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Tumor Biology |
Leukemia Section, Department of Medicine, Biopolymer Facility and Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, New York 14263
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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forms) have been described and
found to block the normal signaling function of the isoforms. We
recently demonstrated STATß isoforms in bone marrow samples from 21
of 27 (78%) acute myeloid leukemia (AML) patients. We sought to
determine the mechanism by which the STATß forms were generated.
Samples from eight newly diagnosed AML patients were studied; four
expressed predominantly STAT, and four expressed predominantly
STATß. The reverse transcription-PCR generated identical products in
the two groups, suggesting that alternate mRNA splicing is not
responsible for the genesis of STATß. Extracts from cells expressing
predominantly STATß incubated with cell extracts from the MO7E cell
line, which expresses predominantly STAT, caused a decrease of the
isoforms and an increase of the ß isoforms, suggesting the
presence of proteolytic activity. This proteolytic activity was:
(a) specific for STAT3 and STAT5, but not for STAT6;
(b) serine dependent; (c) equally present
in nuclear and cytoplasmic fractions of the leukemic blasts; and
(d) different than the activity detected in a murine
hematopoietic cell line. The cleaved ß isoforms retained their
DNA-binding activity. Because expression of truncated STATs may be
involved in blocking differentiation of AML blasts, elucidation of the
regulation of the proteolytic activity may contribute to our
understanding of leukemogenesis. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The existence of different functional isoforms of several STAT proteins
has been documented. Full-length STAT3 (referred to here as STAT3)
has been described as having an isoform (STAT3ß) that is truncated by
55 amino acid residues at the COOH-terminal end (3)
.
STAT3ß has been found in AML blasts in some patients (4
, 5)
. Similarly, STAT5A and STAT5B, originating from ìtwo
separate genes, are described to have at least two isoforms (/ß)
each (6, 7, 8)
. The COOH-terminal sequence that is missing in
the ß-forms of both STAT3 and STAT5 encodes the transcriptional
activation domain (7
, 9 , 10)
. The ß isoforms were shown
to have a transdominant negative effect on gene induction in the STAT
pathway (9, 10, 11, 12)
. If the alternate STATß forms are
expressed, they may inhibit differentiation, which is normally induced
by the forms.
The short forms of STAT proteins (termed ß) are believed to be derived from either alternatively spliced mRNA or proteolytic cleavage. Alternative splicing has been suggested for STAT1 (13) , STAT3 (3) , and STAT5 (14) . However, truncations do not arise only by alternative splicing. Azam et al. (15) were the first to suggest that truncated STAT5 isoforms can be generated by proteolysis. Recently, two groups studying a murine multipotent hematopoietic cell line showed that STAT5ß is produced by a nuclear serine protease (16 , 17) with a molecular weight of 25,000 (17) . To the best of our knowledge, no information exists about a similar activity for STAT3. We sought to determine how the ß isoforms are generated in AML cells. We have previously demonstrated that the majority of AML samples express the ß isoforms, that AML samples containing the STAT3ß isoform also contain the STAT5ß isoform, and that samples with the ß isoforms maintain the same forms on exposure to exogenous cytokines (18) . These data suggest that a common mechanism is responsible for the appearance of the truncated forms in AML blasts. We demonstrate here that the STATß forms in AML cells are generated by a specific serine-dependent proteolytic activity that is different from the activity demonstrated in the murine hematopoietic cell line. This activity has the following characteristics: (a) it modifies both STAT3 and STAT5; (b) it is present in both the cytoplasm and the nucleus; and (c) it migrates with a molecular weight of approximately 40,000.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Studies were approved by the Roswell Park Cancer Institute
Institutional Review Board. Informed consent was obtained from all
patients.
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Cell Collection.
Light-density bone marrow cells were isolated by 1.077
gram/mm3
Ficoll-Hypaque density gradient centrifugation and
either used immediately or cryopreserved under viable conditions, using
standard methodology. Cell viability was verified by trypan blue dye
exclusion.
RT-PCR.
Total RNA was extracted from the cells by RNeasy Minikit (Qiagen Inc.,
Valencia, CA) as recommended by the supplier. The presence of STAT3,
STAT5A, and STAT5B mRNA was determined by RT-PCR using 5' primers
extending from position 2310 to 2336, 3123 to 3142, and 2246 to 2265
and 3' primers extending from position 2508 to 2533, 3621 to 3647, and
2641 to 2650 (5
, 19
, 20)
. Glyceraldehyde-3-phosphate
dehydrogenase was used as an internal standard for mRNA
semiquantitation (21)
. One µg of total RNA was
reverse-transcribed to cDNA using 20 pM random hexamers
(Perkin-Elmer, Branchburg, NJ) in a mixture of 25 mM
MgCl2; 10 x PCR buffer II; and 1
mM deoxyadenosine triphosphate, deoxycytidine triphosphate,
deoxyguanosine triphosphate, and deoxyribosyl thymine triphosphate
(Pharmacia Biotech, Piscataway, NJ); 20 units of RNasin (Promega,
Madison, WI); and 200 units of Moloney murine leukemia virus reverse
transcriptase (Life Technologies, Inc., Gaithersburg, MD). The total
volume of each reaction was 20 µl. The reverse transcription reaction
was first incubated for 10 min at room temperature, reverse transcribed
for 15 min at 42°C, and then denatured for 5 min at 99°C. Each
reaction was placed on ice for 5 min.
PCR was performed in a final volume of 100 µl using 20 µl of the reverse transcriptase mixture containing 20 pM each of the 3' and the 5' PCR primers and 2.5 units of Taq Gold Polymerase (Perkin-Elmer) in its buffer. The amplification was performed for 34 cycles in a Hybaid temperature cycler (National Labnet, Woodbridge, NJ) with initial hot start at 94°C for 5 min, followed by 1 min of denaturation at 94°C, 1 min of annealing at 59°C, 1 min of extension at 74°C, and a final extension period of 7 min. Ten-µl aliquots of this final reaction were analyzed on a 2% agarose gel (FMC Bioproducts, Rockland, ME) that contained 0.01% ethidium bromide in Tris-borate-EDTA electrophoresis buffer.
To determine whether alternatively spliced STAT3 messages are present in samples expressing the full-length and the truncated proteins, RT-PCR reactions were performed with serially diluted RNA preparations initially diluted 1:10 and then further diluted 1:50 and 1:250. Glyceraldehyde-3-phosphate dehydrogenase was used as an internal standard. The STAT PCR products were extracted from the gel with the QIAquick gel extraction kit (Qiagen Inc., Santa Clarita, CA) as recommended by the supplier. The purified DNAs were sequenced with an Applied Biosystems model 373 stretch DNA sequencer (Perkin-Elmer). The data were analyzed with GeneWorks computer program (IntelliGenetics, Campell, CA).
MYC-STAT3 Constructs.
MYC-STAT3 constructs were generated by inserting the rat STAT3 cDNA
into the pSVSPORT1 plasmid (Clontech, Palo Alto, CA; Ref. 22 ). The
NH2 terminus of STAT3 was modified by inserting
six tandem copies of the MYC epitope in-frame (construct generously
provided by Dr. T. Kordula, University of Georgia, Atlanta, GA).
Cell Lines.
COS-1 cells were maintained in MEM supplemented with 5% FCS. FDCP-1
and NIH 3T3 cells (obtained from American Type Culture Collection,
Manassas, VA) were maintained in DMEM with 4 mM
L-glutamine and 4.5 grams/liter glucose. Medium for FDCP-1
cells was supplemented with mouse interleukin 3 as recommended by the
supplier. MO7E cells were maintained in RPMI 1640 with 10% FCS.
Activation of STAT3 and STAT5 was achieved by treating MO7E cells with
TPO (40 ng/ml) for 15 min (23)
.
Transfection.
COS-1 cells were transfected by the DEAE-dextran method
(9)
. After a 36-h recovery, whole cell extracts were
prepared.
Western Blotting.
Western blot analysis was performed as described previously
(18)
. Briefly, whole cell extracts (50–100 µg of
protein) were separated on 7.5% SDS polyacrylamide gels. The proteins
were transferred onto a 0.1 µm nitrocellulose membrane and incubated
with antibodies against the NH2-terminal portion of STAT3,
STAT5, STAT6 (obtained from Transduction Laboratories, Lexington, KY),
STAT3 COOH-terminal-specific antibody (C-20), or MYC (SC-40; Santa Cruz
Biotechnology). The immune complexes were detected by an enhanced
chemiluminescence reaction (Amersham Life Science, Arlington Heights,
IL).
In Vitro Proteolysis of STATs.
MO7E cells containing predominantly the STAT isoforms were lysed
(5 x 106 cells in 45 µl of lysis buffer).
Aliquots of 2.5 µl were mixed with equal volumes of the patients’
cell extracts and then incubated at 25°C for 0, 15, and 30 min.
Sensitivity of the proteolysis to inhibitors was tested as follows:
apyrase (10 units) and calf intestinal alkaline phosphatase (4 units),
both of which are known to hydrolyze ATP, were used to determine
whether the proteolytic activity is ATP dependent (24)
;
PMSF was used at 2–4 mmol/liter, to inhibit serine protease activity
(16)
; calpain I and II inhibitor peptide (BioSource
International, Camarillo, CA) were used at 10 µM and 1
mM to inhibit calpain activity (25)
; and
ALLN (at 12 and 60 µM) and latacystin (at 2 and 10
µM; both from Clabiochem, La Jolla, CA) were used to
inhibit proteasome activity (26)
. Reaction mixtures were
then separated on SDS-polyacrylamide gel and subjected to Western
blotting as described above.
EMSA.
Whole cell extracts were prepared as described previously
(18)
and incubated with 32P-labeled oligomers
corresponding to the binding element for STAT1 and STAT3 (SIE; Ref. 27
)
and STAT5 (TB2; Ref. 28
). The complexes were analyzed by 5% PAGE and
autoradiography. Extracts of TPO-treated MO7E cells served as standard
for activated STAT3 and STAT5. The identity of the STAT proteins
contributing to the gel-shifted bands was determined by antibody
supershift with the anti-STAT3 (C-20) or anti-STAT5 (C-17) monoclonal
antibodies (Santa Cruz Biotechnology).
Cell Fractionation.
To initiate preparation of nuclear and cytoplasmic fractions
(16)
, cells were washed once with PBS containing 1
mmol/liter orthovanadate and then resuspended in a hypotonic buffer
containing 20 mmol/liter HEPES (pH 7.6), 10 mmol/liter KCl, 1
mmol/liter MgCl2, 0.5 mmol/liter DTT, 0.1% Triton X-100,
and 20% glycerol and protease inhibitors (5 µg/ml leupeptin, 5
µg/ml pepstatin A, 200 KIU/ml aprotinin, and 1 mmol/liter sodium
orthovanadate). The cells were then homogenized by 20 strokes in a
glass Dounce homogenizer. The homogenate was centrifuged at
2,000 x g for 5 min. The supernatant
(cytoplasmic fraction) was centrifuged again for 15 min at 14,000 rpm.
The clear lysate was flash frozen in liquid nitrogen. The pelleted
nuclei were resuspended in a hypertonic extraction buffer [20
mmol/liter HEPES (pH 7.9), 0.4 mol/liter NaCl, 1 mmol/liter EDTA, 0.5
mmol/liter DTT, 0.1% Triton-X 100, and 20% glycerol] containing
protease inhibitors (as listed above) for 15 min at 4°C. The extracts
were recovered by centrifugation for 5 min at 14,000 rpm. Aliquots were
frozen in liquid nitrogen and stored at -70°C. The protein
concentrations of nuclear and cytoplasmic extracts were adjusted with
buffer to reflect their cellular ratios. The purity of the cell
fractions was confirmed by Western blot analysis using antiserum
against c-src (Santa Cruz Biotechnology) that is specific for the
cytoplasm.
SE-FPLC.
Two hundred µl of cell extract were loaded onto a superose 6 prep
grade column (Pharmacia Biotech, Uppsala, Sweden). The column was
equilibrated with eluent buffer (HEPES, 40 mM; NaF, 40
mM; Na3VO4, 2 mM;
Na4P2O7, 2 mM; EDTA, 2
mM; EGTA, 2 mM; NaCl, 420 mM) and
calibrated with the following standard proteins: cytochrome
c (Mr 12,500), ovalbumin
(Mr 45,000), monomeric and dimeric bovine serum
albumin (Mr 67,000 and 120,000), and catalase
(Mr 240,000). The eluates were collected
in 500-µl fractions, and each fraction was concentrated 10-fold with
Centricon centrifugal filter devices (YM-10; Millippore, Bedford, MA).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Sample from patient 960421 showed predominantly the isoforms by
Western blot analysis, whereas the sample from patient 970271 expressed
predominantly the ß isoforms (Fig. 1, A and B
,
bottom panels). The samples had the same mRNA species as
demonstrated by RT-PCR, whether they expressed the or the ß
proteins (Fig. 1, A and B
, top
panels). Serial mRNA dilutions did not detect any difference
between the two messages for STAT3 (Fig. 1A)
. STAT5A
and STAT5B each have a single form of message (Fig. 1B)
, and
therefore no analysis of dilutions was performed. Findings were similar
for all other patient samples (data not shown). These results suggest
that the STATß isoforms were generated by proteolytic cleavage.
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have low STAT proteolytic
activity, and patient cells with STATß have high proteolytic
activity. We also considered it conceivable that the samples have a
differential expression of factors that inhibit the constitutively
expressed proteolytic activity. To detect proteolysis, we combined
whole cell extracts from patient samples expressing predominantly
either the or the ß STAT isoforms with extracts from TPO-treated
MO7E cells containing primarily activated isoforms. The reaction
mixtures, after 15 and 30 min of incubation, were analyzed by
immunoblotting for the pattern of STAT proteins (Fig. 2)
. When using an extract from patient cells with isoform, there was no
change in the /ß pattern (Fig. 2, A and B)
.
However, with patient samples containing predominantly the ß
isoforms, there was a decrease in the substrate of MO7E isoforms
and a concomitant increase in the intensity of the ß isoforms (Fig. 2, C and D)
. The pattern was similar for STAT3
and STAT5. The proteolytic activity was specific for STAT3 and STAT5
because the extracts from patient samples with ß isoforms had no
effect on STAT6 of MO7E cells (Fig. 3)
. Essentially identical results regarding changes in STAT protein
patterns were obtained when extracts from MO7E cells exposed to medium
alone (predominantly the latent inactive STAT proteins) or with TPO
(predominantly the activated dimerized STAT proteins were used; data
not shown.
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To study the effect of the proteolytic activity on STAT DNA binding,
EMSA assays were performed as shown in Fig. 4,A and B
. These assays used only TPO-activated
STAT proteins because the nonactivated proteins do not bind to DNA. The
presence of STAT or ß forms in the extracts was identified by
distinct electrophoretic mobility of the DNA-STAT complexes.
Combination of patient cell extract with MO7E extract and incubation
for 30 min produced characteristic modification of EMSA patterns. Cell
extracts from patient samples expressing predominantly the ß isoforms
digested the MO7E-derived isoforms without compromising their DNA
binding activity. For STAT3, the mixture of cell extracts induced the
conversion of the MO7E pattern to the STAT3ß protein that gave
rise to the slower migrating complex (Fig. 4A)
. The slower
migration results from the loss of the acidic COOH-terminal domain of
STAT3. To confirm that the slower migrating band did indeed contain a
COOH-terminal truncated STAT form, an antibody against the
COOH-terminal domain was added to the mixture. No supershift was
detected in any of the MO7E samples incubated with whole cell extracts
from samples expressing predominantly the ß isoforms (data not
shown). In contrast, the antibody shifted the majority of the STAT3 DNA
band when MO7E extract was not treated with leukemic cell extract. For
STAT5, the same mixture showed an additional faster migrating band
(Fig. 4B)
. This faster migrating band was found to contain
STAT5ß that could only form a dimer bound to TB2 element, whereas a
tetramer of STAT5 bound to the same probe was thus responsible for
the slower migrating complex (29)
. As with STAT3, no
supershift was detected with STAT5 when using MO7E samples incubated
with whole cell extracts from samples expressing predominantly the ß
isoforms (data not shown). The appearance of STAT5ß-specific DNA
binding action could be independently confirmed by the binding to the
TB1 oligomer
(18)
,4
an oligonucleotide developed as a substrate specific for the ß forms
of STAT5A and STAT5B (29)
. As shown in Fig. 4C
,
the MO7E-derived activated STAT5 isoforms, when incubated with
extracts from patient samples expressing predominantly the ß
isoforms, were processed to a TB1 complex on EMSA. Control MO7E
extracts treated with whole cell extracts from patients expressing
predominantly the isoforms did not alter the size of STAT3 and
STAT5 or their DNA binding activity (data not shown). Thus, the data
indicate that cells from AML patients expressing predominantly STATß
isoforms contain a proteolytic activity that cleaves STAT forms and
that these proteolytic products retain their DNA binding activity. In
the case of STAT5ß, the truncation may also modify the binding
specificity.
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, PMSF blocked the proteolytic activity from leukemic samples and
prevented the appearance of the STATß isoforms in a dose-dependent
manner. This proteolytic activity was minimally inhibited by high
concentration of calpain I and II peptide inhibitor and was not
dependent on ATP because apyrase or calf intestine alkaline phosphatase
did not have any effect. Also, there was no evidence for a functional
relationship with proteasome activity, because ALLN (Fig. 5)
and
latacystin (data not shown) did not affect STAT conversion. These data
suggest that the proteolytic activity in AML samples is mainly serine
dependent.
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, STAT3ß-generating activity was found in the cytoplasmic and nuclear
fractions of the cells in similar amounts. Similar data were obtained
for STAT5ß (data not shown).
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digestion assay. Our results
demonstrate that whole cell (Fig. 7,A and B)
and nuclear (Fig. 7, C and D)
extracts of FDCP-1 cells had no significant proteolytic
activity on either STAT3 or STAT5 of murine (NIH 3T3 cells) or
human (MO7E cells; data not shown) origin. However, AML blasts
expressing the STATß isoforms and therefore carrying the proteolytic
activity had a prominent effect (Fig. 7, C and D)
on STAT3 and STAT5.
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construct. Due to the MYC epitope added to the
NH2 terminus, this tagged STAT3 can readily be
distinguished from normal proteolysis by physicochemical and
immunological procedures (Fig. 8)
. MYC-STAT3 was expressed in COS-1 cells. Extracts from these COS-1 cells
transfected with MYC-STAT3 construct were then incubated with extracts
from either an AML patient or FDCP-1 cells. No change in MYC-STAT
pattern occurred with extracts from AML patients expressing the STAT
form or with extracts from FDCP-1 cells. However, treatment of COS-1
cell extract with extract from an AML patient expressing STATß caused
a complete conversion of MYC-STAT to STAT3ß (Fig. 8
, Lane
5). This suggests that the assay conditions identified a
proteolytic activity in AML cells distinct from that reported for
FDCP-1 cells.
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. Extracts from AML patients expressing STATß resulted in the
separation of STAT3ß that was collected at a molecular weight between
120,000 and 240,000, and the proteolytic activity converting
MYC-STAT3 to MYC-STAT3ß was collected at a molecular weight of
approximately 40,000. These results support the conclusion that the
proteolytic activity in AML cells is distinct from that in FDCP-1
cells.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The serine-dependent protease described in cell line models was nuclear, had a molecular weight of 25,000, and was tested only against STAT5 (16 , 17) . If one considers nucleus-localized STAT5 to represent activated STAT dimers (30) , then the nuclear protease would have to act on dimerized STAT. In contrast, a cytoplasmic protease would have the ability to cleave STAT proteins in a different form, mainly as a "monomeric" (i.e., latent) STAT. Moreover, in view of our data on AML blasts and the cell lines (15, 16, 17) , it is still unclear whether one enzyme or a family of enzymes with conserved proteolytic activity but differences in substrate specificity accounts for the production of STATß proteins. The minimal inhibition of proteolytic activity by the calpain inhibitor in AML cells suggests the presence of more than one activity. Taken together, one can conclude that the AML-derived protease is different from the one described in the murine hematopoietic cell line.
The role of the STATß proteins and hence the functional consequence
of STAT proteolysis in AML blasts remain unknown. Recent work by
Bromberg et al. (31)
suggests that introducing
a cysteine at the COOH-terminal domain of STAT3 causes the molecule
to dimerize, promote transcription, and induce cell transformation.
STAT3-responsive elements have been identified primarily in
cytokine-responsive genes, including acute phase genes or immediate
early genes (c-fos and JunB). Similarly, STAT5A and STAT5B, which
originate from two separate genes, are described to have at least two
isoforms (/ß) each (6, 7, 8)
. The ß forms were shown
to have a competitive or even transdominant negative effect on gene
induction mediated by the STAT pathway (9
, 10)
. This is
supported by an experimental model in tissue culture (32)
.
In this system, using a murine myeloid hematopoietic cytokine-dependent
cell line, a COOH-terminal truncation of STAT5A resulted in inhibition
of both interleukin 3-dependent proliferation and granulocyte
colony-stimulating factor-dependent differentiation, without induction
of apoptosis. These data suggest that the truncated STATs may have a
role in blocking differentiation and hence promoting survival.
Because the inappropriate appearance of the ß isoforms might have not
only a suppressive effect on the signal transduction pathway
controlling differentiation but also an enhancing effect on
proliferation, controlling ß isoform production can contribute to
leukemogenesis. These mechanisms do not involve only regulated
expression of STAT protease but could conceivably involve the
modulating activity of protease inhibitors. For example, possible
candidates could be serine protease inhibitors (serpins), which belong
to a large family of proteins that include, among others, cytoplasmic
antiproteinase (33
, 34)
and a tumor suppressor called
maspin (35)
that is presumably related to ovalbumin-type
serpin. Recently, bomapin, a novel human serpin, was cloned
(36)
. It was found to be expressed in normal bone marrow
cells and in hematopoietic cells of the monocytic lineage
(37)
. We suggest that cells expressing the STATß
isoforms will have increased levels of the protease and/or reduced
levels of inhibitors. Conversely, cells that express the STAT
isoform should have either no protease or increased levels of a serpin
to prevent the function of the protease. Finally, although it has been
noted that cytokines can activate secretory serpins, this is not known
for intracellular serpins. Because the STAT patterns are part of the
signaling pathways in most hematopoietic and nonhematopoietic cell
types but are under the control of distinct sets of regulators, we
propose that a protease and a protease inhibitor may establish a
yin-yang mechanism in controlling the proliferation and differentiation
of their own. The modification of such a mechanism and its role in AML
remain to be established.
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FOOTNOTES |
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1 Supported in part by a grant from Amgen
(Thousand Oaks, CA), the Tower Foundation (Buffalo, NY), and National
Cancer Institute Grants CA16056 and CA26122. 
2 To whom requests for reprints should be
addressed, at Leukemia Section, Department of Medicine, Roswell Park
Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263. Phone:
(716) 845-8447; Fax: (716) 845-2343; E-mail: meir.wetzler{at}roswellpark.org
3 The abbreviations used are: JAK, Janus Kinase;
STAT, signal transducer and activator of transcription; AML, acute
myeloid leukemia; RT-PCR, reverse transcription-PCR; TPO,
thrombopoietin; EMSA, electrophoretic mobility shift assay; PMSF,
phenylmethysulfonyl fluoride; ALLN,
N-acetyl-Leu-Leu-norleucinal; SE-FPLC, size exclusion-fast
protein liquid chromatography.
4 C. F. Lai and H. Baumann, unpublished
data.
Received 7/26/00. Accepted 12/ 4/00.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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