J Biol Chem, Vol. 273, Issue 47, 31534-31540, November 20, 1998

The Core Binding Factor (CBF)  Interaction Domain and the Smooth Muscle Myosin Heavy Chain (SMMHC) Segment of CBF-SMMHC Are Both Required to Slow Cell Proliferation^*

Wangsen Cao, Neeraj Adya, Martin Britos-Bray, P. Paul Liu, and Alan D. Friedman^§

From the Division of Pediatric Oncology, Johns Hopkins Oncology Center, Baltimore, Maryland 21287 and the NHGRI, National Institutes of Health, Bethesda, Maryland 20892

	ABSTRACT

Top Abstract Introduction Procedures Results Discussion References

We have expressed several variants of core binding factor  (CBF)-smooth muscle myosin heavy chain (SMMHC) from the metallothionein promoter in Ba/F3 cells. Deletion of amino acids 2-11 from the CBF segment, required for interaction with CBF, prevented CBF-SMMHC from inhibiting CBF DNA binding and cell cycle progression. Deletion of 283 carboxyl-terminal residues from the SMMHC domain, required for multimerization, also inactivated CBF-SMMHC. Nuclear expression of CBF(2-11)-SMMHC was decreased relative to CBF-SMMHC. CBF(2-11)-SMMHC linked to a nuclear localization signal still did not slow cell growth. The ability of each CBF-SMMHC variant to inhibit CBF DNA binding and cell proliferation correlated with its ability to inhibit transactivation by an AML1-VP16 fusion protein. Thus, CBF-SMMHC slows cell cycle progression from G₁ to S phase by inhibiting CBF DNA binding and transactivation.

	INTRODUCTION

Top Abstract Introduction Procedures Results Discussion References

The core binding factor (CBF)¹ family is made up of transcription factors that contain a common CBF subunit and one of three CBF subunits: CBF1, AML1 (CBF2), or CBF3 (1-7). CBF increases the affinity of the CBF subunits for DNA, but does not bind DNA directly (4, 7). The CBF subunits contain a domain required for heterodimerization and DNA binding (3, 8).

Translocations involving subunits of CBF are common in acute leukemias (9). Inv(16) is present in 10% of acute myeloid leukemias (AMLs) and encodes CBF-SMMHC, in which CBF is fused to the tail domain of SMMHC (10). t(8;21) is present in 12% of AMLs and encodes AML1-ETO, which includes the DNA-binding domain of AML1 (11). 25% of pediatric B-lineage acute lymphocytic leukemias contain t(12;21), which encodes TEL-AML1 (12, 13).

Each of these "CBF oncoproteins" inhibits CBF activities (14-20). Also, mice expressing CBF-SMMHC or AML1-ETO fail to develop definitive hematopoiesis, just as do mice lacking AML1 or CBF (21-26).

CBF activates the expression of several lymphoid and myeloid genes, suggesting that lack of differentiation accounts for the phenotypes of CBF null mice (27-30). We expressed CBF-SMMHC from the zinc-responsive MT promoter in 32D cl3 myeloid and Ba/F3 B-lymphoid cells (14). Induction of CBF-SMMHC resulted in decreased CBF DNA binding and slowed proliferation during G₁ phase. The differentiation of 32D cl3 cells in response to granulocyte colony-stimulating factor was unaffected. We proposed that initial genetic alterations occur during leukemogenesis that bypass the growth inhibitory effect of CBF-SMMHC, potentiating inhibition of differentiation.

We have now expressed several CBF-SMMHC mutants in Ba/F3 cells. Amino acids 1-165 of the 182-amino acid CBF protein are present in the majority of CBF-SMMHC fusion proteins, although a variant containing only residues 1-133 is present in rare patients (31). Amino acids 1-73 and 102-137 are highly conserved between Drosophila Brother and Big-brother and murine CBF (32). Segment 1-137 is sufficient to strongly increase the affinity of CBF subunits for DNA, whereas segment 1-133 or 1-132 does so only weakly (7, 31-33). Also, deletion of amino acids 1-10, 2-11, 56-94, or 95-133 disrupts binding to CBF in vitro (16, 17).² In addition, CBF-(1-141) rescues hematopoiesis in embryonic stem cells lacking CBF (34). We found that a CBF-SMMHC variant lacking amino acids 2-11 of the CBF segment did not interfere with CBF DNA binding or slow cell proliferation, whereas a variant lacking CBF amino acids 142-165 did.

The tail domain of SMMHC is -helical and consists of multiple, related 28-amino acid regions. One face of the helix is hydrophobic, allowing dimerization, and the other face has alternating positively and negatively charged zones and allows multimerization, which occurs with a 98-amino acid (3.5 repeat) stagger. In addition, SMMHC has a non-helical C terminus required for optimal multimerization (35). Human SMMHC has two isoforms (SMMHC₂₀₄ and SMMHC₂₀₀) that differ in the lengths of their non-helical C termini as a result of alternative splicing (36). Herein we refer to CBF-SMMHC₂₀₀ as INV and CBF-SMMHC₂₀₄ as INVa. INVa is more abundant than INV in M4Eo AML (37).

We demonstrate that INV and INVa have identical effects on Ba/F3 cells. Variants of INV lacking two-thirds or all of the SMMHC segment, and so incapable of oligomerization, did not affect cell growth. Also, the SMMHC segment, in isolation or linked to CBF(2-11), did not inhibit cell proliferation, even when directed to the nucleus. In addition, the ability of each INV mutant to inhibit CBF DNA binding and cell proliferation correlated with its ability to inhibit transactivation.

	EXPERIMENTAL PROCEDURES

Top Abstract Introduction Procedures Results Discussion References

Cell Culture and Transfection-- Ba/F3 cells (38) were maintained in RPMI 1640 medium with 10% heat-inactivated fetal calf serum and 1 ng/ml murine interleukin-3 (R&D Systems). CV-1 cells were maintained in Dulbecco's modified Eagle's medium with 10% calf serum. All cultures contained penicillin/streptomycin. 100 µM zinc chloride was added when indicated. Ba/F3 cells were stably transfected by electroporation as described (14). Single-cell clones were isolated by limiting dilution. Viable cell numbers were determined by enumerating cells that excluded trypan blue dye using a hemocytometer. Incorporation of tritiated thymidine to assess proliferation rate was carried out as described (14). Transient transfection of CV-1 cells and luciferase assays were carried out as described (39).

Plasmids and Oligonucleotides-- pMTINV contains the INV cDNA in the pMTCB6 vector (14). pMTINVa was prepared by ligating the INVa cDNA into pMTCB6 similarly. INV(C283), INV(2-11), CBF(165), and INV(141) cDNAs were prepared by polymerase chain reaction mutagenesis followed by DNA sequencing. The C283 coding sequence ends with 5'-CTCAGATGAAGTGA-3'. These cDNAs were used to construct pMTINV(C283), pMTINV(2-11), pMTCBF(165), and pMTINV(141), respectively. pMTINVa(2-11) was constructed by ligating a C-terminal StuI-XbaI fragment from INVa into similarly digested pMTINV(2-11). pMT-NLS-INV(2-11) was constructed by ligating an oligonucleotide obtained by annealing NLS-A (5'-GATCCGCCGCCACCATGGGGCCAAAAAAGAAGAGAAAGGTAG-3') with NLS-B (5'-AATTCTACCTTTCTCTTCTTTTTTGGCCCCATGGTGGCGGCG-3'), encoding the SV40 T-antigen nuclear localization signal, into BamHI and EcoRI polylinker sites located just upstream of the ATG initiation codon in pMTINV(2-11). The sequence just upstream of the ATG codon reads 5'-GAATTCGGGAAGATG-3', with the EcoRI site and the ATG codon underlined. pMT-NLS-INVa(2-11) was constructed similarly. pMT-HA-SMMHC was constructed by ligating an oligonucleotide obtained by annealing HA-A (5'-CATGGTTGGATACCCCTACGACGTCCCCGACTACGCCGGAGTTGGGGATC-3') with HA-B (5'-TCGAGATCCCCAACTCCGGCGTAGTCGGGGACGTCGTAGGGGTATCCAAC-3'), encoding an ATG initiation codon and the HA epitope, upstream of a C-terminal StuI-XbaI fragment from the INV cDNA in pMTINV. HA-SMMHC retains amino acids 142-165 of CBF. pJ3V-AML1-VP16 was constructed by ligating a SalI-BamHI fragment derived from pMSV-VP16 (40), encoding the VP16 transactivating domain, downstream of the SmaI site at amino acid 216 of murine AML1B, which corresponds to amino acid 189 of human AML1B. pJ3V contains the SV40 promoter. The wild-type and mutant CBF-SMMHC cDNAs were ligated into pGEM/CMV. pTKLUC and p(CBF)₄TKLUC have been described (29).

Western Blot, Gel Shift, and Immunofluorescence Analyses-- Total cellular extracts were subjected to polyacrylamide gel electrophoresis and Western blotting using CBF antiserum as described (14). Gel shift analysis and indirect immunofluorescence were carried out as described (14).

Cross-linking Analysis-- cDNAs in pGEM/CMV or pBS were transcribed and translated using the TNT reticulocyte lysate kit (Promega) in the presence of [³⁵S]methionine (Amersham Pharmacia Biotech) following the manufacturer's instructions. 3 µl of each extract was then diluted to 15 µl with phosphate-buffered saline and exposed to 0.0025% glutaraldehyde at room temperature for 1 h. The reactions were stopped by adding glycine to 192 mM and Tris (pH 6.8) to 25 mM. Samples were then subjected to SDS-polyacrylamide gel electrophoresis and autoradiography.

	RESULTS

Top Abstract Introduction Procedures Results Discussion References

CBF and SMMHC Domains Are Required for INV Activities-- The variants of INV investigated herein are diagrammed in Fig. 1. INV(C283) has a deletion of 283 residues from the 440-amino acid SMMHC segment. INV(2-11) and INVa(2-11) lack amino acids 2-11 of the CBF segment. Deletion of residues 2-11 from INV prevents heterodimerization with AML1B in vitro (16). CBF(165) contains the 165 CBF residues found in INV. Additional variants are described below.

View larger version (42K): [in this window] [in a new window]   Fig. 1.   Diagram of INV and INV variants. INV and INVa are alternatively spliced forms of CBF-SMMHC that differ at their C termini. INV(C283) lacks 283 C-terminal amino acids, which are required for multimerization. INV(2-11) and INVa(2-11) lack CBF amino acids 2-11, which are required to bind CBF subunits. CBF(165) is the CBF segment in INV. INV(141) lacks amino acids 142-165 of the CBF segment, which are dispensable for interacting with CBF subunits. NLS is the SV40 T-antigen nuclear localization signal. HA-SMMHC contains the HA epitope, 24 C-terminal CBF residues, and the entire SMMHC domain.

To verify that the intact SMMHC domain can mediate homodimerization and to determine whether INV(C283) has lost this activity, we expressed several INV variants and AML1B in reticulocyte lysates and subjected them to glutaraldehyde cross-linking (Fig. 2). INVa, INV, and INVa(2-11) contain intact SMMHC domains and dimerized efficiently. CBF, INV(C283), and AML1B did not form dimers.

View larger version (61K): [in this window] [in a new window]   Fig. 2.   Deletion of 283 C-terminal residues from the SMMHC segment of INV prevents its dimerization. The indicated proteins were synthesized in vitro in the presence of [35S]methionine. They were then incubated with (+) or without () 0.0025% glutaraldehyde (glut) for 1 h and analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. Molecular mass markers are indicated.

Clonal Ba/F3 cell lines expressing INV(C283), INV(2-11), or CBF(165) from the MT promoter were obtained by electroporation and G418 selection. Ba/F3 cells were chosen in lieu of 32D cl3 cells for these experiments as it had been much easier to obtain MTINV lines with Ba/F3 cells (14), perhaps because the MT promoter is leakier in 32D cl3 cells. In addition, the effects of INV on CBF DNA binding and cell cycle progression were identical in Ba/F3 and 32D cl3 cells (14). The MT promoter offers the advantage over other inducible systems of higher level expression, and some leakiness is tolerable when expressing a dominant-interfering protein. Expression of these three INV variants ± zinc was assessed by Western blotting (Fig. 3A). INV-3 cells express INV. Expression of each protein was stimulated by zinc to comparable levels. INV and INV(2-11) were detected at 69 kDa; INV(C283) migrated at 35 kDa; and CBF(165) migrated at 18 kDa. Endogenous CBF was evident at 22 kDa.

View larger version (64K): [in this window] [in a new window]   Fig. 3.   Mutation of the CBF interaction domain or truncation of the SMMHC segment prevents inhibition of CBF DNA binding by INV. A, vector-transfected Ba/F3 cells (CB6); a subclone expressing INV (INV-3) (14); and two subclones expressing INV(C283), INV(2-11), or CBF(165) from the MT promoter were cultured with (+) or without () zinc for 6 h. Total cellular proteins were then prepared and subjected to Western blot analysis using CBF antiserum. The position of each expressed protein and of endogenous CBF is indicated. B, nuclear extracts were prepared from CB6, INV-3, INV(C283)-1, INV(2-11)-1, and CBF(165)-1 cells, each cultured with or without zinc for 6 h. These extracts were then subjected to gel shift analysis with a CBF-binding site from the myeloperoxidase gene (upper panels) and a USF-binding site as a loading control (lower panels). Specificity of the CBF complex was confirmed by competition with 50-fold excess unlabeled wild-type (W) or mutant (M) CBF oligonucleotide (upper left panel).

The effect of each of these INV variants on DNA binding by endogenous CBF was assessed by gel shift analysis using a strong CBF-binding site present in the myeloperoxidase gene (Fig. 3B). Zinc did not affect CBF DNA binding in vector-transfected CB6 cells, but induction of INV in the INV-3 cells reduced CBF DNA binding severalfold as described (14). The specificity of the observed gel shift complex was verified by competition with unlabeled wild-type oligonucleotide or a mutant oligonucleotide that does not bind CBF (29). Induction of INV(C283), INV(2-11), or CBF(165) did not affect DNA binding by CBF. Equivalence between paired extracts was verified by gel shift assay with a USF-binding site.

We also expressed INV(141) in two Ba/F3 lines (Fig. 4, left panel). This protein contains the minimal CBF domain required for strongly binding CBF subunits and the entire SMMHC segment present in INV. Induction of INV(141) reduced CBF DNA binding (Fig. 4, right panel).

View larger version (47K): [in this window] [in a new window]   Fig. 4.   Deletion of amino acids 142-165 from the CBF segment of INV does not affect inhibition of CBF DNA binding. Two Ba/F3 subclones expressing INV(141) from the MT promoter were cultured with (+) or without () zinc for 6 h. Total cellular proteins were then prepared and subjected to Western blot analysis using CBF antiserum (left panel). These extracts were then subjected to gel shift analysis with a CBF-binding site and with a USF-binding site (right panels).

The effect of these variants on the proliferation of Ba/F3 cells was assessed by viable cell counting and by tritiated thymidine incorporation (Fig. 5). INV slowed cell proliferation as described (14), whereas INV(C283), INV(2-11), or CBF(165) did not. INV(141) slowed proliferation as effectively as INV. Results are shown for days 2 and 3, as differences were less evident during day 1. These results indicate that both the CBF and SMMHC domains of INV are required to inhibit CBF DNA binding and cell proliferation and suggest that these activities require heterodimerization via the CBF domain and homodimerization via the SMMHC domain.

View larger version (58K): [in this window] [in a new window]   Fig. 5.   Mutation of the CBF interaction domain or truncation of the SMMHC segment prevents inhibition of cell proliferation by INV. The indicated cell lines were seeded at 1-2 × 104 cells/ml with or without zinc. Viable cell numbers were enumerated daily. The cell numbers in the absence of zinc are shown relative to those in the presence of zinc on days 2 and 3 (upper panel; mean ± S.E. of two determinations). These lines were seeded similarly in a 96-well dish. Tritiated thymidine was added 24 or 48 h after zinc addition, and thymidine uptake was quantitated 18 h later, during the second or third day in culture. Thymidine uptake in the absence of zinc is shown relative to the presence of zinc (lower panel; mean ± S.E. of two determinations). Thymidine uptake in CB6 cells was 140,000 cpm, on average, during the third day.

Expression of the SMMHC Domain in the Nucleus Does Not Slow Cell Proliferation-- The subcellular localization of INV(2-11) and of several other INV variants was assessed by indirect immunofluorescence (Fig. 6). INV was detected prominently in the nucleus of Ba/F3 cells in a speckled, rod-like pattern (upper right panel) as described (14). INV(2-11) was detected most prominently in the cytoplasm (second row), as were INV(C283) and CBF(165) (third row).

View larger version (69K): [in this window] [in a new window]   Fig. 6.   Cellular localization of INV and several variants in Ba/F3 cells. Each of the indicated cell lines, expressing variants of INV, were cultured for 6 h in the presence of zinc, cytospun, and subjected to indirect immunofluorescence with CBF antiserum.

To verify that nuclear expression of the SMMHC domain does not slow cell growth, we linked the SV40 T-antigen nuclear localization signal to CBF(2-11) in the context of both SMMHC isoforms. These proteins, NLS-INV(2-11) and NLS-INVa(2-11), were expressed from the MT promoter in Ba/F3 cells. At the same time, we prepared lines expressing INVa and HA-SMMHC. The expression of these proteins in Ba/F3 cells treated with zinc was confirmed by Western blotting with CBF antiserum (Fig. 7). HA-SMMHC retains 24 CBF residues, and so could be detected with this antiserum raised against glutathione S-transferase-CBF (14).

View larger version (66K): [in this window] [in a new window]   Fig. 7.   INVa inhibits CBF DNA binding, but nuclear INV(2-11) or the SMMHC segment alone does not. A, two subclones expressing INVa, NLS-INV(2-11), NLS-INVa(2-11), or HA-SMMHC from the MT promoter were analyzed for expression of the transgene by Western blot analysis as described for Fig. 3A. The position of endogenous CBF is indicated. B, nuclear extracts were prepared from INVa-1, INVa-2, NLS-INV(2-11)-1, NLS-INVa(2-11)-1, HA-SMMHC-1, and parental Ba/F3 cells cultured with (+) and without () zinc for 6 h. These extracts were subjected to gel shift analysis for CBF and USF binding as described for Fig. 3B.

NLS-INV(2-11) was detected most abundantly in the nucleus (Fig. 6, lower left panel), although despite numerous attempts, we never achieved a speckled pattern similar to that detected with INV. Western blot analysis of nuclear and cytoplasmic fractions confirmed that the NLS directed greater than two-thirds of INV(2-11) to the nucleus.³ Strikingly, indirect immunofluorescence detected HA-SMMHC as large bright spots both in the nucleus and cytoplasm, suggesting that the CBF domain of INV limits the extent of its multimerization (Fig. 6, lower right panel).

The ability of these INV isoforms to inhibit CBF DNA binding and to slow cell proliferation was then assessed (Fig. 7B and Fig. 8). INVa inhibited CBF DNA binding and slowed cell proliferation as effectively as INV, whereas the two NLS-INV(2-11) variants were ineffective. HA-SMMHC did not reduce CBF DNA binding upon induction with zinc, relative to USF DNA binding, which was reduced in the zinc-containing extract, and which did not slow cell growth. These results support the conclusion that interaction with CBF subunits is the mechanism whereby INV slows cell proliferation.

View larger version (45K): [in this window] [in a new window]   Fig. 8.   CBF-SMMHC204 inhibits cell proliferation, but nuclear INV(2-11) or the SMMHC segment alone does not. The proliferation of Ba/F3 cells and of each of the lines analyzed in Fig. 7 was assessed as described for Fig. 5 (mean ± S.E. of two determinations).

Inhibition of Transactivation by INV Variants Correlates with Their Inhibition of Proliferation-- To assess the affect of INV variants on transactivation, we employed a reporter containing four CBF-binding sites, derived from the myeloperoxidase gene (29). An internal control was not utilized, as we observed specific inhibition of several viral promoters by INV.³ AML1B activated p(CBF)₄TKLUC 6-fold, and coexpression of INV reduced this activation 3-fold, on average. However, these modest effects did not allow us to identify differences between the INV variants.³

To develop a more reliable assay, we employed a protein containing the AML1B DNA-binding domain and the VP16 transactivating domain. AML1-VP16 did not activate pTKLUC, but activated p(CBF)₄TKLUC 15-fold (Fig. 9). pCMV-INV reproducibly inhibited activation 8-fold. INV(2-11), NLS-INV(2-11), and HA-SMMHC each mildly inhibited activation by AML1-VP16 (1.5-fold). Notably, these three variants each contain an intact SMMHC segment. INV(141) strongly inhibited activation (6-fold), and INV(C283) and CBF(165) were not inhibitory. AML1-ETO was more potent than INV, inhibiting activation 21-fold. Suppression of AML1-VP16 activation by INV and these six variants approximately paralleled their ability to inhibit cell proliferation, suggesting that inhibition of CBF transactivation is the mechanism whereby INV slows cell cycle progression.

View larger version (35K): [in this window] [in a new window]   Fig. 9.   Mutation of the CBF interaction domain or truncation of the SMMHC segment reduces inhibition of CBF transactivation by INV. 750 ng of p(CBF)4TKLUC (shaded bars) or pTKLUC (white bar) was cotransfected by lipofection into CV-1 cells with 50 ng of pJ3V-AML1-VP16 and no additional plasmid (), 50 ng of pCMV-INV, or 50 ng of pCMV expression vectors encoding the indicated INV variants. Cellular extracts were prepared 48 h later and assayed for luciferase activity. The mean ± S.E. from three determinations are shown.

	DISCUSSION

Top Abstract Introduction Procedures Results Discussion References

By expressing INV from the MT promoter in Ba/F3 and 32D cl3 cells, we provide evidence suggesting that lack of hematopoiesis in INV-expressing mice is due to inhibition of cell proliferation during G₁ phase (14). We have now extended these observations by identifying the domains of INV required for inhibition of CBF DNA binding and transactivation and for inhibition of cell proliferation. Deletion of just 10 amino acids from the CBF segment, required for interaction with CBF subunits, prevented each of these activities. Although the resulting protein, INV(2-11), might still interact with other cellular proteins, either via the CBF or SMMHC segment, these interactions were not sufficient to strongly interfere with CBF activities or to slow proliferation. Consistent with this finding, neither the CBF segment nor the SMMHC segment alone was active in these assays. On the other hand, deletion of CBF amino acids 142-165 from INV, which leaves its CBF interaction domain intact, did not prevent its inhibition of CBF DNA binding, transactivation, or proliferation.

INV was expressed most abundantly in the nucleus, whereas INV(2-11) was localized more extensively to the cytoplasm. INV has previously been shown to localize predominantly to the cytoplasm of fibroblastic cells, co-localizing with the cytoskeleton (16, 41). Perhaps increased nuclear expression of INV in hematopoietic cells results from their having less cytoskeletal structure and higher levels of CBF subunits. Using Western blotting, we also detected INV predominantly in the nuclei of M4Eo patient samples (37) and in ME-1 cells,³ which derive from an M4Eo AML (42). We sought to determine whether expressing INV(2-11) more abundantly in the nucleus allows either its CBF(2-11) or SMMHC segment to interact with nuclear proteins and so interfere with cell proliferation. We found that addition of a nuclear localization signal to its N terminus directed INV(2-11) to the nucleus, but did not result in reduced CBF DNA binding or cell growth rate.

We expressed a construct, INV(C283), lacking two-thirds of the SMMHC segment. This protein did not dimerize in vitro or interfere with CBF activities or cell proliferation in vivo. In the future, we intend to more precisely identify the regions of the SMMHC domain required for the activities of INV. Such investigations might eventually lead to the identification of relevant SMMHC-interacting proteins. Herein we demonstrate that both INV and INVa inhibit CBF activities and cell proliferation equally, indicating that their different non-helical C-terminal tails (8 or 34 amino acids) are interchangeable. Interestingly, HA-SMMHC appeared to form much larger intracellular aggregates than INV, suggesting that the CBF segment interferes with, but does not prevent, multimerization via the SMMHC domain.

INV/CBF heterodimers are capable of interacting with CBF-binding sites. INV inhibits activation of the myeloid NP-3 promoter by AML1B (43), indicating that these heterodimers are not transactivating, and we reached the same conclusion using the p(CBF)₄TKLUC reporter. Although this assay is artificial, our results suggest that interaction with CBF subunits and integrity of the SMMHC segment are both required for INV to inhibit CBF-mediated transactivation. INV might inhibit CBF transactivation by sequestering AML1B into multimeric structures away from chromatin. Alternatively, INV/CBF dimers bound to relevant genes might directly interfere with transcription. Which of these mechanisms predominates is unknown, but our recent finding that INV can redirect AML1B to the cytoplasm in NIH 3T3 cells supports the importance of the sequestration model (16).

Phenotypic differences between M2 AML associated with t(8;21) and M4Eo AML associated with inv(16) might in part result from AML1-ETO being a more potent inhibitor of CBF transactivation than INV. AML1-ETO slowed 32D cl3 cell myeloid differentiation, whereas INV did not (14, 43, 44), suggesting that cell proliferation is more sensitive than differentiation to inhibition of CBF activities. In the context of additional genetic hits, inhibition of differentiation might become more evident. Also, we recently found that expression of INV or AML1-ETO in Ba/F3 cells reduces induction of p53 when the cells are treated with DNA-damaging agents (45). This effect was not observed with cells expressing INV(2-11). Thus, inhibition of CBF activities might prevent apoptosis and encourage leukemic progression in premalignant cells. Finally, although we have demonstrated that inhibition of CBF activities accounts for inhibition of cell proliferation by CBF-SMMHC, interactions of the CBF or SMMHC segment with other proteins might contribute to transformation and to the phenotype of M4Eo AML.

	ACKNOWLEDGEMENTS

We thank N. A. Speck for helpful discussion; W. Leung and C. I. Civin for assistance with FACScan analysis; S. W. Hiebert for pCMV-AML1B, pCMV-AML1-ETO, and pJ3V; and K. Yanagisawa for ME-1 cells.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant HL51388.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.

Leukemia Society Special Fellow.

^§ Leukemia Society Scholar. To whom correspondence should be addressed: Johns Hopkins Oncology Center, Rm. 3-109, 600 North Wolfe St., Baltimore, MD 21287. Tel.: 410-955-2095; Fax: 410-955-8897; E-mail: adfrdman{at}jhmi.edu.

The abbreviations used are: CBF, core binding factor; SMMHC, smooth muscle myosin heavy chain; AML, acute myeloid leukemia; MT, metallothionein; NLS, nuclear localization signal; HA, hemagglutinin; CMV, cytomegalovirus; TK, thymidine kinase; LUC, luciferase; USF, upstream response factor.

² N. A. Speck, personal communication.

³ W. Cao, M. Britos-Bray, and A. D. Friedman, unpublished data.

	REFERENCES

Top Abstract Introduction Procedures Results Discussion References

1. Satake, M., Inuzuka, M., Shigesada, K., Oikawa, T., and Ito, Y. (1992) Jpn. J. Cancer Res. 83, 714-722[CrossRef][Medline] [Order article via Infotrieve]
2. Wang, S. W., and Speck, N. A. (1992) Mol. Cell. Biol. 12, 89-102[Abstract/Free Full Text]
3. Bae, S.-C., Yamaguchi-Iwai, Y., Ogawa, E., Maruyama, M., Inuzuka, M., Kagoshima, H., Shigesada, K., Satake, M., and Ito, Y. (1993) Oncogene 8, 809-814[Medline] [Order article via Infotrieve]
4. Wang, S., Wang, Q., Crute, B. E., Melnikova, I. N., Keller, S. R., and Speck, N. A. (1993) Mol. Cell. Biol. 13, 3324-3339[Abstract/Free Full Text]
5. Ogawa, E., Maruyama, M., Kagoshima, H., Inuzuka, M., Lu, J., Satake, M., Shigesada, K., and Ito, Y. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 6859-6863[Abstract/Free Full Text]
6. Ogawa, E., Inuzuka, M., Maruyamna, M., Satake, M., Naito-Fujimoto, M., Ito, Y., and Shigesada, K. (1993) Virology 194, 314-331[CrossRef][Medline] [Order article via Infotrieve]
7. Levanon, D., Negreanu, V., Bernstein, Y., Bar-Am, I., Avivi, L., and Groner, Y. (1994) Genomics 23, 425-432[CrossRef][Medline] [Order article via Infotrieve]
8. Meyers, S., Downing, J. R., and Hiebert, S. W. (1993) Mol. Cell. Biol. 13, 6336-6345[Abstract/Free Full Text]
9. Look, A. T. (1997) Science 278, 1059-1064[Abstract/Free Full Text]
10. Liu, P., Tarle, S. A., Hajre, A., Claxton, D. F., Marlton, P., Freedman, M., Siciliano, M. J., and Collins, F. S. (1993) Science 261, 1041-1044
11. Miyoshi, H., Kozu, T., Shimizu, K., Enomoto, K., Maseki, N., Kaneko, Y., Kamada, N., and Ohki, M. (1993) EMBO J. 12, 2715-2721[Medline] [Order article via Infotrieve]
12. Golub, T. R., Barker, G. F., Bohlander, S. K., Hiebert, S. W., Ward, D. C., Bray-Ward, P., Morgan, E., Raimondi, S. C., Rowley, J. D., and Gilliland, D. G. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 4917-4922[Abstract/Free Full Text]
13. Romana, S. P., Mauchauffe, M., Le Coniat, M., Chumakov, I., Le Paslier, D., Berger, R., and Bernard, O. A. (1995) Blood 85, 3662-3670[Abstract/Free Full Text]
14. Cao, W., Britos-Bray, M., Claxton, D. F., Kelley, C. A., Speck, N. A., Liu, P. P., and Friedman, A. D. (1997) Oncogene 15, 1315-1327[CrossRef][Medline] [Order article via Infotrieve]
15. Liu, P., Seidel, N., Bodine, D., Speck, N., Tarle, S., and Collin, F. S. (1994) Cold Spring Harbor Symp. Quant. Biol. 59, 547-553[Medline] [Order article via Infotrieve]
16. Adya, N., Stacy, T., Speck, N. A., and Liu, P. P. (1998) Mol. Cell. Biol., in press
17. Wijmenga, C., Gregory, P. E., Hajra, A., Schrock, E., Ried, T., Eils, T., Liu, P. P., and Collins, F. S. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 1630-1635[Abstract/Free Full Text]
18. Meyers, S., Lenny, N., and Hiebert, S. W. (1995) Mol. Cell. Biol. 15, 1974-1982[Abstract]
19. Frank, R., Zhang, J., Uchida, H., Meyers, S., Hiebert, S. W., and Nimer, S. D. (1995) Oncogene 11, 2667-2674[Medline] [Order article via Infotrieve]
20. Hiebert, S. W., Sun, W., Davis, J. N., Golub, T., Shurtleff, S., Buijs, A., Downing, J. R., Grosveld, G., Roussel, M. F., Gilliland, D. G., Lenny, N., and Meyers, S. (1996) Mol. Cell. Biol. 16, 1349-1355[Abstract]
21. Castilla, L. H., Wijmenga, C., Stacy, T., Speck, N. A., Eckhaus, M., Marin-Padilla, M., Collins, F. S., Wynshaw-Boris, A., and Liu, P. P. (1996) Cell 87, 687-696[CrossRef][Medline] [Order article via Infotrieve]
22. Okuda, T., van Deursen, J., Hiebert, S. W., Grosveld, G., and Downing, J. R. (1996) Cell 84, 321-330[CrossRef][Medline] [Order article via Infotrieve]
23. Sasaki, K., Yagi, H., Bronson, R. T., Tominaga, K., Matsunashi, T., Deguchi, K., Tani, Y., Kishimoto, T., and Komori, T. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 12359-12363[Abstract/Free Full Text]
24. Wang, Q., Stacy, T., Binder, M., Marin-Padilla, M., Sharpe, A. H., and Speck, N. A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 3444-3449[Abstract/Free Full Text]
25. Wang, Q., Stacy, T., Miller, J. D., Lewis, A. F., Gu, T.-L., Huang, X., Bushweller, J. H., Bories, J.-C., Alt, F. W., Ryan, G., Liu, P. P., Wynshaw-Boris, A., Binder, M., Marin-Padilla, M., Sharpe, A. H., and Speck, N. A. (1996) Cell 87, 697-708[CrossRef][Medline] [Order article via Infotrieve]
26. Yergeau, D. A., Hetherington, A. C., Wang, Q., Zhang, P., Sharpe, A. H., Binder, M., Marin-Padilla, M., Tenen, D. G., Speck, N. A., and Zhang, D.-E. (1997) Nat. Genet. 15, 303-306[CrossRef][Medline] [Order article via Infotrieve]
27. Nuchprayoon, I., Meyers, S., Scott, L. M., Suzow, J., Hiebert, S., and Friedman, A. D. (1994) Mol. Cell. Biol. 14, 5558-5568[Abstract/Free Full Text]
28. Redondo, J. M., Pfohl, J. L., Hernandez-Munain, C., Wang, S., Speck, N. A., and Krangel, M. S. (1992) Mol. Cell. Biol. 12, 4817-4823[Abstract/Free Full Text]
29. Suzow, J., and Friedman, A. D. (1993) Mol. Cell. Biol. 13, 2141-2151[Abstract/Free Full Text]
30. Zhang, D.-E., Fujioka, K.-I., Hetherington, C. J., Shapiro, L. H., Chen, H.-M., Look, A. T., and Tenen, D. G. (1994) Mol. Cell. Biol. 14, 8085-8095[Abstract/Free Full Text]
31. Shurtleff, S. A., Meyers, S., Hiebert, S. W., Raimondi, S. C., Head, D. R., Willman, C. L., Wolman, S., Slovak, M. L., Carroll, A. J., Behm, F., Hulshof, M. G., Motroni, T. A., Okuda, T., Liu, P., Collins, F. S., and Downing, J. R. (1995) Blood 85, 3695-3703[Abstract/Free Full Text]
32. Golling, G., Li, L.-H., Pepling, M., Stebbins, M., and Gergen, J. P. (1996) Mol. Cell. Biol. 16, 932-942[Abstract]
33. Kagoshima, H., Akamatsu, Y., Ito, Y., and Shigesada, K. (1996) J. Biol. Chem. 271, 33074-33082[Abstract/Free Full Text]
34. Miller, J. D., Stacy, T., Bushweller, J. H., and Speck, N. A. (1997) Blood 90, 41a (abstr.)
35. Hodge, T. P., Cross, R., and Kendrick-Jones, J. (1992) J. Cell Biol. 118, 1085-1095[Abstract/Free Full Text]
36. Nagai, R., Kuro-o, M., Babij, P., and Periasamy, M. (1989) J. Biol. Chem. 264, 9734-9737[Abstract/Free Full Text]
37. Liu, P. P., Wijmenga, C., Hajra, A., Blake, T. B., Kelley, C. A., Adelstein, R. S., Baggf, A., Rector, J., Cotelingam, J., Willman, C. L., and Collins, F. S. (1996) Genes Chromosomes Cancer 16, 77-87[CrossRef][Medline] [Order article via Infotrieve]
38. Palacios, R., and Steinmetz, M. (1985) Cell 41, 727-734[CrossRef][Medline] [Order article via Infotrieve]
39. Britos-Bray, M., and Friedman, A. D. (1997) Mol. Cell. Biol. 17, 5127-5135[Abstract]
40. Triezenberg, S. J., Kingsbury, R. C., and McKnight, S. L. (1988) Genes Dev. 2, 718-729[Abstract/Free Full Text]
41. Lu, J., Maruyama, M., Satake, M., Bae, S.-C., Ogawa, E., Kagoshima, H., Shigesada, K., and Ito, Y. (1995) Mol. Cell. Biol. 15, 1651-1661[Abstract]
42. Yanagisawa, K., Horiuch, T., and Fujita, S. (1991) Blood 78, 451-457[Abstract/Free Full Text]
43. Westendorf, J. J., Yamamoto, C. M., Lenny, N., Downing, J. R., Selsted, M. E., and Hiebert, S. W. (1998) Mol. Cell. Biol. 18, 322-333[Abstract/Free Full Text]
44. Ahn, M.-Y., Huang, G., Bae, S.-C., Wee, H.-J., Kim, W.-Y., and Ito, Y. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 1812-1817[Abstract/Free Full Text]
45. Britos-Bray, M., Ramirez, M., Cao, W., Liu, P., Wang, X., Civin, C. I., and Friedman, A. D. (1998) Blood, in press