed to induce intracellular iNOS over-expression, high production of NO, up-regulation of intracellular P53 expression and promotion of cell apoptosis.

Keywords: Bladder cancer; T24 cell line; gene therapy; iNOS gene.

Figure 1

Electropherogram for the identification of rAd-iNOS PCR.

Figure 2

Morphological changes of HEK293 cells packaging adenovirus rAd-Inos. (A) HEK293 cell lines; (B) HEK293 cells infected with Adenovirus.

Figure 3

Expression differences of iNOS and P53 mRNA in cells of each group by RT-PCR. (A) Recombinant adenovirus rAd-iNOS infection group; (B) Negative control group of Adenovirus infection; (C) Blank control group.

Figure 4

Expression differences of iNOS and P53 mRNA in cells of each group by western blot. (A) Recombinant adenovirus rAd-iNOS infection group; (B) Negative control group of Adenovirus infection; (C) Blank control group.

Figure 5

Fluorescence images of double-stained 24 cells from the blank control group (10×10).

Figure 6

Fluorescence images of double-stained 24 cells from the T24 cells with adenovirus infection control (10×10).

Figure 7

Fluorescence images of double-stained 24 cells from the T24 cells with recombinant adenovirus infection (10×10).

Figure 8

Scatter plots of bivariate flow cytometry. (A) denotes blank control group; (B) denotes Adenovirus infection control group; (C) denotes recombinant adenovirus rAd-iNOS infection group.