ABSTRACT
Acute myeloid leukemia (AML) is considered to be a malignancy that is intrinsically resistant to methotrexate (MTX). As compared to acute lymphoblastic leukemia (ALL) blasts, AML blasts, except those of acute monocytic leukemia (AML-M(5)), form fewer amounts of long chain polyglutamate of MTX (MTXPG), when incubated with MTX, thus providing an explanation for their lack of responsiveness to MTX. To explore the novel approach of treatment in patients with AML-M(5), the U937 cell line, which has the monocytic characters, was used. Cell growth inhibition was mearsured by XTT assay after 24 and 48 hours in the continuous presence of various concentrations of MTX ranging from 1 nmol/L to 100 micro mol/L. After 24 hours MTX treatment, the IC(50) value for U937 cells was 0.04 micro mol/L. After 48 hours treatment, the IC(50) was 0.037 micro mol/L and IC(90) was 0.39 micro mol/L. To understand the mechanism of MTX cytotoxicity, the process of cell death was analyzed. A variety of assays, including trypan blue exclusion, flow cytometry, light microscopy (Wright's staining) and DNA fragment electrophoresis, were performed. There were no significant apoptotie changes after shorter exposure of MTX (4 and 6 hours). After 8 hours at various concentrations of MTX treatment ranging from 5 nmol/L to 10 micro mol/L, the percentage of the cells in the pre-G(1) (apoptotic) was 3.2% at 0.1 micro mol/L and it reached a peak of 18.2% at 5.0 micro mol/L. The DNA synthesis in S-phase was inhibited from 41.2% (0.01 micro mol/L) to 19.1% (10 micro mol/L). DNA ladder band, a feature of apoptosis, was observed. The arrest of cell growth and apoptotic properties induced by MTX have lead to its evaluation as a potentially therapeutic agent in the treatment of AML-M(5).