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Folate requirements of methotrexate-resistant human acute lymphoblastic leukemia cell lines
Abstract:
We studied the folate requirements of a human acute lymphoblastic leukemia cell line, MOLT-3, and methotrexate (MTX)-resistant sublines established in vitro. The requirement of pteroylglutamate (PGA) for optimal cell growth was different for each cell line. With increasing MTX resistance, there was progressive increase in PGA requirements, moving the PGA concentration-cell growth curve (dose-response curve) 1 log order of magnitude to the right. The increases in the requirement of 5-methyltetrahydrofolate (5-methyl-THF) by the resistant sublines were more pronounced than PGA requirement, moving the dose-response curve nearly 3 log orders in magnitude to the right. The concentrations in vitro of 5-methyl-THF required for optimal growth of the MTX-resistant sublines far exceeded the normal serum 5-methyl-THF concentrations known in humans. These observations show that MTX-resistant cell established in vitro in culture media containing PGA instead of 5-methyl-THF, a physiological folate, cannot be expected to grow in vivo. The collateral sensitivity of transport-impaired MTX-resistant sublines to 2,4-diamino-5-methyl-6-[(3',4',5'- trimethoxyanilino) methyl] quinazoline (trimetrexate, TMQ) was negated in the absence of PGA. With the addition of 5-methyl-THF, the parent cells became more resistant than the transport-impaired sublines to TMQ These data indicate that the collateral sensitivity of MTX resistant cells to the substituted 2,4-diaminoquinazoline is due to functional folate deficiency by virtue of the impaired transport of folate.
Insights
Methotrexate resistance in leukemia cells increases folate requirements, particularly for 5-methyltetrahydrofolate (5-methyl-THF). This altered folate metabolism impacts drug sensitivity and cell growth, suggesting implications for in vivo therapeutic strategies.
Area of Science:
- Biochemistry
- Cell Biology
- Pharmacology
Background:
- Methotrexate (MTX) is a key antifolate drug used in treating acute lymphoblastic leukemia (ALL).
- MTX resistance can develop in cancer cells, posing a significant clinical challenge.
- Folate metabolism plays a critical role in nucleotide synthesis and cell proliferation, making it a target for cancer therapy.
Purpose of the Study:
- To investigate the specific folate requirements of MTX-resistant human ALL cell lines (MOLT-3).
- To determine how increasing MTX resistance affects the cellular demand for different folate forms, specifically pteroylglutamate (PGA) and 5-methyltetrahydrofolate (5-methyl-THF).
- To explore the relationship between folate metabolism, MTX resistance, and sensitivity to other antifolate drugs like trimetrexate (TMQ).
Main Methods:
- Culturing of MOLT-3 human acute lymphoblastic leukemia cells and their MTX-resistant sublines in vitro.
- Assessing cellular growth responses to varying concentrations of pteroylglutamate (PGA) and 5-methyltetrahydrofolate (5-methyl-THF).
- Evaluating the collateral sensitivity of MTX-resistant cells to trimetrexate (TMQ) under different folate conditions.
Main Results:
- MTX resistance progressively increased the requirement for PGA, shifting dose-response curves rightward.
- Resistant sublines showed a more pronounced increase in 5-methyl-THF requirements, shifting curves nearly 3 log orders rightward.
- In vitro 5-methyl-THF concentrations required by resistant cells exceeded physiological human serum levels.
- Collateral sensitivity to TMQ in transport-impaired MTX-resistant cells was lost without PGA but restored with 5-methyl-THF, indicating functional folate deficiency.
Conclusions:
- MTX-resistant leukemia cells exhibit significantly altered folate metabolism, with a heightened dependence on 5-methyl-THF.
- Cell lines adapted to PGA in vitro may not be viable in vivo due to their inability to utilize physiological folate levels.
- Impaired folate transport contributes to functional folate deficiency, influencing collateral drug sensitivity in MTX-resistant cells.