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NEOPLASIA
From Unité INSERM U417, Hôpital Saint
Antoine, Paris, France; and Department of Microbiology/Immunology,
Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, PA.
A pivotal role has been assigned to Myb in the control of
myeloid cell growth. Although Myb is a target of retinoic acid, little
is known about the mechanisms by which it may contribute to induced
growth arrest in leukemia cells. Indeed, few Myb target genes are known
to be linked to proliferation. Myeloblastin is involved in the control
of proliferation in myeloid leukemia cells. It is expressed early
during hematopoiesis and is a granulocyte colony-stimulating
factor-responsive gene. Myeloblastin can confer factor-independent
growth to hematopoietic cells, an early step in leukemia
transformation. The myeloblastin promoter contains PU.1, C/EBP, and Myb
binding sites, each of which are critical for constitutive expression
in myeloid cells. Inhibition of myeloblastin expression in
leukemia cells growth-arrested by retinoic acid is demonstrated to
depend on Myb down-regulation. Myb is shown to induce myeloblastin
expression and abolish its down-regulation by retinoic acid.
Altogether, the data offer a clue as to how a myeloid-specific
transcriptional machinery can be accessible to regulation by retinoic
acid and point to myeloblastin as a novel target of Myb. This link
between Myb and myeloblastin suggests a previously nonidentified Myb
pathway through which growth arrest is induced by retinoic acid in
myeloid leukemia cells.
(Blood. 2001;97:2449-2456) Retinoic acid (RA) plays a major role in inducing
growth arrest and differentiation of myeloid leukemia cells both in
vitro and in vivo.1-5 However, little is known about those
myeloid genes that respond to RA and are involved in growth control.
Myeloblastin (MBN)6 is a serine protease with a broad
spectrum of proteolytic activity. Inside myeloblastic cells, a main
enzymatic target appears to be the Sp1 transcription factor, which is
physiologically truncated by MBN and is known to contribute to growth
regulation.7 An involvement of MBN in the control of
leukemia cell growth was initially suggested by the fact that its
expression was serum-dependent and down-regulated by growth-inhibiting
inducers such as all-trans retinoic acid
(ATRA).6 Furthermore, down-regulation of MBN expression by
antisense oligodeoxynucleotides inhibited proliferation of
promyelocytic-like leukemia cells.6 MBN is overexpressed in myeloid leukemia cells,8 and several reports suggest
that T-cell responses to MBN peptides can be used for adaptative T-cell therapy in acute and chronic myeloid leukemias.9,10 MBN is a granulocyte colony-stimulating factor (G-CSF) target gene and can
confer factor-independent growth when ectopically overexpressed in an
early hematopoietic cell line.11 This has strongly
reinforced the view that MBN is a key protease involved in the control
of proliferation in normal hematopoietic progenitors and in
contributing to early steps in their transformation.
The Myb family of transcription factors is strongly implicated in the
regulation of cell growth and differentiation. In the murine system,
inappropriate expression of c-Myb clearly contributes to leukemia
transformation.12 The c-Myb promotes proliferation and
blocks differentiation of hematopoietic cells in several experimental models.13,14 Targeting of c-Myb with antisense
oligodeoxynucleotides inhibited hematopoiesis as well as proliferation
of myeloid leukemia cells.15,16 Mice homozygous for the
inactivated c-Myb gene had impaired definitive hematopoiesis with a
drastic decrease in the number of progenitors likely to reflect a
proliferation abnormality.17 Expression of c-Myb declines
in RA-treated myelomonocytic leukemia cells, suggesting that the
retinoic acid receptor (RAR) acts in part by down-regulating c-Myb
expression. This may account for the observation that transcriptional
activation of a Myb-responsive gene can be inhibited by
RA.18 Furthermore, introduction of an exogenous RAR Because MBN is associated with growth control in myeloid leukemia
cells, we have analyzed its transcriptional regulation by RA. For this,
we have cloned the human and murine MBN promoters, which both harbor
binding sites for the PU.1, C/EBP, and Myb transcription factors. Our
data show that these factors are all critical for constitutive MBN
expression and establish MBN as a novel target of Myb, mediating its
regulation by ATRA in leukemia cells. This, together with the fact that
MBN is involved in growth control and can provide factor-independent
growth to hematopoietic cells, suggests that its association with Myb
may play a role in mediating ATRA-induced growth arrest in myeloid
leukemia cells.
Leukemia cell lines culture conditions
Northern blot analysis
Recombinant plasmids Expression vectors for C/EBP (MSV-C/EBP), C/EBP
(MSV-C/EBP), and C/EBP (MSV-C/EBP); for c-Myb
(CMV-c-Myb)22; for PU.1 (pECE-PU.1)23; and
for E1A 13S and E1A 12S Ntdl814 24 were obtained from A. Friedman, B. Lüscher, R. Maki, and P. Whyte, respectively.
A genomic library of SV129 mouse embryonic stem cell DNA was a
gift from P. Chambon. A total of 1 ×106 recombinants were
screened using a 5' probe (85-base pair [bp] NotI-PvuII fragment) of the mouse MBN cDNA kindly
provided by L. Hellman.25 BamHI digestion of a
positive clone and hybridization with the 5'-TCTGGAAGCTACCCATCC-3'
oligonucleotide yielded a 1.1-kilobase fragment that was subcloned and
submitted to automated sequencing (Eurogentec, Seraing, Belgium). A
658-bp human MBN promoter region was subcloned into the pBLCAT6
vector,26 generating the pMBN-658 reporter plasmid. For
constructing the pMBN-194, pMBN-137, and pMBN-91 deletion reporter
plasmids, 194-bp, 137-bp, and 91-bp fragments, respectively, were
generated using PCR and cloned into BamHI/XhoI
sites of the pBLCAT6 vector. The 5' primers were
5'-ATGGATCCGACTTGGGTGGGTGA-3' from positions In vivo expression assays COS-7 cells were transfected using calcium phosphate coprecipitation27 of DNA vectors (adjusted to 14 µg per 9-cm Petri dish with pBluescript carrier DNA). Medium was changed after 16 hours. Cells were harvested after an additional 20-hour culture. Exponentially growing PLB-985 cells were washed twice in serum-free medium, once in Optimem medium, resuspended in the same medium at 15 × 106 cells in 0.5 mL, and electroporated (Gene Pulser, Biorad, Hercules, CA) at 300 V, 960 µF with 20 µg of (1) pBabe Puro28 alone or together with CMV-c-Myb (cells were then cultured for 48 hours prior to selection with 1 µg/mL puromycin [Sigma]); (2) reporter plasmid. Cells transfected with reporter plasmid were cultured in 10 mL of medium for 40 hours. Plasmid (5 µg) containing the luciferase cDNA driven by the cytomegalovirus promoter was an internal control for transfection efficiency. All transfections were performed at least in triplicate with independent template preparations. Assays to measure luciferase29 and chloramphenicol acetylase30 were conducted as described.Electrophoretic mobility shift assays For whole-cell extracts, COS-7 cells were harvested in ice-cold phosphate-buffered saline, pelleted, washed twice in the same buffer, and resuspended in extraction buffer (0.4 M KCl, 20 mM Tris-HCl [pH 7.9], 20% glycerol, 5 mM dithiothreitol, 0.4 mM phenylmethylsulfonyl fluoride, and 2.5 ng each of leupeptin, pepstatin, aprotinin, antipain, and chymostatin [Sigma] per milliliter). After 2 freeze-thaw cycles with liquid nitrogen, the resulting cell lysate was cleared by centrifugation 15 minutes (4°C) at 10 000g and used for electrophoretic mobility shift assays (EMSAs). PLB-985 nuclear extracts were prepared essentially as described31; 2.5 ng each of leupeptin, pepstatin, aprotinin, antipain, and chymostatin (Sigma) per milliliter were added in both lysis and extraction buffers. EMSAs were carried out using PU.1 (human MBN promoter position 84 to
62), 5'-AGCTTAGGCAAAAGGAGGAAGTGGGGACG-3; mut PU.1,
5'-AGCTTAGGCAAAAGGATTAAGTGGGGACG-3'; C/EBP (human MBN promoter position
51 to 36), 5'-AGCTTGGGCATTGGGCAACTCG-3'; mut C/EBP,
5'-AGCTTGGGCGTAGTGGGACTCG-3'; Myb (human MBN promoter position 42 to
22), 5'-ATGGATCCGCAACTCAACGGCCTCTGGCAT-3'; and mut Myb, 5'-ATGGATCCGCAACTCATGGGCCTCTGGCAT-3' oligonucleotides.
Approximatively 0.25 ng (15 000 cpm) of a 32P-5'
end-labeled synthetic double-stranded oligonucleotide was incubated
with 5 µg COS-7 cell lysate or PLB-985 nuclear extracts in the
presence of poly(dIdC) as a nonspecific competitor in 10 mM Tris-HCl
(pH 7.9), 50 mM KCl, 2 mM ethylenediaminetetraacetic acid (EDTA),
0.05% Nonidet P-40, and 0.3 mg/mL bovine serum albumin. After 15 minutes at room temperature, DNA-protein complexes were separated on a
5% polyacrylamide gel (acrylamide:bisacrylamide ratio, 37.5:1) in TBE
(25 mM Tris base, 25 mM boric acid, 0.5 mM EDTA) at 4°C. For
competition experiments, extracts were preincubated with poly(dIdC) and
unlabeled competitor oligonucleotides for 15 minutes before addition of
the probe and further incubation. For antibody supershift assays,
affinity-purified polyclonal anti-PU.1, anti-C/EBP, anti-C/EBP,
anti-C/EBP, or anti-c-Myb antibodies (Santa Cruz Biotechnology,
Santa Cruz, CA) (1 µL) were added to the mixture and incubated for an
extra 30 minutes before loading into the gel.
PU.1, C/EBP, and Myb binding sites are all critical for constitutive MBN promoter activity To assess critical MBN regulatory sequences, we have cloned human and mouse MBN promoter regions (GenBank accession numbers M96628 and AJ007030, respectively). Both promoter sequences had a TATA box and potential binding sites for Myb, C/EBP, and PU.1 (Figure 1A). These sites exhibited consensus sequences in both the mouse and human promoters (Figure 1B). In addition, the human sequence harbored a CG element (Figure 1A). These elements were in agreement with putative binding sites indicated by Sturrock et al,32 except for C/EBP, which was not conserved between human and mouse and fit poorly with the C/EBP consensus sequence (data not shown). Mutation of this site did not modify the MBN promoter activity in transfected myeloid cells (data not shown). In contrast, we have identified another putative C/EBP binding site that differs from the C/EBP consensus sequence only by a G-to-C transition at the fifth position. A similar C/EBP site is present in the granulocyte-macrophage colony-stimulating factor receptor promoter, harbors the same G-to-C transition, and binds
C/EBPs.33 The Myb site is identical to a functional Myb
binding site present in the neutrophil elastase promoter, which binds
c-Myb with much lower affinity than, for example, the mim-1 Myb
site.34
To investigate the mechanisms involved in MBN down-regulation in
ATRA-treated growth-arrested leukemia cells, deletion constructs of the
human MBN promoter (Figure 1Ci) cloned into the pBLCAT6 reporter
plasmid were transfected into myeloblastic PLB-985 cells. Deletion from
position EMSAs were first conducted using total extracts from COS-7 cells
transfected with vectors expressing recombinant PU.1, C/EBP
Deletion fragments of the MBN promoter were not adequate for studying
the respective importance of the C/EBPs and Myb elements because
removal of the PU.1 site resulted in a drastic diminution of the MBN
promoter activity (data not shown). We therefore introduced point
mutations into these sites. Mutation of PU.1, C/EBP, or Myb sites
resulted in a drastic decrease of the promoter activity, indicating
that all 3 sites were critical for the constitutive activity of the MBN
promoter (Figure 2C) and suggesting that their action could be
combinatorial. Indeed, it has been shown that c-Myb requires direct and
specific interaction with the cyclic-AMP response element-binding
protein (CREB)-binding protein (CBP) for its transactivation
function.35,36 Similar observations were made with
PU.137 and C/EBP Inhibition of MBN expression in growth-arrested ATRA-treated leukemia cells is mediated by Myb down-regulation ATRA-induced growth inhibition in differentiating PLB-985 and NB4 leukemia cells (Figure 3Ai,ii, respectively) was accompanied by progressive down-regulation of the MBN messenger RNA (mRNA), which was barely detected at days 4 and 2, respectively (Figure 3Aiii). During these processes, the time at which MBN mRNA expression was inhibited closely paralleled that of c-Myb (Figure 3Aiii). We therefore overexpressed c-Myb in PLB-985 cells to test whether its down-regulation was required for ATRA to down-regulate the MBN promoter. In contrast to the expected ATRA-induced inhibition of MBN promoter activity in PLB-985 cells, overexpression of c-Myb (5 µg plasmid) resulted in an 8-fold induction of the MBN promoter activity (Figure 3Bi), which could no longer be decreased following treatment of the cells with ATRA (Figure 3Bi). To verify that potential ATRA-induced effects on MBN expression were not blunted by saturated levels of MBN as a result of c-Myb overexpression in PLB-985 cells, we used lower levels of engineered c-Myb expression (2 µg), which, following ATRA treatment, resulted in an MBN promoter activity similar to that found in untreated cells transfected with the empty plasmid (Figure 3Bi). This suggested that transfection of 2 µg c-Myb plasmid allowed for a quasi "normal" levels of c-Myb after ATRA treatment. Under these conditions, resistance to ATRA was still detectable. Indeed, while ATRA treatment resulted in a 10.6-fold decrease of the MBN promoter in control cells, a 2.9-fold and 2.4-fold decrease was observed with the same treatment in cells transfected with 1 and 2 µg c-Myb plasmid, respectively (Figure 3Bi). Resistance to ATRA was therefore likely due to exogenous c-Myb expression, which resulted in increased MBN promoter activity. In both untreated and ATRA-treated cells, induction of the MBN promoter activity by c-Myb overexpression and resistance to ATRA were dependent upon the integrity of the Myb binding site (Figure 3Bi). This strongly suggests that the binding of Myb to the MBN promoter is required for its regulation by ATRA. Notably, overexpression of c-Myb resulted in a 3.6-fold induction of the endogenous MBN mRNA (Figure 3Bii).
We then analyzed whether changes in PU.1 and C/EBP binding to their
sites could be responsible for ATRA-induced MBN down-regulation. For
this, EMSAs were conducted out with PLB-985 cell nuclear extracts using
the PU.1 or the C/EBP probe (Figure 3Ci,ii, respectively). ATRA-induced
growth arrest and differentiation of PLB-985 cells were accompanied by
a progressive increased binding to both the PU.1 (Figure 3Ci, left) and
C/EBP (Figure 3Cii, left) probes. Competition assays attested the
specificity of the resulting DNA-protein complexes (Figure 3Ci,ii,
right). The PU.1-retarded complex was specifically supershifted by PU.1
antibodies (Figure 3Ci, right). Incubation with anti-C/EBP
MBN is a myeloid growth-related gene.6,11 Although PU.1, C/EBP, and Myb transcription factors were all critical for constitutive expression of MBN, its down-regulation in leukemia cells growth-arrested by ATRA was dependent upon Myb. This and the fact that overexpression of c-Myb in leukemia cells inhibited down-regulation of MBN by ATRA point to MBN as a novel target of Myb in the context of proliferation. The mechanism by which myeloid-specific genes can be expressed in
immature myeloid cells has been exemplified by the study of neutrophil
elastase, which is regulated as a result of a combinatorial activation
by at least 3 factors, c-Myb, PU.1, and C/EBP, none of which by itself
is myeloid-specific.34 PU.1 and C/EBP are involved in
myeloid-specific gene expression.23,39 The fact that
introduction of mutations in the PU.1 and C/EBP sites of the MBN
promoter resulted in a drastic decrease in its constitutive transcriptional activity is consistent with the observation that MBN
mRNA was markedly reduced in vivo in fetal liver of PU.1 and C/EBP Studies of few genes regulated by Myb have indicated that it frequently acts in cooperation with other transcription factors. Indeed, our finding that functional PU.1, C/EBP, and Myb binding sites exist in the MBN promoter is reminiscent of previous observations with the neutrophil elastase34 and myeloperoxidase promoters. Our data are in agreement with previous work32 that established the importance of PU.1 for MBN promoter activity. However, our results contrast with previous indications32 that C/EBP and c-Myb were unlikely to play a significant role in the expression of MBN. Furthermore, it is of interest that PU.1 appeared to be involved in the regulation of MBN by phorbol myristate acetate (PMA), which decreased PU.1 binding to its site.32 Although there was no evidence that overexpression or change in phosphorylation status of PU.1 would affect PMA-induced MBN transcriptional inhibition, these results are consistent with our observation that the PU.1 site is critical to MBN constitutive activity. Our results indicate that Myb specifically regulates MBN transcriptional inhibition by ATRA. We also found that, in contrast to PMA,32 ATRA up-regulates PU.1 binding to its site though overexpression of PU.1 could not abolish the negative effect of ATRA on MBN promoter activity. Altogether, previous work conducted with PMA32 and our present data suggest that PMA and ATRA down-regulate MBN promoter activity by affecting specific bindings to 2 different sites that are each critical to their constitutive transcriptional expression. Notably, overexpression of PU.1 resulted in a slight decrease in the MBN promoter activity. In PLB-985 cells, we have observed an increased PU.1 binding to its site upon ATRA treatment. In these cells, overexpression of PU.1 appeared to transrepress MBN promoter activity. Therefore, a transrepressor role for PU.1 may contribute to the down-regulation of MBN expression via inhibition of c-Myb transcription during myeloid differentiation.49 Cooperation between myeloid transcription factors for maximal promoter
transactivation has been demonstrated.34,50,51 Recruitment
of CBP, which has a strong histone acetyltransferase activity, by c-Myb
and NF-M potentiated their transcriptional activities by bridging these
2 proteins.35 Our data show that C/EBP It is assumed that leukemic cells descend from a small pool of progenitors with high proliferative activity.52 This suggests that genes that are involved in controlling limited factor-dependent growth in normal hematopoietic progenitors might be continuously expressed, providing factor-independent growth to preleukemic cells. Such genes might be inhibited de novo when leukemia cells are forced into growth arrest by inducers such as ATRA. Our data provide a way by which ATRA can access a myeloid transcriptional machinery in which Myb is instrumental. Myb is known for its pivotal role in controlling cell growth and may therefore provide a way for ATRA to control proliferation in myeloid leukemia cells. Few Myb target genes are known to be linked to proliferation, and little is known about Myb involvement in growth inhibition induced by ATRA in leukemia cells. The fact that MBN, which can confer factor-independent growth to hematopoietic cells,11 is a target of Myb suggests a mechanism by which growth arrest may be induced by ATRA in myeloid leukemia cells.
We thank Drs A. Friedman, B. Lüscher, R. Maki, and P. Whyte for generous gifts of plasmids.
Submitted August 4, 2000; accepted December 21, 2000.
Supported by INSERM and grants from the Association Pour la Recherche sur le Cancer, the Lady Tata Memorial Trust, and the Ligue Nationale Contre le Cancer.
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: Yvon E. Cayre, Unité INSERM U417, Hôpital Saint Antoine, 184 Rue du Faubourg Saint Antoine, 75012 Paris, France; e-mail: cayre{at}st-antoine.inserm.fr.
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A. Burchert, S. Wolfl, M. Schmidt, C. Brendel, B. Denecke, D. Cai, L. Odyvanova, T. Lahaye, M. C. Muller, T. Berg, et al. Interferon-alpha , but not the ABL-kinase inhibitor imatinib (STI571), induces expression of myeloblastin and a specific T-cell response in chronic myeloid leukemia Blood, January 1, 2003; 101(1): 259 - 264. [Abstract] [Full Text] [PDF]
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S. M. Luger, S. G. O'Brien, J. Ratajczak, M. Z. Ratajczak, R. Mick, E. A. Stadtmauer, P. C. Nowell, J. M. Goldman, and A. M. Gewirtz Oligodeoxynucleotide-mediated inhibition of c-myb gene expression in autografted bone marrow: a pilot study Blood, February 15, 2002; 99(4): 1150 - 1158. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
6 M. Differentiation was
assessed by the percentage of cells with cell-associated nitroblue
tetrazolium (Sigma) and cell morphology under light microscopy on
May-Grünwald-Giemsa-stained cytospin slides.
) and ATRA-treated (+ATRA, 
) cells. CAT
activities presented for each pMBN construct were calculated relative
to the pMBN-658. On the right are calculated 
