NIH Public Access: Author Manuscript

5-Aza-2-deoxycytidine Sensitizes Busulfan-resistant Myeloid
Leukemia Cells By Regulating Expression of Genes Involved in

Cell Cycle Checkpoint and Apoptosis
Benigno C. Valdez
1
, Yang Li
1
, David Murray
2
, Paul Corn
3
, Richard E. Champlin
1
, and Borje
S. Andersson
1
1
Department of Stem Cell Transplantation and Cellular Therapy; University of Texas MD Anderson
Cancer Center, Houston, TX, USA
3
Department of Genitourinary Medical Oncology; University of Texas MD Anderson Cancer Center,
Houston, TX, USA
2
Department of Experimental Oncology, Cross Cancer Institute, Edmonton, AB, Canada
Abstract
Busulfan (Bu) is a DNA-alkylating drug used in myeloablative pretransplant conditioning therapy
for patients with myeloid leukemia (ML). A major obstacle to successful treatment is cellular Bu-
resistance. To investigate the possible contribution of DNA hypermethylation to Bu-resistance, we
examined the cytotoxic activity of combined 5-aza-2-deoxycytidine (DAC) and Bu. Exposure of
Bu-resistant B5/Bu250
6
ML cells to 0.5 M DAC resulted in G2-arrest and apoptosis. The observed
G2-arrest was associated with hypomethylation and subsequent expression of epigenetically
controlled genes including p16
INK4A
, activation of the p53 pathway, and phosphorylation of CDC2.
The DAC-mediated apoptosis was partly due to hypomethylation and up-regulation of XAF1, which
resulted in down-regulation of the anti-apoptotic proteins XIAP, cIAP1 and cIAP2. The pro-apoptotic
PUMA and BNIP3 proteins were up-regulated while pro-survival STAT3 and c-MYC were
suppressed. Combination of 0.05 M DAC and 5 g/ml Bu resulted in synergistic cytotoxicity, which
was associated with PARP1 cleavage and activation of caspases 3 and 8, suggesting induction of an
apoptotic response. P53 inhibition in B5/Bu250
6
cells using pifithrin- alleviated these effects,
suggesting a role for p53 therein; this observation was supported by the relative resistance of p53-
null K562 cells to [DAC+Bu] combinations and by the effects of an anti-p53 shRNA on the OCI-
AML3 cell line. We conclude that the synergistic effects of [DAC+Bu] are p53-dependent and
involve cell-cycle arrest, apoptosis induction and down-regulation of pro-survival genes. Our results
suggest that, depending on tumor p53 status, incorporation of DAC might synergistically improve
the cytoreductive efficacy of Bu-based pre-transplant regimen in patients with ML.
Corresponding author: Department of StemCell Transplantation & Cellular Therapy, University of Texas, MD Anderson Cancer Center,
Unit 081, 1515 Holcombe Blvd, Houston, TX 77030 USA, Tel: 713-745-4370, Fax: 713-792-5673, [email protected].
Contributions. B. Valdez contributed to the conception and design of the study, analysis and interpretation of data, and drafted the
manuscript. Y. Li provided technical support and helped in data acquisition. D. Murray helped in data interpretation and writing the
manuscript. P. Corn developed the OCI-AML3 clones and helped in writing the manuscript. R. Champlin provided expertise on the
interpretation and analysis of results and writing the manuscript. B. Andersson was responsible for the research approach, funding,
analysis of data and critical revision of the article. All authors contributed to the final version of the submitted manuscript.
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NIH Public Access
Author Manuscript
Leuk Res. Author manuscript; available in PMC 2011 March 1.
Published in final edited form as:
Leuk Res. 2010 March ; 34(3): 364372. doi:10.1016/j.leukres.2009.08.014.
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Keywords
busulfan; 5-aza-2-deoxycytidine; myeloid leukemia; drug resistance; DNA methylation; cell cycle
signaling; apoptosis
INTRODUCTION
Acquired resistance to anticancer agents is mediated by both genetic and epigenetic
mechanisms [1]. Mutations in the DNA sequences, including amplification of drug-resistance
genes, are known to play a major role in the development of cellular drug resistance [2,3].
Epigenetic changes, including DNA hypermethylation and histone modifications, may play
equally significant roles in resistance development through transcriptional suppression of
genes involved in pro-drug activation, cell cycle checkpoint control, apoptosis, and other
cellular processes [4,5]. DNA methyltransferases (DNMTs), histone deacetylases (HDACs)
and histone methyltransferase have been identified to be responsible for these epigenetic
modifications. Inhibition of these enzymes has been shown to reverse gene silencing by
chromatin remodelling [5,6]. One such inhibitor, 5-aza-2-deoxycytidine (DAC) or decitabine,
is known to inhibit DNMTs which results in DNA hypomethylation and expression of silenced
genes including those involved in apoptosis [7,8]. Facilitation of apoptosis clearly has a
therapeutic potential in various malignancies, and the activity of DAC has been investigated
in patients with hematologic diseases [9]. Based on the favorable results of these studies, DAC
received regulatory approval for the treatment of myelodysplastic syndromes (MDS) and
chronic myelomonocytic leukemia (CMML) in the United States in 2006.
The combination of DAC with other chemotherapeutic agents has been shown to result in
additive or synergistic biological activities and possible reversal of clinical drug resistance
[1013]. Such studies have hitherto been performed with nucleoside analogs and/or DNA-
intercalating agents that are commonly used in antileukemic therapy. There are, however, no
data to support the combination of DAC and DNA-alkylating drugs. DNA-alkylating agent-
based leukemia treatment is typically followed by hematopoietic stem cell transplantation as
an alternative salvage modality for advanced leukemia, and it affords an option for long-term
disease control in patients not responding to conventional chemotherapy. Whether the addition
of DAC to an alkylating agent-based pretransplant regimen will alter DNA methylation status
and translate into a significantly improved long-term disease control is unknown.
We hypothesized that DAC can sensitize alkylating agent-resistant cells to DNA alkylators.
Considering Bu as a prototypic bifunctional alkylating agent used in high-dose pretransplant
conditioning therapy, we utilized a previously established Bu-resistant cell line B5/Bu250
6
[14] to examine the effects of DAC on myeloid leukemia (ML) cells with acquired Bu-
resistance. We demonstrate in the present report that exposure of B5/Bu250
6
cells to DAC
synergistically increases their sensitivity to Bu in a p53-dependent manner, and we describe
possible molecular mechanisms for the synergistic effects of Bu and DAC on Bu-resistant ML
cells.
MATERIALS AND METHODS
Cells and drugs
The Bu-resistant B5/Bu250
6
cells used in this study were established from the human KBM7/
B5 cell line as described previously [14]. B5/Bu250
6
and K562 CML cell lines [15] were
cultured in Iscoves Modified Dulbeccos Medium (IMDM; Sigma-Aldrich, St. Louis, MO)
supplemented with 20% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA) at 37C in a
humidified atmosphere of 5% CO
2
in air. The OCI-AML3, OCI-AML3/empty vector and OCI-
Valdez et al. Page 2
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AML3/p53 shRNA cell lines [16] were grown in RPMI 1640 (Mediatech, Herndon, VA)
supplemented with 10% FBS. Busulfan (Sigma-Aldrich) and 5-aza-2-deoxycytidine (DAC;
Sigma-Aldrich) were both freshly dissolved in dimethyl sulfoxide (DMSO) immediately prior
to cellular drug exposure(s). The final concentration of DMSO in all experiments did not exceed
0.08% (by volume), a level that does not induce differentiation of these cell lines (data not
shown).
Cytotoxicity assay and graphical analysis
Cell suspensions were aliquoted (50 l of 4 10
5
cells/ml) into 96-well plates in the presence
of drug(s) or solvent alone, incubated as above at 37C for 4 d, and analyzed by the 3-(4,5-
dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay [17]. Graphical analyses
including calculations of IC
50
values (the concentration of drug required for 50% growth
inhibition) were done using Prism 5 (GraphPad Software, San Diego, CA). The combination
effects of Bu and DAC were estimated based on the combination index (CI) values [18]
calculated using the CalcuSyn software (Biosoft, Ferguson, MO). This program was developed
based on the median-effect method: CI <1 indicates synergy, CI 1 is additive, and CI >1
suggests antagonism.
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay
Actively growing B5/Bu250
6
cells (2.5 10
5
cells/ml) were exposed to the indicated
concentrations of the drug(s) at 37C for 4 d. Cells were analyzed for apoptosis using the Apo-
Direct
TUNEL assay kit (Millipore, Billerica, MA) according to the procedure provided by
the manufacturer. TUNEL-positive cells were determined by analysis of at least 15,000 cells
using a FACS Calibur instrument (BD Biosciences, San J ose, CA).
Flow-cytometric analysis of cell cycle phase distribution
Cells in logarithmic growth phase (1 10
6
cells/ml) were incubated with the indicated
concentrations of the drug(s) at 37C. Following incubation for 48 h, the cells were centrifuged,
resuspended in 70% ethanol in phosphate-buffered saline (PBS), and fixed at 20C overnight.
Fixed cells were pelleted at 3,000 g at room temperature, washed with PBS, and treated with
0.25 ml of 500 U/ml RNAse A in PBS containing 1.12% sodium citrate at 37C for 30 min.
After addition of 0.25 ml propidium iodide (PI, 50 g/ml) solution, the cells were kept in
subdued light for at least 1 h prior to analysis by flow cytometry. The cellular DNA content of
at least 10,000 cells was analyzed using a BD FACS Calibur instrument and the proportion of
cells in the different phases of the cell cycle was determined using the CellQuest
software
(Becton Dickinson, Franklin Lakes, NJ ).
Real-time PCR
Total RNA was extracted using TRIzol
Reagent (Invitrogen). The high capacity cDNA

Archive kit (Applied Biosystems, Foster City, CA) was used to synthesize cDNA. Real-time
PCR amplification was performed using a SYBR Green-based assay with the 7500 Real Time
PCR System (Applied Biosystems). A dissociation step was included in the amplification, and
only primers with a single dissociation peak were included in the final analysis. The
quantification of gene expression was carried out by comparative C
T
methodology (i.e.,
threshold cycle number above which the increase in fluorescence was logarithmic); the
GAPDH gene was used as an internal control. Primers used for all PCR analyses are listed in
Table 1.
Methylation-specific PCR analysis
Genomic DNA was isolated from the cell lines using a Wizard Genomic DNA Purification kit
(Promega, Madison, WI). Bisulfite modification of the genomic DNA and its purification was
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performed using a MethylDetector kit (Active Motif, Carlsbad, CA). Methylation-specific PCR
(MSP) was performed using 30 ng bisulfite-modified DNA, 0.5 M of each primer (Table 1),
1 mM each of dATP, dCTP, dGTP and dTTP, 5 mM MgSO
4
, and Taq DNA polymerase with
ThermoPol II buffer (New England Biolabs, Ipswich, MA). The amplification method included
initial heating at 95C for 3 min, followed by 30 cycles of 95C for 30 s, 57C or 65C for 30
s depending on the primers (see Table 1), and 72C for 33 s, followed by extension at 72C
for 10 min. MSP products were visualized on a 2.5% agarose gel and DNA band intensities
were analyzed using Quant One software (Bio-Rad, Hercules, CA).
Western blot analysis
Cells were collected by centrifugation, washed with PBS and lysed with cell lysis buffer (Cell
Signaling Technology, Danvers, MA) as recommended by the manufacturer. Total protein
concentrations in cell lysates were determined using a BCA Protein Assay kit (Thermo Fisher
Scientific, Rockford, IL). Western blot analysis was done by separating protein extracts on
polyacrylamide-SDS gels and blotting onto nitrocellulose membranes (Bio-Rad). Immunoblot
analysis by chemiluminescence was done using the SuperSignal West Pico/Dura Substrate
(Thermo Fisher, Waltham, MA) or the Immobilon Western Chemiluminescent HRP Substrate
(Millipore, Bedford, MA). All antibodies, their sources and other relevant information are listed
in Table 2. The -actin protein was used as an internal control.
RESULTS
Bu and DAC combinations have synergistic effects on the survival of B5/Bu250
6
cells
Recent phenotypic characterizations of Bu-resistant B5/Bu250
6
cells demonstrated decreased
expression of several genes known to be epigenetically regulated [14]. We, therefore, sought
evidence that exposure of B5/Bu250
6
cells to DAC might alter gene expression and sensitize
these cells to Bu. Cells were exposed to various concentrations of Bu or DAC alone, or in
combination, and their survival was determined using the MTT assay. B5/Bu250
6
cells have
calculated IC
50
values of 25 g/ml Bu and 0.17 M DAC when continuously incubated with
the individual drug for 4 d (data not shown). Approximately 86% cell survival was observed
using 5 g/ml Bu alone and 83% cell survival using 0.04 M DAC alone (Figure 1A). A more
efficient inhibition of cell survival (down to 57% of control) was observed when the two drugs
were combined at the same concentrations (Figure 1A). When values for cell survival
(logarithmic scale) at three different Bu concentrations were plotted against DAC concentration
(linear scale), the slopes of the three lines increased with Bu concentration, suggesting possible
synergistic effects of Bu and DAC. The possibility of a synergistic cytotoxicity of combined
Bu and DAC was further indicated by analysis using the median-effect method [18]. The
combination indexes (CIs) at fractional effects of 0.30.7 (i.e., 30% 70% cytotoxicity) were
significantly lower than 1.0, suggesting strong synergism in these concentration intervals
(Figure 1B).
Analysis of the isobologram for the different [Bu+DAC] combinations that resulted in 50%
cell survival (Figure 1C) shows that [6 g/ml Bu +0.07 M DAC] would be as cytotoxic for
the Bu-resistant B5/Bu250
6
cells (Figure 1C) as 6 g/ml Bu alone was for the Bu-sensitive
parental B5 cells (i.e., its IC
50
value, data not shown). These results further illustrate that DAC
can sensitize Bu-resistant cells to Bu.
The observed synergistic effects of DAC and Bu using the MTT assay are further validated by
the results obtained using the TUNEL assay, a method for detecting DNA fragmentation as a
result of apoptotic responses [19]. Approximately 1.8% of the cells were positive for the
TUNEL assay in the absence (solvent control) or presence of 0.02 M DAC (Figure 1D).
Exposure of B5/Bu250
6
cells to 15 g/ml Bu for 4 d resulted in 18.3% apoptotic cells and the
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combination of 0.02 M DAC and 15 g/ml Bu increased the level of TUNEL-positive cells
to 30.8% (Figure 1D), which strongly indicates synergistic cytotoxicity.
DAC induces G2-arrest, cell death and DNA hypomethylation of B5/Bu250
6
cells
The observed synergism between Bu and DAC prompted us to determine the molecular
mechanisms underlying these cytotoxic effects. We recently reported that the resistance of B5/
Bu250
6
cells to Bu alone is partly due to their ability to evade drug-induced G2-arrest and
apoptosis [14]. Others have attributed the cytotoxic activity of DAC to its ability to arrest cells
in the G1 and G2-M phases of cell cycle [20]. We therefore examined how DAC alone would
affect the phenotype of B5/Bu250
6
cells. Exposure of cultures to 0.5 M DAC for 48 h and
analysis by flow cytometry showed an ~5-fold increase in cells in G2 phase and an ~5-fold
increase in the sub-G1 fraction, which likely reflects the induction of apoptosis (Figure 2A).
The accumulation of cells in G2 phase is further manifested by a 10-fold increase in G2/G1
and G2/S values (bottom of Figure 2A). These results suggest that DAC exposure results in
alterations in cell cycle checkpoint regulation and in the death of B5/Bu250
6
cells presumably
by apoptosis.
To determine whether DAC affects gene expression in B5/Bu250
6
cells, we initially examined
its effect on the methylation of selected genes known to be epigenetically regulated. Using the
methylation-specific PCR (MSP) methodology [21] we identified the p16
INK4A
and XAF1
genes to be hypermethylated in B5/Bu250
6
cells (Figure 2B). Exposure to DAC for 48 h
resulted in a dose-dependent reversal of the hypermethylation of the p16
INK4A
and XAF1 gene
promoters as suggested by an increasing ratio of unmethylated to methylated (U/M) DNA
(Figure 2B). These changes in the methylation status of the p16
INK4A
and XAF1 promoters
correlated with a dramatic increase in their transcription level as measured by real-time PCR
(Figure 2B, right hand panel). Exposure of B5/Bu250
6
cells to 0.5 M DAC for 48 h resulted
in increased expression of p16
INK4A
and XAF1 by more than 500- and ~70-fold, respectively,
compared with the control cells exposed to solvent alone. Thus exposure of B5/Bu250
6
cells
to DAC results in hypomethylation and transcriptional activation of the p16
INK4A
and XAF1
genes which encode the p16
INK4A
cyclin-dependent kinase (CDK) inhibitor and the pro-
apoptotic XAF1 (XIAP Associated Factor 1) protein, respectively.
To further understand how DAC causes hypomethylation of the p16
INK4A
and XAF1 genes we
examined the status of DNA methyl transferase (DNMT) isozymes which are known to
catalyze DNA methylation. Using real-time PCR analysis, the constitutive expression of
DNMT1, DNMT2, DNMT3A and DNMT3B was found to be 1.5, 1.9, 0.9 and 23-fold higher in
Bu-resistant B5/Bu250
6
cells relative to Bu-sensitive parental B5 cells (data not shown).
Because DNMT1 and DNMT3B are believed to maintain ~95% of the DNA methylation in
human cancer cells [22], we determined how the protein levels of these two isozymes are
affected by DAC. B5/Bu250
6
cells were exposed to various concentrations of the drug, and
protein extracts were analyzed by immunoblotting. Figure 2C shows a dose-dependent
disappearance of the two enzymes in the presence of DAC, which is consistent with studies
using other cell lines [23,24]. The observed higher expression of DNMT3B in B5/Bu250
6
versus B5 cells (23-fold) combined with the previous report that the DNMT3B enzyme
functions principally as a de novo DNA methyltransferase [25] suggest that DNMT3B could
play a significant role in epigenetic regulation of the phenotypic expression of Bu-resistance
in B5/Bu250
6
cells.
DAC alters the expression of several proteins involved in cell cycle regulation, apoptosis
and survival
As noted above, DAC treatment invokes a G2-arrest in B5/Bu250
6
cells (Figure 2A). To
identify the cell cycle regulatory proteins involved in DAC-mediated checkpoint control, we
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examined the expression and phosphorylation status of several key proteins involved in this
response. Exposure of B5/Bu250
6
cells to 0.5 M DAC for 48 h resulted in a dramatic
phosphorylation of the variant H2A histone, H2AX, to its activated form -H2AX (Figure 3A),
suggesting a DNA-damage response that could trigger checkpoint signaling. Downstream in
this signaling cascade is the phosphorylation of the tumor suppressor protein, p53, and its
subsequent induction (by transcriptional transactivation) of the CDK inhibitor protein
p21
WAF1/CIP1
(Figure 3A), an observation consistent with previous studies using this drug in
other tumor cell lines [7, 8]. The up-regulation of p21
WAF1/CIP1
in the presence of DAC is
likely related to the observed phosphorylation of p53 at Ser15 as previously reported [26, 27]
and discussed further below. Although p21
WAF1/CIP1
is known to be capable of triggering G1-
arrest, it also causes G2-arrest by interacting with CDK1/CDC2 [28, 29]. The DAC-induced
increase in p21
WAF1/CIP1
correlates with the observed accumulation of P-CDC2 (Figure 3A),
a known hallmark of G2-M checkpoint activation [30]. Another gene encoding a CDK inhibitor
that was induced by DAC was p16
INK4A
(Figure 2B). The DAC-mediated hypomethylation
and transcription of the p16
INK4A
gene correlates closely with the dramatic increase in
p16
INK4A
protein expression shown in Figure 3A. As will be discussed below, p16
INK4A
is
best known as an activator of the G1 checkpoint, although some studies also indicate a role for
p16
INK4A
at the G2-M checkpoint.
Prolonged arrest of cells in G2 phase may result in apoptosis or mitotic catastrophe and
subsequent cell death. Indeed, protracted exposure (48 h) of B5/Bu250
6
cells to 0.5 M DAC
increased the proportion of cells with sub-G1 DNA content (Figure 2A), which is a strong
indication of cells undergoing apoptosis. Immunoblot analysis was used to identify anti- and
pro-apoptotic proteins affected by DAC (Figure 3B). As shown in Figure 2B, hypomethylation
and transcription of the XAF1 gene were induced by DAC, and Figure 3B shows the
concomitant up-regulation of XAF1 protein expression. The constitutive low expression of
XAF1 (Figure 3B, lane 1) correlated with the low abundance of the unmethylated form of the
XAF1 gene in untreated B5/Bu250
6
cells (Figure 2B, lane 2). XAF1 protein is known to
antagonize the pro-survival activities of the inhibitor of apoptosis (IAP) family of proteins,
such as XIAP, cIAP1 and cIAP2 [31, 32]. Indeed, the increased level of expression of XAF1
protein correlated with decreased levels of its target proteins XIAP, cIAP1 and cIAP2 (Figure
3B). The down-regulation of these pro-survival proteins by DAC was accompanied by up-
regulation of pro-apoptotic proteins such as PUMA (p53-upregulated modulator of apoptosis)
and BNIP3 (BCL2/adenovirus E1B 19kDa interacting protein 3) (Figure 3B). The up-
regulation of PUMA might be attributed to p53 trans-activation [33] and the increase in BNIP3
expression could be due to DNA hypomethylation of its promoter [34].
In addition to proteins involved in apoptosis, we examined the effects of DAC on other pro-
survival factors. Because the STAT3 (signal transducer and activator of transcription 3) protein
is up-regulated in B5/Bu250
6
cells [14], we examined the effects of DAC exposure on the
expression of STAT3 and its target genes. Figure 3B showed a DAC-dose-dependent decrease
in expression of STAT3 protein; a less marked effect was observed at 0.1 M DAC, but an
almost 50% decrease in STAT3 expression was consistently observed at 0.5 M DAC after a
48-h exposure. This down-regulation of STAT3 is consistent with a dramatic decrease in the
expression of c-MYC (Figure 3B), a known downstream target gene of STAT3 [35]. Overall,
the down-regulation of anti-apoptotic and pro-survival proteins and up-regulation of pro-
apoptotic proteins is associated with and presumably contributes to the cytotoxic activity of
DAC towards B5/Bu250
6
cells.
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The synergistic effects of DAC and Bu in B5/Bu250
6
cells include exacerbation of G2-arrest
and apoptosis
The observed G2-arrest and apoptosis described above were obtained at relatively high
concentrations of DAC. We decided to combine DAC and Bu at concentrations much lower
than IC
50
values and determine their cytotoxicity on Bu-resistant B5/Bu250
6
cells. Exposure
of cells to 0.05 M DAC for 48 h resulted in an ~2-fold increase in G2/G1 ratio without a
substantial change in the sub-G1 fraction; exposure to 5 g/ml Bu resulted in an ~4-fold
increase in G2/G1 ratio and small increase in the sub-G1 fraction (Figure 4A). Exposure to the
combination of 0.05 M DAC and 5 g/ml Bu dramatically increased the G2/G1 ratio and sub-
G1 fraction by ~7-fold and ~3-fold, respectively (Figure 4A). This marked increase of cells in
the sub-G1 fraction was also associated with activation of apoptosis as indicated by cleavage
of PARP1 (poly-ADP ribose polymerase-1) and activation (by cleavage) of two key apoptotic
proteins, caspases 3 and 8 (Figure 4B), but not of caspase 2 (data not shown). A less dramatic
increase in cleavage of PARP1, caspase 3 and caspase 8 was observed when B5/Bu250
6
cells
were exposed to either 0.05 M DAC alone or 5 g/ml Bu alone. Thus, combination of the two
drugs resulted in an increased activation of the apoptotic pathway (Figure 4B). These results
suggest that the cytotoxic activity of low concentrations of DAC and Bu can be increased by
combining the two drugs.
The [DAC+Bu]-mediated synergistic effects on myeloid leukemia cells are p53-dependent
As shown in Figure 3, DAC alone induces the phosphorylation of p53 and enhances its
transcriptional transactivation function as suggested by the associated up-regulation of
p21
WAF1/CIP1
and PUMA. Recognizing that p53 plays a critical role in checkpoint signaling,
we examined the effects of combined [DAC+Bu] on the expression and phosphorylation of
p53. Figure 5A shows that whereas 0.05 M DAC alone or 5 g/ml Bu alone induced p53
phosphorylation at Ser15 (Figure 5A, lanes 2 and 4), their combination (lane 5) had an even
more dramatic effect, suggesting that the activation of p53 might play a role in the cytotoxic
activity of combined [DAC+Bu]. We therefore sought to examine the dependence of [DAC
+Bu] cytotoxicity on p53. We used pifithrin- to inhibit the transactivation function of p53 in
B5/Bu250
6
cells and compared the results to those seen in the p53-null K562 CML cell line.
Cells pre-exposed to pifithrin- prior to treatment with the [DAC+Bu] combination were
analyzed by flow cytometry and immunostaining. We used a slightly higher DAC
concentration (0.2 M) in these experiments to elicit more pronounced effects. Exposure of
B5/Bu250
6
cells to 0.2 M DAC and 5 g/ml Bu resulted in a considerable increase of cells
in the sub-G1 compartment (39%, versus 15% in control cells), a response that was inhibited
in a dose-dependent manner by exposing the cells to pifithrin- prior to [DAC+Bu] treatment
(Figure 5B). The sub-G1 fraction was reduced from 39% to 12% in the presence of 20 M
pifithrin-. The [DAC+Bu]-mediated G2-arrest, as measured by the G2/G1 or G2/S ratio, was
alleviated from 0.46 to 0.38 or 0.88 to 0.63, respectively, in B5/Bu250
6
cells pre-exposed to
pifithrin- (bottom of Figure 5B). This observation is consistent with the previously-reported
requirement for p53 and p21
WAF1/CIP1
to sustain G2-arrest in human cells [28]. In contrast,
the p53-null K562 cells showed resistance to the [DAC+Bu] combination as suggested by the
lack of increase in sub-G1 fraction, but interestingly a marked G2-arrest was observed in these
cells (Figure 5B). Pre-exposure of K562 cells to 20 M pifithrin- prior to [DAC+Bu] treatment
did not substantially change the proportion of cells in sub-G1 but further enhanced the G2-
arrest (G2/G1 and G2/S ratios increasing from 0.45 to 0.58 and 0.41 to 0.70, respectively;
Figure 5B).
The results from flow cytometry were consistent with immunostaining studies. Exposure of
B5/Bu250
6
cells to 0.2 M DAC and 5 g/ml Bu dramatically increased the level of
p21
WAF1/CIP1
protein, and pre-exposure of cells to 20 M pifithrin- inhibited this up-
regulation (Figure 5C, left hand panel). This abrogation of p21
WAF1/CIP1
induction implies
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that pifithrin- is an effective inhibitor of p53 transcriptional transactivation in this cell line
under these conditions. Surprisingly, the observed [DAC+Bu]-mediated phosphorylation of
histone H2AX in B5/Bu250
6
cells (Figure 5C) was also abrogated by pifithrin-. Although
exposure of K562 cells to [DAC+Bu] combination also induced the phosphorylation of histone
H2AX, this response was not blocked by pifithrin- (Figure 5C). In addition to blocking
p21
WAF1/CIP1
induction in B5/Bu250
6
cells, the p53 inhibitor pifithrin- also blocked the
[DAC+Bu]-mediated cleavage of PARP1, caspase 9 and caspase 8 (Figure 5C). These
collective results, including the decrease in sub-G1 fraction shown in Figure 5B, suggest that
the [DAC+Bu]-mediated apoptosis of B5/Bu250
6
cells is largely p53-dependent. This
conclusion is further supported by the resistance of p53-null K562 cells to apoptosis when
treated with [DAC+Bu] as shown by lack of increase in sub-G1 fraction (Figure 5B, right hand
panel) and minimal cleavage of PARP1, caspase 8 and caspase 9 (Figure 5C, right hand panel).
The relevance of p53 and p21
WAF1/CIP1
in the maintenance of G2-arrest, as mentioned above,
is further suggested by the observed p53-dependent changes in the level of phosphorylation of
CDK1/CDC2. Pre-exposure of p53-positive B5/Bu250
6
cells to pifithrin- reduced the level
of [DAC+Bu]-induced P-CDK1/CDC2 (Figure 5C), which coincides with the observed
reduction in G2/G1 and G2/S values (Figure 5B). This result was not observed in the p53-null
K562 cells; interestingly, a slight upregulation of P-CDK1/CDC2 was observed when these
cells were exposed to pifithrin- (Figure 5C) with a concomitant increase in G2/G1 and G2/S
ratios (Figure 5B).
To further define the relevance of p53 in [Bu+DAC]-mediated cytotoxicity, we used a
previously established clone of OCI-AML3 cells where p53 was down-regulated using an
shRNA construct [16]. Initial analysis showed that the parental cell line OCI-AML3 was more
resistant to [Bu+DAC] combinations compared with B5/Bu250
6
cells (data not shown).
Exposure of OCI-AML3 and OCI-AML3/empty vector cells to combined 30 g/ml Bu and 4
M DAC for 48 h resulted in G2-arrest and significant cell death as indicated by 4050% cells
in sub-G1; in contrast, OCI-AML3/p53 shRNA cells showed greater resistance to [Bu+DAC]
exposure (Figure 5D). This resistance may be attributed to a lack of p53 expression and
activation of its downstream signaling pathways as suggested by Western blot analysis. Figure
5E shows expression of p53 in the parental and empty vector cell lines but not in OCI-AML3/
p53 shRNA cells. The expressed p53 was phosphorylated at Ser15 when cells were exposed
to [Bu+DAC] and resulted in p21
WAF1/CIP1
expression and PARP1 cleavage. Together, the
results from cell cycle and Western blot analyses underscore the importance of p53-dependent
pathways in mediating the cellular effects of [Bu+DAC] combinations in the ML background.
DISCUSSION
Based on our cellular model for acquired Bu resistance in ML cells, it is apparent from these
findings that DAC can be effectively used to sensitize these Bu-resistant cells to Bu in vitro.
The combination of DAC and Bu over a broad range of concentrations displayed a synergistic
increase in cytotoxicity compared to the individual drugs as evaluated by the median-effect
method. Our results suggest that this synergism can be attributed to the combined efficacies of
these two drugs in inducing cell cycle arrest and apoptosis when administered at low
concentrations.
Since we had already established that Bu alone causes G2-arrest and induces the expression
of pro-apoptotic genes [14], we first evaluated the mechanism of cytotoxicity of DAC alone
in Bu-resistant B5/Bu250
6
cells. Like Bu, DAC induces both G2-arrest and apoptosis in these
cells. The ability of DAC to induce these two cellular responses might be attributed to its
inhibition of DNA methylation and activation of cell cycle checkpoint signaling, similarly to
previous reports for DNA repair responses [36,37]. These two activities DNA
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hypomethylation and cell cycle checkpoint regulation may not be entirely independent of
each other given that the expression of some genes involved in cell cycle regulation is
epigenetically controlled. However, the incorporation of DAC into DNA strands may elicit
responses independent of its DNA-hypomethylating activity. Whether DNA hypomethylation
or cell cycle checkpoint regulation prevails in a given situation in this cell type most likely
depends on factors such as the DAC concentration relative to exposure time.
DAC induces DNA hypomethylation by inhibiting DNA methyltransferases. We observed that
B5/Bu250
6
cells express ~1.5-fold and ~23-fold higher levels of DNMT1 and DNMT3B,
respectively, compared with the Bu-sensitive parental B5 cells. This elevated expression of
DNMT1 and DNMT3B could be a contributory factor to the Bu-resistance of B5/Bu250
6
cells.
Exposure of these cells to 0.5 M DAC for 48 h resulted in decreased levels of both DNMT1
and DNMT3B (Figure 1C), presumably mediated by enhanced protein degradation, which is
believed to be initiated by covalent trapping of the enzymes to DNA-incorporated DAC [23].
This trapping/degradation of DNMTs is expected to result in a reversal of DNA
hypermethylation and re-expression of silenced genes. Among the epigenetically regulated
pro-apoptotic genes that we examined in this context (data not shown), XAF1 showed a
dramatic change in expression in the presence of DAC (Figure 2B). Analysis of the methylation
status of the XAF1 promoter by MSP showed an approximately 4-fold increase in the ratio of
unmethylated to methylated sequences when B5/Bu250
6
cells were exposed to 0.5 M DAC
(Figure 2B).
XAF1 is a pro-apoptotic protein which binds to and antagonizes the anti-apoptotic activities
of several members of the IAP family of proteins, including XIAP, cIAP1 and cIAP2; its
binding to XIAP reverses the anti-caspase activity of XIAP [31] and its interaction with cIAP1
and cIAP2 releases pro-apoptotic Smac/DIABLO from their complexes [32]. Our results are
consistent with these previous reports. Exposure of B5/Bu250
6
cells to DAC resulted in up-
regulation of XAF1 protein and down-regulation of all three of these IAP proteins, i.e., XIAP,
cIAP1 and cIAP2 (Figure 3B).
DNA hypomethylation could also play a role in the DAC-mediated G2-arrest of B5/Bu250
6
cells. Examination of the expression of various cell cycle-regulatory proteins in our cellular
model suggests that DAC controls the cell cycle at various signaling checkpoints. It caused
DNA hypomethylation of the p16
INK4A
gene and up-regulation of the p16
INK4A
protein (Figure
2B), an inhibitor of CDKs which are critical regulators of cell cycle progression. DAC
stimulated the phosphorylation of p53, which was most likely responsible for the up-regulation
of another CDK inhibitor, p21
WAF1/CIP1
(Figure 3A). Although both the p16
INK4A
and
p21
WAF1/CIP1
proteins are known to arrest cells in G1 phase [38], they have been shown to
contribute to the arrest of cells in G2-M phase as well [28, 3941].
Although DAC alone can perturb cell cycle progression, the combination of DAC and Bu at
lower concentrations resulted in a more marked G2-arrest of Bu-resistant B5/Bu250
6
cells
(Figure 4A). We reported previously that Bu causes G2 cell cycle arrest through the CHK2-
CDC25-CDC2 pathway [14]. The [DAC+Bu]-mediated activation of p53 and up-regulation
of p21
WAF1/CIP1
, which eventually inhibits CDC2 [28, 29], may also enhance/maintain G2-
arrest [40, 41]. Together these responses may elicit stronger signals for the activation of cell
cycle checkpoints.
The combination of DAC and Bu also elicited a more efficient activation of the apoptotic
response. The observed phosphorylation of histone H2AX, cleavage of PARP1 and activation
of caspases 3 and 8 are all consistent with DNA-damage responses, which might be caused by
the DNA crosslinking activity of Bu [42] and the incorporation of DAC into DNA strands as
a nucleoside analogue [27]. How inhibition of p53 activity by pifithrin- alleviates the [DAC
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+Bu]-induced phosphorylation of H2AX in B5/Bu250
6
cells remains to be defined, although
the possibility of off-target effects cannot be discounted. Another possibility is that H2AX
phosphorylation is triggered by the attempt of cells to repair such DNA modifications, which
probably results in DNA nicks and single-stranded DNA. It is well known that such repair
events are often p53-dependent [43].
Although further studies are required to dissect the cascade of events and identify specific
caspases involved in [DAC+Bu]-mediated apoptosis, our study using B5/Bu250
6
cells suggests
a major involvement of caspases 3, 8 and 9, but not of caspase 2, in this apoptotic response.
Caspase 3 is a known executioner caspase, but recent reports have shown its role upstream of
the mitochondria [44,45] and may therefore be classified as an initiator caspase.
The cytotoxic activity of combined [DAC+Bu] in ML cells clearly depends on p53 status. The
p53-null K562 cell line exhibited greater resistance to these drugs than the B5/Bu250
6
cell line
as shown by cell cycle analysis (Figure 5B) and cytotoxicity/MTT assay (data not shown).
Inhibition of p53 function and associated abrogation of transactivation of gene expression in
p53-wild type B5/Bu250
6
cells with pifithrin- alleviated the [DAC+Bu]-mediated G2-arrest
and apoptosis (Figure 5B) with a concomitant decrease in P-CDC2 and inhibition of PARP1,
caspase 8 and caspase 9 cleavage (Figure 5C). Similar results were obtained when p53 was
knocked down with shRNA in OCI-AML3 cells (Figure 5D and 5E). As a guardian of
chromosome integrity, it is not unreasonable to imagine that p53 would mediate the effects
caused by incorporation of DAC as a nucleoside analogue into DNA strands or by DNA
crosslinks caused by Bu. These results suggest that analysis of the p53 status of the leukemia
cell population may have predictive value when evaluating the potential benefit of combining
DAC with an alkylating agent-based pretransplant conditioning regimen.
The inclusion of DAC in a Bu-based therapy for ML might not only result in a synergistic
cytoreduction but might also offer additional safety. Our observation that combined 70 nM
DAC +6 g/ml Bu exerts the same degree of cytotoxicity to Bu-resistant cells as 6 g/ml Bu
alone does to Bu-sensitive cells suggests that the proposed strategy might ultimately offer
advantages for treating Bu-resistant ML by overcoming resistance and reducing toxic Bu
exposure, and for treating Bu-sensitive ML by reducing the amount of Bu exposure required
for therapeutic benefit. We should note that the major side effect of DAC therapy in leukemia
patients is severe and prolonged myelosuppression [46]; the major side effects of Bu include
irreversible myelosuppression, hepatic veno-occlusive disease and neurotoxicity [47].
In conclusion, DAC and Bu have partly unique and partly common effects on cell cycle
progression and cell survival. When combined, their cytotoxic effects become synergistic
against Bu-resistant ML cells. Our findings provide a mechanistic explanation for the observed
synergistic cytotoxicity. Based on these findings, we propose that inclusion of DAC in Bu-
based pre-transplant conditioning therapy may increase the cytoreductive efficacy against
myeloid leukemic cell clones with acquired cross-resistance to bifunctional alkylating agents.
Acknowledgments
This work was supported in part by grants fromthe National Institutes of Health (P01 CA055164 and CCSG Core
CA16672), and The Stephen L. and Lavinia Boyd Fund for Leukemia Research.
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Figure 1.
Cytotoxic effects of Bu and DAC, alone or in combination, on B5/Bu250
6
cells. (A) Cells were
incubated for 4 d with the indicated concentrations of Bu or DAC alone, or in combination,
and cell survival was determined by the MTT assay. Values for cell survival (%) on the Y-axis
are in logarithmic scale and were calculated relative to control cells exposed to solvent alone.
(B) Results from the MTT assay were analyzed for synergism by calculating the combination
index (CI) values using the CalcuSyn software. The values in panels A and B are the average
(SE) of five independent experiments. (C) Equipotent combinations of various concentrations
of Bu and DAC that result in 50% cell survival. (D) The synergistic cytotoxicity of [DAC+Bu]
after 4 d was further evaluated by determining the proportion of cells with fragmented DNA
using the TUNEL assay.
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A

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Figure 2.
Effects of DAC on cell cycle phase distribution, DNA methylation and DNA methyltransferase
levels. (A) Representative DNA histograms for B5/Bu250
6
cells following exposure to solvent
or 0.5 M DAC for 48 h prior to staining with propidium iodide and flow cytometry. These
experiments were repeated at least 3 times with very similar results. (B) Left hand panel:
Typical genomic DNA methylation profiles from cells exposed to different concentrations of
DAC for 48 h and analyzed by MSP using primers for methylated (M) and unmethylated (U)
DNA. The numbers at the bottom of each picture refer to the ratio of unmethylated to
methylated DNA (U/M). These experiments were repeated at least 3 times with very similar
results. Right hand panel: mRNA levels of the p16
INK4A
and XAF1 genes as measured by real-
time PCR. The data represent the average (SE) of three independent experiments. (C) Protein
expression of DNMT1 and DNMT3B was determined by immunoblot analysis using extracts
from cells exposed to various concentrations of DAC for 48 h. These experiments were repeated
at least twice, and a typical result is shown.
N
I
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P
A

A
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o
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M
a
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A

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A

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Figure 3.
DAC affects the expression of key cell cycle regulatory (A) and survival (B) proteins. Cells
were exposed to solvent or the indicated concentrations of DAC for 48 h and cell extracts were
analyzed for protein expression by Western blot. Representative results of two experiments
are shown.
N
I
H
-
P
A

A
u
t
h
o
r

M
a
n
u
s
c
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t
N
I
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A

A
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o
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M
a
n
u
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N
I
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A

A
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M
a
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Figure 4.
Synergistic effects of Bu and DAC on Bu-resistant B5/Bu250
6
cells. (A) Typical cell cycle
histograms from flow cytometric analysis of cells exposed to Bu or DAC, alone or combined.
(B) Representative Western blot showing the more efficient activation of the apoptotic response
by [Bu+DAC] combinations than by single drug exposures. These experiments were repeated
at least twice with similar results. [Casp =caspase]
N
I
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-
P
A

A
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M
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N
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A

A
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M
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N
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A

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Figure 5.
Effect of inhibition of p53 using pifithrin- (PF) on cellular responses to Bu and/or DAC. (A)
B5/Bu250
6
cells were exposed to the indicated concentrations of DAC and Bu, alone or
combined, for 48 h prior to Western blot analysis to determine the phosphorylation status of
p53. (B) Cells were or were not pre-exposed to pifithrin- for 24 h then treated with [DAC
+Bu] for 48 h prior to flow-cytometric analysis or (C) Western blot analysis. (D) OCI-AML3
cells were exposed to DMSO solvent or to the indicated concentrations of Bu and DAC for 48
h and analyzed by flow cytometry and (E) Western blot. All panels are representative: in each
case the experiments were repeated at least twice with similar results.
N
I
H
-
P
A

A
u
t
h
o
r

M
a
n
u
s
c
r
i
p
t
N
I
H
-
P
A

A
u
t
h
o
r

M
a
n
u
s
c
r
i
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t
N
I
H
-
P
A

A
u
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h
o
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M
a
n
u
s
c
r
i
p
t
N
I
H
-
P
A

A
u
t
h
o
r

M
a
n
u
s
c
r
i
p
t
N
I
H
-
P
A

A
u
t
h
o
r

M
a
n
u
s
c
r
i
p
t
N
I
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P
A

A
u
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M
a
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t
Table 1
Primers for PCR analysis
Real-time PCR
Gene Forward Reverse Annealing Temp (C)
GAPDH CAACAGCCTCAAGATCATCAGC TCCTAGACGGCAGGTCAGGTC 60
DNMT1 CCGGCCCCGGTTCTT GGACCATGGAGCGCTTGA 60
DNMT2 AGCCCCTGGTCAGGTACTGA CATTGCATATTTTTGTGGATGTACAGA 60
DNMT3A AGAGAGCGGCTGGTGTACGA CTCCCACAGGAGATGCAGATG 60
DNMT3B AAGCTCGCGACTCTCCAAGAG ATCGCCTGTCAAGTCCTGTGT 60
p16 CCCAACGCACCGAATAGTTAC CGCTGCCCATCATCATGAC 60
XAF1 CTCGGTGTGCAGGAACTGTAAA CAGGAACCGCAGGCAGTAA 60
MSP
p16 (Methylated) TTATTAGAGGGTGGGGCGGATCGC GACCCCGAACCGCGACCGTAA 65
p16 (Unmethylated) TTATTAGAGGGTGGGGTGGATTGT CAACCCCAAACCACAACCATAA 65
XAF1 (Methylated) TTTGTAAGAAACGAAATTTAATCGA CCTACCCTTAAAACCCACGAT 57
XAF1 (Unmethylated) TTTGTAAGAAATGAAATTTAATTGA CTCCTACCCTTAAAACCCACAAT 57
N
I
H
-
P
A

A
u
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h
o
r

M
a
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u
s
c
r
i
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t
N
I
H
-
P
A

A
u
t
h
o
r

M
a
n
u
s
c
r
i
p
t
N
I
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-
P
A

A
u
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h
o
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M
a
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t
Table 2
List of primary antibodies, their sources and dilutions
Antigen Source/Cat. # Clone type
*
Dilution
**
BNIP3 Cell Signaling/3769 pAb 2000
Caspase 8 Cell Signaling/9746 mAb 1500
Caspase 9 Cell Signaling/9502 pAb 1500
CDC2 Cell Signaling/9112 pAb 1500
cIAP1 Cell Signaling/4952 pAb 1500
cIAP2 Cell Signaling/7944 pAb 500
Cleaved Caspase 3 Cell Signaling/9661 pAb 1500
c-MYC Cell Signaling/9402 pAb 1500
DNMT1 Santa Cruz Biotech/10222 pAb (goat) 500
DNMT3B Santa Cruz Biotech/10236 pAb (goat) 500
MAPKAPK2 Cell Signaling/3042 pAb 2500
p16 BD Pharmingen/554079 mAb 1000
p21 Upstate Biotech/05-345 mAb 1000
p53 Santa Cruz Biotech/1315 pAb 1000
p38 Cell Signaling/9212 pAb 2500
PARP1 Santa Cruz Biotech/8007 mAb 1000
P-CDC2 (Tyr15) Cell Signaling/9111 pAb 1500
P-MAPKAPK2(Thr334) Cell Signaling/3041 pAb 2500
P-p38(Thr180/Tyr182) Cell Signaling/9211 pAb 2000
P-p53(Ser15) Cell Signaling/9284 pAb 2000
PUMA Cell Signaling/4976 pAb 2000
STAT3 Cell Signaling/9132 pAb 1000
XAF1 Santa Cruz Biotech/19193 pAb (goat) 500
XIAP BD Transduction Lab/610717 mAb 1500
-actin Sigma/A5316 mAb 10000
-H2AX Upstate Biotech/05-636 mAb 1000
*
pAb: polyclonal antibody; used anti-rabbit IgG (or anti-goat as indicated) for secondary antibody fromBio-Rad Lab
mAb: monoclonal antibody; used anti-mouse IgG for secondary antibody fromBio-Rad Lab
**
Fold dilution in PBS with 0.05% Tween 20

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