|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Blood, Vol. 94 No. 3 (August 1), 1999:
pp. 1046-1056
By
From Université Paris 6, Formation de Recherche Claude Bernard,
E 9912 INSERM, and Service d'Hématologie, Hôpital
Hôtel-Dieu, Paris, France.
In adult acute myeloid leukemia (AML), the weight of the
contribution of the combined activity of Pgp and MRP1 to drug
resistance is not known. To address this question, we compared the
activity of these proteins to the in vitro resistance to daunorubicin
(DNR), etoposide, and cytosine arabinoside (Ara-C), using the
calcein-AM uptake and the 3-[4, 5-di-methyl-thiazol-2, 5-diphenyl]
tetrazolium bromide (MTT) assay in 80 adult AML patients. We found no
correlation or only a weak correlation between the in vitro drug
resistance to DNR and etoposide and MRP1 or Pgp expression or function
when tested separately. However, a strong correlation was observed between the simultaneous activity of MRP1 and Pgp (quantified as the
modulation of calcein-AM uptake by cyclosporin A and probenecid) and
the LC50 of DNR (r = .77, P < .0001). This
emphasized the role of these two proteins, not separately, but together
in the resistance to DNR. In contrast, Mvp/LRP expression did not
correlate with the LC50 of DNR. A high level of simultaneous activity
of Pgp and MRP1 was predictive of a poor treatment outcome (for
achievement of CR [P = .008], duration of relapse-free
survival [RFS; P = .01], and duration of
overall survival [OS; P = .02]). In addition, high LC50 of DNR and high LC50 of etoposide together were also predictive of a poor treatment outcome (for duration of RFS [P = .02] and duration of OS [P = .02]). The
unfavorable cytogenetic category was more closely associated with the
combined activity of both MRP1 and Pgp (P = .002) than with
the activity of Pgp or MRP1 separately. This could explain the poor
prognosis and the in vitro resistance to daunorubicin in this group of
patients. These data suggest that treatment outcome may be improved
when cellular DNR and etoposide resistance can be circumvented or
modulated. Modulation of not only Pgp but also MRP1 could be essential
to attain this aim in adult AML.
MULTIDRUG RESISTANCE (MDR) of some
cancers, particularly acute myeloid leukemia (AML), remains a major
obstacle to successful chemotherapy. The best-characterized resistance
mechanism in AML which has been shown to be associated with poor
outcome is mediated by the MDR1 gene and expression of membrane P
glycoprotein.1 But alternative proteins, such as the more
recently recognized multidrug-associated protein (MRP1)2 or
the lung-resistance protein (Mvp/LRP),3 may also contribute
to the resistance to anthracyclines and etoposide in AML. However, the
role of these two proteins are still under discussion.4-10
In several publications, the expression of Pgp did not correlate with
its function of drug efflux.1,11 For this reason,
determining the functional role appears to be more informative than
quantification of MDR proteins. In previous studies, we have shown in
cell lines and in AML that the quantification of
calcein-acetoxymethylester (calcein-AM) uptake (with or without
specific modulator[s] of MRP1 and/or Pgp) can be used to assess the
activity of both MRP1 and Pgp.10,12 Calcein-AM, which is a
substrate of both Pgp and MRP1, becomes fluorescent after the cleavage
of calcein-AM by cellular esterases, producing a measurable fluorescent
derivate calcein in flow cytometry.10,12-16
Despite its association with clinical resistance, MDR1
expression was not correlated with in vitro resistance to daunorubicin (DNR) and etoposide, using the quick and semi-automatized 3-[4, 5-di-methyl-thiazol-2, 5-diphenyl] tetrazolium bromide (MTT) assay, in
several studies.17-19 However, both we and others have
shown that MDR1 and MRP1 gene overexpression emerged in
a sequential manner during selection of different leukemic cell lines
by drugs.20-22 The overexpression of the MRP1 gene
preceded that of the MDR1 gene; afterward, MRP1 and
MDR1 were co-overexpressed. In light of these results,
MRP1 gene overexpression is probably an early event in the
development of drug resistance, and clinical trials that modulated only
Pgp might have limited or no success. In addition, both we and van der
Kolk et al have shown that MRP1 was functional in fresh leukemic blast
cells.10,23 Therefore, it is important to study the
combined activity of Pgp and MRP1. To date, no study has analyzed the
correlations between the simultaneous activity of Pgp and MRP1 and in
vitro and in vivo drug resistance to DNR or etoposide, even though
coexpression of these two proteins is common in adult AML.4
Understanding of these relationships can help unravel the mechanisms of
resistance to anthracyclines and etoposide which are clinically
relevant in adult AML. In addition, such studies can emphasize the
contribution of the combined activity of Pgp and MRP1 in comparison to
other mechanisms.
Therefore, we have studied the contribution of Pgp, MRP1, and Mvp/LRP
expression and Pgp and MRP1 function (using calcein-AM) to the in vitro
resistance to DNR and etoposide and to the in vivo treatment result in
80 adult AML patients.
Patients.
Between July 1995 and December 1997, 80 samples from adult AML patients
(60 de novo and 20 relapsed AML) were successfully tested. The
diagnosis was based on French-American-British (FAB) criteria.24,25 Immunophenotyping was performed by using
flow cytometry. Promyelocytic leukemia (AML3) patients were excluded from the study (because of retinoic acid treatment). For each patient,
several clinical and biological characteristics were analyzed (age,
white blood cell [WBC] count at diagnosis, CD34 expression, and
karyotype). Unfavorable karyotypes were defined as t(9;22) or
abnormalities of chromosomes 5 or 7, abnormalities of 11q2.3 band, or
complex abnormalities. Inversion in chromosome 16 (inv 16) or t(8;21)
indicated good prognosis, and the other karyotypes, including normal,
indicated intermediate prognosis.26 Only untreated de novo
AML patients (60 patients) were analyzed for treatment outcome. De novo
AML patients, in our department, were included in the European
Organization for the Research and Treatment of Cancer
(EORTC) leukemia cooperative group protocols (AML-13 for
patients Level of MDR1, MRP1, and Mvp/LRP mRNA expression.
The level of MDR1, MRP1, and Mvp/LRP mRNA expression measured by
reverse transcriptase-polymerase chain reaction (RT-PCR) was described elsewhere.4,10,27 The variations between
samples in the cDNA synthesis were normalized by their relative
quantities of Levels of Pgp, MRP1, and Mvp/LRP protein expression.
Pgp, MRP1, and Mvp/LRP protein expression was measured by labeling
fresh viable cells with the UIC2, MRPm6, and LRP56 monoclonal antibodies (MoAbs), respectively, and phycoerythrin (PE)-labeled second
antibody as described before.10 The expression of MDR proteins was established with blast cells selected by CD34 antibody (HPCA2 clone; Becton Dickinson, Le Pont de Claix, France)
(two-color assays) or other markers (for example CD33/CD7, CD33/CD2,
CD33/CD19, or CD33/CD22 by three-color assays) whenever possible, or
with physical characteristics only if blast cells did not express
characteristic markers. Fluorescence was analyzed on a FACSORT flow
cytometer (Becton Dickinson). Values were expressed as adjusted for
control, ie, the ratio of MoAb fluorescence/control antibody
fluorescence. We performed this test in 75 of the 80 patients.
Correlations with clinical outcome were largely performed using the
fluorescence ratio as a continuous variable, in accordance with
consensual recommendations.28-30
Functional analysis of Pgp and MRP1 using calcein-AM.
Cells exposed to the nonfluorescent calcein-AM become fluorescent after
the intracytoplasmic cleavage of calcein-AM by cellular esterases which
produced the fluorescent derivate calcein. Both Pgp and MRP1 actively
extruded calcein-AM.12,13 When we measured calcein-AM
uptake by flow cytometry, we assessed the amount of fluorescent calcein
that had been converted from nonfluorescent calcein-AM. When the Pgp
and/or MRP1 proteins were active, less calcein-AM was retained and less
was converted to fluorescent calcein. Therefore, calcein-AM uptake
(with specific modulators of Pgp and/or MRP1) could be used to assess
whether Pgp and/or MRP1 were functional.10,12-15,31,32 In
our previous studies, calcein-AM uptake ± cyclosporin A (CsA)
provided in AML cells a functional test as specific and sensitive as
Rh123 ± CsA,10 the most specific and sensitive Pgp
functional test.15,31 Calcein-AM uptake ± probenecid also provided a functional test for MRP1 in leukemic cells.
Probenecid was used as specific modulator of MRP1 activity.10,12,33
MTT cytotoxicity test.
In vitro sensitivity of cells to DNR, Ara-C, and etoposide was
determined by planting 2 × 105 cells in a 200-µL
growth medium, without any specific growth factor, containing several
dilutions of the drug in 96-well microtiter plates. Each concentration
of drugs was repeated in six wells. After incubation for 3 days at
37°C with 5% CO2, cell viability was determined using
the MTT assay as described by Plumb et al.35 Briefly, 20 µL of MTT (5 mg/mL in PBS) was added to each well and incubated for 6 hours. The medium and MTT were then removed from the wells by
centrifugation, and formazan crystals were dissolved in 200 µL of
dimethyl sulfoxide (DMSO). The absorbance was recorded in a microplate
reader (Model MR5000; Dynatech Laboratories, France) at
the wavelength of 550 nm. The effect of drug on growth inhibition could
be assessed as: % of Growth Inhibition = 1 Statistical analysis.
Clinical and biological factors were investigated for their influence
on remission rate by the Expression and function of MDR variables.
We have performed both mRNA detection by RT-PCR and protein detection
by flow cytometry in 70 of 80 patients. The correlation between RT-PCR
and flow cytometry was good for MRP1 gene expression (r = .87, P < .0001) (Fig 2A). All
the negative samples in RT-PCR (a sensitive technique) had a
fluorescence ratio of MRP1 protein expression
MDR parameters and other in vitro resistance variables.
No statistically significant correlation was found between the level of
Pgp expression and the LC50 of DNR (r = .29, P = .10), etoposide (r = .09, P = .62), and Ara-C (r = .12, P = .37) and between the level of MRP1 expression and the
LC50 of DNR (r = .33, P = .07), etoposide (r = .23, P = .12) and Ara-C (r = .14, P = .50)
(data not shown). There was also no correlation between the level of
Mvp/LRP expression and the LC50 of DNR, etoposide, and Ara-C (data not shown).
MDR parameters and in vivo resistance.
Sixty untreated AML patients were evaluable for clinical
response. Sixty-five percent of patients achieved CR. Variables
influencing CR are shown Table 2. CR rate
significantly decreased with increasing MDR1 gene
expression (0.197 ± 0.022 v 0.092 ± 0.087, P = .04 by RT-PCR; 3.93 ± 2.27 v 2.10 ± 0.59, P = .04 by flow cytometry) and with increasing MRP1 gene
expression (0.756 ± 0.312 v 0.212 ± 0.341, P = .05 by RT-PCR; 1.90 ± 0.43 v 1.38 ± 0.53, P = .05 by flow cytometry). However, CR rate
was not associated with the level of Mvp/LRP expression by both assays
(RT-PCR and flow cytometry) (Table 2). Patients who achieved CR also
had a lower activity of Pgp (1.17 ± 0.27 v 1.55 ± 0.37, P = .05), a lower activity of MRP1 (1.12 ± 0.36 v
1.39 ± 0.22, P = .05) and a lower simultaneous activity of
MRP1 and Pgp (1.35 ± 0.47 v 2 ± 0.54, P = .008)
than patients who did not (Table 2). While the CR rate significantly decreased with increasing LC50 of DNR (0.56 ± 1.32 v 0.29 ± 0.28, P = .05), it was not associated with the level of
LC50 of etoposide and Ara-C. When the threshold of positivity was used
for in vitro MDR variables, we obtained the same results
(Table 3).
Other prognostic factors and correlation with in vitro resistance
variables.
The effect of other well-known variables such as age, cytogenetics, WBC
count at diagnosis, and CD34 expression on clinical response were also
analyzed. RFS and OS were significantly poorer for patients with
unfavorable cytogenetics (P = .04 and P = .01, respectively) and decreased significantly with increasing age (P = .03 and P = .01, respectively) and increasing WBC
(P = .03 and P = .04, respectively) (Table 4). CD34
expression was not a prognostic factor for RFS and OS.
While both MRP1 and Pgp may confer resistance to different families of
drugs in AML, including anthracyclines and etoposide, the relative
importance of these two genes is not known. Several studies reported
the sequential expression of MRP1 and Pgp in drug-selected cell
lines.20-22 At clinically relevant concentrations of
doxorubicin or homoharringtonine, resistance to these drugs was related
to MRP1 overexpression, but not to MDR1 expression in human myeloid
leukemia cell lines.20,22 Only when cell lines were exposed
to drugs for a prolonged time period or selected for relatively
high-level drug resistance did Pgp/MDR1 overexpression become apparent.
Similar findings occur in other cell lines (murine leukemia, small cell
lung cancer cells).21,42,43 In light of these results,
MRP1 gene overexpression is probably an early event in the
development of drug resistance, and MRP1 and Pgp could be coexpressed
in AML cells exposed to pharmacological doses of cytotoxic drugs. In
clinical samples of AML, MRP1 overexpression ranged from 7% to
30%.4,7,8,44-49 MRP1 overexpression was more frequent in
drug-refractory or relapsed patients than in drug-sensitive patients in
one study,4 but not in others.46,47 Similarly,
the coexpression and correlation between MRP1 and MDR1 are under
debate. These contradictory results might be partially caused by
differences in the composition of samples and experimental methods, as
well as differences in the definition of overexpression. To date, few
data on the coexpression of these two genes in AML cells have been
reported, and the combined functionality of these two proteins in
clinical samples has not been studied. We have shown, in previous
studies, that calcein-AM uptake can be used to assess whether MRP1
and/or Pgp are functional and to assess the simultaneous activity of
MRP1 and Pgp in fresh leukemic cells.10,12 In our present
study, 45% of the AML samples studied exhibited a functional activity
of one or both proteins. Taken together, these previous and present
reports suggest that MRP1 and Pgp need to be considered together and
that clinical trials that selectively modulate Pgp are likely to
achieve limited success. Therefore, we analyzed the contribution of the
combined activity of Pgp and MRP1 to in vitro and in vivo resistance to
chemotherapy in AML patients.
Submitted October 9, 1998; accepted March 29, 1999.
Supported in part by a grant from ARC (Grant No. 9637).
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 Ollivier Legrand, MD, PhD,
Hôpital Hôtel Dieu, 1 place du parvis Notre Dame, Service
d'hématologie, 181 Paris Cedex 04, France; e-mail:
olivier.legrand{at}htd.ap-hop-paris.fr.
1.
Marie JP, Zhou DC, Gurbuxani S, Legrand O, Zittoun R:
MDR1/P-glycoprotein in haematological neoplasms.
Eur J Cancer
32A:1034, 1996
2.
Cole SPC, Bhardwaj G, Gerlach JH, Mackie JE, Grant CE, Almquist KC, Kurz EU, Duncan AM, Deeley RG:
Overexpression of a transporter gene in a multidrug-resistant human lung cancer cell line.
Science
258:1650, 1992
3.
Scheper RJ, Broxterman HJ, Scheffer GL, Kaaijk P, Dalton WS, van Heijningen THM, van Kalken GK, Slovak ML, de Vries EGE, van der Valk P, Meijer CJLM, Pinedo HM:
Over-expression of a Mr 110,000 vesicular protein in non-P-glycoprotein-mediated multidrug resistance.
Cancer Res
53:1475, 1993
4.
Zhou DC, Zittoun R, Marie J-P:
Expression of multidrug resistance-associated protein (MRP) and multidrug resistance (MDR1) genes in acute myeloid leukemia.
Leukemia
9:1661, 1995[Medline]
[Order article via Infotrieve]
5.
List AF, Spier CS, Grogan TM, Jonhson C, Roe DJ, Greer JP, Wolff SN, Broxterman HJ, Scheffer GL, Scheper RJ, Dalton WS:
Overexpression of the major vault transporter protein lung-resistance protein predicts treatment outcome in acute myeloid leukemia.
Blood
87:2464, 1996
6.
Leith CP, Kopecky KJ, Godwin J, McConnell T, Slovak ML, Ming-Chen I, Head DR, Appelbaum FR, Willman CL:
Acute myeloid leukemia in the elderly: Assessment of multidrug resistance (MDR1) and cytogenetics distinguishes biologic subgroups with remarkably distinct responses to standard chemotherapy. A Southwest Oncology Group study.
Blood
89:3323, 1997
7.
Leith CP, Kopecky KJ, Chen IM, Slovak M, Head DR, Weick J, Appelbaum FR, Wiilman CL:
Frequency and clinical significance of expression of the multidrug resistance proteins, MDR1, MRP and LRP in acute myeloid leukemia patients less than 65 yrs old. A Southwest Oncology Group study.
Blood
90:389a, 1997 (abstr, suppl 1)
8.
Filipits M, Suchomel RW, Zöchbauer S, Brunner R, Lechner K, Pirker R:
Multidrug resistance-associated protein in acute myeloid leukemia: No impact on treatment outcome.
Clin Cancer Res
3:1419, 1997[Abstract]
9.
Filipits M, Pohl G, Stranzl T, Suchomel RW, Scheper RJ, Jäger U, Geissler K, Lechner K, Pirker R:
Expression of the lung resistance protein predicts poor outcome in de novo acute myeloid leukemia.
Blood
91:1508, 1998
10.
Legrand O, Simonin G, Perrot JY, Zittoun R, Marie J-P:
Pgp and MRP activities using calcein-AM are prognostic factors in adult acute myeloid leukemia patients.
Blood
91:4480, 1998
11.
Bailly JD, Muller C, Jaffrézou JP, Demur C, Gassar G, Bordier C, Laurent G:
Lack of correlation between expression and function of Pgp in acute myeloid leukemia cells.
Leukemia
9:799, 1995[Medline]
[Order article via Infotrieve]
12.
Legrand O, Simonin G, Zittoun R, Marie J-P:
Both P-gp and MRP contribute to drug resistance in AML.
Leukemia
12:1327, 1998[Medline]
[Order article via Infotrieve] (letter)
13.
Homolya L, Hollo Z, Germann UA, Pastan I, Gottesman MM, Sarkadi B:
Fluorescent cellular indicators are extruded by the multidrug resistance protein.
J Biol Chem
268:21493, 1993
14.
Feller N, Broxterman HJ, Wahrer DC, Pinedo HM:
ATP-dependent efflux of calcein by the multidrug resistance protein (MRP): No inhibition by intracellular glutathione depletion.
FEBS Lett
368:385, 1995[Medline]
[Order article via Infotrieve]
15.
Broxterman HJ, Sonneveld P, Feller N, Osenkoppele GJ, Währer DCR, Eekman CA, Schoester M, Lankelma J, Pinedo HM, Löwenberg B, Schuurhuis GJ:
Quality control of multidrug resistance assays in adult acute leukemia: Correlation between assays for P-glycoprotein expression and activity.
Blood
87:4809, 1996
16.
Barrand MA, Bagrij T, Neo SY:
Multidrug resistance-associated protein: A protein distinct from P-glycoprotein involved in cytotoxic drug expulsion.
Gen Pharmacol
28:639, 1997[Medline]
[Order article via Infotrieve]
17.
Chitnis M, Hegde U, Chavan S, Juvekar A, Advani S:
Expression of the multidrug transporter P-glycoprotein and in vitro chemosensitivity: Correlation with in vivo response to chemotherapy in acute myeloid leukemia.
Sel Cancer Ther
7:165, 1991[Medline]
[Order article via Infotrieve]
18.
Noogaard JM, Bukh A, Langkjer ST, Clausen N, Palshof T, Pedersen B, Hokland P:
MDR1 gene expression and drug resistance of AML cells.
Br J Haematol
100:534, 1998[Medline]
[Order article via Infotrieve]
19.
Efferth T, Fabry U, Osieka R:
Apoptosis and resistance to daunorubicin in human leukemic cells.
Leukemia
11:1180, 1997[Medline]
[Order article via Infotrieve]
20.
Slapak CA, Mizunuma N, Kufe DW:
Expression of the multidrug resistance associated protein and P-glycoprotein in doxorubicin-selected human myeloid leukemia cells.
Blood
84:3113, 1994
21.
Brock I, Hipfner DR, Nielsen BS, Jensen PB, Deeley RG, Cole SPC, Sehested M:
Sequential coexpression of the multidrug resistance gene MRP and mdr1 and their products in VP-16 (etoposide)-selected H69 small cell lung cancer cells.
Cancer Res
55:459, 1995
22.
Zhou DC, Ramond S, Viguié F, Faussat AM, Zittoun R, Marie J-P:
Progressive resistance to homoharringtonine in human myeloleukemia K562 cells: Relationship to sequential emergence of MRP and MDR1 gene overexpression and MDR1 gene translation.
Int J Cancer
65:365, 1996[Medline]
[Order article via Infotrieve] |
TOP
ABSTRACT
INTRODUCTION
60 years old, and AML10 for patients <60 years old). In
induction phase, all the patients received a standard dose of cytosine
arabinoside (Ara-C) (100 mg/m2/d × 10 days),
etoposide, and one anthracycline (DNR, idarubicin, or mitoxantrone × 3 days) at random.
2 microglobulin (
[(Absorbance of
Drug-Treated Cells/Absorbance of Untreated Cells) × 100]. The lethal concentration 50% (LC50) was determined as the drug
concentration that resulted in a 50% growth inhibition. Samples were
considered evaluable if the drug-free control wells contained more than
80% of leukemic cells before and more than 70% of leukemic cells
after 3 days of culture. The MTT assay gave reliable results under
these conditions.
2 or Fisher's exact tests for
binary variables and by the Mann Whitney U test for continuous
variables. Correlations among levels of expression of continuous
variables were estimated using the Spearman rank coefficient. The rate
of (1) relapse-free survival (RFS) was measured from
establishment of complete remission (CR) until relapse or death from
any cause, with observation censored for patients last known alive
without report of relapse; and (2) overall survival (OS) was measured
from diagnosis until death from any cause, with observation censored
for patients last known alive. RFS and OS were estimated by the method
of Kaplan and Meier
.05.
Compared by the log-rank test;