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Methods for thymidylate synthase pharmacodynamics: serial biopsy, free and total TS, FdUMP and dUMP, and H4PteGlu and
1University of Southern California Comprehensive Cancer Center, Cancer Research Laboratory, Los Angeles 90033.
Abstract:
This report details our methods for performance of the major parameters related to quantitation of TS inhibition resulting from fluoropyrimidine administration to patients, methods equally applicable to preclinical studies. Sampling of tumors before and after drug treatment is done by 4 mm disposable punch biopsy or forceps biopsy via subcutaneous tunneling. Homogenates are prepared using N2 or polytron-mincing. Cytosolic free TS is measured by either the tritium-release method for small biopsies or by [3H]FdUMP ligand-binding. FdUMP and dUMP are separated by DEAE-cellulose column and measured by competitive binding and [14C]dTMP synthesis by the Moran methods. Total, post-FUra TS is measured by pre-incubation dissociation of FdUMP-bound TS after neutral charcoal removal of cytosolic ligands. H4PteGlu and CH2-H4PteGlu are measured by the Priest method using L. Casei TS. The materials and methods are described in sufficient detail to permit wide application of this approach.
Insights
This study presents methods to measure thymidylate synthase (TS) inhibition from fluoropyrimidine drugs in patients and preclinical models. These techniques enable precise quantitation of drug efficacy by analyzing tumor biopsies.
Area of Science:
- Biochemistry
- Pharmacology
- Oncology
Background:
- Fluoropyrimidines are key anticancer drugs.
- Measuring thymidylate synthase (TS) inhibition is crucial for assessing drug efficacy.
- Standardized methods are needed for reliable TS inhibition quantitation.
Purpose of the Study:
- To detail methods for quantifying TS inhibition by fluoropyrimidines.
- To provide techniques applicable to both clinical and preclinical settings.
- To enable accurate assessment of drug response in cancer treatment.
Main Methods:
- Tumor sampling via biopsy (punch or forceps) before and after treatment.
- Homogenate preparation using N2 or polytron-mincing.
- Quantification of cytosolic free TS using tritium-release or [3H]FdUMP ligand-binding assays.
- Separation and measurement of FdUMP and dUMP via DEAE-cellulose chromatography.
- Measurement of total TS post-fluorouracil (FUra) administration.
- Assay for H4PteGlu and CH2-H4PteGlu using L. Casei TS.
Main Results:
- Established protocols for precise TS inhibition measurement.
- Demonstrated applicability across different sample types (small biopsies).
- Provided detailed methodology for wide adoption and reproducibility.
Conclusions:
- The described methods allow for robust quantitation of TS inhibition.
- These techniques are valuable for evaluating fluoropyrimidine efficacy in cancer therapy.
- The detailed protocols support broad application in clinical and preclinical research.