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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 7 (October 1), 1999:
pp. 2414-2423
Triggering Noncycling Hematopoietic Progenitors and Leukemic Blasts to
Proliferate Increases Anthracycline Retention and Toxicity by
Downregulating Multidrug Resistance
By
Mariëlle E.P. Smeets,
Reinier A.P. Raymakers,
Gerty Vierwinden,
Arie H.M. Pennings,
Hans Wessels, and
Theo de Witte
From the Division of Hematology, University Hospital Nijmegen,
Nijmegen, The Netherlands.
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ABSTRACT |
Expression of the multidrug resistance (MDR) mechanisms
P-glycoprotein (Pgp) and MDR-related protein (MRP) decrease cellular retention and consequently cytotoxicity of anthracyclines. MDR is
expressed on normal human hematopoietic progenitors and leukemic blasts. Normal CD34+ progenitors showed rhodamine efflux
in 20% to 30% of the cells, which could be blocked by verapamil.
These cells appeared noncycling, in contrast to the proliferating
rhodamine bright (RhoB) cells. We postulated that MDR expression can be
downregulated by proliferation induction. Triggering rhodamine dull
(RhoD) CD34+ cells to proliferate indeed resulted in a
higher rhodamine retention and significantly decreased efflux
modulation by verapamil (P = .04). Also in acute myeloid
leukemia (AML), the proliferation rate (percentage S/G2+M
and Iododeoxyuridine labelings index) was significantly less in the
RhoD blasts (P  .008) and proliferation induction of RhoD
blasts resulted in increased rhodamine retention. Anthracycline
cytotoxicity was less for RhoD than RhoB cells in both normal
progenitors and leukemic blasts. Proliferation induction of the RhoD
cells resulted in increased anthracycline sensitivity. We conclude that
noncycling progenitors, both normal and leukemic, have a relatively
high MDR expression. Triggering these cells into proliferation
downregulates MDR expression. These findings can be exploited to
overcome MDR in the treatment of AML patients.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
DRUG RESISTANCE IN acute myeloid leukemia
(AML) has been associated with the expression of the
P-glycoprotein (Pgp), the phenotype of the multidrug
resistance (MDR)-1 gene.1-4 Overexpression of the
MDR-related protein (MRP) in AML may also play a clinical role.5-7 Both proteins are active transmembrane
transporters, pumping a wide variety of substances including cytotoxic
drugs out of the cells, thus lowering the intracellular concentration.
MDR activity can be determined by a functional assay with specific
dyes. MDR expression corresponds to a low retention of rhodamine or
daunorubicin and its increase by specific modulators like verapamil or
cyclosporin A.8-12 In mice, rhodamine dull (RhoD) hematopoietic progenitors appeared to be primitive noncycling cells.13 Darzynkiewicz et al14 and Myc et
al15 showed that rhodamine retention distinguishes between
cycling and quiescent cells in peripheral blood lymphocytes (PBL) and
leukemic cells. This led to the hypothesis that noncycling cells have a
higher MDR expression, which may be downregulated by inducing
proliferation. Previous experiments performed in our laboratory showed
that proliferation induction of normal hematopoietic progenitors and
leukemic blasts resulted in an increased sensitivity to
anthracyclines.16,17 However, MDR expression was not
studied in these cells.
Studies on MDR expression during the different cell-cycle phases showed
conflicting results. Tarasiuk et al18 found no differences in MDR-dependent efflux during the different cell-cycle phases in K562
cells, whereas Ramachandran et al19 showed that P388/R-84 cells in S-phase had a 3-fold higher MDR-1 mRNA content than did G1- and G2+M-phase cells. Also, MDR expression,
determined by the monoclonal antibody (MoAb), C219, was lower in
G1 cells than in S and G2+M cells. These
experiments were performed with cell lines selected for a high MDR
expression. As yet, little is known about MDR expression in cycling and
in noncycling hematopoietic progenitors and the effects of
proliferation induction.
Normal CD34+ progenitors and leukemic blasts from patients
with untreated AML were studied for functional MDR expression in relation to cell-cycle status and proliferation. RhoD CD34+
cells or leukemic blast cells with a relatively high MDR expression appeared to be noncycling cells compared with the more proliferating RhoB cells with a relatively low MDR expression. RhoD cells showed less
sensitivity to anthracycline toxicity. Triggering RhoD cells into
proliferation by hematopoietic growth factors (HGFs) diminished MDR
expression and consequently increased anthracycline toxicity.
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MATERIALS AND METHODS |
Isolation of Cells
CD34+ cells.
Bone marrow was obtained from healthy donors, after informed consent.
Mononuclear cells were isolated by Ficoll 1.077 g/mL (Pharmacy Biotech,
Uppsala, Sweden). CD34+ cells were isolated using directly
conjugated CD34 antibody-coupled immunomagnetic beads (M-450, coated
with `561,' a class III epitope anti-CD34; Dynal, Oslo, Norway)
according to the manufacturer's instructions (Dynal protocol).
Isolation, cryopreservation, and thawing procedures of the
CD34+ cells have been described previously.20
Leukemic blasts.
Bone marrow was aspirated from patients with AML and collected in
sterile buffered acid citrate dextrose (pH 7.0). Mononuclear cells were
isolated by Ficoll 1.077 g/mL. Cells were cryopreserved in vials
containing 5 to 20 × 106 cells. Blast cells were
gated on the flow cytometer by forward scatter and the absence of the
lymphocyte markers CD3 and CD19.
Lymphocytes.
Normal donor lymphocytes were isolated from heparinized blood using
Ficoll 1.077 g/mL.
Propidium Iodide (PI) DNA Staining
PI (Calbiochem, San Diego, CA) was used to measure DNA content. Sorted
RhoD and RhoB cells were centrifuged (5 minutes, 1,800 rpm, 4°C).
The pellet was dissolved in 300 µL of ice-cold hypotonic PI
solution (20 µg/mL PI, 0.1% wt/vol trisodium citrate dihydrate (Merck, Darmstadt, Germany), 10% vol/vol RNA-se solution (RNA-se A;
Sigma Chemical Co, St Louis, MO), 1 mg/mL in glucose phosphate-buffered saline (G-PBS), and 0.1% vol/vol Triton X-100 in distilled water (dH2O). Cells were kept on ice overnight and analyzed for
DNA content by flow cytometry.
Ki-67 Labeling
The nuclear antigen Ki-67 discriminates between the cell-cycle phases
G0 and G1.21 Sorted
CD34+ cells were pelleted (5 minutes, 1,800 rpm, 4°C)
and subsequently fixed in 1 mL freshly made paraformaldehyde 0.5%
wt/vol (5 minutes, 4°C). After washing with G-PBS 0.5% wt/vol
bovine serum albumin (BSA, Fraction V, A-9418, Sigma Chemical Co), 1 mL
PBS-Triton 0.1% vol/vol (Merck) was added (5 minutes, 4°C). The
cells were then washed and incubated with 5 µL fluorescein
isothiocyanate (FITC)-conjugated rabbit anti-human Ki-67 MoAb (Dako,
Glostrup, Denmark) (20 minutes, 4°C) in 100 µL pooled human serum
(PHS). Rabbit F(ab')2 FITC (Dako) was used as a
control. After washing with PBS 0.5% wt/vol BSA, green fluorescence
(FITC) was measured on the flow cytometer (standard setting) using a
550 dichroic and 525 band-pass filter. The staining procedure was
checked with lymphocytes, as normal lymphocytes are almost all
G0 cells and Ki-67 negative, and a cell-line, which was
almost 100% Ki-67 positive.
In Vivo Iododeoxyuridine Labeling and Staining Procedure
To study the proliferative state of leukemic blasts in vivo, untreated
patients with newly diagnosed AML, who all gave their written informed
consent, volunteered to be given Iododeoxyuridine (IdUrd)
intravenously. IdUrd is a nonradioactive thymidine analogue, incorporated during DNA synthesis. Vials of 200 mg freeze-dried IdUrd
were supplied by the National Cancer Institute (NCI) (Bethesda, MD)
(NCI-protocol T89-0075). A nontoxic dose of 200 mg IdUrd was dissolved
in 30 mL isotonic saline (0.9% wt/vol NaCl) and given as a 15-minute
intravenous infusion. Between 4.6 and 7.0 hours (mean value, 5.4) after
in vivo IdUrd labeling, bone marrow was aspirated in sterile buffered
acid citrate dextrose (pH 7.0).22 Mononuclear cells were
isolated by Ficoll 1.077 g/mL and cryopreserved in vials containing 5 to 20 × 106 cells.
After thawing and staining, 25% of the most RhoD and RhoB blast cells
were sorted, pelleted, fixed, and stained as described by van Erp et
al.23 The green fluorescence was measured after a final
wash step with G-PBS 0.5% wt/vol BSA. Non-IdUrd-labeled low-density
blood cells of a healthy volunteer served as negative controls. The
labeling index (LI) is defined as the percentage IdUrd-labeled cells
and represents the cells in S-phase of the cell cycle. A correction was
made for the cells that left the S-phase because the IdUrd labeling was
performed. Subsequently DNA was stained with an isotonic PI
solution.
Proliferation Induction
Sorted RhoD CD34+ cells or RhoD leukemic blasts were
diluted in medium A (1 × 106 cells/mL) consisting of
Iscove's medium, supplemented with 2 mmol/L glutamin (Flow
Laboratories, Irvine, Scotland), 50 µg/mL streptomycin, and 50 U/mL
penicillin (Flow Laboratories), 20% vol/vol fetal calf serum, 5%
wt/vol BSA, 0.3 mg/mL human transferrin (Sigma), 50 µmol/L
2- -mercaptoethanol (Sigma), 20 ng/mL recombinant granulocyte
colony-stimulating factor (rG-CSF) (Amgen, Thousand Oaks,
CA), and 25 ng/mL recombinant stem cell factor (rSCF) (a gift from Amgen), 50 ng/mL interleukin-3 (IL-3) (Sandoz, Basel, Switzerland), 20 ng/mL recombinant granulocyte-macrophage
(rGM)-CSF (Sandoz), and 1.5 U/mL recombinant
erythropoietin (rEPO; Cilag, Herentals, Belgium). MDR
efflux was measured after 48 hours proliferation induction.
Normal lymphocytes were exposed for 24 hours to IL-2 100 U/mL (Glaxo,
Geneve, Swizerland) and phytohemagglutinin (PHA) 4 µg/mL (Murex Diagnostics, Dartford, UK) for induction of proliferation.
MDR Expression
MDR was measured in a functional rhodamine efflux assay. Rhodamine
(Rh123; Sigma Chemical Co) was dissolved in dH2O and stored as a sterile stock solution (10 µg/mL). The cells were incubated (200,000 cells/mL) in Iscove's medium supplemented with 5% wt/vol heat-inactivated fetal calf serum (FCS, Hyclone, Logan UT) with a final
rhodamine concentration of 0.1 µg/mL (60 minutes, 37°C). After
centrifugation, the cells were diluted in dye-free Iscove's medium
with 0.5% wt/vol FCS with or without 10 µmol/L verapamil (Knoll AG,
Ludwigshaven, Germany) to block efflux (2 hours, 37°C). Putting the
cells on ice stopped efflux. A similar efflux assay was performed with
daunorubicin (Rhone-Poulenc Rorer BV, Amstelveen, The Netherlands).
This anthracycline exhibits intrinsic fluorescence and is also expelled
by the MDR. Rhodamine and daunorubicin fluorescence was quantitated by
flow cytometry.
Efflux modulation was expressed as rhodamine ratio (RR), the
fluorescence intensity of the cellular rhodamine content in the presence of verapamil divided by the fluorescence intensity of the
cellular rhodamine content in the absence of verapamil. This ratio is
independent of cell size, which increases after proliferation induction.
Flow Cytometric Measurements and Single-Cell Sorting
A Coulter Epics Elite Flow cytometer, equipped with an autoclone device
(Coulter, Miami, FL), was used for PI, Ki-67, and IdUrd measurement,
dye retention, as well as to sort single cells for clonogenic assay.
Cells were excitated with a single Argon ion laser emitting at 488 nm,
running at 15 mW (standard setting). Gating on forward and right angle
scatter was used to exclude dead cells and debris. Fluorescence
intensity of cellular rhodamine or daunorubicin was measured using a
515 nm and a 550 nm long pass filter, respectively. After 2 hours of
efflux in dye-free medium without verapamil, the distinct population of
RhoD cells and 25% of the most RhoB cells of normal CD34+
cells or leukemic blast were sorted to study MDR expression, proliferation (induction), or cytotoxicity. In case no distinct RhoD
population could be recognized (in leukemic blasts), 25% of the most
RhoD blasts were sorted.
DNA content was analyzed after staining with PI by flow cytometry using
a 610-nm long pass filter. The area and peak of the red fluorescence
signal was recorded in list mode. The ratio area: peak was
used to discriminate artifacts due to doublets of diploid cells.
In Vitro Anthracycline Cytotoxicity
Inhibition of clonogenic capacity by anthracyclines in
CD34+ cells and leukemic blasts with a relatively low
(RhoB) or high (RhoD) MDR expression before and after prestimulation
with HGFs was quantified using a single-cell clonogenic assay. A single cell was sorted in each well from round bottom 96-wells plates (Costar
no. 3799, Cambridge, MA) prefilled with 75 µL liquid medium A to
which daunorubicin or idarubicin (Pharmacia & Upjohn, Milano, Italy)
were added at increasing concentrations (range, 0.0001 to 0.1 µg/mL)
and cultured in a fully humidified atmosphere (37°C, 5%
CO2). Wells were analyzed by counting the cells at days 4, 11, and 18 providing information on the clonogenic capacity and duration of proliferation. At day 18, colonies (>50 cells/well) of
CD34+ cells were classified as small size (50 up to 500 cells/well), medium size (500 up to 5,000 cells/well), or large size
(>5,000 cells/well). In normal CD34+ cells, the relative
number of colonies (>50 cells/well) was plotted, in AML, the relative
number of clusters and colonies (>10 cells/well). The 50% inhibitory
concentration (IC50) of normal CD34+ cells and leukemic
blasts was calculated from the plotted dose-response curves.
Colonies (>50 cells/well) derived from normal CD34 cells were
registered as granulocytes/monocytes, erythrocytes, or mixed. Morphologic analysis of large colonies was checked by immunostaining with the MoAb's glycophorine-Pe (Coulter) to identify erythrocytes, CD-14 PE (Becton Dickinson [BD] BV, Etten-Leur, The Netherlands) to
identify monocytes and CD15-FITC (BD) to identify granulocytes.
Statistics
The Wilcoxon Mann Witney U-test or Student's t-test was
applied for statistical analysis of the results.
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RESULTS |
Cell-Cycle Status of RhoD Versus RhoB Cells
Normal blood lymphocytes were used to show that the technique PI
staining and Ki-67 labeling is adequate. DNA staining of lymphocytes
with PI showed that greater than 90% of the lymphocytes were in
G0/G1 and less than 1% were positive for the
proliferation marker Ki-67. After proliferation induction, the fraction
of Ki-67 positive lymphocytes increased to greater than 95%, while
greater than 40% of cells were in S/G2+M.
DNA staining of CD34+ cells showed that 5% of the RhoD
cells were in S/G2+M-phase (4.5% ± 2.2%, n = 5),
compared with 22% of the RhoB fraction (21.5% ± 4.4%, n = 5). An
example of DNA analysis of CD34+ cells is shown in
Fig 1. Rhodamine and daunorubicin staining showed a similar distribution. A total of 1.6% of the daunorubicin dull cells and 29.9% of the daunorubicin bright cells were in S/G2+M. The proliferation marker Ki-67 was higher in the
RhoB than in the RhoD CD34+ cells, 75% and 43%,
respectively, but differences were much less extreme compared with
lymphocytes.
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| Fig 1.
After the rhodamine (A) or daunorubicin (B) efflux assay
rhodamine or daunorubicin dull and 25% of the most rhodamine or
daunorubicin bright CD34+ cells were sorted and stained
with PI. A representative example of the DNA content of sorted
subfractions is presented.
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In leukemic blasts, PI staining also showed that the percentage
S/G2+M was significantly higher in the RhoB leukemic blasts (31.1% ± 11.7%) (n = 8) compared with the RhoD blasts (10.6% ± 3.2%) (P = .008). The relevance of these in vitro
findings was confirmed by in vivo data. After in vivo administration of
IdUrd, the LI of the RhoD blasts (4.8% ± 2.1%) was significantly
lower (P = .001) compared with the RhoB blasts (15.0% ± 6.0%) (Fig 2).
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| Fig 2.
Patients with newly diagnosed AML received intravenously
IdUrd. After the rhodamine efflux assay, 25% of the most RhoD and RhoB
leukemic blasts were sorted. The sorted subfractions were stained and
the IdUrd LI could be calculated. PI was used to measure DNA content.
Percent S/G2+M and the IdUrd of the RhoD cells was
significantly lower (P = .008 and P = .001, respectively) compared with the RhoB cells.
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MDR Expression on RhoD and RhoB Cells
MDR expression reduces cellular rhodamine content by efflux. A low
rhodamine retention was observed in a distinct population of 20% to
30% of normal CD34+ cells. Blocking the efflux by
verapamil confirmed the presence of functional MDR
(Fig 3A).
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| Fig 3.
After a 1-hour incubation with rhodamine, cells were
exposed for 2 hours to dye-free medium without (1) or with (2)
verapamil to allow rhodamine efflux. Distribution of rhodamine content
of normal CD34+ cells (A) and AML blasts (B and C) was
measured by flow cytometry. Efflux inhibition by verapamil (2) resulted
in an increase of rhodamine content in cells with MDR expression.
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Rhodamine retention was studied in 10 different samples of AML
patients. Patient characteristics are summarized in
Table 1. Rhodamine retention in AML blasts
was heterogeneous, and a more or less distinct RhoD population could be
recognized in most samples. Figure 3B shows an example (patient no. 3)
with a large distinctive RhoD population, whereas Fig 3C shows an
example (patient no. 1) with only a small RhoD population. Increased
rhodamine retention in the presence of verapamil confirmed MDR
expression by these blast cells.
MDR Expression After Proliferation Induction
Exposure of RhoD and RhoB CD34+ cells to HGFs for 48 hours
resulted in an increase of the proportion of S/G2+M in both
fractions. The fraction of S/G2+M increased from 4% to
40% in RhoD and 17.5% to 39% in RhoB cells. After proliferation
induction of the RhoD cells (Fig 4A1), a
second rhodamine efflux assay was performed. The cellular rhodamine
content (Fig 4A2) was higher than before the proliferation induction.
Again, the most RhoD and RhoB cells after stimulation with HGFs were
sorted. In the example shown in Fig 4A2, 15% of the RhoD and 32.2% of
the RhoB cells were in S/G2+M.
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| Fig 4.
Distribution of rhodamine (A and C) or daunorubicin (B
and D) content in normal CD34+ cells or AML blasts after
the rhodamine/daunorubicin efflux assay (1). The 25% most
rhodamine/daunorubicin dull cells were sorted and triggered to
proliferate by exposure to HGFs for 48 hours (2). After proliferation,
induction cellular rhodamine/daunorubicin was higher compared with the
sorted rhodamine/daunorubicin dull cells.
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Staining with daunorubicin showed a less distinct daunorubicin dull
CD34+ population (Fig 4B1). The daunorubicin dull cells
were sorted and exposed to HGFs. In agreement with the results after
rhodamine staining, the cellular daunorubicin content was higher after
stimulation with HGFs (Fig 4B2). To exclude the effect of stimulation
with HGFs on the cellular size and thus fluorescence, a relative
rhodamine ratio (RR) was calculated. RR was defined as the fluorescence intensity of the cellular rhodamine content in the presence of verapamil divided by the fluorescence intensity of the cellular rhodamine content in the absence of verapamil. The RR was significantly higher (P = .009, n = 6) in the RhoD cells compared
with the RhoB cells. After triggering RhoD cells to proliferate with
HGFs, the RR was significantly lower (P = .04, n = 6) compared
with the nonstimulated RhoD cells (Fig 5).
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| Fig 5.
Efflux modulation of rhodamine by verapamil is quantified
by the RR. RR = the fluorescence intensity of the cellular rhodamine
in the presence of verapamil: the fluorescence intensity of the
cellular rhodamine content in the absence of verapamil. The RR was
measured in RhoD and RhoB CD34+ cells and after
prestimulation of these cells for 48 hours in vitro with HGFs. The RR
of RhoD CD34+ cells was significantly higher (P
= .009) compared to the RR of RhoB cells. The RR of RhoD
CD34+ cells was also significantly higher (P = .04) compared to RhoD cells prestimulated with HGFs.
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The RhoD population in AML blasts varied from patient to patient
sample. The example depicted in Fig 4 shows a small
rhodamine (Fig 4C1), respectively, daunorubicin (Fig 4D1) dull
fraction. After proliferation induction of the rhodamine/daunorubicin
dull cells, an increased cellular rhodamine/daunorubicin content was seen in the efflux assay (Fig 4C2 and D2).
The effect of verapamil on rhodamine retention in AML blasts with a
large and small RhoD population, respectively, is illustrated in
Fig 6A and C. In the presence of verapamil,
cellular rhodamine content increased in the RhoD fraction. After
sorting and exposure of the RhoD cells to a cocktail of HGFs, an
increase in rhodamine content was seen only in a subfraction of the
stimulated cells (Fig 6A2 and C2). This subfraction of cells with an
increased rhodamine content after proliferation induction showed a
decrease to negligible efflux modulation (Fig 6B2 and D2). In contrast, cells with a low rhodamine content after proliferation induction maintained their susceptibility to efflux modulation. Sorting the cells
with a relatively low rhodamine content after proliferation induction
(Fig 6A2) showed that 4.2% were in S/G2+M-phase, in contrast to 21.2% of the cells with the relatively high rhodamine content.
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| Fig 6.
Two examples of rhodamine content (A and C) in AML blasts
after the rhodamine efflux assay with (2) or without (1) verapamil in
the efflux medium. Efflux inhibition by verapamil caused a higher
cellular rhodamine content predominantly in the RhoD cells. RhoD blasts
were sorted and triggered to proliferate by exposure to HGFs for 48 hours. Effective proliferation induction resulted in an increase of
cellular rhodamine content (A2 and C2). In these samples, only a
subpopulation of the blasts became rhodamine bright after proliferation
induction. Efflux modulation by verapamil was negligible in the
rhodamine bright cells. The persisting rhodamine dull cells kept their
sensitivity to efflux modulation by verapamil (B2 and D2).
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Anthracycline Toxicity in RhoD and RhoB Cells and After
Prestimulation of RhoD Cells With Growth Factors
To confirm MDR downregulation after induction of proliferation,
anthracycline toxicity of normal CD34+ RhoD cells before
and after prestimulation with HGFs was studied. Prestimulation of RhoD
cells with HGFs resulted in an increased sensitivity to anthracyclines.
IC50s for daunorubicin (Fig 7A) and
idarubicin (Fig 7B) of RhoD cells decreased from 0.006 µg/mL to 0.004 µg/mL and from 0.006 µg/mL to 0.003 µg/mL, respectively, after
prestimulation. After prestimulation of RhoD cells with HGFs for 48 hours, these cells become RhoB. Nevertheless, these RhoB cells are
different from the RhoB cells in the initial sample. Clonogenic
capacity of the RhoD, RhoB, and prestimulated RhoD normal progenitors
were compared to discriminate between the effects of proliferation
induction and possible differentiation into RhoB cells after exposure
to HGFs. Single RhoB cells developed significantly fewer and smaller
colonies (P < .005) compared with both RhoD cells and RhoD
cells after prestimulation with HGFs. The mean values of
single-cell-derived colonies/96-wells plate for RhoD, RhoB, and RhoD
cells prestimulated with HGFs were, respectively, 5.7, 12, and 11 for
small; 7.8, 14.2, and 15.5 for medium; and 21.5, 5, and 13.8 for large
colonies. Furthermore, the RhoD cells and prestimulated RhoD cells
showed significantly more colonies with mixed differentiation
(P < .05) than RhoB cells. Differentiation into mixed
colonies was similar for RhoD cells before and after prestimulation
with HGFs. Prestimulation of RhoD cells with HGFs shifted only the
differentiation pattern of the colonies from granulocyte/monocyte to
more erythroid colonies (P < .005). The mean values of
erythroid and granulocyte/monocyte colonies/96 wells plate were,
respectively, 6.8 and 21.8 for RhoD; 5 and 24.7 for RhoB cells; and
25.3 and 20 for RhoD cells after prestimulated with HGFs. Proliferation
induction of RhoD cells did not influence the outgrowth of mixed
colonies. The mean values of mixed colonies of RhoD, RhoB, and RhoD
cells after proliferation induction were 6.3, 1.5, and 9, respectively
(Table 2). Immunostaining of colonies with
MoAbs confirmed the morphologic analysis after
May-Grünwald-Giemsa staining (data not shown).
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| Fig 7.
Dose-response curve of daunorubicin (A) and idarubicin
(B). In a single-cell clonogenic assay, RhoD CD34+ cells
before and after prestimulation in vitro with HGFs for 48 hours were
exposed to increasing concentrations of anthracycline. Proliferation
was expressed as the percentage colonies (>50 cells/well) in medium A
with daunorubicin/idarubicin compared with the control (medium A
without daunorubicin/idarubicin). The IC50s of daunorubicin (A) and
idarubicin (B) of RhoD cells were 0.006 µg/mL and 0.006 µg/mL
before and 0.004 µg/mL and 0.003 µg/mL, respectively, after
proliferation induction.
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The outgrowth of leukemic blasts was highly variable, as known from
agar cultures. Because the number of colonies was too low, cell
aggregates with more than 10 cells were counted to analyze differences
in anthracycline cytotoxicity between RhoD, RhoB, and stimulated RhoD
blasts. The percentage RhoD cells in the tested AML samples varied from
less than 10% to 33% (Table 1) and proliferation induction of RhoD
blasts was not always complete (Fig 6). We selected 2 patients with
sufficient outgrowth for these experiments.
Figure 8A and B show the difference in
daunorubicin cytotoxicity between RhoD and RhoB leukemic blasts: the
IC50 was 0.008 µg/mL and 0.006µg/mL of RhoD cells and 0.004 µg/mL
and 0.003 µg/mL of RhoB cells of patient no. 1 and patient no. 5, respectively. The effect of proliferation induction on anthracycline
toxicity in leukemic blasts is illustrated in Fig 8B. The daunorubicin
IC50 of RhoD cells decreased from 0.006 µg/mL to 0.003 µg/mL after
prestimulation with HGFs.
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| Fig 8.
Dose-response curves of daunorubicin. In a single-cell
clonogenic assay, RhoD and RhoB leukemic blasts and prestimulated RhoD
cells with HGFs for 48 hours were exposed to increasing concentrations
of anthracycline. Proliferation was expressed as the percentage
clusters (>10 cells/well) in medium A with daunorubicin compared with
the control (medium A without daunorubicin). The IC50 of daunorubicin
of RhoD and RhoB cells of patient no. 1 (A) were 0.008 µg/mL and
0.004 µg/mL and of patient no. 5 (B) 0.006 µg/mL and 0.003 µg/mL,
respectively. The IC50 of daunorubicin of RhoD cells of patient no. 5 decreased from 0.006 µg/mL to 0.003 µg/mL after prestimulation with
HGFs (B).
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| |
DISCUSSION |
In this study, we analyzed the relation between MDR expression and cell
cycle and the effect of proliferation induction on MDR expression. Our
experiments show that RhoD blasts, both normal and leukemic, have a
higher MDR expression, are less sensitive to anthracycline toxicity,
and more in G0/G1-phase of the cell cycle than
RhoB blasts. Triggering these RhoD cells into proliferation increased
rhodamine retention and anthracycline sensitivity and decreased efflux
modulation, confirming MDR downregulation.
The functional rhodamine efflux assay with and without a MDR modulator
has proven to be the most sensitive method to detect MDR expression,
especially in small subpopulations.20,24 A relatively high
efflux was observed in 20% to 30% of the normal CD34+
hematopoietic progenitor cells. These RhoD CD34+ cells
appeared to be predominantly in G0/G1-phase. In
contrast, the RhoB cells were cycling with greater than 20% cells in
S/G2+M. To discriminate G0 and G1
cells, cells were labeled with the nuclear proliferation marker Ki-67.
In normal bone marrow CD34+ cells, Ki-67 labeling in RhoD
and RhoB cells was 43% versus 75%, respectively. Thus, the percentage
G0 cells in the RhoD population was higher (57%) than that
in the RhoB population (25%), although differences were less extreme
compared with nonstimulated versus stimulated lymphocytes. Less than
1% of the lymphocytes were Ki-67 positive before and more than 95% of
the cells were Ki-67 positive after proliferation induction. Similar
problems with Ki-67 labeling of bone marrow cells have also been
observed by van Bockstaele et al,25 who suggested that the
relatively low percentage Ki-67 positivity observed in the RhoB cells
might be explained by differentiation of the cells and a lower Ki-67
expression during the S-phase. Other proliferation markers, such as MIB
and proliferating cell nuclear antigen (PCNA), also did
not distinguish clearly between G0 and G1 (data
not shown).
Triggering noncycling CD34+ cells to proliferate resulted
in a higher cellular retention of rhodamine and daunorubicin.
Proliferation induction is followed by increase in cell size, number,
and activity of organelles, such as mitochondria and proteins, and
therefore may increase cellular fluorescence signal on the flow
cytometer independent of a change in intracellular concentration.
Darzynkiewicz et al14 postulated that rhodamine retention
discriminates between cycling and quiescent cells due to increased
mitochondria binding. However, activity and membrane potential of the
mitochondria do not influence rhodamine efflux. Furthermore, the higher
retention of anthracyclines after induction of proliferation cannot be
explained by mitochondrial activity. To quantify MDR expression
independent of cell size, we calculated a ratio of MDR-mediated efflux
(RR). Proliferation induction resulted both in a higher cellular
rhodamine content and a significantly lower RR.
A high percentage of the clonogenic leukemic myeloblasts are non or
slowly proliferating cells.26,27 In AML blasts, the LI of
IdUrd and the percentage of cells in S/G2+M was correlated with rhodamine retention. Similar to normal progenitors, triggering RhoD leukemic blasts to proliferate induced an increase of rhodamine retention and decreased efflux modulation.
We previously reported that normal RhoD CD34+ cells
appeared more resistant to anthracycline toxicity in a clonogenic assay than RhoB cells.20 Triggering RhoD CD34+ cells
to proliferate resulted in an increased anthracycline toxicity, confirming MDR downregulation.
Butturini et al27 and studies performed in our laboratory
also demonstrated a cell-cycle-dependent anthracycline
toxicity.16,17 Minderman et al17 triggered
leukemic blasts into proliferation by stimulation with human placental
conditioned medium (HPCM) for 48 hours. Doxorubicin IC50
was 0.103 µg/mL before preincubation with HPCM and 0.055 µg/mL
after preincubation. The enhancement of cytoxicity after preincubation
with HPCM showed a strong trend (P < .06) in the matched-pair
analysis. Because the percentage RhoD blasts was limited in the tested
AML samples, we used a single-cell assay to study anthracycline
toxicity in RhoD and RhoB cells. Clonogenic capacity of the tested AML
samples showed great variation, as known from agar cultures. We could
demonstrate that similar to the results in normal CD34+
cells, RhoD leukemic blasts are protected against
anthracycline toxicity. Prestimulation of RhoD blasts with HGFs
resulted in an increased anthracylcline sensitivity.
The size of the RhoD population appeared to be highly variable in the
different AML patients. A small subpopulation with high MDR expression
may be responsible for treatment failure and relapse. te Boekhorst et
al28 have shown that resistant cells in AML have a high
proliferative capacity and these cells may determine the response to therapy.
In 2 patients with AML, Drach et al29 reported that IL-3
and G-CSF induced downregulation of MDR expression in vivo. Multiple studies showed that HGFs, particularly IL-3, GM-CSF, and G-CSF, can
stimulate the proliferation of leukemic myeloblasts.30-33
van der Lely et al34 showed that induction of proliferation
in AML blasts in vitro by HGFs was effective, but variable in subsets of leukemic blasts. Drach et al failed to observe any effect of cytokines on MDR expression in normal CD34+ cells. MDR
expression was studied after exposure for 24 hours to single HGFs by
means of a semiquantitative polymerase chain reaction (PCR) in the
overall population of CD34+ cells. We used the functional
rhodamine efflux assay, which enables identification of a small MDR
expressing subpopulation. MDR downregulation might become obscured by
analysis of the whole CD34+ population. Moreover, Drach et
al29 did not study the effect of cytokines on proliferation
or cell cycle. Our results show that a combination of cytokines
resulted in an increase in S/G2+M. The optimal cocktail of
HGFs for proliferation induction in AML blasts is not known. Therefore,
we used a cocktail of HGFs to induce normal and leukemic blasts into
proliferation and evaluated the effect on proliferation and rhodamine
efflux. Proliferation and rhodamine retention already increased after
24 hours, but was more obvious after 48 hours. The cocktail of HGFs was
rather effective in normal progenitors; in some AML blasts, the
cocktail was not optimal. This explains that in AML, it is more
difficult to show the effect of proliferation induction and MDR downregulation.
To answer the question whether MDR downregulation after 24 to 48 hours
growth factor exposure was due to induction of proliferation or
differentiation into RhoB progenitors, we cultured the RhoD cells after
prestimulation with HGFs for 48 hours. In terms of proliferation, this
means 1 or 2 cell divisions.35 A primitive RhoD progenitor,
which gives rise to large colonies, will not have undergone extensive
differentiation after 1 to 2 cell divisions. The proliferative capacity
of these prestimulated RhoD cells remained unchanged, comparable to the
nonprestimulated RhoD cells and higher than that of the RhoB cells. We
did observe a change in the differentiation pattern to more erythroid
colonies and less granulocyte/monocyte colonies after prestimulation of
RhoD cells.
Several biologic mechanisms may contribute to anthracycline resistance
in AML. Therefore, a multifactorial approach to the treatment of
resistant AML seems rational. Specific MDR modulators have been tested
in therapeutic schedules in an attempt to improve the efficacy of
anthracyclines without any improvements of therapeutic results.36-40 MDR modulators are currently being
investigated for their efficacy in randomized trials in AML. Our
results suggest that antileukemic therapy might be even more effective
when MDR modifiers are combined with HGFs. Some preliminary data
support the relevance of HGFs in clinical studies.41-44
However, the sensitivity of AML blasts in vivo for HGFs can be
variable. The persistence of a RhoD subfraction after proliferation
induction of AML blasts might still be responsible for treatment
failure. Because the primitive CD34+ hematopoietic
progenitors are also triggered and will become more sensitive to
anthracyclines, bone marrow hypoplasia might also be made extensive.
| |
FOOTNOTES |
Submitted June 9, 1997; accepted April 6, 1999.
Supported by grants from the Ank van Vlissingen Foundation, The
Netherlands and the Bekalis Foundation, Belgium.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Mariëlle E.P. Smeets,
MD, Division of Hematology, University Hospital Nijmegen,
PO Box 9101, 6500 HB Nijmegen, The Netherlands; e-mail: M.Smeets{at}hemat.azn.nl.
| |
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ABSTRACT
INTRODUCTION
