Inhibition of thymidylate synthase activity by antisense oligodeoxynucleotide and possible role in thymineless treatment.
Thymidylate synthase (TS) is an important target for chemotherapeutic treatment of cancer. However, efficacy of TS-targeted anticancer drugs is limited by the development of drug resistance as a result of TS gene amplification. In this work, a phosphorothioated antisense oligonucleotide (ODN), designated ATS-2, was used to suppress cellular synthesis of TS. ATS-2 at 0.2 microM concentration was mixed with lipofectin in a charge ratio of 1:1 and was used to treat the human embryonic kidney (HEK) cell line. A reduction of TS mRNA and protein was achieved. Furthermore, a dose-dependent reduction of cumulative viable cells of up to 98% was observed. Flow cytometer analysis of cell cycle progression indicates that ATS-2-treated cells were arrested and went into apoptosis at the S phase, possibly because of thymidine shortage, suggesting that ATS-2 is specifically effective for dividing cells. When used in combination with the anticancer drug FdUrd, ATS-2 exerted a additive inhibitory effect on cellular proliferation. To elucidate the possible role of cellular thymidine kinase (TdR kinase) in ATS-2 treatment, a second cell line, HeLa, was used. Both HEK and HeLa have similar rates of cell division and ODN uptake. In contrast to HEK, which was shown to have very low levels of TdR kinase activity in [(3)H]thymidine incorporation experiments, [(3)H]thymidine incorporation in HeLa was 15-fold greater than that of HEK. We found that HeLa cells were sensitive to FdUrd but were rather resistant to ATS-2. On the contrary, HEK cells were sensitive to ATS-2 but insensitive to FdUrd. Effects of ATS-2 and FdUrd are, therefore, complementary in thymineless treatment too.
Lin
S B
SB
School of Medical Technology, National Taiwan University, Taipei, Taiwan, Republic of China.
Ts'o
P O
PO
Sun
S K
SK
Choo
K B
KB
Yang
F Y
FY
Lim
Y P
YP
Tsai
H L
HL
Au
L C
LC
D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D017209:Apoptosis; D001692:Biological Transport / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004337:Drug Carriers; D004347:Drug Interactions; D005467:Floxuridine / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D020319:Oligodeoxyribonucleotides, Antisense / Q000494:pharmacology*; D010714:Phosphatidylethanolamines; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D016196:S Phase / Q000187:drug effects*; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors* / Q000235:genetics
D000964:Antimetabolites, Antineoplastic; D004337:Drug Carriers; D020319:Oligodeoxyribonucleotides, Antisense; D010714:Phosphatidylethanolamines; D012333:RNA, Messenger; D005467:Floxuridine; C020888:1,2-dielaidoylphosphatidylethanolamine; D013940:Thymidylate Synthase