Determinants of the sensitivity of human small-cell lung cancer cell lines to methotrexate.

Abstract

We hate characterized the determinants of methotrexate (MTX) responsiveness in eight patient-derived cell lines of small-cell lung cancer (SCLC). Clonogenic survival was correlated with factors known to affect sensitivity to drug. NCI-H209 and NCI-H128 were most drug sensitive, with drug concentrations required to inhibit clonogenic survival by 50% with <0.1 M&M MTX. Six cell lines (NCI-H187, NCI-H345, NCI-H60, NCI-H524, NCI-H146, and NCI-N417D) were relatively drug resistant. In all cell lines studied, higher molecular weight MTX-polyglutamates (MTX-PGs) with 3-5 glutamyl moieties (MTX-Glu3 through MTX-Glu5) were selectively retained. Relative resistance to low (1.0 ;M) drug concentrations appeared to be largely due to decreased intracellular metabolism of MTX. Five of the six resistant lines were able to synthesize polyglutamates at higher (10 M&M) drug concentrations, although one resistant cell line (NCI-N417D) did not synthesize higher molecular weight MTX-PGs, even after exposure to 10 MM drug. Two cell lines with resistance to 10 MM MTX (NCI-H146 and NCI-H524) synthesized and retained higher molecular weight MTX-PGs in excess of binding capacity after exposure to 10 MM drug. However, the specific activity of thymidylate synthase in these cell lines was low. MTIX sensitivity in patient-derived cell lines of SCLC requires the ability of cells to accumulate and retain intracellular drug in the form of polyglutamate metabolites in excess of dihydrofolate reductase, as well as a high basal level of consumption of reduced folates in the synthesis of thymidylate.

Methotrexate: pentose cycle and oxidative stress

“…2,12,16 Additionally, MTX has several other eects on cell metabolism. 8, 11±14 Considering such data and also the functional importance of the pentose phosphate pathway for tumour cells, 19, 34 the eects of MTX were also evaluated on some enzymes of this metabolic pathway.…”

“…1,2 Being a high anity inhibitor of dihydrofolate reductase (DHFR), MTX causes depletion of the dihydrofolate pool and indirectly aects the synthesis of thymidilate, suppressing DNA synthesis. 1 In cells, DHFR occurs in elevated concentrations, 3 so, besides the high anity of DHFR for MTX, high levels of freely exchangeable drug are required for a therapeutic response because only a small portion of non-inhibited cellular DHFR ($1 per cent), is sucient to maintain normal pools of reduced folate coenzymes.…”

Resistance to antifolates

“…The major impact is the retention and buildup of appreciable levels of polyglutamyl derivatives of the drug in cells (Fry et al, 1982b;Jolivet et al, 1982;Jolivet and Chabner, 1983) with sustained inhibition of DHFR (Rosenblatt et al, 1978b). The level and rapidity of formation of MTX polyglutamates in tumor cells is an important determinant of the pharmacologic activity of this drug (Fabre et al, 1984;Curt et al, 1985). For other antifolates, this biochemical transformation results not only in retention but also in a marked increase in affinity for their target enzyme(s).…”

Folate pathway gene expression differs in subtypes of acute lymphoblastic leukemia and influences methotrexate pharmacodynamics

“…The ability of cells to form and accumulate MTX polyglutamates (MTXPGs) is well recognized as a determinant of MTX cytotoxicity (3,4). Compared with MTX, MTXPGs are retained longer in cells (5) and compete more avidly with some cellular folate cofactors (e.g., 10-formyl-tetrahydrofolate, 5,10-methylene-tetrahydrofolate, and 5,10-methenyl-tetrahydrofolate), thereby inhibiting target enzymes in biosynthetic pathways that are critical for DNA synthesis, DNA repair, and cell replication (i.e., thymidine synthesis and de novo purine synthesis [DNPS]) (6,7).…”