Four methotrexate (MTX)-resistant sublines of a human squamous cell carcinoma (SCC15) were established in culture by progressive dose escalation. The biochemical basis of resistance was studied. The line with the lowest resistance (R1) had a normal dihydrofolate reductase (DHFR) content but showed decreased MTX transport and polyglutamation. Lines of intermediate resistance (R2 and R3) showed an increased DHFR content and DHFR gene copy number and a defect in MTX transport. The line with the greatest resistance (R4) showed increased DHFR content and gene copy number but nearly normal MTX transport. These results demonstrate that multiple mechanisms of MTX resistance occur in human epithelial cells in culture. We also find evidence of alterations in DHFR gene expression. The MTX-resistant cells were either not cross-resistant or only partly cross-resistant to two lipophilic MTX ester derivatives. These compounds are of potential therapeutic interest for the treatment of MTX-resistant tumors.Methotrexate (MTX) is among the most active known drugs for clinical cancer therapy (1). For example, in patients with head and neck carcinoma, a 60% response is achieved upon initial treatment (2). However, the response is short and dose escalation is limited by the steepness of the dose-response curve for MTX in man. Much is known about the biochemistry of MTX resistance in murine (3-11) and human (12-19) neoplastic cells in culture, but these studies often involve highly resistant lines with little relevance to the clinic. We chose to study MTX resistance in a cell line (SCC15) derived from a patient with a squamous cell carcinoma of the tongue (20). Here we report that MTX resistance in SCC15 cells arises via several mechanisms, including decreased MTX transport, loss of MTX polyglutamation activity, amplification of the genes for dehydrofolate reductase (DHFR), and possible alteration of DHFR gene expression. We also report that lipophilic MTX esters are toxic to SCC15 cells that are resistant to unmodified MTX. MATERIALS AND METHODSCell Culture. Cells were grown in Dulbecco's modified Eagle's medium (DME medium) supplemented with 20% fetal bovine serum, penicillin (100 units/ml), streptomycin (100 ,ug/ml), L-glutamine (2 mM), and hydrocortisone (0.4 gg/ml). Plating efficiency was 10-20%. In cytotoxicity assays, cells were incubated in folate-free improved modified essential medium (IME medium) containing L-glutamine and antibiotics but no fetal bovine serum.Cells. A human head and neck squamous cell carcinoma line (SCC15) was obtained from James Rheinwald (DanaFarber Cancer Institute) (20). To obtain resistant sublines, DME medium containing 20% dialyzed fetal bovine serum was inoculated with cells (2 x 105 per dish), and 24 hr later 50 nM MTX was added. The MTX concentration was increased 1.2-to 2-fold at 1-to 4-week intervals over 13 months. Four resistant sublines were selected: SCC15/R1, 4 months; SCC15/R2, 6 months; SCC15/R3, 10 months; SCC15/R4, 13 months. Doubling times were determined to ensure that the ...
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