Increased sensitivity to the prodrug 5'-deoxy-5-fluorouridine and modulation of 5-fluoro-2'-deoxyuridine sensitivity in MCF-7 cells transfected with thymidine phosphorylase.
Platelet-derived endothelial cell growth factor (PD-ECGF) is identical to human thymidine phosphorylase (dThdPase). The human MCF-7 breast cancer cell line was transfected with the dThdPase cDNA and expressed a 45 kDa protein that was detected with anti-dThdPase antibody. Cell lysates possessed elevated dThdPase activity and cells had up to 165-fold increased sensitivity to the prodrug 5'-deoxy-5-fluorouridine (5'-DFUR) in vitro. Sensitivity to 5-fluorouracil (5-FU) and 5-fluoro-2'-deoxyuridine (5-FUdR) was unchanged. Recombinant dThdPase was shown to catalyse directly the phosphorolytic cleavage of 5'-DFUR to 5-FU. Exogenous thymidine (dThd) reversed the toxicity of 5-FUdR on the parental line (1 microM dThd increased the IC50 value 1000-fold), but the dThd rescue was substantially modulated in the dThdPase-expressing clone 4 (1 microM dThd raised the IC50 value 3-fold). We observed a substantial 'bystander' killing effect when small proportions of dThdPase-expressing cells were mixed with parental MCF-7 cells. dThdPase activity was on average 27-fold higher in breast tumours than in normal breast. The levels of wild-type MCF-7 are similar to the low end of the tumour expression. Thus, in some tumours resistance to 5'-DFUR therapy could be due to low dThdPase activity, and transfection to raise the dThdPase levels within the broad tumour range or above it should markedly enhance sensitivity to the prodrug. These results confirm that dThdPase is a major pathway in the metabolic activation of 5'-DFUR, and the bystander effect suggests that this may be a suitable enzyme for gene therapy-directed enzyme/prodrug activation therapy.
Patterson
A V
AV
ICRF Clinical Oncology Unit, University of Oxford, John Radcliffe Hospital, UK.
Zhang
H
H
Moghaddam
A
A
Bicknell
R
R
Talbot
D C
DC
Stratford
I J
IJ
Harris
A L
AL
D000970:Antineoplastic Agents / Q000494:pharmacology*; D001682:Biological Availability; D001940:Breast / Q000201:enzymology; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000201:enzymology* / Q000235:genetics; D002450:Cell Communication / Q000502:physiology; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D004354:Drug Screening Assays, Antitumor; D005467:Floxuridine / Q000494:pharmacology*; D005472:Fluorouracil / Q000494:pharmacology; D006801:Humans; D011355:Prodrugs / Q000494:pharmacology*; D013936:Thymidine / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology; D013939:Thymidine Phosphorylase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000187:drug effects
D000970:Antineoplastic Agents; D011355:Prodrugs; D005467:Floxuridine; D013939:Thymidine Phosphorylase; D005472:Fluorouracil; C025034:doxifluridine; D013936:Thymidine