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Blood, Vol. 92 No. 3 (August 1), 1998:
pp. 968-980
By
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.
LABORATORY TESTS for chemosensitivity of
leukemia cells can be separated into two major groups: (1) long-term
assays based on clonal growth of the leukemic stem cells, and (2)
short-term assays of the total cell population relying on loss of cell
viability.1-6 Technical and theoretical limitations of
these methods have been reviewed by several
investigators7-10 with the common conclusion that
development of new chemosensitivity assays based on alternative methodologies is needed.
Apoptosis is a distinct mode of cell death that occurs under
physiological conditions and yet can be induced in normal and malignant
cells by low doses of chemical and physical factors. Because
chemotherapeutic agents exert their in vivo antitumor activity by
triggering apoptosis in susceptible tumor cells,11-14 the
in vitro measurement of drug-induced apoptosis should provide a
mechanism-based test for the chemosensitivity of the tumor cells isolated from an individual patient. When malignant cells are exposed
to a chemotherapeutic agent in vitro, apoptosis in the population can
be determined at a specific time by microscopic examination,
electrophoretic separation of DNA fragments,15 or flow
cytometry.16 However, apoptotic cells exist in vitro for a
limited time, after which they promptly disintegrate.17,18 Thus, apoptosis in vitro occurs in a relatively narrow temporal window
such that an accurate determination of apoptosis in a population of
cells requires either frequent determinations or continuous monitoring.
The above-mentioned tests for apoptosis require multiple steps and yet
they permit only one time point per sample to be examined. These
drawbacks make current assays of apoptosis cumbersome and impractical
for clinical use.
We have recently described a microculture kinetic (MiCK) assay for
determining apoptosis in a population of cells.19 In this
method, the cells are cultured in a 96-well microtiter plate and serial
measurements of optical density (OD) are made automatically every 5 minutes over a period of hours to days. By plotting OD as a function of
time, both the extent of apoptosis and its precise timing can be
determined in the cell population. Among the various methods for
determining apoptosis, the MiCK assay is unique for three reasons.
First, the MiCK assay is based on the appearance of the characteristic
membrane distortions termed blebs that precede DNA breakdown, which
is most commonly used to quantitate
apoptosis.17,19 Second, the MiCK assay uses
prolonged monitoring of a cell population by frequent, repeated
measurements without the removal, destruction, or fixation of any part
or the whole of the cell population being examined. Third, once the
cultures are initiated, the MiCK assay is completely automated such
that no further procedures, technician time, or instruments are
required. These unique qualities of the MiCK assay for apoptosis
make it especially useful for determining the in vitro sensitivity of a
leukemia cell population to specific chemotherapeutic agents.
In the present study, the MiCK assay is used to determine the in vitro
chemosensitivities for (1) the human promyelocytic HL-60 and
lymphoblastic CEM leukemia cell lines, and (2) leukemia cells isolated
from the blood or bone marrow (BM) of patients with acute
nonlymphocytic (ANLL) or lymphocytic (ALL) leukemia. We introduce a
unit for measurement of apoptosis in the MiCK assay, the kinetic unit
(KU) of apoptosis, which is correlated with the percentage of
morphologically apoptotic cells within the leukemic population. We
demonstrate that the leukemia cells of the same patient can show
differential sensitivities to drugs with similar mechanisms of action,
ie, the anthracyclines, daunorubicin and idarubicin, and the synthetic
anthracenedione, mitoxantrone. Because these chemotherapeutic
agents are used clinically for the treatment of acute leukemias, the
information provided by the MiCK assay may help in the clinical
decisions about which specific chemotherapeutic agents are most likely
to induce remission in an individual patient.
Cell lines.
Human HL-60 acute promyelocytic leukemia cells (purchased from American
Type Culture Collection, Rockville, MD) and lymphoblastic CEM cells (a
gift of Dr W.T. Beck, St Jude Children's Research Hospital, Memphis,
TN) were maintained in RPMI-1640 medium without phenol red and
supplemented with 10% heat-inactivated fetal bovine serum (FBS;
Hyclone, Logan, UT), 100 U/mL penicillin, and 100 µg/mL streptomycin
(complete medium) in completely humidified air with 5% CO2
at 37°C. The cultures were diluted every third day to a
concentration of 5 × 105 cells/mL. Before use,
exponentially growing cells were obtained, washed with prewarmed
RPMI-1640 medium, and resuspended at various concentrations in complete
medium. Cell counts and viability were determined using a hemocytometer
and trypan blue dye exclusion.
Patient leukemia samples.
Venous blood samples or BM aspirates were obtained with informed
consent from 8 patients with a diagnosis of primary ANLL, 3 patients
with a diagnosis of primary B-lineage ALL, 1 patient with a diagnosis
of T-cell ALL, and 2 patients with biphenotypic acute leukemia before
treatment. Profiles of the patients are summarized in
Table 1. The heparinized samples were
diluted twofold with RPMI-1640 medium and the mononuclear cell
fractions were collected after centrifugation on a Ficoll-Hypaque
gradient (Nicomed Pharma AS, Oslo, Norway). T lymphocytes and monocytes
were removed from the mononuclear fractions using magnetic beads
conjugated with monoclonal antibodies (MoAbs) to CD2 and CD14,
respectively (Dynal, Inc, Lake Success, NY). The resulting leukemia
cell populations contained more than 95% blast cells and had
viabilities exceeding 90%.
MiCK assay for apoptosis.
The MiCK assay for apoptosis was performed as described
previously19 with minor modifications. Cells were suspended
in complete medium and plated in 240-µL aliquots in 96-well
microtiter plates (Corning-Costar, Cambridge, MA). Seeding cell
concentrations were determined in preliminary experiments and varied
depending on the size of the cells. Appropriate dilutions of
daunorubicin (DNR), idarubicin (IDR), or mitoxantrone (MTA) were added
to wells in 10-µL aliquots to yield final concentrations of 0.05, 0.1, 0.5, 1, 2.5, 5, 10, and 20 µmol/L. The microtiter plates were
incubated at 37°C for 30 minutes in a completely humidified
atmosphere of 5% CO2 in air. Next, 50 µL of sterile
mineral oil (Sigma, St Louis, MO) was layered on the top of each
microculture. The microtiter plate was placed in the incubated chamber
of a spectrophotometer (SPECTRAmax 340; Molecular Devices Corp,
Sunnyvale, CA), incubated at 37°C, and the OD at 600 nm was read
every 5 minutes for a period of 24 to 48 hours. The reader was
calibrated to zero absorbance using wells containing only complete
medium without cells. The AUTOmix feature of the reader was used to
shake the microtiter plate before each OD reading.
Data processing.
During the assay, the OD readings are plotted against time providing a
kinetic representation of the drug responses. Apoptosis is graphically
indicated by an "apoptotic curve" with a characteristic portion
showing a steep increase of the OD of the culture
(Fig 1). The slope of this steep increase
in OD is called the maximum kinetic rate (Vmax) and can be calculated
automatically by the computer software (SOFTmax Pro; Molecular Devices)
provided with the microtiter plate reader. We have previously shown
that the value of the Vmax of the apoptotic curve is correlated
directly with the percentage of morphologically apoptotic cells in the culture.19 This automatic determination of the Vmax rate of the apoptotic curve provides an objective and convenient measure of the
extent of apoptosis in cultures. Our observations with a variety of
myeloid and lymphoid leukemia cells have shown 3 hours as the minimal
time that elapses between the initiation of the steep rising portion of
the apoptotic curve and its maximum. Based on these data, the Vmax of
the steep rising portion of the apoptotic curve was always determined
over a 3-hour period. This determination was made by setting the
SOFTmaxPro software to detect Vmax over 36 consecutive OD points which
were taken with a periodicity of 5 minutes. With this setting, the
slopes of the multiple lines derived from the 36 consecutive OD
measurements were calculated over the entire period of the assay and
the steepest one was displayed by the software as Vmax.
Quantitation of apoptosis in cell cultures.
During 48 hours of culture, varying proportions of leukemic cells die
by spontaneous apoptosis. Also, in proliferating cultures, cell number
may increase slightly over the time when the steep OD increase caused
by apoptosis is developed (usually this period lasts from 3 to 10 hours). These spontaneous apoptosis-related changes in cell morphology
and slight increase in cell number can contribute to the rate of the OD
increases of both the drug-treated and control cells. To distinguish
these background OD changes from OD changes due to drug-induced
apoptosis, the Vmax was determined in the control wells (cells in
complete medium with no chemotherapeutic agents added) over the same
period of culture when the steep OD increase was observed in the wells
containing drugs. This Vmax value for the control wells was subtracted
from the Vmax values of the apoptotic curves for each of the
drug-treated wells:
Cell morphology.
Percentages of cells with morphological evidence of apoptosis were
counted on Giemsa-stained cytospin preparation of control and
drug-treated cultures. A total of 200 cells was counted on each
preparation. Apoptotic cells were identified by plasma membrane protrusions, aggregated chromatin, fragmented nuclei, and condensed basophilic cytoplasm.
Fluorescein-conjugated Annexin V (Annexin V-FITC) binding assay.
Labeling of cells with Annexin-V-FITC conjugate was performed using an
Apoptosis Detection Kit (R&D Systems, Minneapolis, MN) in which Annexin
V-FITC, propidium iodide (PI), and binding buffer were included as
standard reagents.20 Flow cytometry was
performed on a FACScan flow cytometer (Becton Dickinson, Mountain View,
CA) with excitation at 488 nm. FITC fluorescence was measured at
515-545 nm and fluorescence of DNA-PI complexes at 565-606 nm. Cell
debris was excluded from analysis by appropriate forward light scatter
threshold setting. Compensation was used wherever necessary.
3H-thymidine (3H-TdR) incorporation assay.
The short-term assay by Dosik et al21 was used to study
effects of chemotherapeutic agents on proliferating activity of HL-60
and CEM cells. Chemotherapeutic agents were added to cultures in a
96-well microtiter plate that was incubated in a
CO2-incubator at 37°C for various periods of time.
During the final 2 hours of incubation, 10-µL aliquots of
3H-TdR (specific activity of 6.7 Ci/mmol; NEN Life Science
Products, Inc, Boston, MA) were added to each well for a final
concentration of 2.5 µCi/mL. Cells were collected on a filter paper
using an automated cell harvester (Scatron Instruments
Inc, Sterling, VA). 3H-TdR incorporation was
measured using a liquid scintillation analyzer (Beckman Instruments
Inc, Irvine, CA) and results were expressed as counts per minute (cpm)
per 103 cells. Data obtained from control and treated
microcultures were used to calculate the percentage of
3H-thymidine incorporation inhibition.
Statistics and graphics.
Polynomial and linear regression analyses of correlations between KU of
apoptosis and percentages of morphologically apoptotic cells in
cultures as well as all graphics and other statistics were performed
using Origin Scientific Software (MicroCal Software, Inc, Northampton,
MA).
Sensitivity limits and optimal cell concentrations.
The MiCK assay monitors OD changes in microcultures caused by the
apoptosis-associated modifications in cell morphology. We have shown
that membrane blebbing is the main determinant of the steep OD
increases in apoptotic cultures.19 In principle, the MiCK
assay should be able to monitor apoptosis in a single cell. In
practice, however, the sensitivity of the MiCK assay is limited by the
technical capabilities of commercially available spectrophotometers which require a certain number of cells undergoing apoptosis to produce
a collective signal that can be detected. An initial cell density that
ensured both consistent growth of immortalized cell lines and
maintenance of slowly dividing or nondividing cells for 48 to 72 hours
and yet provided an adequate signal due to apoptosis-related
morphological changes in the cells is referred to here as an optimal
cell concentration. It follows from our observations that, in the range
of the optimal cell concentrations, a minimum of 5% of cells must
undergo apoptosis to produce a signal which could be "visible"
for the reader. We determined empirically the optimal cell
concentrations for some human myeloid and lymphocytic leukemia cell
lines (HL-60, U-937, AML-2, AML-3, CEM, Raji) and for freshly isolated
human leukemic myeloblasts, lymphoblasts, B-cell chronic lymphocytic
leukemia (CLL), and T-cell CLL cells. The optimal initial
cell concentrations varied fivefold from 3 × 105
cells/mL for HL-60 cells to 1.5 × 106 cells/mL for
B-CLL cells and depended on cell size such that larger cells required
lower initial cell concentrations than smaller cells. The common
criterion used to determine the optimal cell concentration at the
initiation of the culture was the difference between the initial OD of
the control microcultures and the OD of a blank well containing only
complete medium (ODcell KU of apoptosis. The manufacturer's software (SOFTmaxPro; Molecular Devices) is preset to display Vmax for kinetic reactions in milli-optical density units per minute (mOD/min). This often results in extremely small numbers that are cumbersome and inconvenient for analyses. For convenience of presentation, we multiplied the software-determined value of Vmax by 60, thus converting data from mOD/min to mOD/h. Taking into account the Vmax value of the control cultures and normalizing the data with SCDC, the equation for determining the extent of apoptosis in the MiCK assay is:
MiCK assay for apoptosis in HL-60 and CEM cells.
HL-60 and CEM cells were exposed to a wide range of concentrations of
DNR, IDR, or MTA and studied in the MiCK assay for 48 hours. The
maximum concentration of each drug was based on reported peak plasma
concentrations in leukemia patients during induction therapy.22-26 The KU obtained for each drug
was plotted against drug concentration resulting in
dose-response curves (Fig 3A and B). The extent of apoptosis varied
significantly depending on the drug, the drug concentration, and the
cell line tested. In HL-60 cells, IDR and MTA caused maximum apoptosis
at 0.5 µmol/L and 2.5 µmol/L (14.2 and 14.9 KU, respectively;
Fig 3A). This extent of apoptosis
corresponded to approximately 80% morphologically apoptotic cells (Fig
2). Further increases in doses of both drugs were followed by a
decrease of apoptosis in the MiCK assay. A different dose-response
curve was obtained for HL-60 cells exposed to DNR. Here, two maximum
apoptotic responses were seen at 0.1 µmol/L (9.4 KU) and
10 µmol/L (9.5 KU). These may reflect a cell-cycle phase-related
heterogeneity in sensitivities of the subsets of the cell population to
DNR such that more susceptible cells undergo apoptosis at 0.1 µmol/L
DNR while for more resistant cells higher doses of DNR were required to
initiate apoptosis.
Annexin V binding and 3H-TdR incorporation assays.
HL-60 and CEM cells were exposed to the drug concentrations which in
the MiCK assay were found to induce maximum apoptosis in the cells (Fig
3). The time of the drug exposure corresponded to the time at which the
MiCK assay reported a maximum extent of apoptosis in each cell type
(see Fig 9A and B). At the end of the incubation, cells were double
labeled with Annexin V-FITC and propidium iodide (PI) to
distinguish the early apoptotic cells, or studied with the
3H-TdR incorporation assay for antiproliferative effects of
the drugs. Simultaneously, cells were subjected to the MiCK assay of
apoptosis.
MiCK assay for apoptosis in human ANLL and ALL cells.
Freshly isolated leukemic myeloblasts and lymphoblasts were exposed to
increasing concentrations of DNR, IDR, and MTA in a 40-hour MiCK assay.
Apoptotic responses were determined and plotted against drug
concentrations yielding drug-response curves
(Figs 6 and 7).
All three agents induced apoptosis in cells of ANLL patient no. 5 while
only IDR was effective with cells of ANLL patient no. 14 (Fig 6A and
B). The greatest apoptotic responses were seen in cells of patient no.
5 and patient no. 14 at 20 µmol/L IDR (8.2 KU or 53% of apoptotic
cells and 5.6 KU, or 37.6% of apoptotic cells, respectively). By
comparison, in myeloid HL-60 cells a similar extent of apoptosis could
be induced by 0.1 µmol/L of any of the three drugs (Fig 3A). This
indicates that freshly isolated myeloblasts from these two patients
were less sensitive to chemotherapeutic drugs than HL-60 cells.
Timing of apoptosis.
Because the MiCK assay measures apoptosis continuously, it provides a
unique opportunity for timing of the apoptotic process. A typical
apoptotic curve from the MiCK assay of apoptosis is shown (see Fig 9).
The maximum point of the apoptotic curve corresponds to the time when
the greatest percentage of cells form membrane blebs,19 ie,
undergo the initial stage of apoptosis. We have designated the period
of time from the exposure of the cells to an apoptotic stimulus until
the maximum apoptotic OD as the time to maximum apoptotic response
(Tm). If apoptotic stimuli are added to cells shortly
before initiation of the MiCK assay, Tm would correspond to
the time from onset of the assay until the maximum OD. As is seen in
Figure 8, Tm consists of two
components. The first component is the initiation time (Ti)
which is the period between exposure of the cells to an apoptotic
stimulus and the beginning of the steep rising portion of the curve.
The second component is the development time (Td), which is
a period between the beginning of the steep rising portion of the curve
and its apoptotic OD maximum. Figure 9
shows that increases in concentrations of DNR, IDR, and MTA were always
accompanied by shortening of Tm in both leukemia cell lines
and freshly isolated leukemic cells. These shortenings of
Tm resulted from shortening of both Ti and Td (data not shown). Although Ti did not have a
definite lower limit, Td was never less than 3 hours. A
relationship was seen between the degree of sensitivity of cells to a
drug and the difference between Tm at the minimal and
maximal drug concentrations. In cells from both ANLL and ALL patients
exposed to IDR, significant differences between duration of
Tm at minimal and maximal drug concentrations are evident
(Fig 9C through F). This progressive shortening of Tm with
increasing IDR dose corresponds to the effectiveness of IDR in the
induction of apoptosis in cells of both groups of patients (Figs 6 and
7). Conversely, an increase in concentration of MTA had little effect
on Tm in cultures of ANLL patient no. 14 (Fig 9D) and both
ALL patients (Fig 9E and F). The absence of significant changes of the
Tm with increasing MTA dose corresponds to the low
apoptotic response of the cells of these three patients to all tested
concentrations of MTA (Figs 6B and 7A and B).
This study examined the applicability of the MiCK assay of apoptosis
for determining sensitivities of myeloid and lymphoid leukemia cells to
chemotherapeutic agents. The use of apoptosis as an indicator of drug
effectiveness is based on the concept that activation of cellular
self-destructive machinery is the main mechanism by which antitumor
agents exert their therapeutic effects.11-14,27-29
Submitted August 13, 1997;
accepted March 13, 1998.
The authors thank Gail Hermann for her assistance in procurement of
clinical samples, Dr Jim Price for assistance with the Annexin V-FITC
binding assay, and Prof Maurice Bondurant for critical review of the
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Abstract
Introduction
Abstract
ODblank). The
best results of the MiCK assay in terms of its sensitivity and
reproducibility were always achieved when this difference was 0.03 ± 0.01 OD. Before a previously untested cell type was studied in
the MiCK assay for apoptosis, serial dilutions of the cells were
prepared and their ODs compared with the OD of a blank well in single
point measurements at 600 nm. Once a cell concentration producing
ODcell 
) or CEM (
)
cells were exposed to the drugs and distributed in two 96-well
microtiter plates. One plate was placed into an incubated
spectrophotometer and apoptosis was studied in the MiCK assay as
described in Materials and Methods. The second plate was incubated in a
humidified CO2-incubator. At the time points when the MiCK
assay reported maximum apoptosis, cytospin preparations were made from
cell aliquots from appropriate wells of the second plate. Apoptosis was
measured with the MiCK assay in KU and determined microscopically in
Giemsa-stained preparations as described in Materials and Methods. A
total of 50 cell cultures exposed to various inducers of apoptosis were
studied.
, were usually detected
(Fig 
the event which is monitored in the MiCK assay.
on cell death of cultured rat hepatocytes.
Cancer Res
51:2478,
1991
