Co-expression of the herpes simplex virus thymidine kinase gene potentiates methotrexate resistance conferred by transfer of a mutated dihydrofolate reductase gene.
We have previously shown that transfer of a mutated dihydrofolate reductase (DHFR) confers resistance to methotrexate (MTX) to infected cells. We report herein the construction of a retrovirus vector, DC/SV6S31tk, which carries the herpes simplex virus thymidine kinase gene (HSVtk) as well as the mutated Serine 31 DHFR (S31) cDNA. 3T3 cells infected with DC/SV6S31tk are more resistant to MTX than cells infected with DC/SV6S31, which carries the S31 and Neo gene. In DC/SV6S31tk-infected cells, a fraction of cells (20-40%) were more resistant to MTX compared with DC/SV6S31-infected cells, and these cells survived a 5-day exposure to 200 microM of MTX. The mechanism of this augmented resistance is attributed to the salvage of thymidine by HSVtk, as the augmentation is reversed when dialyzed serum is used for cytotoxicity assays. The cells that survive high-dose MTX selection have high levels of expression of S31 DHFR and HSVtk, although copy numbers of the proviral sequences do not increase significantly. Transduction of cells with the DC/SV6S31tk vector also sensitizes cells to ganciclovir (GCV). Co-expression of a metabolically related gene in a retroviral vector to potentiate the resistance imparted by a drug resistance gene may be useful for gene therapy for cancer patients.
Mineishi
S
S
Program of Molecular Pharmacology and Experimental Therapeutics, Memorial Sloan-Kettering Cancer Center, New York, NY, USA.
Nakahara
S
S
Takebe
N
N
Banerjee
D
D
Zhao
S C
SC
Bertino
J R
JR
D016475:3T3 Cells; D000818:Animals; D000964:Antimetabolites, Antineoplastic; D000998:Antiviral Agents / Q000627:therapeutic use; D001854:Bone Marrow Cells; D018920:Coculture Techniques; D019008:Drug Resistance, Neoplasm; D015774:Ganciclovir / Q000627:therapeutic use; D015870:Gene Expression; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D008297:Male; D008727:Methotrexate; D051379:Mice; D008810:Mice, Inbred C57BL; D009154:Mutation; D012190:Retroviridae; D018139:Simplexvirus / Q000201:enzymology*; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D013937:Thymidine Kinase / Q000235:genetics*
D000964:Antimetabolites, Antineoplastic; D000998:Antiviral Agents; D013762:Tetrahydrofolate Dehydrogenase; D013937:Thymidine Kinase; D015774:Ganciclovir; D008727:Methotrexate