Expression of the prodrug-activating enzyme DT-diaphorase via Ad5 delivery to human colon carcinoma cells in vitro

Abstract

Intratumoral injection of recombinant adenoviral type 5 ( Ad5 ) vectors that carry prodrug -activating enzymes like DT -diaphorase ( DTD ) could be used to selectively target tumor cells for chemotherapy. To demonstrate the feasibility of this approach, Ad5 vectors were constructed, which express human DTD minigenes for both wild -type and mutant ( C -to -T change in nucleotide 609 in DTD cDNA ) DTD under the control of the cytomegalovirus ( CMV ) promoter. HT29 human colon carcinoma cells express wild -type DTD, whereas BE human colon carcinoma cells express mutant DTD, have low to undetectable DTD activity, and are 4 -to 6 -fold more resistant to mitomycin C ( MMC ) than HT29 cells. A test of the ability of Ad5 to infect these cells ( using a -galactosidase CMVdriven minigene ) indicated that 90 -100% of BE cells were infected at a multiplicity of infection ( MOI ) of 100, whereas only 15 -40% of HT29 cells were infected at this MOI. Infection of BE cells in vitro with recombinant Ad5 carrying a minigene for wild -type DTD at MOIs of 3 -100 resulted in a progressive increase in DTD activity and a maximal 8 -fold increase in sensitivity to MMC as measured by a colony -forming assay. HT29 cells were sensitized 2 -to 3 -fold following treatment with Ad5.DTD at an MOI of 100. These results indicate that adenovirus -mediated gene transfer and expression of wild -type DTD can sensitize resistant tumor cells to MMC and that this therapeutic strategy may exert a significant bystander effect.

Tailoring heated intraperitoneal mitomycin C for peritoneal metastases originating from colorectal carcinoma: a translational approach to improve survival

“…Sensitivity to MMC is known to be correlated to DT-diaphorase, encoded by the NQO1 gene ( Fitzsimmons et al , 1996 ; Sharp et al , 2000 ; Misra et al , 2002 ), yet the DT-diaphorase overexpressing cell line HT-29 is only intermediately sensitive in our cohort and none of the mRNA products of the MMC activation pathway were significantly correlated to MMC sensitivity. Activation of MMC by DT-diaphorase might be of inferior importance in sensitivity in CRC cell lines, perhaps because of the abundance of other MMC-activating enzymes in human cells that readily transform MMC to its active metabolite ( Cummings et al , 1998 ; Phillips et al , 2000 ).…”

“…One possible way is to inhibit MMC activation as MMC requires enzymatic reduction to become active ( Cummings et al , 1998 ). Several reductases have this capacity, with DT-diaphorase being the most potent and widely researched ( Szybalski and Iyer, 1964 ; Plumb and Workman, 1994 ; Fitzsimmons et al , 1996 ; Misra et al , 2002 ). Absence of this enzyme has been known to lead to increased resistance to MMC in cancer cells ( Fitzsimmons et al , 1996 ).…”