Phenotypic Heterogeneity in Cultured Human Head and Neck Squamous Cell Carcinoma Lines with Low-Level Methotrexate Resistance

Low-level methotrexate (MTX) resistance (<20-fold) was induced by gradual selection pressure in four human head and neck squamous cell carcinoma lines established in culture from biopsies of patients not previously treated with MTX. Each parental and resistant line was characterized with respect to MTX uptake and polyglutamylation, dihydrofolate reductase (DHFR) content, and growth rate. Relative DHFR gene copy numbers and amounts of DHFR-related cytoplasmic messenger RNA were analyzed by plasmid complementary DNA hybridization in a dot blot assay and were correlated with the amount of gene product. The resistant lines were not cloned in order to simulate in vitro the conditions which might exist in an in vivo setting, where multiple resistant subpopulations of cells may be present in a tumor. The study was restricted to cells with tow-level resistance since these are likely to be the clinically most relevant type. Of the four resistant lines characterized, one showed a severe defect in MTX uptake and polyglutamylation, another was a DHFR overproducer with only small changes in uptake and polyglutamylation, a third was likewise a DHFR overproducer but also showed lower MTX uptake, and the fourth was minimally altered except for growth rate. The diversity in resistance phenotype among these cells in vitro suggests that in vivo resistance in patients with head and neck carcinoma who are treated with MTX may similarly involve multiple mechanisms and that further therapeutic intervention using MTX or other antifolates should take this into account.