Cells exposed to antifolates show increased cellular levels of proteins fused to dihydrofolate reductase: A method to modulate gene expression Academic Article uri icon

Overview

MeSH Major

  • Folic Acid
  • Folic Acid Antagonists
  • Gene Expression Regulation
  • Recombinant Fusion Proteins
  • Tetrahydrofolate Dehydrogenase

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.

publication date

  • March 19, 2002

Research

keywords

  • Academic Article

Identity

Language

  • eng

PubMed Central ID

  • PMC122535

Digital Object Identifier (DOI)

  • 10.1073/pnas.062036899

PubMed ID

  • 11891321

Additional Document Info

start page

  • 3400

end page

  • 5

volume

  • 99

number

  • 6