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Phenotypic heterogeneity in cultured human head and neck squamous cell carcinoma lines with low-level methotrexate
Abstract:
Low-level methotrexate (MTX) resistance (less than 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 low-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.
Insights
Low-level methotrexate resistance in head and neck cancer cells can occur through various mechanisms, including altered drug uptake or increased dihydrofolate reductase. Understanding these diverse resistance pathways is crucial for effective antifolate therapy.
Area of Science:
- Oncology
- Pharmacology
- Molecular Biology
Background:
- Methotrexate (MTX) is a key antifolate chemotherapy agent used in treating head and neck squamous cell carcinoma.
- Understanding the mechanisms of low-level MTX resistance is critical for optimizing treatment strategies in patients with head and neck cancer.
- Previous studies have focused on high-level resistance, but low-level resistance is often more clinically relevant.
Purpose of the Study:
- To investigate the mechanisms of low-level methotrexate resistance in human head and neck squamous cell carcinoma (HNSCC) cell lines.
- To characterize the phenotypic diversity of MTX resistance in HNSCC.
- To inform future therapeutic strategies involving MTX and other antifolates.
Main Methods:
- Induction of low-level MTX resistance (<20-fold) in four HNSCC cell lines via gradual selection pressure.
- Characterization of parental and resistant lines for MTX uptake, polyglutamylation, dihydrofolate reductase (DHFR) content, and growth rate.
- Analysis of DHFR gene copy number and messenger RNA levels using plasmid complementary DNA hybridization in a dot blot assay.
Main Results:
- Four distinct resistant cell lines exhibited diverse resistance mechanisms.
- One line showed impaired MTX uptake and polyglutamylation.
- Two lines demonstrated DHFR overproduction, with one also having reduced MTX uptake.
- The fourth line showed minimal alterations, primarily affecting growth rate.
Conclusions:
- Low-level MTX resistance in HNSCC involves multiple, heterogeneous mechanisms.
- The observed diversity in vitro suggests similar complexity in vivo, impacting clinical outcomes.
- Therapeutic strategies using MTX or other antifolates should consider these varied resistance pathways for improved efficacy.