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Methods for Thymidylate Synthase Pharmacodynamics: Serial Biopsy, Free and Total TS, FdUMP and dUMP, and H4PteGlu and CH2-H4PteGlu Assays

  • Colin Paul Spears
  • Bengt G. Gustavsson
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 131)

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, postFUra 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.

Keywords

Homogenization Buffer Ehrlich Ascites Carcinoma Cell Thymidylate Synthetase Ternary Complex Formation Dissociation Buffer 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Plenum Press, New York 1988

Authors and Affiliations

  • Colin Paul Spears
    • 1
    • 2
  • Bengt G. Gustavsson
    • 1
    • 2
  1. 1.Cancer Research LaboratoryUniversity of Southern California Comprehensive Cancer CenterLos AngelesUSA
  2. 2.Department of Surgery Östra HospitalUniversity of GöteborgGöteborgSweden

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