Protein transduction with bacterial cytosine deaminase fused to the TLM intercellular transport motif induces profound chemosensitivity to 5-fluorocytosine in human hepatoma cells.
OBJECTIVE
This study investigates the application of protein based therapeutic suicide enzyme/prodrug approaches providing novel means for both safe and effective local therapeutic regimes in solid tumors.


METHODS
Employing a novel cell permeable peptide, known as the translocation motif (TLM) of hepatitis B virus, E. coli cytosine deaminase (BCDase) suicide fusion proteins were generated.


RESULTS
TLM fusion proteins formed hexamers (as do parental wtBCDase proteins) and retained the specific enzymatic activity of cytosine conversion to uracil also being comparable to parental wtBCDase proteins. However, only BCDase-TLM fusion proteins, but not TLM-BCDase fusion nor parental wtBCDase proteins were found to be taken up to the cytoplasm of target cells as demonstrated both by confocal laser scanning microscopy and cell fractionation. Uptake of BCDase-TLM worked both efficiently and rapidly and was found to be independent from the endosomal pathway. Since BCDase-TLM fusion proteins completely retained their suicide enzymatic activity in the course of translocation across the plasma membrane their usage as profound inducers of chemo-sensitivity to 5-FC strongly is suggested.


CONCLUSIONS
Future therapeutic local application of cell-permeable BCDase-TLM fusion proteins together with a systemic 5-FC prodrug application could result in profound antitumor activities without apparent side effects.
Hillemann
Annett
A
Robert Koch-Institute, Nordufer 20, D-13353 Berlin, Germany.
Brandenburg
Boerries
B
Schmidt
Ulrike
U
Roos
Martin
M
Smirnow
Irina
I
Lemken
Marie-Luise
ML
Lauer
Ulrich M
UM
Hildt
Eberhard
E
D000818:Animals; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular; D045744:Cell Line, Tumor; D002463:Cell Membrane Permeability; D043525:Cytosine Deaminase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology; D019084:Fluorescent Antibody Technique, Indirect; D005472:Fluorouracil / Q000494:pharmacology*; D005784:Gene Amplification; D006515:Hepatitis B virus / Q000378:metabolism; D006801:Humans; D008113:Liver Neoplasms; D008958:Models, Molecular; D011487:Protein Conformation; D021381:Protein Transport; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D043525:Cytosine Deaminase; D005472:Fluorouracil