Blood, Vol. 92 No. 3 (August 1), 1998:
pp. 968-980
Use of the Microculture Kinetic Assay of Apoptosis to Determine
Chemosensitivities of Leukemias
Vladimir D. Kravtsov,
John P. Greer,
James A. Whitlock, and
Mark
J. Koury
From the Department of Medicine, the Division of Hematology and the
Department of Pediatrics, the Division of Pediatric
Hematology/Oncology, Vanderbilt University Medical Center, Nashville,
TN.
Chemotherapeutic agents exert their antitumor effects by inducing
apoptosis. The microculture kinetic (MiCK) assay provides an automated,
continuous means of monitoring apoptosis in a cell population. We used
the MiCK assay to determine the chemosensitivities of the human
promyelocytic HL-60 and lymphoblastic CEM cell lines and leukemia cells
freshly isolated from patients with acute nonlymphocytic (ANLL) or
acute lymphocytic (ALL) leukemias. Continuous monitoring of apoptosis
in the MiCK assay permits determination of the time to the maximum
apoptosis (Tm) and its two components which are initiation
time (Ti) and development time (Td). Duration
of the three timing components of apoptosis varies from hours to days depending on the drug, drug concentration, and type of target cells. In
the MiCK assay, the extent of apoptosis is reported in kinetic units of
apoptosis. Kinetic units are determined by the slope of the curve
created when optical density caused by cell blebbing is plotted as a
function of time. Using the leukemia cell lines, we define the
relationship between kinetic units determined by the MiCK assay and the
percentage of morphologically apoptotic cells in the culture. Flow
cytometry analysis of apoptosis in Annexin-V-fluorescein
isothiocyanate-labeled preparations of HL-60 and CEM cells was also
used to compare with data obtained by the MiCK assay. The feasibility
of the MiCK assay of apoptosis as a chemosensitivity test was confirmed
by its comparison with a 3H-thymidine incorporation assay.
We show that samples from 10 ANLL and ALL patients patients tested for
sensitivity to various doses of idarubicin (IDR), daunorubicin (DNR),
or mitoxantrone (MTA) gave the same percentages of apoptotic cells when
calculated by the MiCK assay as when determined by morphological
analysis. The MiCK assay was used for dose-response analyses of the
sensitivities to IDR, DNR, and MTA of leukemia cells from 4 other
patients (2 ANLL and 2 ALL). The results from both cell lines and
patient samples indicate that ANLL cells are more sensitive than ALL
cells to all three of these chemotherapeutic agents. However, for
individual patients the chemosensitivities varied significantly among
the three chemotherapeutic agents. These varying responses to IDR, DNR,
and MTA indicate that the MiCK assay results can be of potential use in
designing a treatment regimen for a specific patient with acute
leukemia. Among several drugs of presumed similar efficacy, the MiCK
assay can permit the selection of the specific chemotherapeutic agent
that causes the most apoptosis in the patient's leukemic cells.
© 1998 by The American Society of Hematology.
