|
 Previous Article | Table of Contents | Next Article 
Blood, 15 May 2001, Vol. 97, No. 10, pp. 3259-3267
RED CELLS
Short-chain fatty acid derivatives stimulate cell proliferation
and induce STAT-5 activation
Michael S. Boosalis,
Ram Bandyopadhyay,
Emery H. Bresnick,
Betty S. Pace,
Karyn Van DeMark,
Baohua Zhang,
Douglas V. Faller, and
Susan
P. Perrine
From the Departments of Medicine, Pediatrics,
Pharmacology, and Experimental Therapeutics, Cancer Research Center and
Hemoglobinopathy-Thalassemia Research Unit, Boston University School of
Medicine, Boston, MA; the Department of Pharmacology, University of
Wisconsin Medical School, Madison, WI; and the Department of Structural
and Cellular Biology, University of Southern Alabama, Mobile, AL.
 |
Abstract |
Current chemotherapeutic and butyrate therapeutics that induce
fetal hemoglobin expression generally also suppress erythropoiesis, limiting the production of cells containing fetal hemoglobin (F cells).
Recently, selected short-chain fatty acid derivatives (SCFADs) were
identified that induce endogenous  -globin expression in K562 cells
and human burst-forming units-erythroid and that increase
proliferation of human erythroid progenitors and a multilineage interleukin-3-dependent hematopoietic cell line. In this report, -globin inducibility by these SCFADs was further
demonstrated in mice transgenic for the locus control region and the
entire  -globin gene locus in a yeast artificial chromosome and in 2 globin promoter-reporter assays. Conditioned media experiments strongly
suggest that their proliferative activity is a direct effect of the
test compounds. Investigation of potential mechanisms of action of
these SCFADs demonstrates that these compounds induce prolonged
expression of the growth-promoting genes c-myb and
c-myc. Both butyrate and specific growth-stimulatory SCFADs
induced prolonged signal transducer and activator of transcription
(STAT)-5 phosphorylation and activation, and c-cis
expression, persisting for more than 120 minutes, whereas with IL-3
alone phosphorylation disappeared within minutes. In contrast to
butyrate treatment, the growth-stimulating SCFADs did not result in
bulk histone H4 hyperacetylation or induction of
p21Waf/Cip, which mediates the
suppression of cellular growth by butyrate. These findings suggest that
the absence of bulk histone hyperacetylation and p21 induction, but
prolonged induction of cis, myb, myc, and STAT-5
activation, contribute to the cellular proliferation induced by
selected SCFADs.
(Blood. 2001;97:3259-3267)
© 2001 by The American Society of Hematology.
| |
Introduction |
Butyrate analogs have been of interest as potential
therapeutics for the -globin disorders following the demonstration
that butyrate could transcriptionally activate developmentally silenced fetal and embryonic globin genes in many experimental
conditions.1-11 In clinical trials, arginine butyrate
stimulated hemoglobin F (HbF) synthesis to levels above 20% and
induced a nearly 2-fold increase in the amount of
HbF/cell and in proportions of red blood cells expressing HbF
(F reticulocytes) in patients with sickle cell
anemia.10-12 With constant use, however, the activity of
the drug in maintaining substantial increases in HbF-containing
erythrocytes gradually decreased.13 We hypothesized that
this tachyphylaxis was due to the coincident cellular growth-inhibitory
properties of butyrate and developed intermittent or "pulse"
regimens (4 days of use per month) to surmount this
problem.12 Although this regimen proved effective, it also
imposed severe limitations on the amount of drug that can be delivered
to the patient. An orally bioavailable transcriptional inducer of
-globin that does not have cellular growth-inhibitory properties and
could be administered more frequently to stimulate a further increase
in the proportion of HbF-containing cells (F cells) would be
therapeutically advantageous for -globin
disorders.14
Butyrate (at 1-6 mM) inhibits cellular proliferation and entry into S
phase, in a wide variety of cell types,15-22 consistent with a reversible cell cycle arrest at the G1 phase of the
cell cycle.23-27 The precise mechanisms of
butyrate-induced G1 arrest have not been completely
elucidated. Mitogen-stimulated progression through the replicative cell
cycle is mediated by intracellular signal transduction events involving
generation of second messengers, activation of protein kinase cascades,
and gene transcription.28-30 It is probable that
butyrate-induced growth arrest results from perturbation of these
crucial mitogenic signaling events. Indeed, several investigators have
demonstrated effects of butyrate on mitogenic signaling events that are
considered important for normal cell cycle progression. Butyrate blocks
cyclin D1 induction/activation in response to mitogens or
p21Ras, blocks downstream signals generated by
cyclin D1, and induces the cyclin-dependent kinase inhibitor
p21Waf/Cip.31,32 Levels of
p21Waf/Cip are generally low in quiescent cells
but rise in response to mitogenic stimulation or treatment with
butyrate.31
To improve upon arginine butyrate as a therapeutic for induction of the
fetal globin gene (-globin), we designed several short-chain
fatty acid derivatives (SCFADs) and tested them to determine (1) the
types of functional groups that could be substituted for a carbon yet
retain -globin gene-inducing actions and (2) what
substitutions of these types of structures prolong the half-life of the
compound in vivo. To confer resistance to -oxidation and to
-glucuronidation, substitutions with methyl groups, addition of
halogens or methoxy and phenoxy groups, and branched chains, which
result in steric hindrance to cleavage by the enzymes of oxidative
metabolism, have been effective. Three prototype compounds,  -methylhydrocinnamic acid, phenoxyacetic acid, and 2,2 dimethylbutyric acid induce both -globin expression and growth
of erythroid progenitors from normal subjects, hemoglobinopathy
patients, and fetal samples.11,33-35 These
third-generation fatty acid derivatives share some, but not all, of the
biochemical, molecular, and functional properties of the parent
compound, butyrate.11,33-35 For example, these compounds increased fetal globin production but did not inhibit erythroid colony
(burst-forming unit-erythroid, or BFU-E) growth, and certain compounds
even stimulated human BFU-E proliferation beyond that induced by high
concentrations of a panel of hematopoietic growth factors.33 Used in vivo, one compound increases total red
blood cell counts in baboons by 2 g/dL in 2 weeks, despite ongoing
phlebotomy.34-35
To investigate potential mechanisms of action of these compounds,
we analyzed their effects on the 32D cell line, which is dependent upon
interleukin (IL)-3 for growth, undergoes apoptotic cell death in the
absence of IL-3, and survives but does not proliferate when IL-3
concentrations are reduced by 50-fold below the levels required for
maximal proliferation.36-38 No experimental condition or
growth factor has previously been found to abrogate the IL-3 dependency
of this cell line. Under conditions of IL-3 depletion, 32D cells can
also proliferate in response to erythropoietin (EPO) or terminally
differentiate into mature granulocytes in the presence of granulocyte
colony-stimulating factor (G-CSF).36-39 Strikingly, some
of the SCFADs that stimulate -globin expression also supported proliferation of this multilineage cell line and prevented apoptotic cell death when IL-3 was withdrawn.
The molecular basis for the mitogenic or IL-3-sparing activity of
these prototype SCFADs has not yet been established. The growth and
differentiation processes of hematopoietic cells are regulated through
the interaction of several hematopoietic growth factors, including EPO
and IL-3, with their specific cell surface receptors. These cytokine
receptors activate the Janus kinase (JAK) family of tyrosine
kinases.40 The activation of JAKs leads to the recruitment
of a class of latent cytoplasmic transcriptional factors known as
signal transducers and activators of transcription (STATs). After
phosphorylation of the STATs, they dissociate from the receptor, form
heterodimers or homodimers through reciprocal SH2
domain-phosphotyrosine interactions, undergo nuclear translocation, and activate specific genes through binding to specific DNA promoter sequences.40 Both the IL-3 receptor and the EPO receptor
signal through activation of JAK-2 with subsequent activation of
STAT-5.41 STAT-5 activity is normally tightly controlled,
peaking 5 to 30 minutes after stimulation and returning to baseline
within 1 to 2 hours.
We show in this report that certain SCFADs, which induce -globin
expression and stimulate growth of the IL-3-dependent 32D cell
line with kinetics similar to EPOalso induce the expression of the STAT-5-inducible growth-related immediate early genes
c-myc and c-myb and the STAT-5-dependent
transcript c-cis. The growth-stimulatory SCFADs and the
parental compound butyrate augment both the level of STAT-5 activation
and its duration. Unlike butyrate, however, these derivatives have no
obvious histone deacetylase (HDAC)-inhibitory activity and do not
induce p21Waf1/Cip, which
contributes to the cell cycle arrest imposed by butyrate. Thus, the new
compounds share STAT-5 activation and downstream transcript induction
with butyrate but lack its specific growth-inhibitory activities.
| |
Materials and methods |
Reporter gene assays
To determine if the SCFADs act at the level of transcription,
K562 cells stably transfected with a construct containing the 1.4-kilobase (kb) KpnI-BglII fragment of the
human HS2 of the locus control region (LCR) linked to the -globin
promoter and the enhanced green fluorescent protein (EGFP) reporter
gene were used as previously described.42 Because EGFP
messenger RNA (mRNA) has long stability, positive changes average 2- to
4-fold, and weak inducers are not detectable in this
system.43 The cells were cultured in Iscove's modified
Dulbecco's medium, antibiotics, and 2 mM glutamine (Life Technologies,
Rockville, MD) containing 10% fetal calf serum and 100 µg/mL
Hygromycin-B (Sigma, St Louis, MO) as selection for the transfected
construct, and they were treated with 0.5 to 2 mM concentrations of
neutral solutions of the SCFADs for 18 hours. Compounds studied
included butyric acid, -methylhydrocinnamic acid,
3-(3,4-dimethoxyphenyl) propionic acid, 2,2 dimethylbutyrate, and
phenoxyacetic acid (Aldrich Chemical, Milwaukee, WI). GFP fluorescence
was assayed using a Cytofluor 2300 plate reader (Millipore, Bedford,
MA). Each condition was assayed in quadruplicate, and each plate was
analyzed immediately after the addition of the SCFAD to obtain a
baseline (background) measurement. The increase of EGFP fluorescence is
represented in arbitrary fluorescence units.
Selected SCFADs were also tested in a second reporter assay, using
GM979 cells that were stably transfected with a construct containing
the human µLCR linked to the - and -globin gene promoters, each
linked in turn to the Renilla luciferase and the firefly luciferase,
respectively, as previously described.44 GM979 cells were
cultured in RPMI 1640 media (Life Technologies) with 10% fetal calf
serum and G418 (900 µg/mL) (Sigma) alone or with arginine butyrate or
1 of 3 SCFADs described above, at 0.2 to 1 mM concentrations. The 2 luciferases, as reporters for the - and -globin promoters, were
assayed in cell lysates with the Dual Luciferase Reporter Assay System
(Promega, Madison, WI) according to the manufacturer's directions,
using a TD 20/20 Luminometer (Turner Designs, Sunnyvale, CA).
Proliferation of the untreated and the SCFAD-treated GM979 cells was
assessed by enumeration and CellTiter96 (Promega).
Analysis of -globin mRNA in transgenic mice
Transgenic mice containing the LCR and the entire -globin
gene locus in a 240-kb yeast artificial chromosome were used to evaluate -globin inducibility by the SCFADs in vivo.45
The animals were treated either with sodium butyrate (1000 mg/kg/d) continuously administered by a subcutaneous osmotic pump (Alza, Palo
Alto, CA) or with 1 of 3 prototype test compounds, administered as
aqueous solutions in a volume of less than 500 µL, by a single daily
intraperitoneal injection for 5 or 7 days. A derivative of acetic acid,
butyric acid, and propionic acid were each tested in 2 to 3 transgenic
mice. Fifty microliters of blood was collected by retro-orbital
puncture daily for 10 to 13 days, and globin mRNA was assayed by
ribonuclease protection assay, as previously described.45
Control mice received the same volume of normal saline for the same
duration of time. All procedures were performed with approval of the
Institutional Animal Care and Use Committee of the University of
Southern Alabama. Statistical analyses were performed using repeated
measures analysis of variance with the GB STAT software program to
compare the proportions of -globin mRNA obtained in each animal at
baseline to -globin mRNA obtained 3 times sequentially during
treatment with test compounds.
Cell culture conditions
The IL-3-dependent 32D cell line was cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 mg/mL streptomycin, and 2mM glutamine (Life Technologies) in 5%
CO2/95% air at 37°C. These IL-3-dependent cells
required 25 U/mL recombinant murine IL-3 (Biosource International,
Camarillo, CA) for optimal proliferation; a 50-fold lower concentration
(0.5 U/mL) was required to maintain minimal
viability.34,36 Test compounds were dissolved in medium,
neutralized to pH 7.4, and added to the cells at a final concentration
of 1 mM. Cells were withdrawn from 25 U/mL recombinant murine IL-3,
washed, and resuspended in 0.5 U/mL recombinant murine IL-3 (minimal
survival conditions). Recombinant human EPO (3 U/mL) and G-CSF (100 U/mL) (both from Amgen, Thousand Oaks, CA) were also added, and
proliferation was assessed. A cell density of
2.5 × 105/mL to 10 × 105/mL was
maintained by passing the cells into fresh media at 3-day intervals,
with or without the test compounds described above or 2 additional
compounds, 2,2 dimethylmethoxyacetic acid (T. E. Neesby, Fresno,
CA) and DL--amino-n-butyric acid (Sigma), or by
concentrating the cells if apoptosis occurred. Viability was assessed
with trypan blue dye exclusion on days 1, 3, 6, and 9 of culture. The
proportion of viable or apoptotic cells and the fraction of cells in
each phase of the cell cycle were assessed by incubation with propidium
iodide and fluorescence-activated cell sorter
analysis.34
Conditioned media experiments
To evaluate whether effects on cell growth might be mediated
indirectly through autocrine factors produced by the treated cells, 32D
cells were cultured as described above in IL-3 (0.5 U/mL), which
supports minimal survival but not proliferation, and the following
prototype compounds: arginine butyrate, -methylhydrocinnamic acid,
2,2-dimethylbutyrate, 3-(3,4-dimethoxyphenyl) propionic acid, and
phenoxyacetic acid for 48 hours. The cells were pelleted at
500g and resuspended in fresh media with 20 µg/mL
aprotinin without any SCFADs for 24 hours. Media from this cellular
growth were collected, 0.2 µM filtered, and new 32D cells were
cultured in the conditioned media, which were replaced at 3-day
intervals. Proliferation was assessed on days 1, 5, and 8 of culture
using CellTiter96 (Promega). To determine if autocrine
growth-inhibitory proteins were produced by the 32D cells treated with
butyrate, the conditioned media were assayed for tumor necrosis
factor- and interferon- using enzyme-linked immunosorbent assays
(R&D Systems, Minneapolis, MN) according to the
manufacturer's directions.
Northern blot analysis
Expression of several growth-related genes in the presence or
absence of SCFADs was evaluated. Northern blot analysis was performed
on mRNA extracted from 32D cells cultured in the presence or absence of
SCFADs, G-CSF, or EPO under concentrations of low IL-3 (0.5 U/mL), high
IL-3 (25 U/mL), or no IL-3. The methods used for RNA blot analysis have
been described elsewhere.31 Briefly, after exposure to
test compounds for 1 to 11 days, 1 × 107 to
5 × 107 cells were harvested, washed in
phosphate-buffered saline, lysed with RNA denaturing solution (4 M
guanidinium thiocyanate, 25 mM sodium citrate [pH 7], 0.1 M
-mercaptoethanol, and 0.5% N-lauroylsarcosine), and frozen at
 80°C until further processing. RNA was isolated and Northern blots
prepared as previously described.31 DNA probes for
c-myb, c-myc, and -actin were obtained from American Type Culture Collection (Manassas, VA).31 The
c-cis probe was the generous gift of Dr Olivier Hermine, and
the p21 probe was the generous gift of Drs Steven Farmer and
Bert Vogelstein.46 The EcoRI fragment of
c-myb, Pst-1 fragment of c-myc exon-2,
PstI insert of -actin complementary DNA,
HindIII-EcoRI fragment of c-cis, and
EcoRI fragment of p21 clone were radiolabeled
using a random-primer labeling kit (Promega). Globin mRNA
levels were quantitated by densitometric scanning of the autoradiograms
using an LKB-Ultrascan laser densitometer (Pharmacia LKB, Piscataway, NJ).
Western blot analysis of STAT-5 phosphorylation
Cultured in RPMI medium, 32D cells were washed twice and
resuspended at a density of 7.5 × 105/mL with test
compounds at 1 mM overnight. To induce STAT-5, the cells were then
incubated briefly with IL-3 (25 U/mL), and cellular extracts were
prepared at 0, 5, 30, 60, and 120 minutes after IL-3 addition;
1 × 107 cells were lysed with 1 mL ice-cold RIPA buffer
(1% Nonidet P-40 [NP-40], 10% sodium deoxycholate, and 0.1% sodium
dodecyl sulfate [SDS] in phosphate-buffered saline), 100 mM NaF, 2 mM
Na3VO4, 10 µg/mL leupeptin, and 1 mM AEBSF
(ICN Biochemicals, Irvine, CA). Cellular extracts were
immunoprecipitated with 2 µg of a STAT-5 antibody (Santa Cruz
Biotechnology, Santa Cruz, CA) at 4° C for 18 hours. Immune complexes
were recovered by adding 20 µL protein A-agarose beads (Santa Cruz
Biotechnology), and the beads were washed 3 times with 1 × RIPA
buffer and once with 1 × TNE (50 mM Tris-HCl [pH 8.0], 150 mM NaCl,
1 mM Na3VO4, 2 mM ZnCl2, and 1 mM
ethylenediaminetetraacetic acid). Immune complexes were eluted in 40 µL SDS sample buffer by heating at 95°C for 5 minutes, separated on
12% SDS polyacrylamide gels, and transferred to nitrocellulose
membranes (Life Technologies). The membranes were blocked with TBS-T
(20 mM Tris-HCl [pH 7.6], 137 mM NaCl, 0.1% Tween) containing 5%
nonfat dry milk and incubated with an antiphosphotyrosine antibody PY99
(Santa Cruz Biotechnology). Bound antibodies were visualized using the
Luminol reagent (Santa Cruz Biotechnology) according to the
manufacturer's directions. Membranes were incubated at 65°C for 30 minutes with reprobing solution (62.5 mM Tris-HCl [pH 6.8], 0.1 M
-mercaptoethanol, 2% SDS) to strip the antibody and reprobed with
STAT-5 antibody to assess the amount of total STAT-5
protein.31
Analysis of histone H4 acetylation
Acetylation of histone proteins was evaluated in human K562
cells cultured with the test compounds at 2 mM for 18 hours. The nuclei
were isolated in RSB buffer (10 mM Tris-HCl [pH 7.4], 10 mM NaCl, 2 mM CaCl2, 5 mM sodium butyrate) with 0.5% NP-40 on ice for
10 to 15 minutes and washed with RSB without NP-40. For antibody
detection of acetylated histone proteins, isolated nuclei were lysed in
Laemmli's protein loading buffer, and total nuclear proteins were
electrophoresed on an SDS-containing 20% polyacrylamide gel. Resolved
proteins were transferred onto nitrocellulose membrane and subjected to
immunoblotting. Acetylated histone H4 protein was detected as
previously described.31,47,48 An antiacetylated histone H4
with a weak cross-reactivity to acetylated histone H2B rabbit
polyclonal immunoglobulin G (Upstate Biotechnology, Lake Placid,
NY) was used as the primary antibody at 0.75 µg/mL; goat
antirabbit immunoglobulin G (1:1000 dilution) was the
secondary antibody.
| |
Results |
We previously demonstrated that certain SCFADs stimulated
-globin synthesis in human BFU-E above the levels found in untreated cultures from the same subject.33 Furthermore, the same
derivatives increased endogenous -globin mRNA in K562 cells up to
3-fold.34 Although butyrate stimulates -globin
expression at the transcriptional level,49 a
transcriptional mechanism had not previously been studied for the new
SCFADs. A -globin promoter-driven EGFP reporter system was used here
to test for potential transcriptional activation by the
SCFADs.42 EGFP is highly stable, and transcriptional changes in this system average 2- to 4-fold.43 Four
prototype compounds, -methylhydrocinnamic acid, 2,2 dimethylbutyrate, 3-(3,4-dimethoxyphenyl) propionic acid, and
DL--amino-n-butyric acid, all induced EGFP by 2- to 3-fold (Figure
1). Exposure to butyrate for longer than one day consistently decreased EGFP expression, which may result from
its growth-inhibitory and cytostatic effects.
View larger version (62K):
[in this window]
[in a new window]
| Figure 1.
Induction of -globin gene transcription by the
SCFADs.
K562 cells stably transfected with a construct containing HS2 linked to
the -globin gene promoter and the reporter gene EGFP were cultured
with certain SCFADs for 24 hours. The total fluorescence of
2.5 × 105 cells per well was measured in a
cytofluorometric plate reader and is expressed in arbitrary units
including the standard deviation of each condition performed in
quadruplicates. Test compounds were added at 1 mM. C, untreated control
cells; AB, arginine butyrate; 1, -methylhydrocinnamic acid; 2, 2,2 dimethyl butyric acid; 3, 3-(3,4-dimethoxyphenyl) propionic acid; 4, DL-amino-n-butyric acid.
|
|
Both cell proliferation and human -globin expression resulting from
exposure to prototype derivatives were also examined in GM979 cells,
which have extremely low (< 1%) -globin promoter activity
relative to -globin promoter activity.44 Treatment with
butyrate increased -globin by 230% over baseline levels, and
treatment with dimethylbutyrate, -methylhydrocinnamic acid, and
3-(3,4-dimethoxyphenyl) propionate increased -globin promoter activity by 60%, 30%, and 30%, respectively, above control levels. After 2 days of culture, an increase in cell proliferation was demonstrated in GM979 cells treated with -methylhydrocinnamic acid
or dimethylbutyrate of 66% and 50%, respectively, over control growth; cell growth decreased in cells treated with arginine butyrate and 3-(3,4 dimethoxyphenyl) propionic acid by 50% and 20% of control cell number, respectively. These studies were consistent with our
previous findings that 2 of these SCFADs are mitogenic and 1 was
growth-inhibitory, and all induced -globin promoter expression in
this cell line.
To test the activity of the SCFADs in inducing -globin expression in
vivo, human - and -globin mRNA was assayed in transgenic mice
treated with the test compounds. An increase in relative expression of
-globin mRNA from 1.5- to 3.3-fold above baseline was observed in
all 8 animals treated with the SCFADs. The protocol consisted of one
daily treatment of 500 mg/kg/d for 5 to 7 days, and induction was
observed on days 4 to 7 of treatment, with -globin induction
continuing through 10 to 13 days of study, when blood sampling was
discontinued. These responses were similar to responses induced by
treatment with sodium butyrate at 1000 mg/kg/d, which resulted in a
mean 1.9-fold increase in -globin mRNA over baseline, and these
findings were statistically significant (Table
1). These data demonstrate that certain
SCFADs induce expression of the human -globin genes to a similar or
greater degree than does butyrate in vivo. Taken together, these
results from 3 different experimental systems strongly suggest the
-globin induction by the SCFADs is through transcriptional
activation.
After establishing induction of the human -globin gene promoter by
the SCFADs, we next investigated potential effects of the SCFAD on
expression of several growth-related genes, including c-myb
and c-myc. EPO induced these genes in 32D cells in culture (observed only on day 11), and mitogenic concentrations of IL-3 induced
c-myb and c-myc throughout the culture period
(observed on days 1 and 11, Figure 2A).
G-CSF did not support the production of either transcript, with levels
less than in control conditions on day 1. During culture for 11 days
under conditions (0.5 U/mL IL-3) that support only cell survival and
not proliferation, expression of c-myb was reduced to 40%
of control conditions at day 1, and the steady-state transcript levels
of c-myc fell to 25% to 40% of control over the treatment
period (Figure 2A). Culture in the presence of the stimulatory SCFADs
(eg, -methylhydrocinnamic acid, 2,2 dimethylbutyrate, 2,2 dimethylmethoxyacetic acid, phenoxyacetic acid, or
DL--amino-n-butyric acid) induced c-myb levels by 2.5- to
4-fold above control levels within one day and persisted for the
treatment period. C-myc increased 2- to 3-fold
after treatment with the SCFADs to equal that induced by the high
mitogenic concentrations of IL-3 (25 U/mL) and reached relative levels
of up to 3.5-fold in response to phenoxyacetic acid and
-methylhydrocinnamic acid (Figure 2B). Early effects on
c-myc expression (day 1) were observed with 2,2 dimethylbutyrate, phenoxyacetic acid, and -methylhydrocinnamic acid.
In contrast, arginine butyrate had no effect on c-myc or c-myb expression and caused cell death, even in the presence
of 0.5 U IL-3/mL. No viable cells were recovered on day 11 of culture with arginine butyrate.
View larger version (38K):
[in this window]
[in a new window]
| Figure 2.
Relative steady-state levels of
c-myb, c-myc, and -actin transcripts in 32D cells
with hematopoietic growth factors or SCFADs. (A) Cells were
cultured for 11 days in high IL-3 (25 U/mL, proliferating conditions),
low IL-3 (0.5 U/mL, growth-arresting conditions) or no IL-3 (),
death-inducing conditions. SCFADs were tested at 1mM concentrations
with 0.5 U/mL IL-3, growth-arresting conditions. Cells were harvested
for RNA analysis at days 1 and 11. The first lane of each pair
represents RNA from day 1 cultures, the second lane, RNA derived at day
11 (except with arginine butyrate and no IL-3, where RNA was available
only at day 1, with no cells surviving to day 11). Total cellular RNA
was separated by agarose gel electrophoresis, transferred to
nitrocellulose and hybridized with [32P]-labeled probes
specific for c-myc, c-myb, or -actin, as a
control for loading. The autoradiograms are shown here. Experimental
conditions: 25U IL-3, cells cultured in 25 U/mL IL-3; , cultured
without any IL-3; in all the other conditions, cells were cultured in
0.5 U/mL IL-3, which is sufficient for survival, but not proliferation.
The control was 0.5 U IL-3, with 0.5 U/mL IL-3; B, 0.5 U/mL IL-3 plus
1mM arginine butyrate; G-CSF, 0.5 U/mL IL-3 plus 100 U/mL G-CSF; 1, 0.5 U/mL IL-3 plus 1mM methyl hydrocinnamic acid; 2, 0.5 U/mL IL-3 plus
1mM 2,2 dimethyl butyric acid; 3, 0.5 U/mL IL-3 plus 1mM 2-2 dimethylmethoxyacetic acid; 4, 0.5 U/mL IL-3 plus 1mM phenoxyacetic
acid; 5, 0.5 U/mL IL-3 plus 1mM DL--amino-n-butyric acid; EPO, 0.5 U/mL IL-3 plus 3 U/mL erythropoietin. (B) Quantitation of c-myc,
c-myb, and -actin transcript expression levels by densitometric
analysis. The treatment conditions identified by number correspond to
those described in panel A, and a represents mRNA levels from day 1 cultures; b, day 11 cultures; *, no sample available due to death of
the cells by day 11. The data for each transcript are expressed as
relative to the levels of that specific transcript found in RNA from
day 11 cultures under control conditions (low IL-3, 0.5 U/mL), which
were arbitrarily given a value of one. The dashed line in each graph
represents the levels of c-myc, c-myb, or
-actin transcripts in the cells at day 11 under control
conditions. The conditions were the same as in part A, and conditions
3-10 had 0.5U/mL IL-3 in addition to 1mM of the SCFA derivatives or
cytokines added. 1, 25 U/mL IL-3; 2, no IL-3; 3, arginine butyrate; C,
control had 0.5 U/mL IL-3 alone; 4, 100U/mL G-CSF; 5, methylhydrocinnamic acid; 6, 2,2 dimethylbutyric acid; 7, 2,2 dimethylmethoxyacetic acid; 8, phenoxyacetic acid; 9, -amino-n-butyric acid; 10, 3U/mL erythropoietin.
|
|
To determine whether the proliferative effects of the SCFADs on 32D
cells were associated with activation of any of the second messengers
involved in signaling pathways of IL-3, we examined the activation of
STAT-5 in 32D cells treated with arginine butyrate, 3 stimulatory
derivatives, or the nonmitogenic SCFAD 3-(3,4 dimethoxyphenyl) propionic acid. IL-3 induces activation of STAT-5 through tyrosine phosphorylation. Pretreatment of 32D cells with arginine butyrate or
the mitogenic SCFADs resulted in increased and sustained levels of
phosphorylation of the STAT-5 proteins after IL-3 stimulation, as
assessed by immunoblotting using an anti-pTyr antibody. Sixty minutes
after IL-3 stimulation, 32D cells pretreated with the SCFADs showed
significantly higher levels of phosphorylation of STAT-5 than did the
control cells, and this phosphorylation was sustained for 2 hours,
compared with the more transient phosphorylation induced by IL-3 alone
(Figure 3A,B). Treatment with the fourth derivative, 3-(3,4 dimethoxyphenyl) propionic acid, did not result in
cellular proliferation, as occurred with the other SCFADs, and
phosphorylation of STAT-5 was not observed in cells treated with this
compound for the same duration. The lack of both a mitogenic effect and
STAT-5 activation by this growth-inhibitory compound is consistent with
STAT-5 activation being directly related to cell proliferation induced
by the mitogenic SCFADs.
View larger version (46K):
[in this window]
[in a new window]
| Figure 3.
Activation of STAT-5 by treatment with SCFADs.
(A) Immunoblot analysis of total STAT-5 protein and phosphorylated
STAT-5 protein in 32D cells. 32D cells were deprived of IL-3 for 18 hours, in the presence of various SCFADs, and then stimulated with 25 U/mL IL-3. Total cellular protein extracts harvested at 0, 5, 30, 60, and 120 minutes after IL-3 stimulation were treated with anti-Stat5
antibody, and the immunoprecipitated complexes were separated by
SDS-PAGE and transferred to nitrocellulose. Filters were probed first
with an antiphosphotyrosine, developed, stripped, and subsequently
probed with an anti-STAT-5 antibody and developed. The figure is an
autoradiogram of the ECL exposures. Treatments included Control (C);
arginine butyrate at 1mM (AB); phenoxyacetic acid at 1mM (1); methylhydrocinnamic acid at 1mM (2); 2,2 dimethyl butyric acid at 1mM
(3); and 3-(3,4-dimethoxyphenyl) propionic acid at 1mM (4). (B)
Densitometric quantitation of the films of the ECL immunoblots,
expressed as arbitrary units, after subtraction of background and
normalization for total Stat5 protein present in the sample. The dashed
lines in all bar graphs indicate the level of phosphorylated STAT-5
protein remaining in the IL-3-only control at each specific time
point. C, control; AB, arginine butyrate at 1mM; 1, phenoxyacetic acid
at 1mM; 2, -methyl hydrocinnamic acid at 1mM; 3, 2,2 dimethyl
butyric acid at 1mM; and 4, 3-(3,4-dimethoxyphenyl) propionic acid
at 1mM.
|
|
Cis (cytokine-inducible SH2-containing protein) is one of
the STAT-5 target genes in hematopoietic cells. Northern blot analysis demonstrated that treatment with mitogenic (25 U/mL, data not shown) or
0.5 U/mL levels of IL-3 resulted in induction of c-cis mRNA
in 32D cells within 1 hour of exposure; a profound decline in
expression was observed by 3 hours after exposure to the growth factor.
After exposure to either butyrate or 2 growth-stimulatory SCFADs,
c-cis was induced by 2-fold compared with the cells under control culture conditions, and expression persisted beyond 3 hours in
cells treated with butyrate and the stimulatory SCFADs (Figure
4).
View larger version (39K):
[in this window]
[in a new window]
| Figure 4.
Northern blot analysis of
c-cis expression in 32D cells with and without
treatment with the SCFA derivatives. After the addition of 0.5 U/mL IL-3 at time zero, mRNA was prepared from the 32D cells at 0, 1, and 3 hours, as labeled above each lane. In the control lane (IL-3) 0.5 U/mL IL-3 was added at time 0, and in the EPO lane 3 U/mL was added at
time 0 after starvation overnight. AB lanes had a concentration of 1 mM
arginine butyrate; 1, 1 mM methylhydrocinnamic acid; 2, 1 mM 2,2 dimethylbutyric acid; the cells were starved overnight in these
concentrations and at time zero 0.5 U/mL IL-3 was
added.
|
|
Conditioned media experiments demonstrated no inhibitory effects on
proliferation from cells treated with butyrate or stimulatory effects
from conditioned media obtained from cells treated with the SCFADs.
Further investigation of potential autocrine production of
growth-inhibitory proteins in media conditioned from butyrate-treated cells did not detect the presence of 2 common cellular growth inhibitors, tumor necrosis factor-, and interferon-. These
findings strongly suggest that the observed effects on cell growth are directly mediated by the test compounds and butyrate.
Butyrate and other SCFADs are known inhibitors of HDACs, and their
effects on both gene transcription and cell growth are often attributed
to this biochemical activity. To determine if the growth-stimulatory
SCFADs also possess global HDAC inhibitory activity, cells treated with
4 different mitogenic SCFADs were examined for bulk histone H4
acetylation by immunoblot analysis using a specific antiacetylated
histone H4 antibody. Acetylated histone H4 was only detected after
butyrate treatment and was not observed in untreated cells or in cells
treated with the SCFADs (Figure 5). This
represents a major difference from the activity of butyrate, although
it cannot be entirely ruled out whether these compounds have a very
weak inhibitory activity or inhibit a subset of cellular histone
acetylases that do not contribute to bulk histone acetylation. A second
mechanism whereby butyrate has been demonstrated to inhibit cell growth
involves induction of p21Waf/Cip expression. We
therefore compared the activity of butyrate and the mitogenic SCFADs on
p21Waf/Cip expression. Butyrate treatment
strongly induced p21Waf/Cip mRNA in 32D cells
within one day of exposure; the SCFADs had no effect on p21 transcript
levels (Figure 6). A schema of these findings, in relation to established cell cycle regulators, is illustrated in Figure 7.
View larger version (31K):
[in this window]
[in a new window]
| Figure 5.
Histone deacetylase inhibitory activity of butyrate
compared to SCFA derivatives.
Immunoblot analysis of total nuclear proteins from K562 cells separated
on SDS-PAGE, transferred to nitrocellulose, and immunoblotted with an
antibody to acetylated histone H4. Cells were left untreated (C) or
treated for 18 hours with the following test compounds at 1 mM:
arginine butyrate (B); methyl hydrocinnamic acid (1); 2,2 dimethylbutyric acid (2); phenoxyacetic acid (3); or
3-(3,4-dimethoxyphenyl) propionic acid (4).
|
|
View larger version (45K):
[in this window]
[in a new window]
| Figure 6.
Northern blot analysis of p21 expression in 32D cells
with and without treatment with SCFADs.
The mRNA was prepared from cells cultured for 1 day. p21 expression was
induced 3-fold with arginine butyrate (AB) compared to control (C) 0.5 U/mL IL-3 alone. In contrast, the short-chain fatty acid derivatives,
2,2 dimethylbutyric acid (1); -methylhydrocinnamic acid (2); DL-
amino-n-butyric acid (3); phenoxyacetic acid (4); and 2,2 dimethylmethoxyacetic acid (5) were not significantly different from
the control cells treated with 0.5 U/mL IL-3 alone.
|
|
View larger version (40K):
[in this window]
[in a new window]
| Figure 7.
Schema of signaling pathways.
Butyrate induces components of both signaling pathways, growth
inhibitory and growth stimulatory. Treatment of 32D cells with the
selected short-chain fatty acid derivatives results in activation of
the growth-stimulatory signaling events shown.
|
|
| |
Discussion |
Butyric acid and the analog phenylbutyrate inhibit cell growth,
and administration of butyrate as a therapeutic must accordingly be
limited to 4 days per month in sickle cell disease.12 We have demonstrated that selected SCFADs can stimulate hematopoietic cell
growth and abrogate requirements for the multipotential growth factor
IL-3. In this report, we have extended the study of the growth-stimulatory effects of specific SCFADs. We demonstrate here that
these derivatives further stimulate the activity of a chromosomal
-globin promoter in 2 reporter assays and in vivo in an animal model
to a degree similar to that observed with treatment with butyrate. The
compounds also induce the growth-related genes c-myb
and c-myc and a STAT-5 target gene, c-cis.
Although these 2 activities are molecularly distinct, the combination
of a likely transcriptional induction of -globin expression by these
compounds and their stimulatory effects on erythropoiesis should result in an increase in F cells that is severalfold greater than that achievable by arginine butyrate, as similar dosing restrictions should
not be required if these compounds were administered as therapeutics.
The SCFADs do not appear to stimulate mitogenesis though autocrine
mechanisms: Hematopoietic progenitor cells that do not respond to IL-3
or EPO still respond to the SCFADs, and conditioned media experiments
did not yield any evidence of indirect growth stimulation by
SCFAD-conditioned media or growth inhibition by arginine
butyrate-conditioned media. Our finding that the magnitude and
duration of STAT-5 phosphorylation is enhanced by both the growth-stimulatory SCFADs and by butyrate, but not by a nonmitogenic SCFAD, strongly suggests that STAT-5 may be a mediator of the IL-3-sparing effects of these agents. There appears to be some specificity to the action of the growth-stimulatory SCFADs for STAT-5.
There are 6 STAT family members with redundancy in the ability of these
members to activate similar profiles of genes when ectopically
expressed.44,50,51 We have previously found that STAT-2 is
not phosphorylated in response to the SCFADs (or in response
to EPO or IL-3). Although knockout studies indicate that STAT-5
activation may not be required for IL-3 signaling in mice, use of a
dominant-negative STAT-5 (Tyr694Phe, deficient in DNA binding) has
established the need for STAT-5 activation in IL-3 and EPO
signaling in the 32D cells studied here.50-53 Regardless of whether it is required in IL-3 signaling, STAT-5 activation clearly
suffices to induce the proliferation of IL-3 or GM-CSF-dependent primary cells and cell lines.50,54-57
Butyrate or the SCFADs do not increase levels of STAT-5 protein. The
phosphorylation status of the STATs is the only known regulator of
their DNA binding and transcriptional activity.55 Thus,
the augmentation of STAT-5 activity by stimulatory SCFADs could be due
to (1) increases in the activity of STAT-5 kinases, (2) alteration of
phosphorylated STAT-5 half-life,58 or (3) changes in the
activity of the phosphatase that inactivates STAT-5. Regarding the
first potential mechanism, the best-characterized STAT kinases are the
JAKs,40 and no changes were found in JAK activation with
these compounds (unpublished data, 1998). With respect to the
second possible mechanism, butyrate has been reported to inhibit the
26S proteosome and induce accumulation of ubiquitinated proteins,
promoting their degradation.59 However, in preliminary studies no effect of SCFADs was found on general proteosome inhibition, making this mechanism less likely. Regarding the third possible mechanism, the nuclear phosphatase responsible for dephosphorylating and inactivating the STATs is not yet known.60,61 The
growth-stimulatory SCFADs do not structurally resemble
phosphatase-inhibitors, however, and butyrate itself has been reported
to activate, not inhibit, a cellular phosphatase.62
The possibility that the SCFADs might act by "augmenting" a
subthreshold cytokine response can also be considered. For example, insulin-like growth factor-1 has been shown to augment EPO-induced proliferation of an EPO/IL-3-dependent cell line by enhancing phosphorylation of STAT-5.52,53 The mechanism appears to
be via enhancement of EPO activation of STAT-5 through independent activation of a Ras-mediated pathway by insulin-like growth factor-1. The 32D cells studied here require a very low level of IL-3
(submitogenic) for viability (to prevent apoptosis) and for mitogenic
responses to EPO or the SCFADs, and IL-3 in this role may act as a
"survival factor" rather than a growth factor. We did not detect
any growth stimulation effects from media conditioned by the SCFADs. It
is possible, though, that the SCFADs are somehow augmenting this subthreshold IL-3 survival signal into a mitogenic signal.
These studies demonstrated that 2 known target genes of STAT-5,
c-myc and c-myb,55,63 are activated
in cells treated with the stimulatory SCFADs and were not activated by
a derivative that did not stimulate cell proliferation. These genes are
induced by multiple mitogenic stimuli. The c-myb
transcription factor has dual functions in hematopoietic cells in that
it regulates both genes that prevent apoptosis and genes involved in
cellular proliferation.63,64 The ectopic expression of
c-myb can prevent growth arrest of differentiating
hematopoietic cells. Overexpression of c-myb in 32D cells
blocks the ability of these cells to terminally differentiate,
resulting in indefinite growth in the presence of G-CSF.64
Notably, butyrate is as effective at enhancing STAT-5 phosphorylation
and c-myb and c-cis as are the SCFADs, yet
butyrate is clearly not growth-stimulatory. In fact, although butyrate arrests normal cells in G1, it induces apoptosis in
transformed cells within 4 hours after G1-S
arrest.58,65-77 Thus, the activities of butyrate that
cause cell cycle arrest may be problematic when the drug is used in
patients with hemoglobinopathies unless it is given intermittently,
which, in turn, limits the amount of -globin and the proportions of
F cells that can be induced.
Analysis of the mechanistic differences between butyrate and the
growth-stimulatory SCFADs may shed some light on the mechanisms whereby
butyrate arrests cells. Transitions between different phases of the
cell cycle are regulated by the activities of cyclin-dependent kinases
(CDKs). CDK activities are, in turn, dependent upon the expression of
appropriate cyclin proteins.30,78,79 Thus, progression through G1 and into S phase requires the sequential
expression of cyclins D, E, and A and concomitant activation of their
CDK partners. The CDK inhibitor, p21Waf1/Cip, which
negatively regulates cell cycle progression in G1, is induced to high levels in response to multiple antiproliferative stimuli.46,80,81 Butyrate-treated cells express high
levels of p21Waf/Cip transcripts relative to untreated
control cells.31,82 The marked induction of p21 by
butyrate, in contrast to the lack of induction by the growth-promoting
SCFADs, may explain why these compounds activate STAT-5,
myb, and myc, yet only butyrate suppresses growth.
Because of the known activity of butyrate as an HDAC inhibitor, this
activity has been proposed as the mechanisms of p21Waf/Cip
gene induction and the subsequent G1 cell cycle arrest.
Nucleosomal histone acetylation is determined by the opposing actions
of acetyltransferase and deacetylase enzymes.83-86 The
charge neutralization of core histones is thought to induce a chromatin
structure transition that increases accessibility of nucleosomal DNA to
sequence-specific DNA-binding proteins. By inhibiting deacetylases with
butyrate or with trichostatin A,47 acetyltransferases act
unopposed, leading to global histone hyperacetylation. Yet, despite the
"global" changes in acetylation, most cells exhibit only a
reversible growth inhibition and trichostatin A is reported to alter
the expression of only 2% of cellular genes.87
Histone H4 and its acetyl forms are used as indicators of global
histone acetylation. In the absence of butyrate, most H4 is a
nonacetylated and monoacetylated species. After treatment with 0.5 to 5 mM butyrate or the potent inhibitor of HDAC, trichostatin A,
tetra-acetylated H4 is the major isoform among the H4 species. Sodium
or arginine butyrate, phenylbutyrate, and phenylacetate are all strong
inhibitors of HDACs, inducing bulk H4 acetylation, and all show
antiproliferative properties, causing G1 arrest in normal
cells. In contrast to butyrate, none of the growth-promoting SCFADs
tested induced bulk H4 hyperacetylation. Although these compounds might
still theoretically inhibit a subset of HDACs that have gene-specific
effects not detected by measuring bulk histone acetylation, the
findings strongly suggest a correlation between bulk HDAC-inhibitory
activity and cell cycle arrest. This finding also dissociates global
histone hyperacetylation from stimulation of -globin gene
transcription. Until this time, the small molecules known to stimulate
-globin expression have been HDAC inhibitors that strongly induce
bulk histone hyperacetylation or chemotherapeutic agents that suppress
the bone marrow.88
In summary, these findings demonstrate that selected SCFADs that induce
-globin gene expression, growth-related gene expression, and cell
proliferation lack 2 activities of butyrate that are associated with
cell growth arrest. These candidates for HbF-enhancing therapeutics and
related stimulatory SCFADs should not require restricted administration
to avoid inhibiting erythropoiesis and thus have potential to produce
several-fold more F cells in vivo than do butyrate or phenylbutyrate.
These derivatives also have potential as oral therapeutics for
stimulating erythropoiesis in anemias of other etiologies.
| |
Acknowledgments |
We thank Drs Olivier Hermine, Steven Farmer, and Bert Vogelstein
for generously providing probes and Dr George Stamatoyannopoulos for
generously providing GM979 cells.
| |
Footnotes |
Submitted June 29, 2000; accepted January 16, 2001.
Supported by National Institutes of Health grants DK-52962, HL-61208,
and CA-084193.
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.
Reprints: Susan P. Perrine, Boston University School of
Medicine, 715 Albany St K-701, Boston, MA 02118;
sperrine{at}medicine.bu.edu.
| |
References |
1.
Burns LJ, Glauber JG, Ginder GD.
Butyrate induces selective transcriptional activation of a hypomethylated embryonic globin gene in adult erythroid cells.
Blood.
1988;72:1536-1542 |
Top
Abstract
Introduction