Cytosine deaminase adenoviral vector and 5-fluorocytosine selectively reduce breast cancer cells 1 million-fold when they contaminate hematopoietic cells: A potential purging method for autologous transplantation

摘要: Ad.CMV-CD is a replication incompetent adenoviral vector carrying cytomegalovirus (CMV)-driven transcription unit of the cytosine deaminase (CD) gene. The CD in this catalyzes deamination nontoxic pro-drug, 5-fluorocytosine (5-FC), thus converting it to cytotoxic drug 5-fluorouracil (5-FU). This prodrug activation system has been proposed for use selectively sensitizing breast cancer cells, which may contaminate collections autologous stem cells products from patients, toxic effects 5-FC, without damaging reconstitutive capability normal hematopoietic cells. could conceivably kill even nondividing because levels 5-FU generated by are 10 30 times that associated with systemic administration 5-FU. incorporation into mRNA at these high sufficient disrupt processing and protein synthesis so die starvation. To test if sensitizes we exposed primary explants human mammary epithelial (HMECs) established cell (BCC) lines MCF-7 MDA-MB-453 90 minutes. produced 100-fold sensitization 48 hours exposure 5-FC. We next tested selectivity BCC. When peripheral blood mononuclear (PBMCs), collected patients during recovery phase conventional dose chemotherapy-induced myelosuppression, were minutes serum-free conditions, little or no detectable conversion 5-FC was seen after doses In contrast, 70% converted agent when (BCCs) same followed hours. All BCC shown be sensitive infection vectors recombinant containing reporter gene betagalactosidase (Ad.CMV-betagal). less than 1% CD34-selected their more immature subsets, such as CD34+CD38- CD34(+)CD33- subpopulations, positive Ad.CMV-betagal vector, judged fluorescence-activated sorting (FACS) analysis, under conditions did not commit early precursor maturation. artificial mixtures BCCs days more, greater 1 million fold reduction number BCCs, measured colony-limiting dilution assays, observed. reconstituting marrow 5-FU-treated male donor mice incubated then either 4 vitro before transplantation 14 immediately vivo. There significant decrease persistence female irradiated recipients up 6 months transplantation. These observations suggest adenovirus-mediated transfer Escherichia coli pro-drug potential strategy reduce level contaminating used autografts patients.