Induction of thymidine phosphorylase expression by Taxol does not enhance Furtulon sensitivity in a cisplatin-resistant human ovarian carcinoma cell line.
OBJECTIVE
Thymidine phosphorylase (TP) can metabolize the prodrug 5'-deoxy-5-fluorouridine (Furtulon) to 5-fluorouracil (5-FU) and 5'-deoxy-D-ribose-1-phosphate. Furthermore, TP may enhance the toxicity of the active drug 5-FU by the transfer of 2'-deoxyribose 1-phosphate, so producing 5-fluoro-2'-deoxyuridine. This product can form 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) through the action of thymidine kinase, and FdUMP in turn can inhibit thymidylate synthase (TS), leading to reduced thymidylate formation and subsequent inhibition of DNA synthesis. The aim of this study was to determine whether the expression of TP is associated with the sensitivity to Furtulon, using a single-cell clone of the human ovarian carcinoma cell line, KF-28, and KFr13, a cisplatin-resistant subline derived from KF28 cells. Next, if lower TP expression correlated with decreased sensitivity to Furtulon, we planned to investigate the possibility that increased TP expression induced by Paclitaxel (Taxol) exposure might increase the sensitivity to Furtulon.


METHODS
Cell growth was evaluated by MTT assay. TP and TS expression were assessed by RT-PCR and Western blot analysis.


RESULTS
Cell growth was significantly inhibited compared to the control at 10 (p < 0.0001), 100 (p < 0.0001) and 1000 microM (p < 0.0001) of Furtulon after 24 hours Furtulon treatment in KF28. However, cell growth was significantly inhibited only at 1000 microM (p < 0.0001), in KFr13. The expression of TP was observed only in KF28 in our PCR condition. Next, Western blot analysis confirmed that TP protein levels in KF28 were markedly elevated compared to those in KFr13. TP gene expression emerged at 72, 96 and 120 hours after 0.5 nM Taxol (about twenty percent of IC50; 2.61 +/- 0.06 nM) exposure to KFr13 in our PCR condition. The level of TP gene expression was the highest at 120 hours Taxol exposure. Similarly, Western blot analysis showed that the TP protein level of KFr13 cells 120 hours after Taxol exposure (KFr13/120 hours Taxol exposure) was elevated compared to the control. Cell growth did not significantly differ between KFr13 and KFr13/120 hours Taxol exposure cells. KFr13 and KFr13/120 hours Taxol exposure cells were incubated with 0-1000 microM Furtulon for 24 hours-168 hours. Furtulon sensitivity of KFr13/120 hours Taxol exposure cells was not found to be significantly enhanced compared to that of KFr13 cells at any of the indicated times. The level of TS gene expression assessed by RT-PCR in KFr13 and KFr13/120 hours Taxol exposure cells was significantly lower than that in KF28 cells (p < 0.001). Moreover, higher protein level expression of TS was noted in KF28 cells compared to KFr13 or KFr13/120 hours Taxol exposure cells.


CONCLUSIONS
Our results suggest that lower expression of TP is not a critical determinant in the development of resistance to Furtulon in the cisplatin-resistant human ovarian carcinoma cell line, KFr13. The clinical relevance of these observation remains to be established.
Hata
Kohkichi
K
Department of Obstetrics and Gynecology, Shimane Medical University, Izumo 693-8501, Japan. hata31@shimane-med.ac.jp
Osaki
Mitsuhiko
M
Kanasaki
Haruhiko
H
Nakayama
Kentaro
K
Fujiwaki
Ritsuto
R
Ito
Hisao
H
Miyazaki
Kohji
K
D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D001711:Biotransformation / Q000187:drug effects; D015153:Blotting, Western; D002277:Carcinoma / Q000473:pathology*; D002455:Cell Division / Q000187:drug effects; D002945:Cisplatin / Q000494:pharmacology*; D019008:Drug Resistance, Neoplasm; D004790:Enzyme Induction / Q000187:drug effects; D005260:Female; D005467:Floxuridine / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D010051:Ovarian Neoplasms / Q000473:pathology*; D017239:Paclitaxel / Q000494:pharmacology*; D011355:Prodrugs / Q000378:metabolism / Q000494:pharmacology*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D012334:RNA, Neoplasm / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013939:Thymidine Phosphorylase / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000201:enzymology
D000972:Antineoplastic Agents, Phytogenic; D011355:Prodrugs; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D005467:Floxuridine; D013939:Thymidine Phosphorylase; D017239:Paclitaxel; D002945:Cisplatin; C025034:doxifluridine