Deoxyribonucleotide metabolism in hydroxyurea-resistant V79 hamster cells

Area of Science:

• Cell Biology
• Biochemistry
• Molecular Biology

Background:

• Hydroxyurea is a chemotherapeutic agent that inhibits ribonucleotide reductase.
• Understanding drug resistance mechanisms is crucial for cancer therapy.
• Deoxyribonucleotide metabolism plays a key role in DNA synthesis and repair.

Purpose of the Study:

• To investigate the mechanisms of hydroxyurea resistance in V79 hamster cells.
• To determine the impact of increased ribonucleotide reductase activity on deoxyribonucleotide metabolism.
• To elucidate the role of allosteric regulation and substrate cycles in drug resistance.

Main Methods:

• Continuous culture of V79 cells with stepwise increasing hydroxyurea concentrations.
• Cloning of resistant cell lines (V79/H0.4 and V79/H4).
• Measurement of soluble ribonucleotide reductase activity, in situ enzyme activity, and deoxyribonucleotide pool levels (dATP, dTTP).
• Analysis of substrate cycles involving pyrimidine deoxyribonucleosides and their phosphates.

Main Results:

• Resistant cell lines (V79/H4) showed a 20-fold increase in soluble ribonucleotide reductase activity.
• In situ enzyme activity was only moderately elevated (1.3-fold).
• Steady-state levels of dATP and dTTP were significantly increased in resistant cells.
• Increased deoxyribonucleotide pools likely inhibit ribonucleotide reductase via allosteric feedback.
• Surplus deoxyribonucleotides were excreted as thymidine and deoxycytidine.

Conclusions:

• Hydroxyurea resistance is maintained by a balance between increased enzyme synthesis and allosteric inhibition.
• Substrate cycles facilitate the excretion of excess deoxyribonucleotides, preventing feedback inhibition.
• These findings extend the understanding of ribonucleotide reductase regulation and drug resistance.