Effects Of Recombinant Adenovirus-mediated ARE/SUZ12-regulated TK/GCV On Chronic Myelogenous Leukemia Blast Crisis Cells

Objective Clinically, blast phase chronic myeloid leukemia remainsa challenge, with a dilemma for the lack of therapeutic agent that providesremission. In CML-BC cells, Nrf2was widely demonstrated to beactivated, which can function to stimulate the transcriptional activation ofthe cytoprotective and detoxification genes by recognizing antioxidantresponse elements (ARE) of the genes. Moreover, Pizzatti et al. reportedSUZ12was overexpressed in CML-BC cells, due to the activation ofSUZ12promoter by the constitutively active WNT pathway, includingWNT11and WNT5A. Therefore, in this study, we explored to eliminateCML-BC cells using adenoviral (Ad) vectors expressing HSV-TK systemunder the dual control of specific SUZ12promoter and ARE element.Methods (1) Three fragments were designed for SUZ12corepromoter sequences, based on bioinformatics databases and literatures, andthe promoter activity were tested by luciferase report system, respectively.The recombinant adenoviral plasmids were produced using the AdEasy system based on the constructed shuttle plasmids, includingpAdtrack-SUZ-TK, pAdtrack-ARE/SUZ-TK, pAdtrack-SUZ-Luc andpAdtrack-ARE/SUZ-Luc, and the recombinant replication-deficientadenoviruses were packed in293cells.(2) After transfecting Ad-AS-Luc,Ad-S-Luc or Ad-control into K562、 K562-G01、KCL22, with HepG2cells as control, luciferase assay was conducted to evaluate the promoteractivity of composite element. After transfecting Ad-AS-TK, Ad-S-TK orAd-control into CML-BC cells and HepG2, the killing effect of theARE/SUZ12dual specific suicide gene system was assessed by CCK8，Wright’s staining，Annexin V flow cytometry analysis。Results (1) Among the three sequences in the upstream of SUZ12promoter, the-2.3kb fragment demonstrated a stronger activity.Ad-AS-Luc, Ad-S-Luc, Ad-AS-TK and Ad-S-TK adenoviruses weresuccessfully packed in293cells, by recombining the AdEasy system withthe constructed shuttle plasmids, including pAdtrack-SUZ-TK,pAdtrack-ARE/SUZ-TK, pAdtrack-SUZ-Luc andpAdtrack-ARE/SUZ-Luc.(2) After incubation CML-BC cells withAd-AS-Luc, Ad-S-Luc, Ad-control, ARE/SUZ12combined device showeda stronger promoter activity. Similarly, after incubation CML-BC cellswith Ad-AS-TK, Ad-S-TK, Ad-control, TK/GCV system under theregulation of ARE/SUZ12significantly inhibited the proliferation andinduced the apoptosis. Conclusion This study demonstrated that ARE/SUZ12-dual targetingHSV-TK/GCV therapy was effective in killing CML-BC cells, whichmight be an attractive therapeutic approach for curing CML-BC patients inthe future.