CYTOSINE DEAMINASE ADENOVIRAL VECTOR AND 5-FLUOROCYTOSINE SELECTIVELYREDUCE BREAST-CANCER CELLS 1 MILLION-FOLD WHEN THEY CONTAMINATE HEMATOPOIETIC-CELLS - A POTENTIAL PURGING METHOD FOR AUTOLOGOUS TRANSPLANTATION

Ad.CMV-CD is a replication incompetent adenoviral vector carrying a cy tomegalovirus (CMV)-driven transcription unit of the cytosine deaminas e (CD) gene. The CD transcription unit in this vector catalyzes the de amination of the nontoxic pro-drug, 5-fluorocytosine (5-FC), thus conv erting it to the cytotoxic drug 5-fluorouracil (5-FU). This adenoviral vector prodrug activation system has been proposed for use in selecti vely sensitizing breast cancer cells, which may contaminate collection s of autologous stem cells products from breast cancer patients, to th e toxic effects of 5-FC, without damaging the reconstitutive capabilit y of the normal hematopoietic cells. This system could conceivably kil l even the nondividing breast cancer cells, because the levels of 5-FU generated by this system are 10 to 30 times that associated with syst emic administration of 5-FU. The incorporation of 5-FU into mRNA at th ese high levels is sufficient to disrupt mRNA processing and protein s ynthesis so that even nondividing cells die of protein starvation. To test if the CD adenoviral vector sensitizes breast cancer cells to 5-F C, we exposed primary explants of normal human mammary epithelial cell s (HMECs) and the established breast cancer cell (BCC) lines MCF-7 and MDA-MB-453 to the Ad.CMV-CD for 90 minutes. This produced a 100-fold sensitization of these epithelial cells to the effects of 48 hours of exposure to 5-FC. We next tested the selectivity of this system for BC C. When peripheral blood mono nuclear cells (PBMCs), collected from ca ncer patients during the recovery phase from conventional dose chemoth erapy-induced myelosuppression, were exposed to the Ad.CMV-CD for 90 m inutes in serum-free conditions, little or no detectable conversion of 5-FC into 5-FU was seen even after 48 hours of exposure to high doses of 5-FC. In contrast, 70% of 5-FC was converted into the cytotoxic ag ent 5-FU when MCF-7 breast cancer cells (BCCs) were exposed to the sam e Ad.CMV-CD vector followed by 5-FC for 48 hours. All of the BCC lines tested were shown to be sensitive to infection by adenoviral vectors when exposed to a recombinant adenoviral vector containing the reporte r gene betagalactosidase (Ad.CMV-beta gal). In contrast, less than 1% of the CD34-selected cells and their more immature subsets, such as th e CD34(+)CD38(-) or CD34(+)CD33(-) subpopulations, were positive for i nfection by the Ad.CMV-beta gal vector, as judged by fluorescence-acti vated cell sorting (FACS) analysis, when exposed to the adenoviral vec tor under conditions that did not commit the early hematopoietic precu rsor cells to maturation. When artificial mixtures of hematopoietic ce lls and BCCs were exposed for 90 minutes to the Ad.CMV-CD vector and t o 5-FC for 10 days or more, a greater than 1 million fold reduction in the number of BCCs, as measured by colony-limiting dilution assays, w as observed. To test if the conditions were damaging for the hematopoi etic reconstituting cells, marrow cells collected from 5-FU-treated ma le donor mice were incubated with the cytosine deaminase adenoviral ve ctor and then exposed to 5-FC either for 4 days in vitro before transp lantation or for 14 days immediately after transplantation in vivo. Th ere was no significant decrease in the reconstituting capability of th e male marrow cells, as measured by their persistence in female irradi ated recipients for up to 6 months after transplantation. These observ ations suggest that adenovirus-mediated gene transfer of the Escherich ia coli cytosine deaminase gene followed by exposure to the nontoxic p ro-drug 5-FC may be a potential strategy to selectively reduce the lev el of contaminating BCCs in collections of hematopoietic cells used fo r autografts in breast cancer patients. (C) 1998 by The American Socie ty of Hematology.