From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
NEOPLASIA
Cholesterol synthesis and import contribute to protective cholesterol increments
in acute myeloid leukemia cells
Deborah E. Banker, Sasha J. Mayer, Henry Y. Li, Cheryl L. Willman, Frederick R. Appelbaum, and Richard A. Zager
Cholesterol levels are abnormally increased in many acute myeloid leukemia
(AML) samples exposed in vitro to chemotherapy. Blocking these acute cholesterol
responses selectively sensitizes AML
cells to therapeutics. Thus, defining the
molecular mechanisms by which AML
cells accomplish these protective cholesterol increments might elucidate novel
therapeutic targets. We now report that
the levels of mRNAs encoding the cholesterol synthesis-regulating enzyme, 3-hydroxy-3-methylglutaryl coenzyme A reductase, and the cholesterol-importing low-
density lipoprotein (LDL) receptor were
both increased by daunorubicin (DNR) or
cytarabine (ARA-C) treatments in almost
three fourths of cultured AML samples.
However, less than one third of AML
samples significantly increased LDL accumulation during drug treatments, suggesting that de novo synthesis is the primary
mechanism by which most AML cells
increase cholesterol levels during drug
exposures. LDL increments were not correlated with cholesterol increments in
ARA-C–treated AML samples. However,
LDL and cholesterol increments did corre-
late in DNR-treated AML samples where
they were measured, suggesting that a
subset of AMLs may rely on increased
LDL accumulation during treatment with
particular drugs. Our data suggest that
cholesterol synthesis inhibitors may improve the efficacy of standard antileukemia regimens, but that for maximum benefit, therapy may need to be tailored for
individual patients with leukemia. (Blood.
2004;104:1816-1824)
© 2004 by The American Society of Hematology
Introduction
In normal cells, cholesterol is synthesized via the mevalonate
pathway and is also derived from circulating low-density lipoprotein (LDL) complexes via receptor-mediated endocytosis and
lysosomal processing (for reviews, see Goldstein and Brown1 and
Allayee et al2). Cellular cholesterol is essential to membrane
structure and membrane protein function, and its homeostasis is
achieved by complex feedback regulation of LDL receptors
(LDLRs) and of key enzymes of the mevalonate pathway, including 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCoAR) and squalene synthase (SS). Leukocytes from healthy
individuals show increased cholesterol synthesis when deprived of
lipoprotein and show decreased synthesis when exposed to complete serum or purified cholesterol-LDL.3,4 This homeostatic feedback is accomplished by coordinate transcriptional control of
cholesterol-regulating genes. For example, both HMG-CoAR and
LDLR mRNA levels are increased in normal leukocytes when
HMG-CoAR activity is inhibited by lovastatin.5
On the other hand, cholesterol homeostasis is abnormal in acute
myeloid leukemia (AML) cells. Both cholesterol synthesis and
LDL processing are hyperactive in cultured AML cells,6-8 and the
transcription of HMG-CoAR and LDLR genes is apparently not
coordinated in AML cells.5 In addition, exogenous cholesterolLDL does not reduce these processes as it does in normal
leukocytes.3,4 Consistent with data showing that AML cells import
abnormally high levels of LDL-cholesterol, hypocholesterolemia is
common in patients with de novo AML but rarely seen when the
same patients achieve remission.9
We have recently shown that cholesterol levels acutely increase
in AML cells that are treated in vitro with sublethal doses of
radiation or chemotherapeutics.10 We also showed that inhibiting
cholesterol synthesis at the first committed step of the mevalonate
pathway with an HMG-CoAR inhibitor (mevastatin) or at the final
committed step of cholesterol synthesis with an SS inhibitor
(zaragozic acid A), or blocking LDL import by serum deprivation,
all radiosensitize and chemosensitize AML cells, and that exogenous LDL protects AML cells from the heightened cytotoxicity of
combination treatments with a therapeutic drug plus mevastatin.
We have shown that normal bone marrow cells do not make these
same adaptive cholesterol responses and are relatively insensitive
to statins, as others have also shown.11-13 Therefore, we hypothesize
that AML cells require abnormally high levels of cholesterol for
their survival, that acute cholesterol responses protect AML cells
and contribute to therapy failures in patients with AML, and that
cholesterol responses might be a rational target for the development
of new antileukemia therapies.
Elucidating mechanisms by which AML cells acutely increase
cholesterol to reduce drug sensitivity could help target new
therapeutic strategies. For example, if AMLs primarily increase
cholesterol by new synthesis, then it would be appropriate to
pursue various cholesterol synthesis inhibitors as chemosensitizers
From the Fred Hutchinson Cancer Research Center, Seattle, WA; and
University of New Mexico Cancer Research Facility, Albuquerque, NM.
An Inside Blood analysis of this article appears in the front of this issue.
Submitted February 2, 2004; accepted April 22, 2004. Prepublished online as
Blood First Edition Paper, May 25, 2004; DOI 10.1182/blood-2004-01-0395.
Supported by National Institutes of Health grants R21-CA89491 (D.E.B.) and
UO1-CA32102 (C.L.W.) and a Chuck Griffin Memorial Scholarship from the
Leukemia and Lymphoma Society TR 6079-02 (D.E.B.).
1816
Reprints: Deborah E. Banker, Clinical Research Division, FHCRC, 1100
Fairview Ave N, D1-100, PO Box 19024, Seattle, WA 98109; e-mail:
[email protected].
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2004 by The American Society of Hematology
BLOOD, 15 SEPTEMBER 2004 䡠 VOLUME 104, NUMBER 6
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BLOOD, 15 SEPTEMBER 2004 䡠 VOLUME 104, NUMBER 6
CHOLESTEROL SYNTHESIS AND IMPORT PROTECT AML CELLS
in anti-AML regimens. Mouse xenograft analyses14 and preliminary clinical investigations15-18 have suggested that statins might
have anticancer activities, and one trial has shown that statins can
improve the efficacy of standard therapy in hepatocarcinoma.19 On
the other hand, if AMLs primarily use imported LDL-cholesterol to
survive chemotherapy, then regimens that block LDL accumulation
or include LDL-formulated drugs would be supported, as others
have already suggested.5 Finally, if different AMLs rely differentially on synthesis versus import processes, or if different drug
treatments differentially affect these processes, then it might be
necessary to develop robust laboratory tests to distinguish the
mechanisms by which particular AMLs make cholesterol responses
so that these laboratory findings could be used to tailor therapies for
individual patients. Therefore, we have begun characterizing the
mechanisms by which individual AML samples mount protective
cholesterol increments. We now report the results of analyses in
which we measured the levels of cholesterol-regulating mRNAs
and of LDL accumulation in AML cells that were cultured with or
without sublethal doses of the standard chemotherapeutic agents,
daunorubicin (DNR) or cytarabine (ARA-C), because those drugs
represent different classes of therapeutics that kill cells by different
mechanisms and may therefore affect cholesterol metabolism by
different mechanisms.
Table 1. Relative drug sensitivites and cholesterol and RNA assays
performed with samples from AML patients
Materials and methods
Cell culture
Human NB4, KG1a, and HL-60 AML cells were obtained from the
American Type Culture Collection (Rockville, MD). NB4 and KG1a cells
were cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 5% heat-inactivated bovine calf serum (BCS; Hyclone, Logan,
UT). HL-60 cells were cultured in Iscove modified Dulbecco medium
supplemented with 10% cosmic calf serum (CCS; Hyclone). Human
hepatocyte TAMH cells20 were received from Dr D. Hockenbery at the Fred
Hutchinson Cancer Research Center (FHCRC) and were cultured in
Dulbecco modified Eagle medium/Ham F-12 medium (Invitrogen) with 5
␮g/mL insulin, 5 ␮g/mL transferrin, 5 ng/mL selenium, 100 nM dexamethasone, and 10 mM nicotinamide (all additives from Sigma, St Louis, MO).
Drug treatments and assays were performed as described herein for primary
cell analyses, except as noted in “Results” or figure legends.
Cryopreserved primary AML bone marrow samples were obtained
from the Southwest Oncology Group (SWOG) cell repository (Dr C.
Willman; Albuquerque, NM) and the Children’s Oncology Group
(COG) AML Reference Laboratory (Dr I. Bernstein; Seattle, WA) all
with appropriate patient consent, as approved by the institutional review
boards of the FHCRC, SWOG, and COG. Twenty AML primary cell
samples thawed with 38% to 89% viability (median, 70%) and were
resuspended into Iscove media supplemented with 20% CCS, 100
ng/mL human interleukin 3 (Biosource, Camarillo, CA), 100 ng/mL
human stem cell factor (Biosource), 100 U/mL penicillin (Invitrogen),
and 100 ␮g/mL streptomycin sulfate (Invitrogen). Primary cells were
allowed to recover for 5 to 6 hours and in some cases dead cells were
removed by centrifugation through Ficoll-Hypaque (density, 1.077) if
thaw viabilities were less than 80%. Cells were then incubated for 18 to
24 hours in culture medium without drugs or with 0.05 ␮M DNR (ICN
Biomedicals, Aurora, OH), 0.2 ␮M ARA-C (Sigma), 12.5 ␮M mevastatin (Sigma), or a combination of statin plus DNR or ARA-C at the same
doses. As we previously demonstrated,11,21 the drug doses used were
relatively nontoxic to primary AML cell samples during 18 to 24 hours
of incubation whether viabilities were determined by trypan blue
staining or flow cytometry (Tables 1 and 2). Neither cholesterol, LDLR
RNA, HMG-CoAR RNA, SS RNA, nor LDL accumulation data
correlated with viability indices (P ⬎ .15 for all).
1817
Relative
Via ⴙ DNR
Relative Via
ⴙ ARA-C
Chol
Semi-Q
RT-PCR
Quant
RT-PCR
Adult 1
1.05
1.00
x
ND
ND
Adult 2
0.89
0.91
x
ND
ND
Adult 3
1.01
1.04
x
x
x
Adult 4
0.92
0.95
x
ND
ND
Adult 5
0.86
0.87
x
x
x
Adult 6
0.95
0.84*
x
x
x
Adult 7
0.98
0.99
x
x
x
Adult 8
0.88
1.01
x
x
x
Adult 9
0.81*
1.07
x
x
x
Adult 10
0.97
0.94
x
ND
ND
Adult 11
0.79*
0.92
x
x
x
Adult 12
0.90
1.00
ND
x
x
Adult 13
0.97
0.97
x
x
x
Pediatric 1
0.93
1.03
x
x
x
Pediatric 2
0.99
1.01
x
x
x
Pediatric 3
0.88
0.94
x
x
ND
Pediatric 4
1.00
0.91
x
x
x
Pediatric 5
1.00
0.91
x
x
x
Pediatric 6
0.94
1.00
x
x
x
Pediatric 7
0.83*
0.88
x
ND
ND
AML sample
No.
19
15
14
Mean
20
0.92
20
0.96
—
—
—
SEM
0.02
0.01
—
—
—
Twenty Ficoll-purified primary AML cell samples were analyzed for viability,
cholesterol content, LDLR and HMG-CoAR RNA levels in semiquantitative, multiplex
RT-PCR and quantitative, real-time RT-PCR after cells were cultured for 18 to 24
hours with and without drug treatments (DNR, 0.05 ␮M; ARA-C, 0.2 ␮M). Viability
data represent trypan blue-negative cell fractions and are expressed relative to
untreated controls for each sample. The number of samples analyzed and the mean
relative viabilities are shown, as are SEMs; x represents assays that were performed
with individual AML samples (see data in Figure 2).
Via indicates viability; chol, cholesterol, Semi-Q, semiquantitative, multiplex
RT-PCR; Quant, quantitative, real-time RT-PCR; and ND, assays not performed.
*Indicates viabilities that were reduced more than 15% by drug treatments.
Cellular cholesterol assay
As in our published report,10 we measured cellular cholesterol levels with
the Amplex Red assay (Molecular Probes, Eugene, OR), a fluorometric
technique in which cholesterol is oxidized into a ketone and hydrogen
peroxide, which then reacts stoichiometrically with the Amplex Red reagent
(10-acetyl-3,7-dihydroxyphenoxazine) in the presence of horseradish peroxidase to form the fluorescent compound resorufin. To perform this assay,
5 ⫻ 105 cells were plated in wells of 24-well plates and exposed to drugs for
18 to 24 hours; viable cell fractions were determined by trypan blue
staining. Both viable and nonviable cells were washed in phosphatebuffered saline (PBS), resuspended at 2000 cells/␮L in reaction buffer, and
dispensed into wells of a 96-well tissue culture plate (Falcon/Becton
Dickinson, Franklin Lakes, NJ) with 50 ␮L Amplex Red working solution
added to each well, per the manufacturer’s instructions (Molecular Probes).
After incubations for 90 minutes at 37°C, protected from light, fluorescence
was measured on a CytoFluor II fluorescent plate reader (PerSeptive
Biosystems, Framingham, MA) using an excitation wavelength of 530 nm
and an emission wavelength of 590 nm. A cholesterol standard curve was
determined for each plate using a cholesterol standard (Sigma) diluted at
various concentrations in Amplex Red reaction buffer.
RT-PCR
The expression levels of LDLR, HMG-CoAR, and SS mRNAs were
evaluated in AML cells by semiquantitative, multiplex reverse transcriptionpolymerase chain reaction (RT-PCR) with glyceraldehyde phosphate dehydrogenase (GAPDH) internal RNA controls and by quantitative, real-time
RT-PCR with ␤2-microglobulin RNA signals used as standardization
controls. For both types of RNA analyses, approximately 106 cells were
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1818
BLOOD, 15 SEPTEMBER 2004 䡠 VOLUME 104, NUMBER 6
BANKER et al
Table 2. Relative drug sensitivities and LDL accumulation assays
performed with samples from AML patients
Relative
Via ⴙ MEV
Relative
Via ⴙ DNR
Relative Via
ⴙ ARA-C
Adult 1
0.94
1.15
1.17
x
Adult 2
0.79*
0.89
0.91
x
Adult 3
0.95
1.01
1.04
x
Adult 4
1.00
0.92
0.95
x
Adult 5
0.99
1.01
1.00
x
Adult 6
0.78*
0.92
0.95
x
Adult 7
1.01
0.98
0.99
x
Adult 8
0.94
0.88
1.01
x
Adult 9
0.88
0.81*
1.07
x
Adult 10
0.90
0.97
0.94
x
Adult 11
0.93
0.90
1.00
x
Adult 12
ND
ND
ND
ND
Adult 13
0.94
0.97
0.97
x
Pediatric 1
1.00
0.93
1.03
x
Pediatric 2
0.97
0.99
1.01
x
Pediatric 3
0.89
0.88
0.94
x
Pediatric 4
0.94
1.00
0.91
x
Pediatric 5
0.94
1.00
0.91
x
Pediatric 6
0.99
0.94
1.00
x
Pediatric 7
1.03
0.83*
0.88
AML sample
No.
19
19
19
LDL
Quantitative, real-time RNA analyses were also performed, using
standard techniques.22 The RT reaction used 0.5 ␮g RNA and 400 U
Superscript II, per the manufacturer’s instructions (Invitrogen), with 50
minutes at 42°C and 15 minutes at 70°C. ␤2-Microglobulin and NB4 RNAs
were serially diluted into yeast RNA for RT reactions. PCR was performed
with 5 U Platinum Taq DNA polymerase (Invitrogen,), 2 ␮L cDNA, and the
passive reference dye Rox. Taq polymerase was activated by heating for 5
minutes at 95°C, and PCR amplification began with cycles of 15 seconds at
95°C followed by 1 minute at 60°C times 40. The following primers and
probes were used: ␤2-microglobulin, forward 5⬘-CATTCGGGC-CGAGATGTC-3⬘ and reverse 5⬘-CTCCAGGCCAGAAAGAGAGAGTAG-3⬘; probe,
x
19
Mean
0.94
0.95
0.99
—
SEM
0.02
0.02
0.02
—
Ficoll-purified primary AML cells were analyzed for viability and LDL accumulation in flow cytometry assays performed after cells were cultured for 18 to 24 hours
with and without drug treatments (MEV, 12.5 ␮M; DNR, 0.05 ␮M; ARA-C, 0.2 ␮M).
Viability data represent flow cytometry assessments of live cell fractions in forward by
side light-scatter plots (Figure 3A) and are expressed relative to untreated controls for
each sample. The number of samples analyzed and the mean relative viabilities are
shown, as are SEM values; x represents assays that were performed with individual
AML samples (see data in Figure 5).
MEV indicates mevastatin; other abbreviations are explained in Table 1.
*Indicates viabilities that were reduced more than 15% by drug treatments.
untreated or treated with the drug, RNA extractions were performed using
TRIZOL reagent, and oligo(dT)–primed cDNA was synthesized from total
cellular mRNA by RT using Superscript II per the manufacturer’s instructions (Invitrogen). A 1-␮g mRNA input was added for AML cell sample
assays or serially diluted mRNA inputs were added for standard curve
production. In all cases, PCR products were sequenced to confirm identity.
For multiplex, semiquantitative mRNA analyses, LDLR, HMG-CoAR,
and SS primers were multiplexed one each with GAPDH primers so that
GAPDH RNA levels could be used as a normalization factor for the
measured levels of LDLR, HMG-CoAR, and SS RNAs. The intronspanning primers used for PCR amplification and their size of PCR product
were: LDLR, forward 5⬘-CAATGTCTCACCAAGCTCTG-3⬘ and reverse
5⬘-TCTGTCTCGAGGGGTAGCTG-3⬘; HMG-CoAR, forward 5⬘TTACTCCTTGGTGATGGGAGCTTG-3⬘ and reverse 5⬘-TCCTGTCCACAGGCAATG-TAGATG-3⬘; SS, forward 5⬘-CCACTTTGGCTGCCTGTTAT-3⬘ and reverse 5⬘-CCT-AAACCGTGGCACTGAAT-3⬘; and GAPDH,
forward 5⬘-GTCTTCACCACCATGGAGAAG-3⬘ and reverse 5⬘-GCTTCACCACCTTCTTGATGTCATC-3⬘. PCR amplification was performed
using Platinum Taq DNA polymerase per the manufacturer’s instructions,
with cycles of 1 minute at 94°C, 1 minute at 55°C, and 1 minute at 72°C
(Invitrogen). LDLR, HMG-CoAR, and SS primer pairs were optimized to
ensure amplification in the linear range for both products (30, 29, and 28,
respectively), and 2 ␮L GAPDH primers were added with 23 cycles
remaining. PCR products were run on a 2% agarose gel and visualized by
scanning the ethidium bromide–stained gel on a Typhoon 8600 Imager
(Amersham Biosciences, Piscataway, NJ) using a 532-nm laser line and a
610-bandpass (BP) filter. Band densities were quantified using the software
ImageQuant version 5.2 (Amersham Biosciences). The reproducibility of
the multiplex RT-PCR assays was verified in repeated, independent assays
of RNAs from untreated and mevastatin-treated NB4 cells (Figure 1).
Figure 1. The abundance of RNAs encoding cholesterol import (LDLR) and
cholesterol synthesis-regulating (HMG-CoAR, SS) proteins are substantially
increased by mevastatin treatments in NB4 AML cells. NB4 cells were incubated
for 18 to 24 hours with 12.5 ␮M mevastatin, total cellular mRNAs were prepared from
untreated and treated cells, and oligo(dT)–primed cDNAs were synthesized. For
semiquantitative RT-PCR, LDLR intron-spanning primer pairs were multiplexed with
GAPDH primers using conditions optimized to ensure amplification in the linear range
for both products, as determined by image analysis of ethidium bromide-stained
agarose gels (A-B). Arrows indicate 3-␮L cDNA inputs. (C) For real-time RT-PCR,
␤2-microglobulin DNA and NB4 cell line RNAs were serially diluted into yeast RNA
and processed to create standard curves. Quenched, fluorescent internal probes
allowed the amount of PCR amplicon created to be quantitated per cycle as
fluorescence on an ABI Prism 7700 detector. Standard curves were generated for
␤2-microglobulin using plasmid DNA and for ␤2-microglobulin, LDLR, and HMGCoAR using the NB4 cell line. Standard curves are shown that plot the cycle number
at which signals were detected above threshold (y-axis) against the starting
quantities of ␤2-microglobulin plasmid (10-108 copies/␮L) diluted into yeast RNA
(x-axis) or against the starting quantity of NB4 cell RNA (0.01-100 ng/␮L). (D)
Methods similar to those used in panels A and B were used to optimize HMG-CoAR
and SS RT-PCRs, as described more completely in “Materials and methods.” When
standardized to GAPDH signals and normalized to untreated signals, mean LDLR
and HMG-CoAR signals were significantly increased in mevastatin-treated samples
(5 independent assays), and mean SS signals (n ⫽ 3) were also increased. (E) RNA
quantities were normalized based on ␤2-microglobulin levels in real-time PCR, as
shown in panel C, and LDLR and HMG-CoAR mRNA levels in mevastatin-treated and
untreated NB4 cells are expressed relative to the untreated NB4 cell line controls.
Means are plotted in panels C and E, and error bars represent SEM. Wilcoxon rank
sum tests were used to compare means of treated and untreated AML samples along
with 2-tailed tests of significance.
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BLOOD, 15 SEPTEMBER 2004 䡠 VOLUME 104, NUMBER 6
CHOLESTEROL SYNTHESIS AND IMPORT PROTECT AML CELLS
5⬘-CCGTGGCCTAGC-TGTGCTCGC-3⬘); LDLR, forward 5⬘-ACAGAGGATGAGGTCCACATTTG-3⬘ and reverse 5⬘-AGATGT-TCACGCCACGTCATC-3⬘; probe, 5⬘-CTGACCATCTGTCTGCCGTCCTGGTT-3⬘), and
HMG-CoAR, forward 5⬘-GGACGCAACCTTTATATCCGTTTC-3⬘ and
reverse 5⬘-GTACAATAGTTACCACTAACG-GCTAGAATC-3⬘; probe, 5⬘AAGTGCTTTCTCTGTACCCTTTGAAATCATGTTCA-3⬘. The ␤2-microglobulin, LDLR, and HMG-CoAR sequence-specific forward and reverse
primers were synthesized by Qiagen (Alameda, CA). Sequence-specific
probes were synthesized by Synthegen (Houston, TX) to include the FAM
reporter dye at the 5⬘ end and the TAMRA quencher group at the 3⬘ end. The
amount of PCR amplicon created per cycle was quantitated as unquenched
fluorescence on an ABI Prism 7700 Sequence Detector (PE Applied
Biosytems, Wellesley, MA).
Human ␤2-microglobulin DNA was cloned using the TOPO TA cloning
kit (Invitrogen), and ␤2-microglobulin normalization curves were produced
by serially diluting ␤2-microglobulin DNA into yeast RNA (at 108-102
copies/␮L) for RT prior to PCR amplification. RNAs produced from
mevastatin-treated NB4 cells were serially diluted in yeast RNA (⬃0.01100 ng/␮L) to create standard curves for ␤2-microglobulin, LDLR, and
HMG-CoAR RNA analyses. The starting quantity of sequence-specific
RNA in patient samples was determined by extrapolating from these
standard curves. DNA amplification and detection was prevented by using
primers and probes that spanned introns.
AML samples, and Pearson correlations were calculated, with 95%
confidence intervals and 2-tailed tests of significance, to assess possible
correlations of cholesterol increments with increments in specific RNA
levels or with LDL accumulation increments.
LDL accumulation assays
Bodipy-LDL fluorescence is neither quenched at high concentration nor
reduced by acidic pH as in the lysosomes to which LDL-bound LDLRs
traffic, so it can be used to quantify intracellular LDL accumulation
(Molecular Probes). To measure the capacity of cells to accumulate LDL,
5 ⫻ 105 cells were incubated overnight with or without drugs, and then
exposed to warm 0.05% trypsin (Invitrogen) for 5 minutes before washing
once with warm RPMI media to remove any exogenous LDL. Cell samples
were then divided, and 1 aliquot was incubated with 10 ␮g/mL LDL-bodipy
(Molecular Probes) in RPMI, whereas the second aliquot was incubated
with 10 ␮g/mL LDL-bodipy plus 210 ␮g/mL (21-fold excess) unlabeled
LDL (Calbiochem, San Diego, CA) in RPMI, to distinguish LDLR-specific
binding. In some assays, 3% sodium azide was added to inhibit LDLR
recycling. Cells were incubated in the dark at 4°C or 37°C for the time
specified, and then washed once in cold RPMI media, and fixed in 2%
formaldehyde before flow cytometry. Surface LDLRs were quantified after
washing cells twice with PBS/2% BCS and then incubating samples with
either 1 ␮g/mL mouse anti–human LDLR (C7; Maine Biotechnology
Services, Portland, ME) or 1 ␮g/mL isotype control mouse IgG2b (Sigma)
for 10 minutes at room temperature, washing twice with cold PBS/2% BCS,
and then incubating with anti–mouse IgG-fluorescein isothiocyanate (FITC;
1:64 dilution; Sigma) for 10 minutes at room temperature in the dark. Cells
were washed twice again and kept cold before flow cytometry. At least
10 000 cells were analyzed with a Becton Dickinson (Mountain View, CA)
FACSCalibur bench-top flow cytometer, using light-scatter gating to
distinguish AML blasts in patient samples and mononuclear cells in bone
marrow samples from healthy individuals, and to exclude dead cells from
the analyses. LDL-bodipy and FITC signals were recognized in the
FL1 detector.
LDLR internalization and processing is energy dependent, and bodipy
fluorescence was accumulated to a substantially higher level in NB4 cells at
37°C than at 4°C, and in the absence of mitochondrial respiration-inhibition
as compared to the presence of the respiration inhibitor, sodium azide (data
not shown). Bodipy-LDL accumulation was also dependent on dose and
time, as expected. We found that 10 ␮g/mL bodipy-LDL was subsaturating
in NB4 cells that showed increasing bodipy fluorescence across 15-minute
to 1-hour incubation periods, whereas KG1a cells required nearly 3 hours to
accumulate similar levels of bodipy-LDL as accumulated by NB4 cells in 1
hour (data not shown).
Statistical analyses
Instat3 software (GraphPad, San Diego, CA) was used to perform Wilcoxon
matched pairs tests that compare means of treated and untreated aliquots of
1819
Results
Many primary AML samples mount cellular cholesterol
increments during treatments with standard therapeutic agents
We previously showed that AML cell viabilities are generally
unaffected by in vitro treatments with DNR or ARA-C at doses that
reproduce peak plasma concentrations reported for patients with
AML undergoing induction chemotherapies, whereas normal bone
marrow cells are typically more chemosensitive in the same in vitro
assays.21 In considering mechanisms that promote this relative
AML chemoresistance, we recently found that a majority of AML
cells mount cholesterol increments during in vitro treatments with
0.05 ␮M DNR or 0.2 ␮M ARA-C, and that blocking these acute
cholesterol responses increases AML chemosensitivity.10 To begin
determining how AML cells increase cholesterol to survive drug
treatments, and to address whether DNR and ARA-C have similar
or different effects on cholesterol metabolism, we analyzed AML
samples after in vitro treatments with 0.05 ␮M DNR or 0.2 ␮M
ARA-C, rather than a DNR plus ARA-C drug combination that
would mimic a standard anti-AML regimen. Consistent with our
previous report, these DNR and ARA-C doses were minimally
cytotoxic (Table 1), and cholesterol increments were not correlated
with drug-reduced viabilities. As before,10 we used a fluorometric
assay to measure cellular cholesterol and found that a majority of
primary AML cell samples (n ⫽ 19) showed cholesterol increments after treatment with 0.05 ␮M DNR or 0.2 ␮M ARA-C.
Eleven (58%) of 19 samples significantly increased cholesterol
levels during ARA-C or DNR treatments; 9 (48%) showed
cholesterol increments after DNR treatments (P ⫽ .0007) and 8
(42%) showed cholesterol increments after ARA-C treatments
(P ⫽ .004). There was significant overlap in these groups such that
6 of 8 samples that showed cholesterol increments after ARA-C
treatment also responded to DNR (r ⫽ 0.49, P ⫽ .03), suggesting
that particular AMLs are more likely to mount cholesterol increments during treatments with different damaging agents.
Drug treatments can significantly increase the abundance of
LDLR and HMG-CoAR mRNA in AML cells
To begin elucidating whether synthesis or import mechanisms
contribute to cholesterol increments in drug-treated AMLs, we
developed RT-PCR assays to measure the levels of mRNAs that
regulate cholesterol synthesis (HMG-CoAR, SS) and LDL import
(LDLR). We first tested our ability to detect biologically relevant
mRNA increments using NB4 cells as an AML cell line model
because we had already shown that NB4 cells are sensitive to
mevastatin and substantially increase cholesterol during DNR and
ARA-C treatments.10,11 We compared mRNA levels in NB4 cells
before and after treatments with mevastatin because cholesterolregulating gene transcription increased by cholesterol synthesis
inhibition.5 We prepared polyadenylated mRNAs from NB4 cells
and performed semiquantitative RT-PCR analyses in which LDLR,
HMG-CoAR, or SS mRNA-specific signals were normalized to
GAPDH mRNA-specific signals (Figure 1A-B), as more completely described in “Materials and methods.” The levels of LDLR
mRNA and HMG-CoAR mRNA were reproducibly and significantly increased by statin treatments in 5 independent NB4 cell
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1820
BANKER et al
assays (Figure 1D). Levels of SS mRNA were also consistently
increased in 3 assays, but SS mRNA increments were not statistically significant, due to the smaller sample size. We also found that
SS mRNA levels were rarely or only minimally increased by drug
treatments in 15 primary AML samples in which cellular cholesterol levels had already been measured and for which sufficient
RNA was available. However, significant LDLR mRNA increments were measured in 13 (87%) AML samples after DNR or
ARA-C treatments and HMG-CoAR mRNA increments were
measured in 10 (75%) drug-treated AML samples (data not shown).
To more quantitatively assess LDLR and HMG-CoAR RNA
increments, we developed real-time RT-PCR assays, using ␤2microglubulin RNA levels as normalization controls. Experimental
details are described in “Materials and methods.” RT-PCR results
were linear across at least a 4-log range of input RNAs (Figure 1C
and data not shown), and relative LDLR and HMG-CoAR RNA
levels were reproducibly and significantly higher in 5 independent
assays of mevastatin-treated NB4 cells as compared to untreated
NB4 cells (Figure 1E). RNA increments determined in multiplex,
semiquantitative and real-time, quantitative RT-PCR assays were
very similar for both LDLR (2.12 ⫾ 0.15, 2.29 ⫾ 0.23) and
HMG-CoAR (2.09 ⫾ 0.24, 2.06 ⫾ 0.17), documenting our ability to accurately detect specific RNA increments with both of
these assays.
Additional mRNA materials were available for real-time RTPCR analyses of 14 of the 15 AML samples in which cellular
cholesterol levels had been measured and semiquantitative RTPCR analyses had been performed. Substantial LDLR mRNA
increments were measured in 5 drug-treated AMLs (36%; DNR
increment range, 1.16-2.10; ARA-C increment range, 1.02-2.68)
and HMG-CoAR mRNA increments were measured in 14 AMLs
(100%) after DNR (increment range, 1.05-1.88) or ARA-C treatments (increment range, 1.03-2.49). Drug-treated LDLR and
HMG-CoAR RNA levels were significantly greater than untreated
levels in these responsive samples, except for LDLR RNA levels
after ARA-C treatments, due to smaller sample number (Figure
2A). LDLR RNA increments were significantly correlated after
DNR versus ARA-C treatments (r ⫽ 0.92; P ⫽ .03). In addition,
LDLR and HMG-CoAR RNA increments were significantly correlated in ARA-C–treated AML samples that showed LDLR RNA
increments (r ⫽ 0.92; P ⫽ .003). However, LDLR RNAs were not
consistently increased in ARA-C–treated AML samples that showed
HMG-CoAR RNA increments; 4 AML samples showed HMGCoAR RNA increments but did not show LDLR RNA increments.
We used data from quantitative RT-PCR analyses to ask whether
LDLR and HMG-CoAR RNA increments correlated with cholesterol increments in the same primary AML cell samples. We found
that increments were significantly correlated in ARA-C–treated
AML samples in which LDLR mRNA increments were measured
(4 samples with both measurements, r ⫽ 0.99, P ⫽ .008), and
positively correlated in DNR-treated AML samples (5 samples,
r ⫽ 0.80), but not significantly (Figure 2B). Increments were also
significantly correlated in ARA-C–treated AML samples in which
HMG-CoAR mRNA increments were measured (7 samples,
r ⫽ 0.88, P ⫽ .005), and correlated in DNR-treated AMLs (8
samples, r ⫽ 0.45), but not significantly.
LDL accumulation can be accurately measured
in flow cytometry assays
Unfortunately, no commercially available antibody specifically
recognizes HMG-CoAR protein with enough sensitivity to
accurately distinguish protein levels in different primary cell
BLOOD, 15 SEPTEMBER 2004 䡠 VOLUME 104, NUMBER 6
Figure 2. The abundance of LDLR and HMG-CoAR RNAs is frequently increased in primary DNR- and ARA-C–treated AML cell samples. (A) Specific RNA
levels were measured by real-time RT-PCR in aliquots of 14 primary AML samples,
and significant LDLR mRNA increments and HMG-CoAR mRNA increments were
measured in a majority of AML samples after DNR and ARA-C treatments, except for
measured LDLR RNA increments that were not significant, in association with a
smaller sample number. Error bars represent SEM, and Wilcoxon rank sum tests
were used to compare means of treated and untreated AML samples along with
2-tailed tests of significance. Bold horizontal line refers to mean. (B) Real-time
RT-PCR data were used to show that cholesterol increments were positively
correlated with both LDLR RNA increments (r ⫽ 0.99; 95% CI, 0.67-0.99) and
HMG-CoAR RNA increments (r ⫽ 0.88; 95% CI, 0.43-0.98) in ARA-C–treated AMLs,
whereas neither LDLR RNA increments nor HMG-CoAR RNA increments induced by
DNR were significantly correlated with cholesterol increments (LDLR r ⫽ 0.809; 95%
CI, ⫺036-0.98; HMG-CoAR r ⫽ 0.45, 95% CI ⫺0.92-0.36).
samples, and HMG-CoAR activity assays require larger cell
numbers than are readily available from clinical AML samples
(data not shown). Therefore, we have not yet determined
whether HMG-CoAR RNA increments produce increased HMGCoAR activity or protein expression. However, because the
abundance of LDLR mRNA was increased in AML samples by
both DNR and ARA-C treatments and LDLR RNA increments
were correlated with cholesterol increments, and because hyperactive LDL import might have therapeutic utility,9,23-25 we asked
whether LDLR activity was commonly increased in AML cells
during in vitro drug treatments. To address this question, we
developed a multiparameter flow cytometry assay using a
fluorescent, bodipy-LDL derivative. AML cells were incubated
for 1 hour at 37°C with 10 ␮g/mL bodipy-LDL with or without
20-fold excess unlabeled LDL (Figure 3A) to estimate the
LDLR-specific binding fraction, as more fully described in
“Materials and methods.”
Because we had previously shown that NB4, HL60, and
KG1a AML cells are differentially sensitive to mevastatin and
differentially mount cholesterol increments during DNR and
ARA-C treatments,10,13 we used these AML cell lines to begin
assessing the variability of baseline LDL accumulation in AML
cells. Because liver cells are known to accumulate especially
high levels of LDL,1,2 we also measured LDL accumulation in
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BLOOD, 15 SEPTEMBER 2004 䡠 VOLUME 104, NUMBER 6
CHOLESTEROL SYNTHESIS AND IMPORT PROTECT AML CELLS
1821
Figure 3. Differential LDL accumulation can be accurately measured using flow cytometry assays that can also distinguish LDL accumulation increased by
mevastatin or DNR in NB4 and KG1a AML cells. (A) Cells were incubated in the dark at 37°C to allow LDL binding and internalization and LDLR recycling, and LDL-bodipy
signals were recognized by flow cytometry, after gating to exclude dead cells and nonblasts in analyses of primary AML cell samples, and using 20-fold molar excess unlabeled
LDL to distinguish LDLR-specific binding, as more completely described in “Materials and methods.” (B) Untreated cells of AML cell lines showed reproducibly different levels of
LDL accumulation, and these were lower than accumulation levels in TAMH liver cells. (C) LDL accumulation was increased in NB4 AML cells by mevastatin (MEV), in a
dose-dependent manner (2.5 ␮M and 6.25␮M MEV data shown here), and to a similar degree as anti-LDLR antibody binding was increased. Mean LDL accumulation
increments are plotted for panels C and D, and error bars represent SEM. Bold horizontal lines indicate the mean.
TAMH hepatocytes. As expected, LDL accumulation was consistently and substantially higher in TAMH hepatocytes as compared to AML cells (Figure 3B). Consistent with differential
mevastatin sensitivity, HL60 and KG1a cells consistently accumulated lower levels of LDL than NB4 cells. Thus, our flow
cytometry assay can reproducibly measure different levels of
LDL accumulation in different cell types and in AML cell lines
that are differentially sensitive to the cytotoxic effects of
cholesterol synthesis inhibition.
AML cell lines show increased LDL accumulation after statin
treatments and supra-additive increases after statin
plus drug cotreatments
LDL processing is increased by statin treatments in normal
leukocytes and AML cells.6-8 We found, as expected, that bodipyLDL accumulation was increased in NB4 cells after treatments
with sublethal doses of mevastatin. Similar statin-induced increments could be measured using the well-characterized anti–human
LDLR monoclonal antibody, C7 (Figure 3C), suggesting that
increased LDL accumulation in statin-treated cells corresponds to
increased surface expression of LDLR. KG1a AML cells consistently showed even larger LDL accumulation increments after
statin treatments, whereas HL60 AML cells reproducibly showed
smaller statin-induced increments than NB4 cells (Figure 4 and
Table 3).
Having documented that we could accurately measure constitutive differences and acutely increased levels of LDL accumulation,
we next asked whether DNR and ARA-C treatments that induce
cholesterol increments also induce LDL increments in AML cells.
We found NB4 and KG1a cells significantly increased LDL
accumulation after mevastatin treatments and that NB4 cells
mounted small but significant LDL accumulation increments
during DNR treatments, but that LDL accumulation was not
increased by DNR in other cell lines (Figure 4). Cholesterol
increment-inducing ARA-C treatments did not induce LDL accumulation increments in any of the AML cell lines analyzed. We
hypothesized that statin cotreatments might force cholesteroldependent cells to measurably increase LDL import. Consistent
with this idea, NB4 and KG1a AML cells showed significantly
supra-additive LDL accumulation after statin plus DNR cotreatments. However, these cells did not show supra-additive LDL
accumulation after statin plus ARA-C treatments, and HL60 cells
did not show LDL increments after either statin plus drug
cotreatment.
LDL accumulation is consistently increased in primary AML
cell samples by mevastatin treatments, but DNR and ARA-C
responses are less common
Because NB4 cells accumulated higher levels of bodipy-LDL than
other AML cell lines and showed significant LDL accumulation
increments after mevastatin or DNR treatments, NB4 cells were
used as interassay controls for analyses of primary AML cell
Figure 4. LDL accumulation is differentially affected in AML cell lines by
nontoxic treatments with mevastatin, DNR, and ARA-C, and DNR plus statin
treatments can be used to uncover the ability of AML cells to mount LDL
increments when new cholesterol synthesis is blocked. LDL accumulation was
measured in untreated NB4, KG1a, and HL60 AML cells, and in the same cells after
various drug treatments, as described more completely in “Materials and methods.”
Wilcoxon rank sum tests were used to compare means of treated and untreated AML
samples, with 2-tailed tests of significance. LDL accumulation was significantly
increased by 6.25 ␮M mevastatin (MEV) treatments in NB4 (P ⫽ .0001) and KG1a
(P ⫽ .02) cells. LDL accumulation was significantly increased by DNR (P ⫽ .001) but
not ARA-C (P ⫽ .27) in NB4 cells. LDL accumulation was not increased by DNR or
ARA-C in KG1a cells, but DNR plus mevastatin (⫹D⫹M) cotreatments produced LDL
accumulation increments that were supra-additive (P ⫽ .05 with DNR only, P ⫽ .06
with MEV only) compared to increments measured in KG1a cells treated with statin or
DNR alone. HL60 cells did not show increased LDL accumulation after mevastatin,
DNR, or ARA-C treatments, and combination treatments did not produce supraadditive increments. Mean LDL accumulation increments are plotted, and error bars
represent SEM. Bold horizontal lines indicate mean.
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1822
BLOOD, 15 SEPTEMBER 2004 䡠 VOLUME 104, NUMBER 6
BANKER et al
Table 3. Correlations for RNA increments
Table 4. Correlations for LDL accumulation
No. increments
r2
P
LDLR RNA ⫹ DNR
5
.64*
.57
LDLR RNA ⫹ DNR
LDLR RNA ⫹ ARA-C
4
.98†
.008
LDLR RNA ⫹ ARA-C
HMG.RNA ⫹ DNR
7
.29*
.21
HMG.RNA ⫹ DNR
HMG.RNA ⫹ ARA-C
8
.76†
.005
HMG.RNA ⫹ ARA-C
6
*with chol ⫹ DNR.
†with chol ⫹ DNR
samples. Eighteen of 19 AML samples that had been analyzed in
cholesterol assays were available for LDL accumulation assays.
LDL accumulation in untreated AML samples was variable, but
consistently lower than in NB4 interassay controls (AML mean,
0.21 ⫾ 0.05, relative to NB4 standards).
Nineteen AML samples (100%) showed significant LDL increments after mevastatin treatments (Figure 5), showing that primary
AML cells can increase LDL accumulation during 18 to 24 hours of
in vitro drug treatments. However, only 7 (39%) of 18 AML
samples significantly increased LDL accumulation after DNR or
ARA-C treatments. Four of these samples (22%) increased LDL
accumulation during DNR and ARA-C treatments (r ⫽ 0.54,
P ⫽ .025), suggesting that particular AMLs are more likely to
increase LDL import during treatments with either cytotoxic drug.
Seven additional AML samples (39%) showed supra-additive LDL
increments when treated with mevastatin plus DNR or mevastatin
plus ARA-C, and 3 of these (17%) showed supra-additive LDL
increments after treatment with mevastatin plus DNR and mevastatin plus ARA-C (r ⫽ 0.75, P ⫽ .0004). However, LDL increments
were significantly correlated with cholesterol increments only in
DNR-treated and DNR/statin cotreated AML samples (Figure 5 and
Table 4), suggesting that LDL increments may contribute to
cholesterol increments in a subset of AMLs treated with particular
drugs, including DNR.
Discussion
We previously showed that cells in many AML samples acutely
increase cellular cholesterol levels during in vitro radiation or
chemotherapy exposures and are sensitized to therapeutics by
agents that block cholesterol synthesis or block LDL-cholesterol
import.10 Our current study confirms that many AMLs increase
intracellular cholesterol levels during drug treatments, shows that
the cholesterol increments produced during DNR and ARA-C
treatments are often associated in individual AML samples, and
suggests that a subset of AML is most likely to mount cholesterol
increments after treatments with therapeutic agents. Based on these
No. increments
5
r2
P
.88*
.01
5
.85†
.03
6
⫺.18*
.73
⫺.21†
.68
*with chol ⫹ DNR.
†with chol ⫹ ARA-C.
data, we hypothesize that acute cholesterol responses contribute to
therapy failures in particular AML patients. To enhance the
development of new cholesterol-focused, anti-AML strategies on
the most relevant molecular targets, we investigated the mechanisms by which protective cholesterol increments might be achieved
in AML cells. We used both real-time, quantitative RT-PCR and
multiplex, semiquantitative RT-PCR assays to measure the levels
of mRNAs that encode cholesterol synthesis-regulating enzymes
(HMG-CoAR and SS) and the LDL-cholesterol receptor (LDLR),
and measured LDL accumulation in primary AML cell samples that
were untreated or treated with the standard chemotherapeutic
agents, DNR and ARA-C.
Whether specific RNA levels were assessed relative to GAPDH
or ␤2-micorglobulin RNA levels, our analyses showed that the
levels of both LDLR and HMG-CoAR mRNAs were increased in
many AML samples by ARA-C and DNR treatments, suggesting
that both cholesterol synthesis and LDL-cholesterol import processes are acutely stimulated in AML cells by relevant drug
treatments. In fact, LDLR and HMG-CoAR RNA increments were
positively correlated in ARA-C–treated AML cells that showed
LDLR RNA increments. However, LDLR and HMG-CoAR mRNA
increments were not correlated in DNR-treated cells, and 4 samples
showed HMG-CoAR RNA increments that did not show LDLR
RNA increments after drug treatments. In addition, multiplex
RT-PCR assays showed that SS mRNA levels were relatively
unchanged in AMLs treated with either DNR or ARA-C. Our data
are consistent with other data showing that cholesterol-regulating
gene expression is frequently discordant in AML cells,5 and suggest
that treatments with certain particular therapeutic agents increase
the levels of particular cholesterol-regulating mRNAs, whereas the
levels of other cholesterol-regulating mRNAs are rarely influenced
by any drug treatment.
In our study, LDLR and HMG-CoAR mRNA increments were
significantly correlated with cholesterol increments in AML samples
treated with ARA-C but not DNR, suggesting that particular drugs
are more likely to induce cholesterol responses in AML cells and
that specific mRNA responses might have prognostic utility, if
cholesterol increments decrease chemotherapeutic efficacy as our
Figure 5. LDL accumulation is consistently and significantly increased by mevastatin treatments in primary AML cell samples, but
less frequently increased by DNR or ARA-C treatments, although
LDL increments are significantly correlated with cholesterol increments in DNR-treated AMLs. (A) All 18 AML cell samples showed
significant LDL increments after mevastatin treatments, but only 5 of these
samples significantly increased LDL accumulation after DNR and only 6
samples significantly increased LDL accumulation after ARA-C treatment.
Mean LDL accumulation increments are plotted, error bars represent SEM,
and Wilcoxon rank sum tests were used to compare means of treated and
untreated aliquots of AML samples. Bold horizontal line indicates mean. (B)
Cholesterol increments were positively correlated with LDL accumulation
increments in DNR-treated (r ⫽ 0.93; 95% CI, 0.34-0.99) and DNR plus
statin cotreated AML samples (r ⫽ 0.92; 95% CI, 0.21-0.99), but not in
ARA-C–treated (r ⫽ ⫺0.18; 95% CI, ⫺0.87 to 0.74) or ARA-C plus statin
cotreated AML samples (r ⫽ ⫺0.21; 95% CI ⫺0.87 to 0.72).
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BLOOD, 15 SEPTEMBER 2004 䡠 VOLUME 104, NUMBER 6
CHOLESTEROL SYNTHESIS AND IMPORT PROTECT AML CELLS
previously published in vitro data suggest.10 Others have shown
that AML cells can increase LDL processing after statin treatments
without measurably increasing LDLR mRNA levels and have
concluded that AML cells regulate cholesterol levels by abnormally
active posttranscriptional mechanisms.5 Our data are consistent
with this idea but show instead that LDLR mRNA increments do
not necessarily produce higher LDLR expression and activity. In
either case, posttranscriptional regulation of cholesterol may
contribute to the adaptive responses mounted by a subset of AMLs
treated with particular antileukemia agents. This posttranscriptional mechanism will be the focus of future laboratory studies
because such studies might elucidate new, leukemia-selective
therapeutic targets.
Increased LDLR mRNA levels in AML cells were commonly
measured in our multiplex and real-time RT-PCR assays of DNRand ARA-C–treated AML samples, and hyperactive LDLR might
provide therapeutic targets in AML.23-25 Therefore, we used a flow
cytometry assay to ask how frequently LDL accumulation increases in primary AML cells during in vitro drug treatments. We
found that LDL accumulation increased in every AML sample and
in every cell line analyzed after mevastatin blockade of cholesterol
synthesis. NB4 AML cells also significantly increased LDL
accumulation during DNR treatments. Thus, AML cells can acutely
increase LDL accumulation in response to in vitro drug treatments,
and LDL accumulation increments can be measured in our flow
cytometry assay. Cellular LDL levels were increased in 7 (39%) of
the 18 AML cell samples that were exposed to DNR or ARA-C in
our analyses, and supra-additive LDL increments were measured in
an additional 7 AMLs when mevastatin was added to DNR or
ARA-C treatments to prevent new cholesterol synthesis. Thus,
more than three fourths of AML samples (78%) were able to
acutely increase LDL accumulation during chemotherapy, which is
very nearly the same fraction of AMLs (84%) that mounted
cholesterol increments in our study, further supporting the idea that
many AMLs require acute cholesterol increments to survive
chemotherapy and that increased LDL import can contribute to
cellular cholesterol increments in AMLs.
LDL accumulation increments were correlated with cholesterol
increments in DNR-treated AML cells, but not in ARA-C–treated
AML cells, suggesting that LDL import may be differentially used
by AML cells to accomplish cholesterol increments, according to
particular drug treatment. Larger studies are needed to test this
idea, but if our preliminary findings are confirmed, serum LDL
modulation or LDL-formulated therapeutics might be beneficial in
particular AML patients. Others have already shown that LDL (and
lipid emulsions resembling the lipid portion of LDL) is concentrated by leukemic blasts in patients and have suggested that LDL
might be used as a carrier for cytotoxic drugs to improve
therapeutic indices in AML and in other cancers.9,23-25
Approximately two thirds of the AML samples that we analyzed
did not measurably increase LDL accumulation in the absence of
statin cotreatments, and approximately one fourth did not increase
LDL accumulation even during statin plus drug cotreatments. In
addition, our real-time RT-PCR data suggest that HMG-CoAR
RNA levels are more frequently increased in AML cells by drug
treatments than are LDLR RNA levels. Although it is a formal
possibility that we were unable to measure small LDL accumulation increments that were nonetheless sufficient to produce critical
cholesterol increments in drug-treated AML cells, our data suggest
that a majority of AML cells rely primarily on new cholesterol
synthesis to mount protective cholesterol increments. We have not
yet determined whether HMG-CoAR protein expression or activity
increments significantly correlate with cholesterol increments in
drug-treated AML cell samples, and future studies should test the
utility of HMG-CoAR RNA measures as predictors of clinical drug
response. However, our published data10,11 and the data herein
combine to strongly support the idea that cholesterol synthesisinhibiting drugs might improve the efficacy of antileukemia
regimens for many patients with AML, as other have also
suggested.12,14 Further, our data suggest that laboratory tests that
accurately identify cholesterol-dependent AMLs, and identify
the precise mechanism by which particular AMLs mount
protective cholesterol increments, may have value in tailoring
antileukemia therapies.
Larger laboratory studies are needed to confirm these ideas, but
clinical trials testing the safety and chemosensitizing efficacy of
statins in standard antileukemia regimens seem warranted. We have
shown that lymphoma and myeloma cells also mount cholesterol
increments during in vitro treatments with relevant chemotherapeutics and are statin sensitive (data not shown). Others have shown
that, like AML cells, astrocytoma and glioblastoma cells, gastric
adenocarcinoma cells, hepatocarcinoma cells, colon carcinoma
cells, neuroblastoma cells, and hairy cell leukemia cells are
sensitive to statins.16-18,26-28 A phase 1 trial showed that pravastatin
plus 5-fluorouracil therapy was well tolerated and that pravastatin
significantly improved the median survival of patients with hepatocarcinoma from 9 months to 18 months.19 Therefore, our findings
may have broad significance for the development of new, effective
anticancer regimens.
1823
Acknowledgment
The authors are grateful to Dr Derek Stirewalt for his help in
developing the quantitative RT-PCR assays used in this work.
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2004 104: 1816-1824
doi:10.1182/blood-2004-01-0395 originally published online
May 25, 2004
Cholesterol synthesis and import contribute to protective cholesterol
increments in acute myeloid leukemia cells
Deborah E. Banker, Sasha J. Mayer, Henry Y. Li, Cheryl L. Willman, Frederick R. Appelbaum and
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