Mouse 3T6 cells that overproduce glutamine synthetase

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Mouse 3T6 cells were adapted to grow without glutamine.
• Glutamine synthetase is a key enzyme in glutamine metabolism.

Purpose of the Study:

• To investigate gene amplification as a mechanism for drug resistance.
• To understand the regulation of glutamine synthetase expression.

Main Methods:

• Exposure of cells to increasing concentrations of methionine sulfoxime.
• Measurement of glutamine synthetase activity.
• Immune precipitation and in vitro translation assays.
• Analysis of chromosome structure.

Main Results:

• Methionine sulfoxime-resistant cells exhibited a 75-fold increase in glutamine synthetase activity.
• Increased enzyme activity correlated with enhanced biosynthesis and mRNA levels.
• Drug resistance was associated with double minute chromosomes, suggesting gene amplification.

Conclusions:

• Methionine sulfoxime selects for cells with amplified glutamine synthetase genes.
• Gene amplification is a mechanism for adapting to metabolic stress.
• This study provides evidence for gene amplification in drug resistance.