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Modulation of antifolate cytotoxicity by metabolites from dying cells in a lymphocyte clonal assay
J M Hughes1, A deFazio, M H Tattersall
1Department of Cancer Medicine, University of Sydney, NSW, Australia.
Abstract:
A lymphocyte clonal assay developed to quantitate in vivo somatic cell mutations at the hypoxanthine-guanine phosphoribosyltransferase locus was modified in order to study resistance to methotrexate. Even though nucleoside-free culture conditions were used methotrexate was not lethal to lymphocytes plated into micro-wells at greater than 10(2) cells/well. HPLC analysis of supernatants from wells plated initially with 10(4) cells/well in 100 microM methotrexate revealed the presence of micro-molar levels of hypoxanthine and thymidine by the 5th and 8th day of culture respectively. When lymphocytes were plated at less than or equal to 10(2) cells/well in nucleoside free medium, methotrexate was cytotoxic and micro-molar levels of thymidine together with hypoxanthine protected lymphocytes cultured under these conditions from toxicity. Modulation of nucleic acid antimetabolite cytotoxicity by nucleosides and bases has been recognised for some years. Nucleoside free culture conditions have been advocated for studying cellular sensitivity to antifolates to avoid such interfering factors. However our results indicate that metabolites from dying or damaged cells can prevent methotrexate cytotoxicity, further complicating the development of a suitable clonogenic assay for investigating antifolate sensitivity.
Insights
Developing a methotrexate resistance assay for lymphocytes is complicated. Metabolites from dying cells can protect lymphocytes from methotrexate, interfering with accurate sensitivity testing.
Area of Science:
- Immunology
- Cell Biology
- Pharmacology
Background:
- Lymphocyte clonal assays are used to study somatic cell mutations.
- Methotrexate (MTX) is an antifolate drug used in cancer therapy.
- Studying cellular sensitivity to antifolates requires controlled culture conditions.
Purpose of the Study:
- To modify a lymphocyte clonal assay for studying methotrexate resistance.
- To investigate the cytotoxicity of methotrexate under different cell densities and culture conditions.
Main Methods:
- A lymphocyte clonal assay was adapted to study methotrexate resistance.
- High-performance liquid chromatography (HPLC) was used to analyze culture supernatants.
- Lymphocytes were cultured under nucleoside-free conditions at varying cell densities.
Main Results:
- Methotrexate was not lethal to lymphocytes at densities >10^2 cells/well, even in nucleoside-free media.
- HPLC analysis revealed hypoxanthine and thymidine release from dying cells.
- At densities ≤10^2 cells/well, methotrexate was cytotoxic, but protected by hypoxanthine and thymidine.
Conclusions:
- Metabolites released from dying cells can interfere with methotrexate cytotoxicity.
- Nucleoside-free conditions alone do not prevent confounding factors in antifolate sensitivity assays.
- The presence of metabolites complicates the development of reliable lymphocyte-based clonogenic assays for antifolate sensitivity.