Retroviral transduction of a mutant dihydrofolate reductase-thymidylate synthase fusion gene into murine marrow cells confers resistance to both methotrexate and 5-fluorouracil.
Gene transfer-based myeloprotection strategies against chemotherapy require the development of effective drug resistance genes or gene combinations. Our laboratory has previously generated drug-resistant mutants of dihydrofolate reductase (DHFR F/S) and thymidylate synthase (TS G52S) for myeloprotection against methotrexate (MTX) and 5-fluorouracil (5-FU), respectively. For the purpose of conferring dual myeloprotection against both MTX and 5-FU, we have generated two retroviral constructs encoding both DHFR F/S and TS G52S as a fusion protein (DHFR F/S-TS G52S) or as individual proteins from a bicistronic gene. The DHFR F/S-TS G52S fusion protein is functional and exhibits kinetic properties similar to that of the individual mutant enzymes. NIH 3T3 cells and mouse bone marrow progenitors retrovirally transduced with the fusion DHFR F/S-TS G52S cDNA provided similar levels of resistance to MTX and 5-FU as cells expressing the individual mutant enzymes and higher levels of resistance to MTX than cells expressing DHFR F/S from the 3' end of a bicistronic gene. As MTX and 5-FU are used in combination therapy for diseases such as breast and colon cancer, this fusion gene may be useful in the clinic to reduce myelosuppressive toxicity associated with this drug combination.
Capiaux
Gina M
GM
Graduate School of Medical Sciences, Cornell University, New York, NY 10021, USA.
Budak-Alpdogan
Tulin
T
Takebe
Naoko
N
Mayer-Kuckuk
Philipp
P
Banerjee
Debabrata
D
Maley
Frank
F
Bertino
Joseph R
JR
D016475:3T3 Cells; D000818:Animals; D015153:Blotting, Western; D001854:Bone Marrow Cells / Q000187:drug effects* / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004351:Drug Resistance; D005472:Fluorouracil / Q000633:toxicity*; D015870:Gene Expression; D005822:Genetic Vectors; D008297:Male; D008727:Methotrexate / Q000633:toxicity*; D051379:Mice; D008810:Mice, Inbred C57BL; D009154:Mutation; D014176:Protein Biosynthesis; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D012190:Retroviridae / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013762:Tetrahydrofolate Dehydrogenase / Q000032:analysis / Q000235:genetics*; D013940:Thymidylate Synthase / Q000235:genetics*; D014161:Transduction, Genetic
D011993:Recombinant Fusion Proteins; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase; D005472:Fluorouracil; D008727:Methotrexate