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NEOPLASIA
The FA/BRCA pathway is involved in melphalan-induced DNA interstrand
cross-link repair and accounts for melphalan resistance in multiple myeloma cells
Qing Chen, Pieter C. Van der Sluis, David Boulware, Lori A. Hazlehurst, and William S. Dalton
Melphalan, a DNA cross-linker, is one of
the most widely used and effective drugs
in the treatment of multiple myeloma (MM).
In this report, we demonstrate that enhanced interstrand cross-link (ICL) repair
via the Fanconi anemia (FA)/BRCA pathway contributes to acquired drug resistance in melphalan-resistant myeloma cell
lines, and disruption of this pathway reverses drug resistance. Using the alkaline comet assay (single-cell gel electrophoresis), we observed that melphalan-
resistant cells have reduced ICL formation
and enhanced ICL repair compared with
melphalan-sensitive cells. Cell-cycle studies demonstrated that enhanced ICL repair released cells from melphalan-induced cell-cycle delay. Using siRNA to
knock down FANCF in 8226/LR5 and U266/
LR6 drug-resistant cells demonstrated a
direct relationship between ICL repair capacity and drug sensitivity. Overexpression of FANCF in 8226/S and U266/S drugsensitive cells partially reproduced the
drug-resistant phenotype. These data
show that enhanced DNA repair via the
Fanconi anemia/BRCA pathway is involved in acquired melphalan resistance.
Our findings provide for a new target to
enhance response to DNA cross-linking
agents in cancer treatment. (Blood. 2005;
106:698-705)
© 2005 by The American Society of Hematology
Introduction
DNA cross-linking agents, including melphalan, induce interstrand
cross-links (ICLs) and are considered as important drugs in cancer
treatment.1 Unfortunately, although most patients respond to standard- and high-dose melphalan therapy, essentially all patients
relapse due to acquired drug resistance. We previously reported that
acquired melphalan resistance is associated with reduced melphalaninduced DNA cross-links, and elevated levels of glutathione.2
Cross-resistance to other DNA cross-linkers (such as cisplatin,
nitrogen mustard, and radiation) was observed in melphalanresistant myeloma cells.
More recently, we reported that the acquired melphalanresistant 8226/LR5 myeloma cell line consistently expressed
different genes compared with the parental drug-sensitive
8226/S cell line.3 Results showed significant increases in the
expression of FANCF and RAD51C, which are involved in the
Fanconi anemia (FA)/BRCA pathway and ICL repair,4,5 in
melphalan-resistant cells compared with drug-sensitive cells.
Fanconi anemia has at least 11 complementation groups, and 9
Fanconi anemia genes (FANCA,6 FANCB,7 FANCC,8 FANCD1,9
FANCD2,10 FANCE,11 FANCF,12 FANCG,13 and FANCAL14) have
been cloned. Studies have shown that Fanconi proteins participate
in cell-cycle control,15 regulation of detoxification,16 and survival
signal transduction.17-20 Of 9 known Fanconi proteins, 7 (A, B, C,
E, F, G, L) form a heteromeric complex and monoubiquitinate
FANCD2, which, in turn, interacts with well-known DNA-damageresponse proteins, including ataxia telangiectasia (ATR), BRCA1,
Nijmegen breakage syndrome 1 (NBS1), and RAD51, known to be
involved in DNA ICL repair.4,21-24 Disruption of the FA/BRCA
pathway results in chromosome instability and hypersensitivity to
DNA cross-linking agents.4
Several recent reports suggest that enhanced ICL repair
contributes to melphalan resistance. It has been reported that the
rate of removal of ICL is associated with melphalan resistance in
leukemic cells.25 In another study, similar results were reported
for primary multiple myeloma specimens.26 These investigators
compared the formation and repair of ICL in plasma cells from
melphalan-naive and treated myeloma patients, and found that
in vitro sensitivity to melphalan in plasma cells correlated with
ICL repair capacity.26
In this study, we show that melphalan-resistant myeloma cell
lines have elevated gene expression involving the FA/BRCA
pathway, reduced formation of ICLs, and enhanced removal of
ICLs. Enhanced ICL repair capacity reduced melphalan-induced
growth inhibition. Furthermore, silencing FANCF in drugresistant cells with siRNA reversed drug resistance, and, conversely, overexpression of FANCF in drug-sensitive cells enhanced
cell survival following melphalan treatment. These data show
that the FA/BRCA pathway mediates ICL repair and contributes
to acquired melphalan resistance in myeloma cell lines. We
propose that disruption of the FA/BRCA pathway may prevent
acquired resistance to melphalan, and possibly other DNA
cross-linking agents, and improve treatment outcome for myeloma and other cancers.
From the Department of Interdisciplinary Oncology and Experimental
Therapeutics Program, Biostatistics Core Facility, H. Lee Moffitt Cancer Center
& Research Institute at the University of South Florida, Tampa, FL.
Biology Core, Flow Cytometry Core, Microarray Core, and Molecular Imaging
Core Facilities at the H. Lee Moffitt Cancer Center & Research Institute.
Submitted November 9, 2004; accepted March 27, 2005. Prepublished
online as Blood First Edition Paper, March 31, 2005; DOI 10.1182/
blood-2004-11-4286.
Supported by Multiple Myeloma Research Foundation (Q.C.), the Peninsula
Myeloma Research Foundation (W.S.D.), and National Institutes of Health
grant CA76292 (W.S.D.). Supported in part by the Microscopy Core, Molecular
698
Reprints: William S. Dalton, Experimental Therapeutics Program, H. Lee
Moffitt Cancer Center & Research Institute, 12902 Magnolia Dr, Tampa, FL
33612; 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.
© 2005 by The American Society of Hematology
BLOOD, 15 JULY 2005 䡠 VOLUME 106, NUMBER 2
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BLOOD, 15 JULY 2005 䡠 VOLUME 106, NUMBER 2
Materials and methods
Cell lines and drugs
The RPMI 8226 and U266 human multiple myeloma cell lines were
obtained from the American Type Culture Collection (Rockville, MD). Cell
lines were grown in RPMI 1640 medium (CellGro; MediaTech, Herndon,
VA) supplemented with 10% heat-inactivated fetal bovine serum (FBS;
Omega Scientific, Tarzana, CA), 1% penicillin/strepromycin, and 100 mM
L-glutamine (Gemini Bio-Products, Calabasas, CA). The melphalanresistant cells 8226/LR5 and U266/LR6 were passaged weekly in media
containing 5 ␮M or 6 ␮M melphalan, respectively.2
Methyl-thiazol tetrazolium (MTT) cytotoxicity assay
Cells were seeded at 8000 to 25 000 cells/well in 96-well plates (Becton
Dickinson, Lincoln Park, NJ). To establish a dose response to melphalan (Sigma,
St Louis, MO), cells were incubated with melphalan for 96 hours in 2-fold serial
dilutions ranging from 1 ⫻ 10⫺4 M to 3.9 ⫻ 10⫺7 M. The melphalan-induced
growth inhibition assays were performed as previously described.27
Melphalan-induced apoptosis
Cells were continuously treated with 50 ␮M or 100 ␮M melphalan or
vehicle control (acid-ethanol) for 20 hours. Annexin V–fluorescein isothiocyanate (FITC) (Biovision, Palo Alto, CA) staining was used to measure
apoptotic cells as described previously.28
Real-time quantitative reverse-transcriptase–polymerase chain
reaction (RT-PCR)
Total RNA was extracted using the RNeasy Mini Kit (Qiagene, Valencia,
CA) and used for cDNA synthesis (Invitrogen first-strand cDNA synthesis
kit; Invitrogen, Frederick, MD). Expression of 11 genes involved in the
FA/BRCA pathway, BRCA1, BRCA2, FANCA, FANCC, FANCD2, FANCE,
FANCF, FANCG, FANCL, RAD51, and RAD51C, was analyzed using a
custom-designed microfluid card (ABI, Foster City, CA). The geneexpression level was normalized using the endogenous control gene
GAPDH and the relative gene-expression level was determined using 2-(–delta
delta C (T)) (⌬⌬CT) methods.29 Real-time PCR reactions were performed using
ABI 7900 Sequence Detection System (ABI).
Western blot analysis
Cells were washed twice with ice-cold PBS, and resuspended in ice-cold
lysis buffer (50 mM Tris [tris(hydroxymethyl)aminomethane]–HCl, pH
7.40, 0.1% nonidet P40 [NP40], 1 M NaCl) supplemented with protease and
phosphatase inhibitors (10 ␮g/mL aprotinin, 25 ␮g/mL leupeptin, 10
␮g/mL pepstatin A, 2 mM phenylmethylsulfonyl fluoride [PMSF], 0.1 M
NaP2O4, 25 mM NaF, and 2 mM sodium orthovanadate). After sonication, the
lysates were quantified using Biorad reagent (Biorad, Hercules, CA). Lysates
(50 ␮g) were separated by 4% to 12% NuPage gel (Invitrogen) and transferred to
polyvinylidenefluoride (PVDF) membrane. The antibodies used for the Western
blotting were raised against FANCF (Santa Cruz Biotechnology, Santa Cruz,
CA), FANCD2 (Novus Biologicals, Littleton, CO), and ␤-actin (Sigma).
Alkaline comet assay
The alkaline comet assay was used to detect melphalan-induced DNA
cross-links in drug-sensitive (8226/S and U266/S) and drug-resistant
(8226/LR5 and U266LR6) myeloma cells as described previously.3 To
examine dose-response effect, 2 ⫻ 105 cells were treated with 25 ␮M, 50
␮M, and 100 ␮M melphalan or vehicle control for 2 hours, washed in PBS,
and cultured in drug-free medium for another 3 hours. Samples were then
irradiated at 9 Gy (MARK I model 68A irradiator; J. L. Sheperd and
Associates, San Fernando, CA), and alkaline comet assay was performed
according to the manufacturer’s instructions (Travegene, Gaithersburg,
MD). Per slide, 50 images were randomly captured by fluorescence
microscopy and images were quantified using Loats Associates comet
FANC/BRCA AND MELPHALAN RESISTANCE IN MYELOMA
699
analysis software (Loats Associates, Westminster, MD). The percent
cross-linking was calculated as previously described3: Relative crosslinking ⫽ (1 ⫺ [(comet tail moment drug treated ⫺ comet tail moment
control)/(comet tail moment 9 Gy ⫺ comet tail moment control)]) ⫻ 100.
To examine the ICL removal, 8226/S, 8226/LR5, U266/S, and U266/
LR6 were incubated in 25 ␮M, 50 ␮M, 25 ␮M, or 60 ␮M melphalan,
respectively, or vehicle control for 2 hours, after which they were cultured
in drug-free medium for various times. Cells were collected at 8 hours and
23 hours after a 2-hour drug treatment to perform the comet assay. The data
shown are the means of 3 independent experiments (n ⫽ 50 images for each
dose of each independent experiment).
Cell-cycle analysis
Propidium iodide (PI; Sigma) staining was used to measure dose-response
effects of melphalan on cell-cycle progression. Both 8226/S and 8226/LR5
were treated with 10 ␮M and 25 ␮M melphalan for 2 hours and cultured in
drug-free medium, and cells were collected at 24 and 48 hours. Cells were
then fixed with 70% ethanol at 4°C overnight; resuspended with 500 ␮L
PBS containing 25 ␮g/mL propidium iodide (PI) and 1.25 mg/mL Rnase A
(Invitrogen); and incubated at 37°C for 30 minutes in the dark. Bromodeoxyuridine (BrDU)/PI staining was performed as previously described to
evaluate cell-cycle progression over time following exposure to melphalan.30 Briefly, at specified time points following a 2-hour drug treatment,
cells were incubated with 10 ␮M bromodeoxyuridine (BrDU; Sigma) at
37°C for 30 minutes, fixed in 70% ethanol, denatured (2 M HCl), and
neutralized (0.1 M sodium borate). Cells were then stained with anti-BrDU
FITC antibody (BD Pharmingen, San Diego, CA); resuspended with
500 ␮L PBS containing 25 ␮g/mL propidium iodide (PI) and 1.25 mg/mL
Rnase A; and incubated at 37°C for 30 minutes in the dark. Nuclear staining
was analyzed using a FACScan flow cytometer (Becton Dickinson, San
Jose, CA). Watson (Pragmatic, Ashland, OR) model from FlowJo 4.4.4
software was used for DNA content analysis.
Transient transfection of FANCF or siRNA to inhibit FANCF
FANCF siRNA (catalog no. M-014206-00) and cyanin 3 (Cy3)–labeled
Luciferase GL2 siRNA (catalog no. D-001110-01-05) were purchased from
Dharmacon (Chicago, IL). FANCF was digested from the cDNA clone
(kindly provided by Dr Grover C. Bagby, Oregon Health and Science
University [OHSU] Cancer Institute, Portland, OR) by NotI and BamHI
(NewEngland Biolab, Beverly, MA), gel purified using Qiagene kit, cloned
into pQCXIP vector (BD Biosciences Clontech, San Jose, CA), and
confirmed by sequencing. For transient expression, cell lines were transfected by electroporation using Nucleofector (Amaxa, Gaithersburg, MD).
Briefly, 8226 and 8226/LR cells and U266 and U266/LR6 cells were
resuspended in Nucleofector solution V and R (Amaxa), respectively, to the
final concentration of 5 ⫻ 107 cell/mL. For each transfection, 8 ␮g plasmid
or siRNA duplex (final concentration 1 ␮M) was mixed together with
5 ⫻ 106 cells. The A20 and T16 programs were used for 8226 and
8226/LR5 transfection and U266 and U266/LR6 transfection, respectively.
Cells were then cultured in 5 mL of 37°C prewarmed medium.
Statistical analysis
Analysis of variance (ANOVA) was used to compare ICL formation in
drug-sensitive and -resistant cells. All other statistical comparisons were
made using the Student t test.
Results
Resistant cells have less melphalan-induced growth inhibition
and apoptosis
MTT and apoptosis assay were used to compare melphalan
sensitivity in the drug-sensitive (8226/S, U266/S) and drugresistant (8226/LR5, U266/LR6) myeloma cell lines. Similar to our
previously published reports,2,3 melphalan-resistant cells 8226/
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CHEN et al
Figure 1. Melphalan-resistant myeloma cells have less drug-induced growth
inhibition and apoptosis compared with melphalan-sensitive cells. (A) MTT
assay. Median inhibitory concentration (IC50) values are the mean of 3 independent
experiments and SD. Student t test was used for statistical analysis. *P ⬍ .05. (B)
Apoptosis assay. Melphalan treatment causes less apoptosis in drug-resistant
8226/LR5 and U266/LR6 cells compared with drug-sensitive 8226/S and U266/S
cells. The mean values and standard deviations from a representative experiment
performed in triplicate are shown. Student t test was used for statistical analysis.
*P ⬍ .05.
LR5 and U266/LR6 have increased survival (Figure 1A) and
decreased drug-induced apoptosis (Figure 1B) compared with their
drug-sensitive parental cells 8226/S and U266/S.
Melphalan-resistant myeloma cells have enhanced expression
of the FA/BRCA pathway
Using the Affymetrix oligonucleotide microarray (Affymetrix,
Santa Clara, CA) to examine gene expression profile (GEP), we
recently reported that the melphalan-resistant myeloma cell line
8226/LR5 showed significant increases in the expression of the
FANCF and RAD51C genes and reduced DNA cross-links compared with the drug-sensitive 8226/S cells.3 Because the Af-
BLOOD, 15 JULY 2005 䡠 VOLUME 106, NUMBER 2
fymetrix HG-133A chip does not include the FANCD2 and FANCL
genes, we used a custom-designed real-time PCR microfluid card
to examine 11 genes of the FA/BRCA pathway in 8226/S and
U266/S myeloma cells and their drug-resistant variants, 8226/LR5
and U266/LR6, respectively. The results revealed that expression
of multiple components of the FA/BRCA pathway were upregulated in drug-resistant 8226/LR5 and U266/LR6 cells compared with drug-sensitive 8226/S and U266/S cells, respectively
(Figure 2A). The expression levels of BRCA1, BRCA2, FANCA,
FANCC, FANCF, FANCL, and RAD51C were at least 2-fold
increased in 8226/LR5 compared with 8226/S. The expression of
FANCL in U266/LR6 is at least 3-fold higher than U266/S
drug-sensitive cells (Figure 2A).
The full integrity of the upstream FA complex promotes
monoubiquitination of FANCD2 following DNA cross-linker treatment, therefore formation of the monoubiquitinated FANCD2
(FANCD2-L) isoform has been used to clinically diagnose FA
patients.31 To determine whether the upstream portion of the
FA/BRCA pathway is intact, we examined FANCD2-L at various
time points after drug treatment in the 4 cell lines. The results
revealed that the expression of FANCD2-L was significantly
increased within 2 hours of melphalan treatment (Figure 2B), and a
maximum increase of FANCD2-L occurred within 24 hours after
melphalan treatment (Figure 2B lower panel). These data suggest
that proteins involved in the upstream FA complex (FANCA, B, C,
E, F, G, L) are functionally complete in all 4 cell lines. Data also
suggest that increased formation of FANCD2-L may be due to the
enhanced expression of the FA complex proteins, and elevated
levels of the FA/BRCA pathway may contribute to enhanced ICL
repair capacity in drug-resistant cells. To examine this possibility,
we compared DNA ICL formation and removal in drug-sensitive
and -resistant myeloma cells.
Melphalan-resistant cells have reduced DNA ICL formation and
enhanced ICL removal compared with drug-sensitive cells
We recently reported that, within 2 hours following drug exposure,
melphalan-induced cross-links were significantly reduced in the
Figure 2. Enhanced expression of the FA/BRCA
pathway in melphalan-resistant 8226/LR5 and U266/
LR6 cells. (A) Real-time RT-PCR was performed using
ABI low-density array card. Fold values were obtained by
externally standardizing against identical amplifications
in drug-sensitive 8226/S cells and by internally standardizing against GAPDH in each cell line. Data shown are
mean value and SD. (B) FANCD2-L relative expression
was elevated after melphalan treatment. Experiments
were repeated 3 times. (Top) Representative blots are
shown. ␤-Actin blot served as loading control in all blots.
(Bottom) The FANCD2-L/S ratio was quantified using
densitometry. The mean values and standard deviations
from 3 independent experiments are shown. Student t
test was used for statistical analysis. *P ⬍ .05.
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BLOOD, 15 JULY 2005 䡠 VOLUME 106, NUMBER 2
drug-selected 8226/LR5 cell line.3 In this study, using alkaline
comet assay, we compared the kinetics of DNA ICL formation and
removal between melphalan-sensitive (8226/S, U266/S) and melphalan-resistant (8226/LR5, U266/LR6) cells. When cells were
treated with 25 ␮M, 50 ␮M, and 100 ␮M melphalan, cross-links
were significantly reduced in 8226/LR5 and U266/LR6 compared
with drug-sensitive 8226/S and U266/S cells, respectively (Figure
3A-B, ANOVA P ⬍ .05). To examine the ICL removal, melphalantreated cells were examined at 10-hour and 25-hour time points.
Similar amounts of ICL were induced in drug-sensitive and
drug-resistant cells at the 10-hour time point, when 8226/S,
8226/LR5, U266/S, and U266/LR6 were treated with 25 ␮M, 50
␮M, 25 ␮M, and 60 ␮M melphalan, respectively (Figure 3C). At
25 hours after melphalan treatment, 37% and 22% of ICLs were
removed in 8226/LR5 and U266/LR6 cells, respectively, while no
significant ICL removal was observed in drug-sensitive 8226/S and
U266/S cells (Figure 3C, t test P ⬍ .05).
Melphalan has less effect on cell-cycle progression in 8226/LR5
cells compared with drug-sensitive 8226/S cells
To measure dose-response effects of melphalan on cell-cycle
progression, both 8226/S and 8226/LR5 cells were treated with 10
M or 25 ␮M melphalan for 2 hours, cultured in drug-free medium,
and collected at 24 hours and 48 hours. Serial DNA histograms
revealed that within 24 hours, high-dose melphalan treatment (25
␮M) resulted in a greater accumulation of both 8226/S and
8226/LR5 cells in early S phase compared with low-dose (10 ␮M)
treatment (Figure 4A). High-dose melphalan (25 ␮M)–treated cells
FANC/BRCA AND MELPHALAN RESISTANCE IN MYELOMA
701
required more time to eliminate the melphalan-induced G2 block,
and fewer cells were released from the drug-induced growth
inhibition, compared with low-dose melphalan–treated 8226/S and
8226/LR5 cells (Figure 4A). However, the 8226/LR5 cells showed
less growth inhibition compared with 8226/S cells, when cells were
treated with either 10 ␮M or 25 ␮M melphalan (Figure 4A).
BrDU/PI staining was performed to evaluate cell-cycle progression
over time following drug exposure (Figure 4B). Within 12 hours
after 10-␮M melphalan treatment, 8226/S cells predominantly
accumulated in early S phase (S1, S2) compared with 8226/LR5
cells, which accumulated in late S phase (S3, S4) (Figure 4B-C).
Figure 4D shows further detail regarding S phase cell-cycle
accumulation at 24 hours using FlowJo 4.4.4 analysis. At the
48-hour post–10-␮M melphalan treatment time point, the DNA
content of 8226/LR5 cells accumulated at G2/M and G1 phase,
while the DNA content of the drug-sensitive 8226/S cells accumulated at late S and G2/M phases and less G1 accumulation was
observed. By the 72-hour time point, 8226/LR5 cells had recovered
from the drug-induced cell-cycle delay; whereas, the drug-sensitive
8226/S cells remained arrested in the G2/M phase (Figure 4B).
Overexpression or silencing of FANCF shows a direct
relationship among FANCF expression, DNA repair capacity,
and drug sensitivity to melphalan
To further examine the contribution of the FA/BRCA pathway to
ICL repair and melphalan resistance, we transiently overexpressed
FANCF in 8226/S and U266/S cells, and knocked down FANCF in
8226/LR5 and U266/LR6 cells. Overexpression of FANCF demonstrated by Western blot analysis in both drug-sensitive myeloma
cell lines enhanced cell survival following melphalan treatment
(Figure 5A). Conversely, knockdown of FANCF in melphalanresistant 8226/LR5 and U266/LR6 cells reversed drug resistance
(Figure 5B). We also performed the alkaline comet assay to
determine whether knocking down FANCF was sufficient to reduce
DNA ICL repair in the drug-resistant cell line. As shown in Figure
5C, a reduction in FANCF reduced the repair capacity of the
8226/LR5 and U266/LR6 cell lines. Taken together, these data show
that despite increased expression in multiple genes in the FA/BRCA
pathway, knocking down FANCF is sufficient to reverse, at least
partially, the acquired drug-resistant phenotype in 8226/LR5 and
U266/LR6 myeloma cells.
Discussion
Figure 3. Melphalan-resistant cells have reduced ICL formation and enhanced
ICL removal. (A) The alkaline comet assay was used to detect melphalan-induced
cross-links. When cells were treated with vehicle control without irradiation, no comet
tail moment was observed. When cells were irradiated, the comet tail moment was
increased to 50. (B) Within 3 hours following a 2-hour exposure to 25 ␮M, 50 ␮M, and
100 ␮M melphalan, fewer ICL formations were observed in 8226/LR5 and U266/LR6
compared with 8226/S and U266/S, respectively. *P ⬍ .05 (ANOVA). (C) Similar
amounts of cross-links are formed in drug-sensitive and drug-resistant cells at 10
hours by increasing the dose of melphalan treatment for 8226/LR5 (50 ␮M) and
U266/LR6 (60 ␮M) compared with 8226/S (25 ␮M) and U266/S (25 ␮M), respectively.
Within 24 hours after treatment, the remaining ICLs in drug-resistant 8226/LR5
(30.4%) are significantly less than the percentage of drug-sensitive 8226/S cells
(55.2% ICLs) within 10 hours. The remaining ICLs in U266/LR6 cells (54.9%) is
significantly less compared with U266/S cells (75.6%). Student t test was used for
statistical analysis. *P ⬍ .05. Error bars denote standard deviation.
DNA cross-linking agents, including melphalan, are important in
the treatment of multiple myeloma and other cancers. The precise
mechanisms contributing to melphalan resistance in multiple
myeloma cells are poorly understood. This study demonstrates that
overexpression of FA/BRCA pathway genes contributes to acquired resistance to melphalan for 2 multiple myeloma cell lines. In
addition, this study provides evidence that the FA/BRCA pathway
contributes to drug resistance via enhanced ICL repair, and release
of cells from melphalan-induced growth inhibition.
We previously reported that acquired melphalan-resistant myeloma cells were cross-resistant to irradiation and also crossresistant to other DNA cross-linkers, such as cisplatin and nitrogen
mustard.2 This information, together with our recently published
microarray data,3 led us to hypothesize that the FA/BRCA pathway
may be involved in repair of melphalan-induced ICL and contributes to the drug-resistant phenotype. Using a custom-designed
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CHEN et al
BLOOD, 15 JULY 2005 䡠 VOLUME 106, NUMBER 2
Figure 4. 8226/LR5 cells release earlier from melphalan-induced cell-cycle inhibition compared with drug-sensitive 8226/S cells. (A) Flow cytometric analysis of
cell-cycle phases. DNA histograms of untreated and melphalan-treated 8226/S (top) and 8226/LR5 cells (bottom) show the dose-response effects of melphalan on cell-cycle
progression. Representative data are shown. (B) Time course analysis of cell-cycle progression in 8226/S and 8226/LR5 cells with and without melphalan treatment. The dot
plots depict BrDU incorporation (S phase cells) detected with an FITC anti-BrDU antibody on the y-axis, and propidium iodide (PI) to detect DNA content on the x-axis. The
distribution of DNA content is represented by the inset histograms. Shown is 1 of 3 representative experiments. 8226/LR5 cells are able to progress through the cell cycle
following melphalan exposure; whereas 8226/S cells arrest at G2/M (72 hours). (C) Quantitative analysis of the delay of progression through the cell cycle. To study cell-cycle
progression delay in early and late S phases, DNA content was divided into 6 sections and quantified as G1, S1, S2, S3, S4, and G2 phase using FlowJo 4.4.4 software Watson
(Pragmatic) model. Within 12 hours following melphalan treatment, 8226/S and 8226/LR5 cells significantly accumulated at early S phase compared with non–drug-treated
cells. (D) At 24 hours after melphalan treatment, 8226/S cells significantly accumulated at S2 compared with 8226/LR5, while 8226/LR5 cells significantly accumulated at late S
phase (S3) compared with 8226/S cells. The mean values and standard deviations from 3 independent experiments are shown. Student t test was used for statistical analysis.
*P ⬍ .05.
real-time PCR microfluid card, we examined 11 genes involved in
the FA/BRCA pathway. Expression of these genes was greater in
drug-resistant cells compared with their drug-sensitive counterparts; however, greater differences were observed between the
8226 sensitive and resistant cells compared with the U266 sensitive
and resistant cells. This may, in part, be due to the higher basal
expression of FANC/BRCA genes in U266/S cells compared with
8226/S cells. The expression data correlated well with enhanced
cell survival and reduced apoptosis following drug treatment. We
observed that multiple components of the FANC/BRCA pathway
were up-regulated in drug-resistant cells compared with drugsensitive cells. These data suggest that genes involved in this
pathway may be coregulated, and further studies are warranted to
delineate the transcriptional regulation of this pathway.
Real-time PCR data showed at least 2-fold increase of FANCA,
FANCC, FANCF, and FANCL in 8226/LR5 compared with 8226/S
cells. Increased expression of FANCD2-L isoform in 8226/LR5
compared with 8226/S cells indicated that the upstream FA
complex might facilitate FANCD2-L formation. In comparison,
while some of the FA genes were less changed in U266/LR6
compared with the 8226/S-8226/LR5 pair, the increased posttranslational modification of FANCD2 may be related to the 3-fold
increase of FANCL, which has E3 ubiquitin ligase activity. Within
24 hours after melphalan treatment, the FANCD2-L/S ratio isoform
in 8226/LR5 cells was significantly increased compared with
drug-treated 8226/S cells.
We also observed a significant delay in cell-cycle progression
within 24 hours after melphalan treatment with most of the cells
accumulating in S phase. This observation is consistent with the
observation reported by Rothfuss and Grompe.21 They noticed that
in human fibroblasts, FANCD2-L is specifically expressed during
the S phase after drug treatment.21 It has also been reported that the
FANCD2-L/S ratio increased within 4 hours after treatment with
photoactivated psoralen-inducing DNA ICLs, and a 3-fold increase
of FANCD2-L occurred within 24 hours after melphalan treatment.21 Since the FANCD2 monoubiquitination event is important
for normal cellular recovery from exposure to DNA cross-linkers,32
our data suggest that the increased formation of the FANCD2-L
isoform, which is activated by melphalan-induced ICL during the
S phase, is also essential for melphalan resistance in myeloma
cell lines.
It has been suggested that the ICL-induced stalled replication
fork is the major signal that activates the FA pathway and induces
G2/M arrest in FA cells.21-23,33,34 In human myeloma cell line 8226,
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BLOOD, 15 JULY 2005 䡠 VOLUME 106, NUMBER 2
FANC/BRCA AND MELPHALAN RESISTANCE IN MYELOMA
703
Figure 5. Overexpression of FANCF in 8226/S and U266/S reduced melphalan response, whereas silencing FANCF in 8226/LR5 and U266/LR6 enhanced melphalan
response, respectively. (Ai) MTT assay. Overexpression of FANCF in drug-sensitive 8226/S and U266/S cells enhanced cell survival. The data are presented as percent
survival above control cells. The experiment was repeated at least 3 times. Representative results are shown. (ii) IC50 is the mean of 3 independent experiments and SD.
Student t test was used for statistical analysis. *P ⬍ .05. The experiments were repeated 3 times. (iii) Empty vector–transfected 8226-PQC and U266-PQC cells served as
controls for FANCF-transfected 8226-FANCF and U266-PQC cells. ␤-Actin blot served as loading control. (Bi) Apoptosis assay. Transfection of siFANCF partially reversed
melphalan resistance in 8226/LR5 and U266/LR6 cells. Annexin-V–FITC staining was used. The percentage of annexin-V–FITC⫹ cells is labeled, and specific apoptosis has
been calculated. The mean values and standard deviations from a representative experiment performed in triplicate are shown. Student t test was used for statistical analysis.
*P ⬍ .05. (Biii) Western blot analysis of FANCD2 and FANCF showed reduced protein expression in 8226/LR5 and U266/LR6 cells transfected with siFANCF compared with
cells transfected with siLuc as a control. ␤-Actin blot served as loading control. (C) Capacity to repair ICLs was reduced in FANCF knockdown LR5 and LR6 cells compared with
control siLuc-transfected cells, LR5 siLuc (top) and LR6 siLuc (bottom), respectively. The mean values and standard deviations from 3 independent experiments are shown.
Student t test was used for statistical analysis. *P ⬍ .05.
melphalan treatment induces a cell-cycle progression delay and
G2/M arrest.35 Although equivalent doses of melphalan induce
more ICL in drug-sensitive 8226/S cells compared with drugresistant 8226/LR5 cells, a 2-fold increase in FANCD2-L was
observed in 8226/LR5 compared with 8226/S (Figure 2B, 25-hour
time point). Because only the monoubiquitinated FANCD2
(FANCD2-L) can be translocated to DNA damage foci to perform
repair function, our data suggest that the efficiency of FANCD2-L
formation is important for drug resistance. This efficiency in
FANCD2-L formation in drug-sensitive and -resistant cell lines
may be due to enhanced expression of upstream FA complex.
Approximately one third of the DNA-melphalan adducts are
DNA interstrand cross-links.36 By studying gene-specific formation
of DNA monoadducts and interstrand cross-links, Souliotis et al37
reported that maximum monoadducts were formed within 2 hours
as a result of rapid binding of melphalan to nucleophilic site
(N7-guanine) in one DNA strand, whereas DNA ICLs accumulate
slowly and reach maximal levels within 8 hours through binding of
a second chloroethyl group to another N7-guanine site in the cDNA
strand. In our study, we were able to induce equivalent maximum
ICLs in drug-sensitive and -resistant cells by increasing the
melphalan dose exposure for drug-resistant 8226/LR5 and U266/
LR6 cells compared with 8226/S and U266/S drug-sensitive cells.
Interestingly, within 24 hours after melphalan treatment, approximately 37% and 22% of the ICLs were removed in the 8226/LR5
and U266/LR6 cells, respectively, while the drug-sensitive 8226/S
and U266/S cells showed no significant removal of ICLs in this
time frame. Our results strongly suggest that the FA/BRCA
pathway contributes to the removal of ICLs during this time period.
Furthermore, we demonstrated that the capacity to remove ICLs
was significantly reduced in FANCF knockdown 8226/LR5 and
U266/LR6 cells compared with control 8226/LR5 siLuc and
U266/LR6 siLuc cells. Taken together, these data show that
FANC/BRCA is involved in melphalan-induced ICL repair and
contributes to drug resistance.
Hypermethylation of FANCF has been reported in ovarian, oral,
lung, and cervical cancers,38-41 and demethylation of FANCF restores
the FA/BRCA pathway in ovarian tumors leading to cisplatin resistance.38 In this study, we examined the promoter methylation status of
FANCF in both 8226/S and 8226/LR5 cells. Although there is a 2-fold
increase of FANCF expression in 8226/LR5 compared with 8226/S, no
changes in cytosine-phosphate-guanosine (CpG) methylation were
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704
BLOOD, 15 JULY 2005 䡠 VOLUME 106, NUMBER 2
CHEN et al
found (data not shown). Mechanisms associated with increased expression of FANCF in 8226/LR5 cells are undergoing further study.
We have shown that silencing FANCF in 8226/LR5 and
U266/LR6 cells partially reversed drug resistance. We also showed
that overexpression of FANCF in drug-sensitive cells could
enhance drug resistance. It has been reported that FANCF plays an
important role in stabilizing subunits of the FA complex and
contributes to the proper function of this pathway.42 It has also been
shown that the cellular level of FANCA, FANCC, and FANCG
depends on the expression of FANCF.43 Overexpression of FANCF
in parental 8226 and U266 cell lines induced less resistance
compared with acquired melphalan-resistant cell lines, suggesting
that other members of the FANC/BRCA complex contribute to
melphalan resistance. Taniguchi et al reported that forced expression of FANCF in cisplatin-sensitive ovarian parental cells conferred a relative cisplatin resistance, however the resistance is less
than the acquired cisplatin-resistant cells.38 Their data also show
that overexpression of FANCF in acquired cisplatin-resistant cells
increased resistance to cisplatin.38 They suggested that acquired
cisplatin-resistant cells might have additional mechanisms involved in cisplatin resistance beyond the re-expression of FANCF.
For example, overexpression of FANCC has been reported to
inhibit FAS-induced apoptosis.44 This suggests that FA proteins
might have other functions beyond assembly of the complex and
participate in the drug-resistant phenotype. Taken together, we
suggest that repeated exposure to DNA cross-linking agents
increases expression of multiple components of the FA pathway,
which contribute to the acquired drug resistance. Further investigations will analyze the role of individual FA proteins to the overall
melphalan-resistance phenotype in myeloma cells.
Studying cell-cycle kinetics to diagnose Fanconi anemia patients has been reported.45 Exposing peripheral blood mononuclear
cells ex vivo to DNA cross-linker treatment induces a significant G2
block in patients with FA.45 In our study, we observed that 10-␮M
melphalan treatment induces a cell-cycle progression delay in S
phase within 24 hours and a G2 block within 48 hours. It has been
shown that fibroblasts from FA patients require 3 times longer to
recover from the ICL-induced cell-cycle arrest compared with
fibroblasts from healthy subjects.33 In 8226/S cells, removal of
melphalan-induced ICL is required for cells to progress from G2
block.35 We observed a higher degree of G2/M blockade in
drug-sensitive 8226/S cells compared with 8226/LR5 cells following melphalan treatment. We also observed that 8226/LR5 cells
require less time for cell-cycle progression from G2/M to the G1
phase compared with 8226/S cells; this is related to increased
expression of the FA/BRCA pathway genes and successful removal
of ICLs in 8226/LR5 cells compared with 8226/S cells. Overall, our
data demonstrate that the FA/BRCA pathway contributes to melphalan
resistance via enhanced ICL repair and reduced ICL-induced
growth arrest via release of cells from G2/M block.
In summary, to our knowledge, our study is the first to show that
enhanced expression of the FA/BRCA pathway is involved in
melphalan-induced DNA ICL repair and is an important mechanism for acquired melphalan resistance in myeloma cells. This
pathway also likely contributes to cross-resistance to other DNA
cross-linking agents and irradiation. We propose that the FA/BRCA
pathway represents a new target for preventing acquired drug
resistance and improving cancer treatment.
Acknowledgments
We thank Dr Grover C. Bagby (OSHU Cancer Institute, Portland,
OR) and Dr Zhi-Wei Li (H. Lee Moffitt Cancer Center, Tampa, FL)
for helpful comments.
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2005 106: 698-705
doi:10.1182/blood-2004-11-4286 originally published online
March 31, 2005
The FA/BRCA pathway is involved in melphalan-induced DNA
interstrand cross-link repair and accounts for melphalan resistance in
multiple myeloma cells
Qing Chen, Pieter C. Van der Sluis, David Boulware, Lori A. Hazlehurst and William S. Dalton
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