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Ribonucleotide diphosphate reductase from human metastatic melanoma
K U Schallreuter1, T E Elgren, L S Nelson
1Department of Dermatology, University of Hamburg, Germany.
Melanoma Research
|December 11, 1992
Summary
Human melanoma cells possess a unique ribonucleoside diphosphate reductase (RR) enzyme that is not inhibited by hydroxyurea but is affected by fotemustine. This enzyme utilizes specific substrates and effectors, and its activity involves distinct free radicals.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Human melanoma metastases exhibit high ribonucleoside diphosphate reductase (RR) activity.
- This RR shows specific substrate and effector preferences, differing from other known RRs.
Purpose of the Study:
- To characterize the activity and properties of RR in human melanoma metastases.
- To investigate the enzyme's kinetics, cofactor requirements, and response to inhibitors and free radical traps.
- To explore the potential of fotemustine as an inhibitor of melanoma RR.
Main Methods:
- Cell-free extracts from melanoma metastases were used to assay RR activity.
- Enzyme kinetics were studied using guanosine diphosphate (GDP) as substrate and deoxythymidine triphosphate (dTTP) as effector.
- Electron paramagnetic resonance (EPR) spectroscopy was employed to detect free radicals.
- Inhibition studies were performed using hydroxyurea and fotemustine.
Main Results:
- Melanoma RR showed high activity with GDP/dTTP but no activity with CDP/ATP.
- Enzyme activity required magnesium or calcium, with melanotic tumors exhibiting a time lag.
- Unlike other RRs, melanoma RR was resistant to hydroxyurea but sensitive to fotemustine (IC50 = 10(-4) M).
- EPR detected two distinct free radicals associated with the enzyme: one in the resting state and another during catalysis.
Conclusions:
- Human melanoma RR possesses unique biochemical properties, including resistance to hydroxyurea.
- The enzyme's catalytic mechanism involves at least two distinct free radicals.
- Fotemustine demonstrates inhibitory potential against melanoma RR, suggesting a possible therapeutic application.