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Blood First Edition Paper, prepublished online August 28, 2014; DOI 10.1182/blood-2014-03-564302
Induction of fetal hemoglobin through enhanced translation efficiency of
γ-globin mRNA
Cynthia K. Hahn1 and Christopher H. Lowrey1-3
1
Department of Pharmacology and Toxicology, Geisel School of Medicine at Dartmouth,
Hanover NH
2
Department of Medicine, Dartmouth-Hitchcock Medical Center, Lebanon NH
3
Norris Cotton Cancer Center, Dartmouth-Hitchcock Medical Center, Lebanon NH
Correspondence: Christopher H. Lowrey, MD, Section of Hematology/Oncology, DartmouthHitchcock Medical Center, One Medical Center Dr, Lebanon, NH 03756
E-mail: [email protected]
Running Title: Salubrinal Regulates the Translation of γ-Globin
Scientific review category: Red Cells, Iron, and Erythropoiesis
1
Copyright © 2014 American Society of Hematology
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KEY POINTS:
1. HbF induction by salubrinal is not mediated through changes in globin mRNA stability, mRNA
cellular localization, or HbA levels.
2. Translation efficiency of γ-globin mRNA is increased during stress recovery following
salubrinal enhanced eIF2α phosphorylation.
ABSTRACT
Fetal hemoglobin (HbF) induction can ameliorate the clinical severity of sickle cell
disease and β-thalassemia. We previously reported that activation of the eukaryotic initiation
factor 2α (eIF2α) stress pathway increased HbF through a post-transcriptional mechanism. In
this study, we explored the underlying means by which salubrinal, an activator of eIF2α
signaling, enhances HbF production in primary human erythroid cells. Initial experiments
eliminated changes in globin mRNA stability or cellular location and reduction of adult
hemoglobin (HbA) as possible salubrinal mechanisms. We then determined that salubrinal
selectively increased the number of actively translating ribosomes on γ-globin mRNA. This
enhanced translation efficiency occurred in the recovery phase of the stress response as
phosphorylation of eIF2α and global protein synthesis returned toward baseline. These findings
highlight γ-globin mRNA translation as a novel mechanism for regulating HbF production and as
a pharmacologic target for induction of HbF.
INTRODUCTION
Induction of fetal hemoglobin (HbF) is an effective therapeutic strategy for βhemoglobinopathies.1-5 Most studies have focused on understanding transcriptional regulation
of hemoglobin switching to discover new mechanism-based therapeutic approaches to γ-globin
2
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gene activation.6 However, there is data indicating that HbF may also be post-transcriptionally
regulated.7-10
Recently, we identified the eukaryotic initiation factor 2α (eIF2α) pathway as a critical
post-transcriptional regulator of HbF.11 We found that Increasing eIF2α phosphorylation (peIF2α) increased HbF protein without changing γ/(γ+β) mRNA levels. The most dramatic posttranscriptional HbF induction was elicited by salubrinal (Sal), a pharmacologic enhancer of peIF2α, which increased HbF up to 4.5-fold without altering globin mRNA levels, cellular
differentiation, or total hemoglobin content.
Here, we investigate the post-transcriptional
mechanism of HbF induction when eIF2α is activated by Sal.
METHODS
Cell Culture and Chemicals
K562 cells were maintained in RPMI medium (Cellgro) with 10% FBS and 1% penicillinstreptomycin. CD34+ cells were obtained from the University of Washington or Dr. Patrick
Gallagher (Yale Medical School) using IRB-approved protocols. Cultures were maintained as
described in Sankaran et al.12 Salubrinal (Sal-003; Tocris) and actinomycin-D (Sigma) were
dissolved in DMSO and puromycin (Sigma) was dissolved in PBS.
mRNA and Protein Analyses
RNA isolation, cDNA synthesis, qPCR, hemoglobin HPLC, and western blotting were
performed as previously described (see Supplementary Methods).11 Transcript levels were
calculated by the method of Larionov et al.13 relative to GAPDH expression. PCR primers are
listed in Table S1.
3
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Lentiviral Infection
Vectors and virus were generated as previously described.11 The sequence targeted by
HBB shRNA was 5’-TGGCCCATCACTTTGGCAAAG-3’ and infections were performed on days
8 and 9. GFP was monitored by flow cytometry for transduction efficiency (range 90-95%).
RESULTS AND DISCUSSION
To investigate Sal’s mechanism, we used an erythroid primary cell differentiation
system12 in which Sal was applied at doses previously shown to increase p-eIF2α and reduce
protein translation.11 First, we assessed if Sal changed γ- and β-globin mRNA stability.
Cells
were treated with Sal in combination with actinomycin D, an inhibitor of transcription, and
compared to actinomycin D treatment alone. During a 60-hour timecourse, Sal did not change
the relative half-life of γ- or β-globin mRNA (Figure 1A). Next, we determined if changes in γ- or
β-globin mRNA cellular localization could explain Sal’s ability to increase HbF. Cytoplasmic and
nuclear RNA fractions were compared as cytoplasmic-to-nuclear mRNA ratios. Sal treatment
did not alter the cellular location of γ- or β-globin when compared to the control (Figure 1B),
indicating that changes in mRNA transport were not sufficient to account for the difference in
HbF after Sal treatment.
Previously, we observed that Sal treatment not only increased HbF, but concomitantly
reduced HbA.11 We questioned if reducing HbA was sufficient to increase HbF. Decreased βglobin translation or inhibition of β-globin chain association with α-globin could reduce HbA,
thereby allowing α-globin chains to preferentially complex with γ-globin chains. To test this
hypothesis, we used a short-hairpin RNA targeting β-globin that reduced protein levels by ~50%
(Figure 1C). This was an appropriate level of knockdown since Sal only modestly reduced HbA
in previous experiments. When equal amounts of hemoglobin were analyzed by HPLC, β-globin
knockdown increased %HbF by 2-fold (4% to 8%) and %HbA2 by 5-fold (2% to 10%) and
4
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decreased HbA from 94% to 82% (Figure 1D). However, the absolute hemoglobin content per
cell was reduced by ~50% with β-globin knockdown. When total hemoglobin levels per cell
were calculated, HbF was unchanged (Figure 1E). In contrast, total HbA2 was increased, a
finding consistent with elevated HbA2 levels seen in β-thalassemia intermedia patients.14 These
results confirm that reducing HbA is not sufficient to increase HbF, but does increase HbA2.
We next tested whether Sal affected globin translation. Using polysome profiling, we
confirmed that Sal treatment for 6 hours reduced protein translation, as indicated by a shift in
mRNA from the polysomes to the 80S peak (Figure 2A). This coincided with an increase in peIF2α, which reduces translation initiation15 (Figure 2B). However, quantification of γ- and βglobin mRNA isolated from pooled polysome fractions revealed that Sal did not significantly
change the translation efficiency of either mRNA after 6 hours (Figure 2C).
We then questioned if translation was changed during stress recovery, which has been
reported for other mRNAs.16, 17 After 24 hours of Sal treatment, p-eIF2α recovered to control
levels (Figure 2B) and the polysome profile from Sal treated cells was less shifted to the 80S
fraction (Figure 2D). During this recovery phase, the translation efficiency of γ- and β-globin
was increased as indicated by a significant shift to higher ribosome occupancy (Figure 2E). In
contrast, β-actin translation efficiency remained unchanged and ATF4 mRNA shifted slightly, but
not significantly, to the polysome fractions.
Together, these results suggest that globin
translation efficiency is specifically increased 24 hours after Sal treatment. When the ratio of
heavy-to-light polysomes was compared, Sal increased the γ-globin ratio by 2.9-fold (p=0.019)
but only increased the β-globin ratio by 1.6-fold (p=0.025) (Figure 2F), indicating γ-globin mRNA
was preferentially translated. Since our previous results showed that two Sal treatments over
five days increased HbF up to 4.5-fold,11 smaller changes in translation efficiency would be
enhanced by multiple treatments to account for the observed increase in HbF.
5
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However, the steady-state number of ribosomes on a transcript is not necessarily a
measure of active translation.18 To confirm that changes in globin translation efficiency were
due to the addition of functional ribosomes, we co-treated cells with Sal and puromycin.
Puromycin requires peptidyl-transferase activity to be incorporated into nascent peptides and
causes premature ribosome release, differentiating stalled from active ribosomes.19 At 24 hours
post-Sal treatment, puromycin caused disruption of polyribosomes and increased the 80S peak
in Sal treated and control cells (Figure 2G). Puromycin also produced a shift to lower ribosome
occupancy of γ- and β-globin transcripts (Figure 2H).
Notably, puromycin eliminated the
difference in translation efficiency observed between Sal treated and control cells (Figures 2HI).
These results confirm that Sal enhances globin translation efficiency at 24 hours as
evidenced by a greater number of actively translating ribosomes occupying the transcripts.
To further implicate enhanced protein translation as a mechanism responsible for
increased HbF after Sal treatment, we utilized the K562 cell line. We first confirmed that Sal
increased γ-globin protein levels without changing γ-globin mRNA at 24 hours as seen in
primary cells (Figures S1A-B). Next, we used the non-radioactive SUnSET method to analyze
protein synthesis.20 In this method, low doses of puromycin are incorporated into
neosynthesized peptides20-22 and monitored by flow cytometry to deduce protein translation
rates.
The puromycin fluorescence was not significantly changed after 24 hours of Sal
treatment, confirming similar levels of total protein synthesis (Figure S1C). In control
experiments,
the
fluorescence
of
cells
pre-incubated
with
the
translation
inhibitor,
cycloheximide, was reduced by 70%. We then analyzed cells dual-stained for puromycin and
HbF and found that Sal significantly increased HbF fluorescence by 22% (Figure S1D).
Additionally, immunocytochemistry indicated a similar subcellular localization of puromycin and
HbF (Figure S1E), suggesting that nascent γ-globin translation enhances HbF production by
Sal.
6
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In summary, we have determined that post-transcriptional induction of HbF by Sal is the
result of enhanced γ-globin mRNA translation that occurs during the recovery phase of the peIF2a stress response. This provides an explanation for previous observations where HbF
induction exceeded increases in γ-globin mRNA.9, 10 Our results suggest that control of γ-globin
mRNA translation is an important mechanism for regulating HbF production that could be used
to benefit β-hemoglobinopathy patients.
ACKNOWLEDGEMENTS
The authors thank Dr. Patrick Gallagher for kindly supplying CD34+ cells; Dr. Lionel
Lewis and Bernie Beaulieu for assistance with Hb HPLC; Dr. Elizabeth McCoy and Dr. Alexei
Kisselev for guidance regarding polysome profiling; and Dr. Edwin Hahn, Dr. Rodwell Mabaera,
Dr. Rachel West, and Emily Schaeffer for valuable discussions.
This work was supported by the National Institutes of Health (NHLBI grant HL73442 to
C.H.L. and NIDDK grant F30DK094540 to C.K.H.) and by the Knights of the York Cross of
Honour and the Royal Order of Scotland, Masonic charitable organizations.
AUTHORSHIP
Contribution: C.K.H. designed and performed the research, analyzed and interpreted the
data, and wrote the manuscript; and C.H.L. designed and oversaw the research, analyzed and
interpreted the data, and wrote the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
REFERENCES
1.
Platt OS, Brambilla DJ, Rosse WF, et al. Mortality in sickle cell disease. Life expectancy
and risk factors for early death. N Engl J Med. Jun 9 1994;330(23):1639-1644.
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2.
Nuinoon M, Makarasara W, Mushiroda T, et al. A genome-wide association identified the
common genetic variants influence disease severity in β0-thalassemia/hemoglobin E.
Hum Genet. Mar 19 2010;127(3):303-314.
3.
Platt OS, Orkin SH, Dover G, Beardsley GP, Miller B, Nathan DG. Hydroxyurea
enhances fetal hemoglobin production in sickle cell anemia. J Clin Invest. Aug 1
1984;74(2):652-656.
4.
Ley TJ, DeSimone J, Noguchi CT, et al. 5-Azacytidine increases gamma-globin
synthesis and reduces the proportion of dense cells in patients with sickle cell anemia.
Blood. Aug 1 1983;62(2):370-380.
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Lowrey CH, Nienhuis AW. Brief report: treatment with azacitidine of patients with endstage beta-thalassemia. N Engl J Med. Sep 16 1993;329(12):845-848.
6.
Sankaran VG, Orkin SH. The switch from fetal to adult hemoglobin. Cold Spring Harb
Perspect Med. Jan 1 2013;3(1):a011643.
7.
Russell JE. A post-transcriptional process contributes to efficient γ-globin gene silencing
in definitive erythroid cells. Eur J Haematol. Dec 1 2007;79(6):516-525.
8.
Chakalova L, Osborne CS, Dai Y, et al. The Corfu deltabeta thalassemia deletion
disrupts gamma-globin gene silencing and reveals post-transcriptional regulation of HbF
expression. Blood. Mar 1 2005;105(5):2154-2160.
9.
Weinberg RS, Ji X, Sutton M, et al. Butyrate increases the efficiency of translation of
gamma-globin mRNA. Blood. Feb 15 2005;105(4):1807-1809.
10.
Mabaera R, Greene MR, Richardson CA, Conine SJ, Kozul CD, Lowrey CH. Neither
DNA hypomethylation nor changes in the kinetics of erythroid differentiation explain 5azacytidine's ability to induce human fetal hemoglobin. Blood. Jan 1 2008;111(1):411420.
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11.
Hahn CK, Lowrey CH. Eukaryotic initiation factor 2alpha phosphorylation mediates fetal
hemoglobin induction through a post-transcriptional mechanism. Blood. Jul 25
2013;122(4):477-485.
12.
Sankaran VG, Menne TF, Xu J, et al. Human fetal hemoglobin expression is regulated
by
the
developmental
stage-specific
repressor
BCL11A.
Science.
Dec
19
2008;322(5909):1839-1842.
13.
Larionov A, Krause A, Miller W. A standard curve based method for relative real time
PCR data processing. BMC Bioinformatics. Jan 1 2005;6:62.
14.
Higgs DR, Engel JD, Stamatoyannopoulos G. Thalassaemia. Lancet. Sep 9 2011.
15.
Harding HP, Novoa I, Zhang Y, et al. Regulated translation initiation controls stressinduced gene expression in mammalian cells. Molecular Cell. Nov 1 2000;6(5):10991108.
16.
Ma S, Bhattacharjee RB, Bag J. Expression of poly(A)-binding protein is upregulated
during recovery from heat shock in HeLa cells. FEBS Journal. Dec 12 2008;276(2):552570.
17.
Preston AM, Hendershot LM. Examination of a second node of translational control in
the unfolded protein response. Journal of Cell Science. Sep 15 2013;126(18):42534261.
18.
Darnell Jennifer C, Van Driesche Sarah J, Zhang C, et al. FMRP Stalls Ribosomal
Translocation on mRNAs Linked to Synaptic Function and Autism. Cell. Jul 1
2011;146(2):247-261.
19.
Azzam ME, Algranati ID. Mechanism of puromycin action: fate of ribosomes after
release of nascent protein chains from polysomes. Proc Natl Acad Sci USA. Dec 1
1973;70(12):3866-3869.
20.
Schmidt EK, Clavarino G, Ceppi M, Pierre P. SUnSET, a nonradioactive method to
monitor protein synthesis. Nat Meth. Apr 22 2009;6(4):275-277.
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21.
Starck SR, Green HM, Alberola-Ila J, Roberts RW. A general approach to detect protein
expression in vivo using fluorescent puromycin conjugates. Chem Biol. Jul 1
2004;11(7):999-1008.
22.
Aviner R, Geiger T, Elroy-Stein O. Genome-wide identification and quantification of
protein synthesis in cultured cells and whole tissues by puromycin-associated nascent
chain proteomics (PUNCH-P). Nat Protoc. Mar 6 2014;9(4):751-760.
FIGURE LEGENDS
Figure 1. Salubrinal does not change mRNA stability, mRNA cellular localization, or total
HbA to induce HbF.
(A) Neither γ-globin nor β-globin relative mRNA half-life is changed in cells treated with Sal in
combination with 5 μg/ml actinomycin D versus actinomycin D alone. Expression is reported as
fold change relative to the untreated control (Untx) on day 15 prior to treatment. Actinomycin D
and 6 μM Sal were applied on day 15 simultaneously. mRNA expression was quantified using
primers located at either the 5’- or 3’-end of each mRNA. Error bars represent ± SEM of three
independent experiments.
(B) Sal does not change the cytoplasmic to nuclear ratio of γ- or β-globin mRNA compared to
the untreated control. Cells were treated with 6 μM Sal on days 15 and 18. Cytoplasmic and
nuclear RNA was isolated on days 15, 18, and 20, mRNA expression for γ- and β-globin was
quantified in each compartment using primers that spanned at least one exon-exon junction,
and the cytoplasmic to nuclear ratio was compared. Error bars represent ± SEM of four
independent experiments.
10
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(C) Western blotting shows shHBB causes 50% knockdown of β-globin protein compared to
shCTRL. Protein lysates were taken on days 13, 15, and 17 of differentiation after infections on
days 8 and 9. GAPDH was used as a loading control.
(D) HPLC was performed on day 20 of culture to assess the proportions of HbF, HbA, and HbA2
in shCTRL and shHBB infected cells. These HPLC traces from a representative experiment
reveal that shHBB enhances the %HbF and %HbA2 compared to shCTRL.
(E) β-globin knockdown does not increase total HbF, but enhances total HbA2, After HPLC
analysis, the area under the curve for HbF, HbA, and HbA2 was corrected for the total
hemoglobin concentration in 2x106 cells. Total amounts of HbF, HbA, and HbA2 are depicted as
fold change relative to the shCTRL. Error bars represent ± SEM of three independent
experiments. P values were determined using an unpaired two-tailed t-test. NS = not significant;
* P < 0.05; ** P < 0.001.
Figure 2. Salubrinal selectively increases the translation efficiency of γ-globin mRNA.
(A) Representative polysome profile on day 15 after 6 hours of 12 μM Sal treatment reveals a
polysome to monosome shift, indicative of halted translation initiation and reduced translation.
(B) Western blotting shows that p-eIF2α is increased at 3 and 6 hours after 12 μM Sal treatment
on day 15 relative to an untreated control. At 24 hours post-treatment, p-eIF2α levels are similar
between the two conditions. Total eIF2α and β-Actin are used as loading controls.
(C) After 6 hours of treatment with 12 μM Sal, γ- and β-globin translation efficiency is unchanged
relative to the untreated control. Polysome profiling fractions were pooled into 7 larger fractions
11
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containing ribosome free, 40S/60S, 80S, or polysome bound RNA ranging from lower to higher
ribosome occupancy. γ- and β-globin mRNA was quantified in each pooled fraction and
normalized to an exogenous luciferase mRNA control. The percent of total mRNA for each
fraction was calculated. Error bars represent ± SEM of three independent experiments.
(D) After 24 hours of 12 μM Sal treatment, the representative polysome profile on day 16 shows
a slight polysome to monosome shift.
(E) 12 μM Sal significantly increases the translation efficiency of γ- and β-globin mRNA after 24
hours, whereas β-actin and ATF4 translation is not significantly changed. mRNA was quantified
in each pooled fraction, normalized to an exogenous luciferase control, and the percent of total
mRNA found in each fraction was calculated. Error bars represent ± SEM of three independent
experiments. P values were determined using an unpaired two-tailed t-test. * P < 0.05; ** P <
0.01.
(F) At 24 hours, Sal increases translation efficiency of γ-globin to a greater extent than β-globin.
Percentage of mRNA in the lightest two polysome fractions was compared to the percentage of
mRNA in the heaviest two polysome fractions using a heavy-to-light polysome ratio. Error bars
represent ± SEM of three independent experiments. P values were determined using an
unpaired two-tailed t-test. * P < 0.05.
(G) The representative polysome profile at 24 hours shows ribosome dissociation and a
dramatic shift to the monosome peak when 12 μM Sal and the untreated control are incubated
with 1 mM puromycin.
12
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(H) Puromycin treatment after 24 hours of 12 μM Sal shifts the percentage of γ- and β-globin
mRNA to lighter polysome fractions to the same extent as in the untreated control. Error bars
represent ± SEM of two independent experiments.
(I) Puromycin reduces the heavy-to-light polysome ratio to the same extent in 12 μM Sal treated
and untreated cells. Error bars represent ± SEM of two independent experiments.
13
Figure 1
A
50
0
12
24
36
48
D
50
0
60
0
12
24
100
24
36
48
0
60
0
12
24
60
60
Cyto/Nuc Ratio
-globin mRNA
Cyto/Nuc Ratio
-globin mRNA
80
40
20
16
48
60
18
HbF
40
D13
+
-
-
+
+
16
-
-
+
D17
+
-
HbA2
HbF
HbA
HbA2
shHBB
20
18
Day
D15
HbA
shCTRL
0
20
Untx
Sal 6 μM
Day
C
36
Hours
-
+
20
E 1.5
shHBB
β-globin
GAPDH
1.5
NS
3
*
**
shCTRL
1.0
0.5
0.0
shCTRL
shHBB
1.0
Total HbA2
12
Total HbA
0
80
0
60
50
Hours
B
48
100
50
0
36
Hours
% β-globin mRNA
3’-biased primers
% γ-globin mRNA
3’-biased primers
Hours
Total HbF
0
Untx + Act D
Sal 6 μM + Act D
100
% β-globin mRNA
5’-biased primers
% γ-globin mRNA
5’-biased primers
100
0.5
0.0
shCTRL
shHBB
2
1
0
shCTRL
shHBB
Figure 2
B
40S 60S 80S
3 hr
Polysomes
-
120
6 hr
-
+
+
-
+
24 hr
-
-
+
-
+
Untx
+
Sal 12 μM
p-eIF2α
6hUntx
eIF2α
β-Actin
40
20
20
160
24h Untx
Free
Light
Heavy
Polysomes Polysomes
Untx
Sal 12 μM
60
*
*
40
20
40
**
0
40
60
30
Free
80
Light
Heavy
Polysomes Polysomes
% ATF4 mRNA
24 hours
80S
80
Free
0
40S/60S
120
% β-Actin mRNA
24 hours
*
20
*
24h Sal 12 μM
40
Light
Heavy
Polysomes Polysomes
60
80
% β-globin mRNA
24 hours
Polysomes
% γ-globin mRNA
24 hours
40S 60S 80S
80S
0
Free
E
D
Absorbance 254nm
40
0
0
200
Untx
Sal 12 μM
60
40S/60S
40
60
% β-globin mRNA
6 hours
% γ-globin mRNA
6 hours
80
40
20
40S/60S
C
80
40S/60S
Absorbance 254nm
6h Sal 12 μM
80S
160
80S
A
Light
Heavy
Polysomes Polysomes
20
10
0
150
24h Untx + puro
24h Sal 12 μM + puro
*
1.0
0.5
-globin
80S
Free
Untx
Sal 12 μM
1.5
0.0
H
0.15
Untx
Sal 12 μM
0.05
0.00
-globin
-globin
% β-globin mRNA
24 hours
Untx + Puro
40
20
Sal 12 μM + Puro
40
20
80S
Free
80S
Light
Heavy
Polysomes Polysomes
40S/60S
0
0
Free
0
-globin
60
60
40S/60S
40
Light
Heavy
Polysomes Polysomes
0.10
100
% γ-globin mRNA
24 hours
Absorbance 254nm
200
*
I
Heavy/Light
Polysome Ratio
Polysomes
Heavy/Light
Polysome Ratio
40S 60S 80S
80S
Free
2.0
250
40S/60S
F
G
Light
Heavy
Polysomes Polysomes
40S/60S
0
0
Light
Heavy
Polysomes Polysomes
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Prepublished online August 28, 2014;
doi:10.1182/blood-2014-03-564302
Induction of fetal hemoglobin through enhanced translation efficiency of γ
-globin mRNA
Cynthia K. Hahn and Christopher H. Lowrey
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