Characterization of the Pools of 5,10-Methylenetetrahydrofolates and Tetrahydrofolates in Xenografts of Human Colon Adenocarcinoma1

The method for measuring polyglutamate forms of CH2-H4PteGlu and ILtPteGlu, by entrapment in ternary complexes with |6-'H|5-fluoro-2'deoxyuridylate and Lactobacillus casei thymidylate synthase has been characterized. Results demonstrated that (a) the relationship between concentration of ( 11 -1U'M ,ln and complex isolated on nondenaturing gels was dependent upon the number of glutamyl residues, and an alternative method for data analysis has been presented, (b) the relation ship was linear over a 100-fold change in concentration, (c) formation of isolatable complex was time dependent, (¡I)noncovalent complexes formed with l'it-<.In . could be isolated only at concentrations consider ably higher than those required for < 11.-H ,I"H'<,lu, ,„ and (e) endogenous deoxyuridylate would be unlikely to interfere significantly with the assay. The distribution of polyglutamates of ( 'I I.-) I,I'u-(¡Inand the combined pools of <'I I..-II41V<¡Inplus H4PteGlu were subsequently examined in three human colon adenocarcinoma xenografts. In each tumor, the pentaglutamate of ( 11 114l'k'(.lu and 11,I'M.lu was the most prominent species, followed by the hexaglutamate, constituting 68 to 92% of the ( 11.-I Ijl'li (. In pool, and >93% of the combined pools. A small percent age of di-, tri-, and tetraglutamates were also detected. Using a catalytic assay, the combined pool of <'I I..-11,1'M .1« and 11,l'u-<.In was estimated in the range of 0.5 to 2.7 MMin cell water, and for CH2-H4PteGlu, from 185 nM to 1.7 /IM. Using thymidylate synthase purified from colon adenocarcinoma HxVRCs, < II -II4l'u<.ln. (where the subscript digit attached to the glutamate portion equals the number of glutamate resi dues) stabilized the covalent ternary complex at > 200-fold lower concen tration in comparison to CH2-HtPteGIUi. Data indicated that in each colon tumor, the concentrations of CH2-K,PteGlu„or CH2-H4PteGlu„ plus I Ijl'lt-i .ln„ were suboptimal for the interaction of 5-fluoro-2'-deoxyuridylate with thymidylate synthase, and would predict for relatively transient inhibition of thymidylate synthase after treatment with 5fluorouracil. These data support therapeutic modulation to increase the concentration of ( 11:-1U'M.lu,, in the treatment of colon adenocarcinonias with 5-fluorouracil.