Enforced expression of cytosolic 5'-nucleotidase I confers resistance to nucleoside analogues in vitro but systemic chemotherapy toxicity precludes in vivo selection.
OBJECTIVE
Retroviral transfer of cDNA sequences that confer drug resistance can be used to protect against chemotherapy-induced hematopoietic toxicity and for the selective expansion of gene-modified cells. To successfully expand genetically engineered cells in vivo, an appropriate balance must be achieved between systemic toxicity induced by the selecting agent and the expansion of modified cells.


METHODS
In this study, we investigate retroviral transfer of cytosolic 5'-nucleotidase I (cN-I) for protection and selection of gene-modified cells when treated with 2-chloro-2'-deoxyadenosine (2-CdA) and 5-fluorouracil (5-FU) alone and in combination. We also attempt to design a treatment strategy for the potential in vivo selection of cN-I-modified cells by administering 5-FU to mice prior to 2-CdA treatment.


RESULTS
Our results show that cN-I can be transferred by recombinant retroviruses, and that enforced expression of cN-I protects murine fibroblast and hematopoietic progenitor cells from the cytotoxic effects of 2-CdA and/or 5-FU. Furthermore, we show that the combined administration of 5-FU and 2-CdA potentiates hematopoietic stem cell toxicity. However, the treatment also results in severe myelosuppression.


CONCLUSIONS
These results show that while cN-I provides both protective and selective benefits to gene-modified cells in vitro, selection requires a treatment strategy that is likely too toxic to consider cN-I as an in vivo selectable marker.
Gray
Tasha
T
Division of Animal Resources, Emory University, 2015 Uppergate Drive, Room 420, Atlanta, GA 30322, USA.
Morrey
Erin L
EL
Gangadharan
Bagirath
B
Sumter
Takita F
TF
Spychala
Jozef
J
Archer
David R
DR
Spencer
H Trent
HT
D015720:5'-Nucleotidase / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001854:Bone Marrow Cells / Q000187:drug effects; D002470:Cell Survival; D017338:Cladribine / Q000494:pharmacology*; D003600:Cytosol / Q000201:enzymology; D018432:Drug Resistance, Multiple; D005472:Fluorouracil / Q000494:pharmacology*; D051379:Mice; D008815:Mice, Inbred Strains; D041681:NIH 3T3 Cells; D012190:Retroviridae / Q000235:genetics; D014161:Transduction, Genetic
D000970:Antineoplastic Agents; D017338:Cladribine; D015720:5'-Nucleotidase; D005472:Fluorouracil