Protein-Programmed Accumulation of Yeast Cytosine Deaminase in Cancer Cells in Response to Mock-Hypoxia

ACS Synth Biol. 2019 May 17;8(5):948-954. doi: 10.1021/acssynbio.9b00036. Epub 2019 Apr 23.

Abstract

One limitation of gene-directed enzyme prodrug therapy (GDEPT) is the difficulty in selectively and efficiently transducing cancer cells with the gene encoding a prodrug-converting enzyme. To circumvent this issue, we sought to move the selectivity from the gene delivery level to the protein level. We developed fusion proteins of the prodrug-activating enzyme yeast cytosine deaminase (yCD) and the oxygen-dependent degradation domain (ODDD) of HIF-1α, a domain that regulates the accumulation of HIF-1α in an oxygen-dependent manner. We called these HOPE fusions for HIF1-α Oxygen-dependent degradation domain/Prodrug-converting Enzyme. The HOPE fusions were designed to selectively accumulate in cells experiencing hypoxia and thus selectively cause conversion of the prodrug 5-fluorocytosine (5-FC) to the chemotherapeutic 5-fluorouracil (5-FU) where oxygen levels are low (e.g., at the center of a tumor). Consistent with our hypothesis, HT1080 fibrosarcoma cells transduced with HOPE fusion genes exhibited increased fusion protein accumulation and increased sensitization to 5-FC in mock-hypoxia.

Keywords: GDEPT; HIF-1α; enzyme/prodrug therapy; protein engineering; yeast cytosine deaminase.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Cell Hypoxia*
  • Cell Line, Tumor
  • Cell Survival / drug effects
  • Cobalt / toxicity
  • Cytosine Deaminase / genetics*
  • Fluorouracil / pharmacology
  • Humans
  • Hypoxia-Inducible Factor 1, alpha Subunit / chemistry
  • Hypoxia-Inducible Factor 1, alpha Subunit / genetics
  • Plasmids / genetics
  • Plasmids / metabolism
  • Protein Domains / genetics
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / pharmacology
  • Saccharomyces cerevisiae / enzymology*
  • Saccharomyces cerevisiae Proteins / genetics*

Substances

  • Hypoxia-Inducible Factor 1, alpha Subunit
  • Recombinant Fusion Proteins
  • Saccharomyces cerevisiae Proteins
  • Cobalt
  • Cytosine Deaminase
  • cobaltous chloride
  • Fluorouracil