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Blood, Vol. 93 No. 3 (February 1), 1999:
pp. 1067-1074
Differential Methotrexate Resistance in Childhood T- Versus
Common/PreB-Acute Lymphoblastic Leukemia Can Be Measured by an In Situ
Thymidylate Synthase Inhibition Assay, But Not by the MTT Assay
Marianne G. Rots,
Rob Pieters,
Gert-Jan L. Kaspers,
Christina H. van Zantwijk,
Paul Noordhuis,
Rob Mauritz,
Anjo J.P. Veerman,
Gerrit Jansen, and
Godefridus J. Peters
From the Departments of Pediatric Hematology/Oncology and Medical
Oncology, University Hospital Vrije Universiteit, Amsterdam, The
Netherlands.
Methotrexate (MTX) is not cytotoxic to patient-derived acute
lymphoblastic leukemia (ALL) cells in total-cell-kill assays, such as
the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay, putatively due to the rescue effects of hypoxanthine and
thymidine released from dying cells. This was mimicked by a diminished
methotrexate (MTX) cytotoxicity for the cell lines HL60 and U937 in the
presence of hypoxanthine, thymidine, or lysed ALL cells. However,
enzymatic depletion or inhibition of nucleoside membrane transport did
not result in MTX dose-dependent cytotoxicity in patient samples.
Alternatively, a thymidylate synthase inhibition assay (TSIA), based on
inhibition of the TS-catalyzed conversion of 3H-dUMP to
dTMP and 3H2O, correlated with the
MTT assay for antifolate sensitivity in four human leukemia cell lines
with different modes of MTX resistance. For 86 ALL patient samples,
TSI50 values after 21 hours exposure to MTX were not
different between T- and c/preB-ALL (P = .46). After 3 hours
incubation with MTX followed by an 18-hour drug-free period, T-ALL
samples were 3.4-fold more resistant to MTX compared with c/preB-ALL
samples (P = .001) reflecting the clinical differences in MTX
sensitivity. TSI50 values correlated with MTX accumulation
(r =  .58, P < .001). In conclusion,
the TSIA, but not the MTT assay, can measure dose-response curves for
MTX in patient-derived ALL cells and showed relative MTX resistance in
T-ALL compared with c/preB-ALL.
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