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In vitro drug sensitivity of cells from children with leukemia using the
MTT assay with improved culture conditions
R Pieters, AH Loonen, DR Huismans, GJ Broekema, MW Dirven, MW Heyenbrok, K Hahlen and AJ Veerman
Department of Pediatrics, Free University Hospital, Amsterdam, The
Netherlands.
The knowledge about drug resistance in childhood leukemias and acute
lymphoblastic leukemia (ALL) in general is limited. This is because of the
lack of a suitable in vitro drug sensitivity assay, which is in part due to
low in vitro ALL cell survival. We recently adapted the highly efficient
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay
to test cells from ALL patients and showed that its results were comparable
with those of the DiSC assay, up to now the most valid but laborious assay.
In this study, in vitro drug sensitivity was assessed in cells from 82
children with leukemia, 79 of whom had ALL, with the MTT assay. Dose
response curves were obtained for 6-mercaptopurine, 6-thioguanine (6-TG),
prednisolone (Pred), daunorubicin (DNR), vincristine (VCR), cytosine
arabinoside (Ara-C), L- asparaginase (L-Asp), mafosfamide, and mustine. A
cytotoxic effect of methotrexate could be detected in only a few cases.
Large interindividual differences in drug sensitivity were detected.
Compared with leukemia cells from newly diagnosed patients, leukemia cells
from relapsed patients were significantly more in vitro resistant to 6-TG,
Pred, Ara-C, mafosfamide and mustine but not to DNR, VCR, and L-Asp.
Improvements of culture medium and methods to increase MTT reduction were
studied. From 10 components tested, addition of insulin and bovine serum
albumin to serum-containing medium improved ALL cell survival. Addition of
succinate did not increase the amount of MTT reduction. We conclude that
the in vitro MTT assay highly facilitates large-scale studies on drug
resistance of ALL patients that can lead to rational improvements in
existing treatment protocols.
Volume 76,
Issue 11,
pp. 2327-2336,
12/01/1990
Copyright © 1990 by The American Society of Hematology
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