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Methotrexate decreases thymidine kinase activity
1Laboratory for Experimental Oncology, Indiana University School of Medicine, Indianapolis 46202-5200.
Abstract:
MTX cytotoxicity is not fully explained by its well-known inhibition of dihydrofolate reductase activity which leads to a decrease in the dTMP synthase reaction, since TdR kinase which converts TdR to dTMP could readily circumvent MTX action through this salvage activity. TdR kinase is of particular significance, since in various types of carcinoma cells its activity is orders of magnitude higher than that of dTMP synthase. To throw light on this problem, we tested the hypothesis that the impact of MTX treatment might in fact involve an inhibition or decrease in TdR kinase activity. Injection in rat of MTX (i.p.) decreased TdR kinase activity in a time- and dose-dependent fashion in liver (t1/2 = 46 h; IC50 = 95 mg/kg), bone marrow (t1/2 = 10 h; IC50 = 5 mg/kg) and rapidly growing transplantable hepatoma 3924A (t1/2 = 56 h; IC50 = 5 mg/kg). Injection in rat of cycloheximide (15 mg/kg, i.p.), an inhibitor of protein biosynthesis, rapidly decreased TdR kinase activity in the hepatoma (t1/2 = 3.6 h); activities of other purine and pyrimidine synthetic enzymes, dTMP synthase, IMP dehydrogenase, GMP reductase and GMP synthase, declined at a markedly slower rate (t1/2 = 11, 11.6, 12 and 22 h, respectively). MTX, by curtailing purine and pyrimidine biosynthesis, limits product of TdR kinase which is more sensitive to unopposed protein degradation than other enzymes of nucleic acid biosynthesis. TdR kinase is a newly discovered target of MTX treatment.
Insights
Methotrexate (MTX) cytotoxicity involves more than dihydrofolate reductase inhibition. MTX treatment also decreases thymidine (TdR) kinase activity, a newly identified target, impacting cancer cell proliferation.
Area of Science:
- Biochemistry
- Pharmacology
- Oncology
Background:
- Methotrexate (MTX) is a chemotherapy drug whose cytotoxicity is primarily attributed to dihydrofolate reductase (DHFR) inhibition.
- DHFR inhibition leads to decreased thymidylate synthase (dTMP synthase) activity, crucial for DNA synthesis.
- Thymidine (TdR) kinase, a salvage enzyme, can bypass DHFR inhibition by converting TdR to dTMP, potentially limiting MTX efficacy, especially in cancer cells with high TdR kinase activity.
Purpose of the Study:
- To investigate the hypothesis that MTX treatment may also inhibit or decrease TdR kinase activity.
- To identify TdR kinase as a potential novel target of MTX action.
- To elucidate the mechanisms underlying MTX-induced cytotoxicity beyond DHFR inhibition.
Main Methods:
- MTX was administered intraperitoneally (i.p.) to rats at various doses and time points.
- TdR kinase activity was measured in liver, bone marrow, and hepatoma tissues.
- Cycloheximide, a protein biosynthesis inhibitor, was used to assess enzyme stability and degradation rates.
- Activities of other nucleic acid biosynthesis enzymes were also quantified.
Main Results:
- MTX treatment dose- and time-dependently decreased TdR kinase activity in liver, bone marrow, and hepatoma.
- Cycloheximide administration led to a rapid decrease in TdR kinase activity in hepatoma, with a half-life of 3.6 hours.
- Other purine and pyrimidine synthetic enzymes showed slower declines in activity following cycloheximide treatment.
- MTX's effect on purine and pyrimidine biosynthesis limits TdR kinase substrates, making it susceptible to protein degradation.
Conclusions:
- TdR kinase is a newly identified target of MTX treatment.
- MTX reduces TdR kinase activity, contributing to its cytotoxic effects.
- The increased sensitivity of TdR kinase to protein degradation, influenced by MTX-induced purine and pyrimidine depletion, is a key mechanism.