Resistance to cytarabine and gemcitabine and in vitro selection of transduced cells after retroviral expression of cytidine deaminase in human hematopoietic progenitor cells

Abstract

Overexpression of the detoxifying enzyme cytidine deaminase (CDD) renders normal and leukemic hematopoietic cells resistant to cytarabine (1-b-D-arabinofuranosylcytosine), and studies on murine cells have suggested transgenic CDD overexpression as a way to reduce the substantial myelotoxicity induced by the deoxycytidine analogs cytarabine and gemcitabine (2 0 ,2 0 -difluorodeoxycytidine). We now have investigated CDD (over-)expression in the human hematopoietic system. Retroviral gene transfer significantly increased the resistance of CDD-transduced cord blood and peripheral bloodderived progenitor cells for doses ranging from 20 to 100 nM cytarabine and 8-10 nM gemcitabine. Protection was observed for progenitors of erythroid as well as myeloid differentiation, though the degree of protection varied for individual drugs. In addition, significant selection of CDD-transduced cells was obtained after a 4-day culture in 30-100 nM cytarabine. Thus, our data demonstrate that overexpression of CDD cDNA results in significant protection of human progenitors from cytarabineas well as gemcitabine-induced toxicity, and allows in vitro selection of transduced cells. This strongly argues for a potential therapeutic role of CDD gene transfer in conjunction with dose-intensive cytarabine-or gemcitabine-containing chemotherapy regimen.

Nucleoside-catabolizing Enzymes in Mycoplasma-infected Tumor Cell Cultures Compromise the Cytostatic Activity of the Anticancer Drug Gemcitabine

“…Previous research also demonstrated a decreased activity of dCyd analogues such as gemcitabine and cytarabine (araC; an antitumor drug that is also subject to deamination) in Cyd deaminaseoverexpressing human tumor cells (40,41) and progenitor cells (42). The activity of such antitumor molecules could be counteracted by co-administration of THU (40).…”

Gene transfer of cytidine deaminase protects myelopoiesis from cytidine analogs in an in vivo murine transplant model

“…14 To generate high-titer ecotropic producer cell lines, supernatant from these PG13 producer clones was used for transduction of FNX-eco cells. 17 FNX-eco cells (3 ϫ 10 4 ) were cultured in 6-well plates in DMEM supplemented with 10% FCS, 2 mM L-glutamine, 100 U/mL penicillin and 100 U/mL streptomycin (P/S), and 20 mM HEPES (all from Sigma-Aldrich, Taufkirchen, Germany), and were incubated with 1 mL supernatant for 4 hours in the presence of 8 g/mL Polybrene (1,5-dimethyl-1,5-diazaundecamethylene polymethobromide, hexadimethrine bromide; Sigma-Aldrich) for 3 times on 3 consecutive days.…”

“…13 More recently, our group has investigated CDD gene transfer into primary human hematopoietic cells demonstrating significant resistance to cytarabine and gemcitabine as well as successful in vitro selection of transduced progenitor cells following retrovirally mediated gene transfer and cytarabine application. 14 Studies investigating CDD gene transfer in relevant murine in vivo models, however, are scarce and so far have failed to define the exact myeloprotective potential of this approach. Although one study demonstrated long-term expression of the CDD transgene in the hematopoietic system but did not address in vivo hematoprotection, 15 successful treatment of a lymphoma cell line with a combination of methotrexate and cytarabine in NOD/SCID mice given transplants with drug-resistant murine bone marrow was demonstrated in another model.…”

A ubiquitous chromatin opening element prevents transgene silencing in pluripotent stem cells and their differentiated progeny

“…CDD represents a clinically highly relevant CTX-R gene, as overexpression of this enzyme protects lymphohematopoietic cells from deoxycytidine analogs such as cytosine-arabinoside (1-b-D-arabinofuranosylcytosine, Ara-C), the most effective single-agent in the treatment of acute leukemias. Myeloprotection as well as chemoselection by hCDD gene transfer has been established in murine and human clonogenic progenitor cells [21][22][23] as well as in murine in vivo bone marrow transplant models [24][25][26]. However, in leukemias, HSC-based myeloprotective gene therapy approaches always carry the risk of inadvertent transduction of malignant cells.…”