MOLECULAR MECHANISMS OF RESISTANCE TO ANTIFOLATES, A REVIEW

Methotrexate (MTX) is a clinically important antifolate that has been used in combination with other chemotherapeutic agents in the treatmen t of malignancies including acute lymphocytic leukemia, osteosarcoma, carcinomas of the breast, head and neck, choriocarcinoma and non-Hodgk in's lymphoma. The primary target of MTX is the enzyme dihydrofolate r eductase (DHFR) which catalyzes the reduction of folate and 7,8-dihydr ofolate to 5,6,7,8-tetrahydrofolate. Understanding of MTX action has r evealed how cells acquire resistance to this drug. The four known mech anisms of MTX resistance are a decrease in the uptake of the drug, a d ecrease in the retention of the drug due to defective polyglutamylatio n or an increase in polyglutamate breakdown, an increase in the enzyme activity and a decrease in the binding of MTX to DHFR. The molecular basis for some of these mechanisms has been elucidated in MTX resistan t cell lines; in particular the occurrence of gene amplification resul ting in increased DHFR and point mutations resulting in altered DHFR w ith reduced affinity for MTX. Cloning of the human folylpolyglutamate synthase gene and the reduced folate transport gene have been reported recently and should facilitate the identification of the molecular ba sis of these resistant phenotypes. DHFR protein has been shown to regu late its synthesis by exerting an inhibitory influence on its own tran slation. Addition of MTX relieves this inhibition thus providing a pos sible molecular explanation for the rapid rise in DHFR activity noted in some cells after MTX administration. Alterations in genes involved in regulating the cell cycle such as cyclin D1 and the retinoblastoma (Rb) gene have also been shown to influence cellular response to MTX. Overexpression of cyclin DI in HT1080, a human fibrosarcoma cell line, results in decreased MTX sensitivity. The molecular basis of this obs ervation is under investigation. Abnormalities in the Rb gene may also have profound effects on MTX sensitivity. Rb interacts with the famil y of transcription factors called E2F reducing transcription of genes that contain E2F binding sites in the promoter regions e.g. DHFR. When Rb is deleted or rendered nonfunctional levels of ''free'' or unbound E2F are high resulting in enhanced transcription of genes such as DHF R. This results in increased DHFR protein and may lead to MTX resistan ce. As the knowledge regarding mechanisms of resistance increases newe r approaches to circumvent such resistance or to target resistant cell s can be undertaken.