title: Cells exposed to antifolates show increased cellular levels of proteins fused to dihydrofolate reductase: a method to modulate gene expression.
abstract: Human cells exposed to antifolates show a rapid increase in the levels of the enzyme dihydrofolate reductase (DHFR). We hypothesized that this adaptive response mechanism can be used to elevate cellular levels of proteins fused to DHFR. In this study, mouse cells transfected to express a green fluorescent protein-DHFR fusion protein and subsequently exposed to the antifolate trimetrexate (TMTX) showed a specific and time-dependent increase in cellular levels of the fusion protein. Next, human HCT-8 and HCT-116 colon cancer cells retrovirally transduced to express a DHFR-herpes simplex virus 1 thymidine kinase (HSV1 TK) fusion protein and treated with the DHFR inhibitor TMTX exhibited increased levels of the DHFR-HSV1 TK fusion protein and an increase in ganciclovir sensitivity by 250-fold. The level of fusion protein in antifolate-treated human tumor cells was increased in response to a 24-h exposure of methotrexate, trimetrexate, as well as dihydrofolate. This effect depended on the antifolate concentration and was independent of the fusion-protein mRNA levels, consistent with this increase occurring at a translational level. In a xenograft model, nude rats bearing DHFR-HSV1 TK-transduced HCT-8 tumors and treated with TMTX showed, after 24 h, a 2- to 4-fold increase of fusion-protein levels in tumor tissue from treated animals compared with controls, as determined by Western blotting. The fusion-protein increase was imaged with positron-emission tomography, where a substantially enhanced signal of the transduced tumor was detected in animals after antifolate administration. Drug-mediated elevation of cellular DHFR-fused proteins is a very useful method to modulate gene expression in vivo for imaging as well as therapeutic purposes.
authors.affiliation: Molecular Pharmacology and Therapeutics Program, Department of Surgery, Nuclear Medicine Service, Radiochemistry/ Cyclotron, and Preparative Synthesis Chemistry Core Facilities, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.
authors.forename: Philipp
authors.initials: P
authors.lastname: Mayer-Kuckuk
authors.forename: Debabrata
authors.initials: D
authors.lastname: Banerjee
authors.forename: Sundeep
authors.initials: S
authors.lastname: Malhotra
authors.forename: Mikhail
authors.initials: M
authors.lastname: Doubrovin
authors.forename: Marian
authors.initials: M
authors.lastname: Iwamoto
authors.forename: Tim
authors.initials: T
authors.lastname: Akhurst
authors.forename: Julius
authors.initials: J
authors.lastname: Balatoni
authors.forename: William
authors.initials: W
authors.lastname: Bornmann
authors.forename: Ronald
authors.initials: R
authors.lastname: Finn
authors.forename: Steven
authors.initials: S
authors.lastname: Larson
authors.forename: Yuman
authors.initials: Y
authors.lastname: Fong
authors.forename: Juri
authors.initials: J
authors.lastname: Gelovani Tjuvajev
authors.forename: Ronald
authors.initials: R
authors.lastname: Blasberg
authors.forename: Joseph R
authors.initials: JR
authors.lastname: Bertino
mesh_terms: D000818:Animals; D003110:Colonic Neoplasms / Q000235:genetics / Q000378:metabolism / Q000628:therapy; D004305:Dose-Response Relationship, Drug; D004790:Enzyme Induction / Q000187:drug effects; D005492:Folic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D005493:Folic Acid Antagonists / Q000494:pharmacology*; D015774:Ganciclovir / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects*; D018259:Herpesvirus 1, Human / Q000201:enzymology / Q000235:genetics; D006801:Humans; D008727:Methotrexate / Q000494:pharmacology; D051379:Mice; D009368:Neoplasm Transplantation; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D013937:Thymidine Kinase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D014055:Tomography, Emission-Computed; D014161:Transduction, Genetic; D014183:Transplantation, Heterologous; D016597:Trimetrexate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
chemical_list: D005493:Folic Acid Antagonists; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C010920:dihydrofolate; D005492:Folic Acid; D013762:Tetrahydrofolate Dehydrogenase; D013937:Thymidine Kinase; D015774:Ganciclovir; D016597:Trimetrexate; D008727:Methotrexate