Methenyltetrahydrofolate synthetase prevents the inhibition of phosphoribosyl 5-aminoimidazole 4-carboxamide ribonucleotide formyltransferase by 5-formyltetrahydrofolate polyglutamates.

Methenyltetrahydrofolate synthetase (EC 6.3.3.2) catalyzes the irreversible ATP and Mg2+-dependent transformation of 5-formyltetrahydrofolate (N5-HCO-H4-pteroylglutamic acid (PteGlu] to 5,10-methenyltetrahydrofolate. The physiological function of this reaction remains unknown even though it is potentially involved in the intracellular metabolism of the large doses of N5-HCO-H4-PteGlu (leucovorin) administered to cancer patients. We have tried to elucidate methenyltetrahydrofolate synthetase's physiological role by examining the consequences of its inhibition in MCF-7 human breast cancer cells by the folate analog 5-formyltetrahydrohomofolate (fTHHF), a potent competitive inhibitor with a Ki of 1.4 microM. fTHHF inhibited MCF-7 cell growth with an IC50 of 2.0 microM during 72-h exposures, and this effect was fully reversible by hypoxanthine but not thymidine, indicating specific inhibition of de novo purine synthesis. A correlation was observed between increases in intracellular N5-HCO-H4-PteGlu concentrations following fTHHF and cell growth inhibition. De novo purine synthesis was inhibited at the second folate-dependent enzyme, phosphoribosyl aminoimidazole-carboxamide formyltransferase (AICAR transferase; EC 2.1.2.3), as determined by aminoimidazole carboxamide rescue and azaserine inhibition studies. N5-HCO-H4-PteGlu pentaglutamate was a potent inhibitor of purified MCF-7 cell AICAR transferase with a Ki of 3.0 microM while the monoglutamate was not an inhibitor up to 10 microM and fTHHF was only weakly inhibitory with a Ki of 16 microM. These findings suggest that methenyltetrahydrofolate synthetase activity is needed to prevent de novo purine synthesis inhibition by N5-HCO-H4-PteGlu polyglutamates.

Gas permeability of the film of block and graft copolymers of polydimethylsiloxane and poly(gamma-benzyl L-glutamate).

A-B-A type block copolymers of poly(gamma-benzyl L-glutamate) (PBLG, A segment) and polydimethylsiloxane (PDMS, B segment) and PDMS (trunk)-PBLG (branch) graft copolymers were synthesized, and the permeation of oxygen in water and the permeation of oxygen and carbon dioxide in the dry state were investigated. The gas permeation coefficient (P) increased with increasing content of PDMS. However, PCO2/PO2 values of copolymer films were in the range 6-9, i.e. larger than 5.4 for PDMS film. The oxygen permeation in water suffered from the interfacial resistance, which was reduced by the hydrolysis of film surface. The Arrhenius plot of the gas permeation coefficient in the dry state of the block copolymer B showed a turning point at about 40 degrees C. This temperature is close to beta-peak temperature (39 degrees C) and may be ascribed to the molecular motion of the PBLG segment. Transmission electron microscopy showed that one of the block copolymer films (PDMS 46 mol%) appears to have PDMS segments dispersed in the PBLG matrix (island-in-sea structure) and one of the graft copolymer films (PDMS 58 mol%) appears to take a lamellar structure. The gas permeation across the graft copolymer film appears to occur through the continuous PDMS phase, leading to a near-negligible activation energy in this process.

Conjugation of methotrexate to poly (L-lysine) as a potential way to overcome drug resistance.

Methotrexate (MTX) was conjugated through a carbodiimide-catalyzed reaction to poly (L-lysines) of various molecular sizes at a ratio of approximately one molecule per 27 lysyl residues. These conjugates were tested on cultured, Chinese hamster, ovary cells known to be drug resistant because of a deficient methotrexate transport. The cellular uptake of conjugated drug far exceeded the uptake of free drug in both drug sensitive and resistant lines. The conjugated drug inhibited the growth of the transport-deficient cells at concentrations at which free drug had no effect. The conjugate failed to inhibit dihydrofolate reductase in vitro. This and other evidence indicate that the strong pharmacologic effect of the MTX-poly(Lys) conjugate is due to the intracellular--presumably intralysosomal--hydrolysis of its polymeric backbone followed by the release inside the cell of an active form of MTX. This conclusion is supported by data obtained with conjugates using poly(D-lysine) as a carrier. This optical isomer is not susceptible to common proteolytic enzymes and MTX-poly(D-Lys) has not growth inhibitory effect whatever on either transport proficient or deficient CHO-cells. MTX-poly(L-lys) can thus be seen as a lysosome-activated drug.

Formation of methotrexate polyglutamates in rat hepatocytes.

Polyglutamate derivatives of [3H]methotrexate (MTX) were detected in freshly isolated rat hepatocytes in suspension within 15 min after exposure to the folate analog. The rate of polyglutamate synthesis remained constant for at least one hr, and the polyglutamate derivatives accounted for an increasing proportion of the intracellular radiolabel with time. After initial exposure to 1 micron [3H]MTX, polyglutamate derivatives of Mtx continued to be synthesized even after the extracellular [3H]-MTX concentration had been reduced 20-fold. Prolonged exposure of hepatocytes in primary culture to 1 micron [3H]MTX resulted in the formation of longer-chain polyglutamate derivatives of MTX. The present studies demonstrate another important biosynthetic capacity of the freshly isolated hepatocyte and suggest the usefulness of this system for studying the mechanism of, and controlling factors in, the synthesis of polyglutamate derivatives of MTX. The ramifications of the formation of MTX polyglutamates on drug cytotoxicity in general and hepatotoxicity in particular are considered.

Treatment of a murine cytomegalovirus infection with exogenous interferon, polyinosinic-polycytidylic acid, and polyinosinic-polycytidylic acid-poly-L-lysine complex.

Treatment of a cytomegalovirus infection of mice with exogenous murine interferon did not alter final mortality or mean day of death. Pretreatment with two interferon inducers significantly reduced mortality, but treatment initiated after infection was not effective.