Increased ribonucleotide reductase activity in hydroxyurea-resistant mosquito cells

Area of Science:

• Biochemistry
• Molecular Biology
• Insect Cell Culture

Background:

• Hydroxyurea is a known inhibitor of ribonucleotide reductase.
• Developing drug-resistant cell lines is crucial for understanding resistance mechanisms.

Purpose of the Study:

• To establish and characterize a hydroxyurea-resistant Aedes albopictus insect cell line.
• To investigate the molecular basis of hydroxyurea resistance in this cell line.

Main Methods:

• Incremental exposure of unmutagenized Aedes albopictus cells to increasing hydroxyurea concentrations.
• Selection and characterization of resistant clonal populations.
• Analysis of cell morphology, growth rate, and chromosome number.
• Protein overproduction analysis using (35)S[methionine/cysteine] labeling.
• Measurement of ribonucleotide reductase activity.

Main Results:

• Established a hydroxyurea-resistant Aedes albopictus cell line (HU1062) 40-fold more resistant than wild-type.
• Resistant cells exhibited altered morphology and a decreased growth rate (45 h doubling time).
• Resistant cells remained diploid with a modal chromosome number of 6.
• Clone HU1062 overproduced a protein consistent in size with the M2 subunit of ribonucleotide reductase.
• Ribonucleotide reductase activity was approximately 10-fold higher in resistant cells.

Conclusions:

• This is the first reported hydroxyurea-resistant insect cell line.
• The resistance is associated with the overproduction of the M2 subunit of ribonucleotide reductase.
• This cell line serves as a valuable tool for studying drug resistance mechanisms in insects.