Mammalian mutants genetically altered in CTP synthetase activity

Area of Science:

• Biochemistry
• Cell Biology
• Pharmacology

Background:

• Cytotoxic nucleosides are utilized in cancer chemotherapy.
• Understanding resistance mechanisms to these drugs is crucial for improving treatment efficacy.

Purpose of the Study:

• To investigate the mechanisms underlying resistance to 5-fluorouracil in a newly isolated mouse lymphoma cell line (FURT-1A).
• To characterize the biochemical alterations responsible for the observed drug resistance phenotype.

Main Methods:

• Isolation of a 5-fluorouracil-resistant cell clone (FURT-1A) from wildtype mouse lymphoma cells.
• Comparative growth rate experiments assessing sensitivity to various nucleosides.
• Measurement of intracellular nucleoside triphosphate accumulation.
• Metabolic flux analysis using [3H]-uridine.
• Kinetic analysis of CTP synthetase activity.

Main Results:

• FURT-1A cells exhibited cross-resistance to thymidine, deoxyguanosine, 5-fluorouridine, and arabinosylcytosine.
• These resistant cells showed decreased accumulation of corresponding nucleoside triphosphates from exogenous sources.
• Elevated cytidylate nucleotide pools were observed in FURT-1A cells, impairing nucleoside salvage.
• A 2-fold enhanced conversion of UTP to CTP was detected, linked to CTP synthetase activity refractory to CTP inhibition.

Conclusions:

• The resistance phenotype in FURT-1A cells is attributed to elevated cytidylate pools.
• Loss of allosteric inhibition of CTP synthetase by CTP is the likely genetic basis for these altered cellular properties.
• This study provides insights into nucleoside metabolism and drug resistance mechanisms in lymphoma cells.