From the Department of Chemistry, Columbia
University, New York, New York 10027, the § Program of Cell
Biology, Memorial Sloan-Kettering Cancer Center, New York, New York
10021, the ¶ Max Delbrück Center of Molecular Medicine,
13122 Berlin-Buch, Germany, and the
Program of Molecular
Biology, Memorial Sloan-Kettering Cancer Center,
New York, New York 10021
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ABSTRACT |
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The ability of a novel class of hybrid polar
compounds (HPCs) to induce differentiation and consequent cessation of
proliferation of transformed cells has led to their development as
potential chemotherapeutic agents in the treatment of cancer.
Suberoylanilide hydroxamic acid (SAHA) is a prototype of a family of
hydroxamic acid based compounds (SAHA-like HPCs) that can, at
micromolar concentrations, induce a variety of transformed cell lines
to differentiate. The mechanism of action of the HPCs is not entirely understood. Searching for a cellular target of the SAHA-like HPCs, we
synthesized a photoaffinity labeling reagent structurally based on
SAHA, and probed for SAHA-binding proteins in murine erythroleukemia (MEL) cells. Photoaffinity labeling in cell free extracts identified a
32-kDa protein (p32) that was specifically labeled by the photoaffinity reagent. Cell fractionation assays localized p32 to the P100 fraction. p32 was partially purified and identified by mass spectrometry as the
40 S ribosomal protein S3. Expression of epitope-tagged S3 in bacterial
lysates followed by photoaffinity labeling confirmed its specific
labeling. Identification of a cytodifferentiation agent target may shed
light on the mechanism by which the SAHA-like HPCs exert their
antitumor effects.
Hybrid polar compounds have been previously identified as potent
inducers of differentiation in murine erythroleukemia
(MEL)1 cells and in a wide
variety of other transformed cells (1, 2). Hexamethylene bisacetamide
(HMBA; Table I) is the prototype HPC, able to induce differentiation
and, in some instances apoptosis, in culture at low millimolar
concentrations (3-6). Recently we reported on a class of hydroxamic
acid based HPCs of which suberoylanilide hydroxamic acid (SAHA; Table
I) is the prototype. These SAHA-like HPCs induce a variety of
transformed cell lines to differentiate or undergo apoptosis at low
micromolar concentrations (3, 7). Thus these agents have been
recognized as potentially useful in the treatment of cancer, and are
currently being studied at the National Cancer Institute.
In MEL cells, a model system we have used to study these agents, SAHA
exerts its effects on the cell cycle. Shortly after exposure to SAHA, a
transient prolongation of the initial G1 phase of the cell
cycle is observed, and modulation in expression of a number of proteins
involved in regulating cell cycle progression occurs (3). Over the next
48 h of culture with SAHA, there is progressive recruitment of
most of the cells to differentiate, accompanied by accumulation of both
total and underphosphorylated retinoblastoma gene product (pRB), the
onset of globin synthesis, and permanent G1 arrest.
Extensive structure-activity studies have shown that to achieve high
potency, SAHA-like HPCs must meet strict structural requirements. The
hydroxamic acid is crucial for achieving micromolar activity (SAHA;
Table I). The optimal spacer length spanning the two polar sites is six
methylenes, and only certain substitutions are tolerated on the benzene
ring. These structural requirements suggest a specific binding
interaction with a cellular target, rather than a nonspecific interaction.
Photoaffinity labeling is widely used for investigation of
receptor-ligand interactions (11). We probed for the cellular target(s)
of SAHA-like HPCs by designing a photoaffinity labeling reagent
([3H]498; Table I)
structurally based on SAHA. Photoaffinity labeling in cell free
extracts has identified a 32-kDa protein (p32), which was subsequently
identified by mass spectrometry as the 40 S ribosomal protein S3. We
show that S3 can be specifically labeled in bacterial lysates. Our
results are consistent with S3 being a target of the SAHA-like HPCs.
Synthesis (Fig. 1)
Suberic Acid Monomethyl Ester (4-Amino-3,5-diiodoaniline) Amide,
4--
Carbonyl diimidazole (1.12 mmol) was added at 0 °C to
a 50-ml round-bottomed flask containing 15 ml of dry THF and 1.11 mmol of suberic acid monomethyl ester. After stirring at 0 °C for 30 min,
then at room temperature for 2 h, 1.13 mmol of
2,6-diiodo-4-aminoaniline 3 in 20 ml of THF were added
dropwise through an addition funnel and the reaction was stirred at
room temperature overnight. Then the THF was evaporated in
vacuo and the residue was partitioned between
CHCl3/H2O. The organic layer was evaporated,
and the product was purified by silica gel chromatography (5%
MeOH/CHCl3), to yield 0.337 g (57%) of 4.
1H NMR: Suberic Acid (4-Amino-3,5-diiodoaniline) Amide, 5--
A
solution of 0.62 mmol of 4 and 3.17 mmol of
LiOH·H2O in 30 ml of 3:1 MeOH/H2O was heated
to 60 °C, until TLC showed no presence of the starting material. The
MeOH was removed in vacuo, and the aqueous residue was
diluted, filtered, and acidified to pH 5 with 1 M HCl. The
resulting precipitate was filtered and recrystallized from MeOH to
afford 0.2655 g (82.7%) of 5. 1H NMR: Suberic (O-TBDPS) Hydroxamic Acid (4-Amino-3,5-diiodoaniline)
Amide, 6--
A solution of 0.616 mmol of carbonyl diimidazole
in 10 ml of THF was added to a solution of 0.385 mmol of 5 in 20 ml of THF, and the reaction was stirred overnight at room
temperature. Then, 0.914 mmol of H2NO-TBDPS were added, and
the reaction was stirred at room temperature overnight. The THF was
removed, and the reaction mixture was purified over silica gel (10%
MeOH/CHCl3), then by preparative TLC, using the same
solvent mixture. The product was washed with ether to afford 0.202 g
(53%) of 6. 1H NMR: Suberic Hydroxamic Acid (4-Amino-3,5-diiodoaniline) Amide,
7--
A solution of 77 mg of 6 in 15 ml of THF was
chilled to 0 °C, and 0.2 ml of a 25% solution of tetrabutyl
ammonium fluoride in THF were injected. The solution was stirred for
5 h, the first 10 min at 0 °C, then at room temperature. Then 6 ml of H2O/EtOAc (1:1) were added, and the THF was removed
in vacuo. The solid in the organic layer was filtered,
washed with ether and dried. This afforded 49.5 mg (97%) of
7. 1H NMR: Suberic Hydroxamic Acid (4-Amino-3,5-ditritioaniline)
Amide, 8--
The following was performed by the NEN Life
Science Products custom tritium labeling service. A suspension of 4 mg
of Pd-C (10% Pd) in 3 ml of methanol was mixed with 10 mg of
Na2CO3 in H2O, cooled down over an
ice/salt bath, and flushed with 3H2 gas (1 atm). Then, a prechilled 10-mg solution of 7 in MeOH was
injected. The tritiation was carried out for 2-3 h. Then, the catalyst
was filtered and the solvent was removed by distillation. The product
(0.0188 mmol) had a specific activity of 40.42 Ci/mmol. 300 mCi were
dissolved in MeOH (0.00742 mmol; ~2 mg) and used in the subsequent
step without further purification.
Suberic Hydroxamic Acid (4-Aminoaniline) Amide,
8a--
Compound 8a was synthesized by catalytic
hydrogenation of 7, using the above procedure.
Suberic Hydroxamic Acid (4-Azido-3,5-Ditritioaniline) Amide,
[3H]498--
A solution of 18.8 mg of
8a in 7 ml of MeOH was cooled to 0 °C over an ice bath. A
chilled solution of 0.00742 mmol of radioactive 8 (300 mCi)
in 4 ml of MeOH was added, followed by a solution of 0.1 ml of HCl in 1 ml of H2O. Then a solution of 0.14 mmol of
NaNO2 in 0.5 ml of H2O was added to the
reaction mixture over a 5-min period, and the reaction was stirred for 45 min at 0 °C, until a positive result to I2/starch
paper was obtained. Sulfamic acid (0.093 mmol) was added, and the
reaction was stirred for 15 min. A solution of 0.145 mmol of
NaN3 in 0.2 ml of H2O was injected, and the
reaction was allowed to warm to room temperature. After 15 min, 2 ml of
EtOAc were added and the reaction mixture was concentrated by rotary
evaporation. The residue was partitioned between 10 ml of 1:1
EtOAc/H2O, and the EtOAc layer was evaporated to dryness,
to afford 11.6 mg (50%) of [3H]498
(specific activity 4 Ci/mmol).
Suberic Hydroxamic Acid (4-Azidoaniline) Amide,
498--
Compound 498 was synthesized from
8a using the above procedure for the synthesis of
[3H]498, without the addition of
8.
Cell Culture
MEL DS19/Sc9 cells, derived from 745A cells, were maintained in
minimal essential medium supplemented with 10% (v/v) fetal calf serum,
penicillin, and streptomycin and incubated at 37 °C in 95% air, 5%
CO2 atmosphere. Cultures were initiated at a cell density
of 1 × 105 cells/ml, and all experiments were
performed with cells in logarithmic growth phase. Induction of
differentiation, cell density, and benzidine reactivity were determined
as described (12).
Preparation of Whole Cell Extracts
An aliquot of 1.6 × 107 MEL cells was washed
twice with phosphate-buffered saline and lysed with 1 ml of lysis
buffer (250 mM NaCl, 50 mM Hepes/KOH, pH 7.0, 0.1% Nonidet P-40, 50 mM NaF, 5 mM EDTA, 0.1 mM Na3VO4, 50 µg/ml PMSF, 1 µg/ml leupeptin, 1 µg/ml aprotinin) at 0 °C for 30 min. The
lysed cells were centrifuged at 14,000 rpm (Beckman 5402 microcentrifuge) for 15 min. The supernatant was stored at Cell Fractionation by Differential Centrifugation
Cell fractionation was performed according to the method
previously described by van't Hof et al. (13) with
modifications. Briefly, 2 × 108 MEL cells were washed
twice with phosphate-buffered saline and suspended in 1 ml of hypotonic
buffer (10 mM Tris-HCl, pH 7.4, 0.2 mM
MgCl2) at 0 °C for 15 min, then Dounce homogenized (1 stroke). After the addition of 200 µl of 1.25 M sucrose
and 2 µl of 0.5 M EDTA, the mixture was centrifuged at
1,000 × g for 10 min at 4 °C. The supernatant (S1)
was removed. The pellet (P1) was resuspended in 1 ml of a mixture of
0.25 M sucrose, 10 mM Tris-HCl, pH 7.4, 1 mM EDTA and centrifuged at 1,000 × g for
10 min at 4 °C. The combined S1 supernatants were centrifuged at
10,000 × g for 10 min at 4 °C (SS-34 rotor). The
supernatant (S10) was centrifuged for 45 min at 100,000 × g in a Beckman 70.1 Ti. The pellets (P10 and P100) were
suspended in 100 µl of 10 mM Tris-HCl, pH 8.0. The
supernatant (S100) was used as the cytosolic extracts.
Photoaffinity Labeling in Cell-free Extracts
Cell preparations were incubated in 10 mM Tris-HCl,
pH 8.0, with or without nonradioactive 498 for 1 h at
4 °C. [3H]498 was added to the
final indicated concentration with the final protein concentration at 2 mg/ml. The samples were incubated for an additional 2.5 h,
followed by 20 min of UV irradiation at 4 °C (Rayonet photochemical
reactor lamp, Southern New England Ultraviolet Co., maximum light
intensity at 253.7 nm). The UV irradiation experiments were carried out
by maintaining the distance of 5 cm between the sample and light
source. After irradiation, the samples were subjected to protein
purification or solubilized in SDS sample buffer and subjected to
SDS-PAGE. Gels were normalized for protein unless otherwise stated.
Gels were enhanced (EN3HANCE, NEN Life Science Products),
dried, and mounted on x-ray film for fluorography.
Detergent Extraction
For Triton X-100 extraction, P100 fractions were incubated for
30 min at 4 °C with Csk buffer (10 mM PIPES, pH 6.8, 100 mM KCl, 2.5 mM MgCl2, 1 mM CaCl2, 0.3 M sucrose, 1% Triton
X-100, 1 mM Na3VO4, 10 µg/ml
aprotinin, 10 µg/ml leupeptin, 1 mM PMSF) at a final
protein concentration of 5 mg/ml. For Zwittergent 3-16 extractions,
Triton X-100-resistant fractions (PTx) were incubated for
30 min at 4 °C with extraction buffer (20 mM Tris-HCl,
pH 8.0, 0.1% Zwittergent 3-16, 150 mM NaCl, 5 mM NaF, 1 mM EDTA, 1 mM
Na3VO4, 10 µg/ml aprotinin, 10 µg/ml
leupeptin, 1 mM PMSF) at a final protein concentration of
0.5-1 mg/ml. The samples were centrifuged at 100,000 × g in a Beckman TL-100 desktop ultracentrifuge (TLA 100.2 rotor) for 1 h at 4 °C, and the supernatants were removed. For
Triton X-100 extraction, PTx fractions were resuspended in 10 mM Tris-HCl, pH 8.0, for further analysis, or
solubilized in SDS sample buffer. For Zwittergent 3-16 extraction, the
supernatants (STx+zw) were precipitated with 10%
trichloroacetic acid, washed twice with ice-cold ether, and solubilized
in SDS sample buffer. The samples were subjected to SDS-PAGE as
described above. For mass spectrometric analysis, after
electrophoresis, proteins were transferred to a nitrocellulose membrane
and stained with Amido Black.
Protein Digestion
The 32-kDa band was excised from the nitrocellulose blot and
processed for internal sequence analysis as described (14, 15).
Briefly, in situ digestion was done using 0.1 µg of
trypsin (modified sequencing grade; Promega, Madison, WI) in 10 µl of 100 mM NH4HCO3 (supplemented with
0.5% Zwittergent 3-16) for 2 h at 37 °C. The resulting peptide
mixture was then loaded onto a 2-µl bed volume of Poros 50 R2
(PerSeptive, Framingham, MA) reversed-phase beads (sized to be between
40 and 60 µm, and slurry packed into an Eppendorf gel-loading tip),
washed with 20 µl of 5% MeCN, 0.1% formic acid, and stepwise eluted
in 4 µl of 16% (and then with 4 µl of 30%) MeCN, 0.1% formic
acid; the two resulting fractions are designated "16% pool" and
"30% pool."
Mass Spectrometry
Each peptide pool was analyzed twice by matrix-assisted
laser-desorption/ionization (MALDI) time-of-flight (TOF) mass
spectrometry (MS), in the presence and absence of peptide calibrants
(15). Aliquots (0.5 µl) were deposited on the probe surface, mixed
with Electrospray ionization (ESI) MS was done on an API 300 triple
quadrupole instrument (PE-SCIEX; Thornhill, Canada), modified with an
injection adaptable fine
ionization source (JaFIS) as described (16).
Needle voltage ranged from 600 to 1350 V depending on the application.
The voltages for the orifice and the curtain plate were set at 5 and
350, respectively. Q1 scans were collected using a 0.5-atomic mass unit
step size, and a 3-ms dwell time over a mass range from 400 to 1400 atomic mass units; scans were averaged for statistical analysis, and Q1
resolution was set such that the charge state of singly, doubly, and
triply charged ions could be ascertained. For operation in the MS/MS mode, Q1 was set to transmit the complete isotopic envelope of the
parent. All spectra were averaged with a 0.5-Da step size and a 3-ms
dwell time for 5 min over the mass range of the singly charged
m/z. Q3 resolution was set such that the charge state of the
fragment ions could be distinguished. Collision energies, as well as
collision assisted dissociation gas pressures, were optimized
individually for each peptide as to obtain the best MS/MS spectra.
Selected, "major" mass values (combined from the 16% and 30%
peptide pools, but restricted to 900 atomic mass units Construction of FLAG-tagged Ribosomal Proteins S3 in Bacteria
The mouse ribosomal protein S3 cDNA was obtained by reverse
transcriptase-PCR using total cellular RNA isolated from MEL cells. Reverse transcriptase-PCR, RNA isolation, and molecular cloning were
carried out according to Ausubel et al. (20). The primers are designed according to DNA sequences for S3 in GenBank (Accession no. X76772) with additional sequences at 5' end (lowercase letters
below) that contain restriction enzyme sites (HindIII for
the 5' end primers and BglII for the 3' end primers). The sequences of these primers are as follows: 5' end primer, 5'-ccc aag
ctt ATG GCG GTG CAG ATT TCC AAG-3'; 3' end primer, 5'-ata aga tct CCA
GAT GCA GCT CGC CAA GAC-3'. PCR products were ligated into pGEM-T
vector (Promega) and verified by DNA sequencing. Inserts from clones
with the correct DNA sequences were released from the vector by
HindIII and BglII digestion, gel-purified, and
ligated into HindIII- and BglII-digested
pFLAG-MAC vector (Kodak). Transformation, confirmation of FLAG fusion
junction, preparation of bacterial lysates, and analyses of FLAG-S3
fusion protein were done according to manufacturer's instruction
(Kodak). Bacterial lysates containing no fusion protein and FLAG-S3
were subjected to photoaffinity labeling as described above.
Immunoblotting Analysis
Photoaffinity-labeled samples were subjected to SDS-PAGE and
transferred to a nitrocellulose membrane. Expression of S3 was detected
by rabbit polyclonal anti-S3 antibody and horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (21). The
signal was detected by the enhanced chemiluminescence system (Pierce).
Design of Photoaffinity Labeling Reagent
[3H]498--
We designed a
photoaffinity labeling reagent structurally based on SAHA (Table
I). The phenyl azide moiety was chosen as the photoactivatable group for two reasons. (a) The utility
of phenyl azides as photoaffinity labeling reagents is well known and
documented (22). Upon photolysis at 254 nm, azido groups generate a
highly reactive nitrene that can bind to many cellular components.
(b) The photoaffinity labeling reagent maintains its activity as a potent inducer of differentiation. We have previously synthesized a SAHA analog (498; Table I), and found that incorporation of an azido group in the para position of the benzene ring does not interfere with the differentiation activity (Fig. 2).
Tritium labeling was chosen over other isotopes since bulky substituents such as iodine resulted in a marked decrease in the potency of the inducer.
Synthesis--
The synthesis of
[3H]498 (Fig.
1) involved introduction of tritium by
catalytic dehalogenation of suberic hydroxamic acid
(4-amino-3,5-diiodoaniline) amide 7, followed by conversion of the amine to an azide. Radiolabeling had to precede azide formation because of the instability of the azide group to the reduction conditions used to introduce tritium.
Diiodination of 4-nitroaniline 1 with iodine monochloride
(23) gave rise to 2,6-diiodo-4-nitroaniline 2, which was
reduced with SnCl2 (24) to yield 2,6-diiodo-4-aminoaniline 3. Activation of suberic acid monomethyl ester with carbonyl diimidazole followed by coupling to 3 gave rise to the ester
4. The ester was hydrolyzed with LiOH to yield the carboxylic acid 5 (25). The t-butyldiphenylsilyl
protected hydroxamic acid 6 was prepared by activation of
5 with carbonyl diimidazole, followed by reaction with the
silyl protected hydroxylamine. Deprotection with tetrabutylammonium fluoride gave rise to hydroxamic acid 7. Compound
7 was catalytically tritiated with
3H2/Pd-C in
methanol/H2O/Na2CO3 to yield
8 (specific activity 40 Ci/mmol). Compound 8 was
diluted 10-fold with nonradioactive analog 8a, diazotized,
and treated with sodium azide to yield suberic hydroxamic acid
(4-azido-3,5-ditritioaniline) amide ([3H]498, specific activity 4 Ci/mmol).
Induction of Differentiation--
At 3.5 µM,
498 induces on the average 50% of the cells to
differentiate (Fig. 2). In addition, cell
growth is inhibited by more than 50% (data not shown). In comparison,
SAHA induces on the average about 70% of the cells to differentiate at
2.5 µM (Fig. 2), inhibiting cell growth by more than 50%
(data not shown). Our preparation of
[3H]498 was found to have a level
of activity similar to that of the nonradioactive analog 498 (data not shown).
Photoaffinity Labeling Studies in Cell-free Extracts--
We asked
initially whether [3H]498
specifically binds any cellular proteins. We separated specific from
nonspecific interactions by competition with excess nonradioactive
analog 498. Whole cell lysates were prepared from MEL cells,
incubated with [3H]498 in the
presence or absence of nonradioactive 498 and irradiated.
Protein samples were solubilized in SDS sample buffer, separated by
SDS-PAGE, and visualized by fluorography. We observed (Fig.
3A) one specifically
radiolabeled protein of 32 kDa (p32) and several nonspecifically
labeled proteins. Furthermore, we specifically labeled p32 in the T24
human bladder carcinoma cell line and the ARP 1 human myeloma cell line
(data not shown). SAHA-like HPCs induce T24 cells to differentiate (7)
and ARP 1 cells to undergo apoptosis (7). Thus p32, has been identified as a SAHA-like HPC-binding protein.
To determine the cellular distribution of p32, we prepared P10, P100,
and S100 fractions from MEL cells and assayed for the presence of p32.
Fig. 3B shows that p32 localizes to the P100 fraction, and
is the most abundant protein detected by the label in this fraction.
To establish that binding to p32 occurs at the same concentration range
at which SAHA and [3H]498 induce
differentiation, we followed the specific binding of
[3H]498 to p32 by incubating P100
fractions with different concentrations of the compound in the presence
or absence of excess 498. [3H]498
can be detected specifically bound to p32 in a
dose-dependent manner (Fig. 3C), and at the
concentration in which it is active as an inducer of differentiation.
Furthermore, we performed a dose-response competition experiment, by
incubating P100 fractions with 5 µM
[3H]498 and varying the
concentration of 498. The labeling of p32 by
[3H]498 can be inhibited in a
dose-dependent manner with increasing concentrations of
498 (Fig. 3D).
Protein Purification and Mass Spectrometric Analysis--
We
partially purified p32 by sequential extraction taking advantage of
solubility differences between p32 and other cellular components. P100
fractions were extracted with buffer containing 1% Triton X-100,
partitioning p32 into the detergent-resistant fraction
(PTx). PTx fractions were extracted with buffer
containing 0.1% Zwittergent 3-16, partitioning p32 into the detergent
soluble fraction (STx+zw) (Fig.
4A). Silver staining (Fig.
4B) confirmed the partial purification of p32.
Proteins were separated by SDS-PAGE, transferred to nitrocellulose
membrane, and stained with Amido Black. The band corresponding to p32
was excised from the blot and subjected to MALDI-reflectron TOF (reTOF)
MS and continuous flow ESI (JaFIS) triple quadrupole MS/MS (Fig.
5). Both types of MS analyses
independently identified p32 as the 40 S ribosomal protein S3.
Photoaffinity Labeling of Ribosomes and of FLAG-S3 in
Bacteria--
To further confirm the identity of p32, we labeled a
preparation of ribosomes from dog pancreas (26). p32 was specifically labeled in this preparation (Fig. 6).
Moreover, p32 was the most abundantly labeled band detected in the
ribosome preparation. To confirm that S3 is labeled by
[3H]498, we expressed FLAG-S3 in
bacteria. Photoaffinity labeling of bacterial lysates revealed that S3
is specifically labeled by [3H]498
(Fig. 7A). Western blots using
S3 antiserum (21) (Fig. 7B) confirmed the expression of S3
in the bacteria. These results suggest that S3 is the SAHA-binding
protein that was detected in cell-free extracts.
We have previously reported on a class of HPCs that are highly
potent inducers of differentiation and/or apoptosis in transformed cells compared with the prototype HPC, HMBA (2). Hydroxamic acid-based
HPCs, of which SAHA is the prototype, have been shown to induce
differentiation of MEL cells and other transformed cell lines at
micromolar concentrations.
The strict structural requirements that define a high potency inducer
of differentiation led us to hypothesize that there is a specific
receptor to which these agents bind in the cell. We have used a
photoaffinity labeling reagent, structurally based on SAHA, in a search
for SAHA-binding proteins. Using this approach, we have identified the
ribosomal protein S3 as a target of the SAHA-like HPCs. The
identification of a SAHA-binding protein provides evidence of a direct
physical interaction between the SAHA-like HPCs and this cellular target.
S3 is located on the external surface of the 40 S ribosomal subunit and
plays an important role in protein synthesis (21). It contributes to
the domain where translation is initiated; it can be cross-linked to
initiation factors eIF2 (27) and eIF3 (28), and is directly involved in
ribosome-mRNA-aminoacyl tRNA interactions during translation
(29).
A growing body of evidence indicates that individual ribosomal proteins
and changes in their expression, participate in and modulate a variety
of cellular activities (30, 31). Several recent reports implicate
ribosomal proteins and other components of the translational machinery
as regulatory mediators of growth, proliferation, and neoplastic
changes (32-36).
Ribosomal protein S3 appears to possess apurinic/apyrimidinic
endonuclease activity that strongly implicate it in DNA repair functions (32, 37-39). It contains an activity that cleaves
8-oxaguanine residues and 2,6-diamino-4-hydroxy-5-formamidopyrimidine
residues of damaged DNA (38), an associated apurinic/apyrimidinic lyase activity that cleaves phosphodiester bonds via a Deregulation of the DNA repair machinery by an S3-related mechanism may
contribute to tumorigenesis. Xeroderma pigmentosum group D patients,
who show an increase in the frequency of sunlight-induced skin
carcinomas and malignant melanomas (40), have been shown to lack an
endonuclease activity that has been identified as S3 (32). Moreover, S3
mRNA is overexpressed in colorectal cancer (41).
Recently we reported that SAHA-like HPCs (but not HMBA), are potent
inhibitors of histone deacetylase (HDAC) activity (7). The inhibition
of the enzyme has been demonstrated both in cell-free extracts and in
intact cells. Using photoaffinity labeling, we have recently obtained
preliminary results suggesting that there is a direct and specific
binding interaction in cell-free extracts between the SAHA-like HPCs
and purified HDAC. 3 Direct
binding of HPCs to HDAC may contribute to the mechanism by which
hydroxamic acid-based HPCs induce cell differentiation.
The identification of more than one potential target for the HPCs
suggests that active agents may exert effects on more than one
biological pathway in inducing terminal differentiation. The hypothesis
that inducer-mediated commitment to terminal differentiation is a
multistep process is supported by earlier studies (42, 43) with HMBA.
We have shown (42) that dexamethasone (an inhibitor of inducer-mediated
MEL cell differentiation) did not suppress early events in the process
of commitment, but inhibited later steps involving initiation of cell
division and globin mRNA synthesis. In other studies, Nomura
et al. (43) reported that erythroid differentiation in MEL
cells is a synergistic result of at least two distinctive and
fundamentally different intracellular reactions: one originating from
the inhibition or cessation of DNA replication and the other involving
a transmembrane reaction triggered by inducing agents such as HMBA or
dimethyl sulfoxide (another known inducer of differentiation in MEL
cells). It is therefore plausible that SAHA targets more than one
protein to induce MEL cell differentiation. The identification of S3
and HDAC as targets for the HPCs may allow the rational design of more
potent compounds that will be effective in treating cancer. Further
studies of these targets may contribute to the understanding of the
control of cell proliferation and differentiation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.2 (m, 4H), 1.5 (m, 4H), 1.5 (m, 2H), 2.3 (t,
2H), 2.3 (t, 2H), 3.6 (s, 3H), 4.84 (s, 2H), 7.9 (s, 2H), 9.6 (s, 1H).
1.2-1.3 (m, 4H), 1.4-1.6 (m, 4H), 2.2 (m, 4H), 4.8 (s, 2H), 7.9 (s,
2H), 9.6 (s, 1H), 12.0 (s, 1H).
1.1 (s, 9H), 1.0-1.2
(m, 4H), 1.2-1.4 (m, 2H), 1.4-1.5 (m 2H), 1.9(t, 2H), 2.2 (t, 2H),
4.8 (s, 2H), 7.3-7.4 (m, 6H), 7.6-7.7 (m, 4H), 7.9 (s, 2H), 9.8 (s,
1H), 10.6 (s, 1H).
1.2-1.3 (m, 4H),
1.4-1.6 (m, 2H), 1.9 (t, 2H), 2.2 (t, 2H), 4.8 (s, 2H), 7.9 (s, 2H),
8.6 (s, 1H), 9.6 (s, 1H), 10.4 (s, 1H).
80 °C
and used as whole cell extracts.
-cyano-4-hydroxycinnamic acid solution (MALDI-Quality;
Bruker-Daltonics, Billerica, MA) on the plate, and allowed to dry at
room temperature; calibrants were diluted from concentrated stocks and
mixed to yield 12.5 fmol of each per 0.2-µl volume of the same
solvent prior to mixing with the analytes. MALDI-TOF mass spectra were acquired on a REFLEX III (Bruker-Franzen, Bremen, Germany) instrument equipped with a 337-nm nitrogen laser, a gridless pulsed-extraction ion
source, and a 2-GHz digitizer. The instrument was operated in reflector
mode; 25-kV ion acceleration, 26.25-kV reflector, and
1.4-kV
multiplier voltages were used. Ion extraction was done 200 ns after
each laser irradiance by pulsing down the source extraction lens to
17.7 kV from its initial 25-kV level to give appropriate time-lag focus
conditions at the detector. Spectra were obtained by averaging multiple
signals; laser irradiance and number of acquisitions (typically
100-150) were operator-adjusted to yield maximal peak deflections,
derived from the digitizer as TOF data and displayed in real time as
mass spectra using a SPARC station 5 (Sun Microsystems, Mountain View,
CA). After recalibration with internal standards, monoisotopic masses
were assigned for all prominent peaks, and a peptide mass list generated.
m/z
3,000 atomic mass units) from the MALDI-TOF
experiments were arbitrarily taken to search a protein
non-redundant data base (NRDB; European Bioinformatics Institute,
Hinxton, UK) using the PeptideSearch (17) algorithm. A molecular mass
range of up to 300 kDa was covered, with a mass accuracy restriction of
30 ppm or better, and a maximum of one missed cleavage site allowed per peptide. After a tentative identification was made, as many as possible
of the experimental masses were fitted to the listed sequence
(monoisotopic mass values), allowing for maximal 0.1-Da discrepancy.
MS/MS spectra from the ESI triple quadrupole analyses were inspected
for uninterrupted y" ion series using the "find higher Aas" routine
of the BioToolbox (PE-SCIEX) software; the resultant information
(2-6-amino acid partial sequence, plus corresponding precursor and
fragment ion masses) was semi-automatically transferred, by way of a
custom AppleScript (Apple Computer, Cupertino, CA), to the SequenceTag
(18) program and used as a search string, with a 2-Da mass error
restriction. In case less than three y" ions could be tagged, this
limited information was taken (together with precursor ion mass) to
search the data base using the PepFrag protein identification program
from the PROWL resource
(19).2 Any protein
identification thus obtained was verified by comparing the
computer-generated fragment ion series of the predicted tryptic peptide
with the experimental MS/MS data; this also allowed the discrimination
of true from false positives in case more than one protein was retrieved.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Photoaffinity labeling reagent is a structural analog of the parent
compound
SAHA
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Fig. 1.
Synthesis of photoaffinity labeling reagent
[3H]498.
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Fig. 2.
Induction of differentiation of MEL
cells. Cells were cultured with no inducer (C), SAHA
(2.5 µM), or 498 (3.5 µM) for 5 days, after which the percentage of benzidine-positive staining cells
was determined.
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Fig. 3.
Photoaffinity labeling in cell free
extracts. A, MEL whole cell extracts were incubated
with [3H]498 with (+) or without
( ) a 30 fold excess of 498. Proteins were solubilized in
SDS sample buffer and separated by SDS-PAGE. Gels were enhanced and
fluorographs were obtained by exposure to film for 1-3 days.
B, P10, P100, and S100 fractions were isolated by
differential centrifugation and analyzed as described in A.
C, P100 fractions were incubated with the indicated amounts
of [3H]498 with (+) or without (
)
600 µM 498, and analyzed as described in
A. D, P100 fractions were incubated with 5 µM [3H]498 without
(
) or with 0, 5, 10, 20, 40, 50, 150, or 500 µM
498, and analyzed as described in A.
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Fig. 4.
Partial protein purification of p32 by
sequential extraction. P100 fractions were extracted with Csk
buffer containing 1% Triton X-100. Following centrifugation at
100,000 × g, the insoluble fraction (PTx)
was extracted with buffer containing 0.1% Zwittergent 3-16. The
soluble fraction (STx+zw) was isolated after centrifugation
at 100,000 × g for 1 h. The partial purification
of p32 was followed by monitoring its labeling with
[3H]498 (A) and by
silver staining (B).
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Fig. 5.
Mass spectrometric analyses. The tryptic
digest mixture was passed over an RP micro-tip and the peptides
batch-fractionated into a 16% and 30% pool. Each pool was
individually analyzed by MALDI-reTOF MS (16% fraction shown in
panel A; 30% fraction in panel
B) and by continuous flow ESI (JaFIS) triple quadrupole
MS/MS (Q1 scan of 30% fraction shown in panel
C); only the relevant portions of the spectra are shown.
Both types of MS analysis served to independently identify this 32-kDa
protein as 40 S ribosomal protein S3 (SwissProt P23396). MALDI-reTOF
mass spectra were obtained by averaging 150 scans under constant
irradiance. The calibrants (CAL) and the 18 most prominent
peaks (from both pools combined) are labeled in panels
A and B; the corresponding m/z values
were taken to query a non-redundant protein sequence data base (NRDB)
for pattern matches, using the PeptideSearch program. With a
requirement of 14 matches out of 18, at a mass accuracy of 80 ppm or
better, and a maximum of two missed cleavage site per peptide, a single
protein was retrieved (51.8% sequence coverage). The ESI-MS (Q1)
spectrum of the 30% fraction (obtained by a JaFIS-generated continuous
flow of 4 nl/min, and averaging 100 scans; panel
C) contained several peaks corresponding to those observed
by MALDI-reTOF mass analysis of the same pool (panel
B). One peptide (24843+;
panel C) was then selected, by appropriate tuning
of Q1, for collision-induced dissociation and subsequent analysis of
fragment spectra (in Q3), as shown in panel D. A
short sequence was assigned, based on the presence of a contiguous y"
ion series, enabling positive identification of RS3 by SequenceTag
(oxidized peptide Mr 2484 ± 2;
[446.0]DVYYN[1100.8])-based searching of the NRDB.
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Fig. 6.
p32 is specifically labeled in a dog pancreas
ribosome preparation. Ribosomes were labeled with
[3H]498 in the presence (+) or
absence ( ) of 498. The labeling of p32 was observed after
exposure of enhanced gels for 2 days.
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Fig. 7.
A, lysates of bacteria expressing
FLAG-S3 or control vector and P100 fraction of MEL cell lysate were
subjected to photoaffinity labeling using [3H]498 as
described under "Experimental Procedures." The FLAG-S3 (*) has a
slower mobility than S3 (**) due to the FLAG sequences at the N
terminus. B, a parallel gel was subjected to immunoblot
analysis as described under "Experimental Procedures" using anti-S3
antibody.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
elimination (37), and a DNA deoxyribophosphodiesterase activity that removes sugar-phosphate residues from DNA substrates containing 5' and 3'
incised apurinic/apyrimidinic sites (39).
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ACKNOWLEDGEMENTS |
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We thank Dr. Martin Wiedmann and Dr. Klaus Van Leyen for helpful discussions and for kindly providing ribosome preparations. We are grateful to Lynne Lacomis, Mary Lui, Anita Grewal, and Scott Geromanos for help with mass spectrometric analysis, and to Matthias Mann for the PeptideSearch and SequenceTag programs.
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FOOTNOTES |
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* These investigations were supported in part by Grant CA-0974823 (to P. A. M. and R. A. R.) from the National Cancer Institute, Grant GM18754-35A1 (to R. B.) from the National Institute of Health, Grant BDI-9420123 (to P. T.) from the National Science Foundation, Core Grant P30 CA08748 from the National Cancer Institute, and grants from the Japan Foundation for the Promotion of Cancer Research Fund and the Dewitt Wallace Fund for Memorial Sloan-Kettering Cancer Center.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed: Box 86, Memorial Sloan Kettering Cancer Center, 1275 York Ave., New York, NY 10021. Tel.: 212-639-6568; Fax: 212-639-2861; E-mail: v-richon{at}ski.mskcc.org.
2 The PepFrag protein identification program from the PROWL resource is available via the World Wide Web (http://prowl. rockefeller.edu/PROWL/pepfragch.html).
3 Y. Webb and V. Richon, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: MEL, murine erythroleukemia; HPC, hybrid polar compound; SAHA, suberoylanilide hydroxamic acid; HMBA, hexamethylene bisacetamide; THF, tetrahydrofuran; NRDB, non-redundant data base; PMSF, phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis; PIPES, 1,4-piperazinediethanesulfonic acid; JaFIS, injection adaptable fine i source; HDAC, histone deacetylase; PCR, polymerase chain reaction; MALDI, matrix-assisted laser-desorption/ionization; TOF, time-of-flight; reTOF, reflectron TOF; MS, mass spectroscopy; ESI, electrospray ionization; TBDPS, t-butyldiphenylsilyl.
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