Clinical Focus

  • Plasmacytoma
  • Multiple Myeloma - Medical Oncology
  • Hematology
  • Cancer > Hematology
  • Plasmacytoma - Hematology
  • Plasmacytoma - Medical Oncology
  • Multiple Myeloma

Academic Appointments

Administrative Appointments

  • Chief, Division of Hematology, Stanford University School of Medicine (2004 - Present)

Professional Education

  • Fellowship:Stanford University School of Medicine (1990) CA
  • Fellowship:Stanford University School of Medicine (1989) CA
  • Residency:Stanford University School of Medicine (1984) CA
  • Internship:Stanford University School of Medicine (1982) CA
  • Board Certification: Medical Oncology, American Board of Internal Medicine (1987)
  • Board Certification: Internal Medicine, American Board of Internal Medicine (1984)
  • Medical Education:Stanford University School of Medicine (1981) CA
  • MD, Stanford University, Medicine (1981)
  • PhD, Stanford University, Biophysics (1981)

Research & Scholarship

Current Research and Scholarly Interests

We are studying the transcriptional deregulation of several oncogenes in human hematologic malignancies.

Activation of c-myc in Burkitt's lymphoma: We are examining the transcriptional deregulation of the translocated c-myc gene and the silencing of the normal c-myc gene in Burkitt's lymphoma. We are looking at the c-myc promoter and regions of the immunoglobulin locus that are responsible for the deregulation of the translocated c-myc gene. Several model systems have been designed.

Activation of bcl-2 in hematologic malignancies: In lymphomas with the t(14;18) translocation, the bcl-2 gene is translocated to the immunoglobulin locus and expressed at high levels. We are studying the mechanism of activation at a molecular level in human lymphoma tissue. We have also developed a model system to determine which region of the immunoglobulin locus is responsible for the activation of the translocated bcl-2 gene.

Expression profiling in ALL: We are examining gene expression profiles in acute lymphoblastic leukemia in response to chemotherapeutic agents.


2013-14 Courses

Postdoctoral Advisees

Graduate and Fellowship Programs


Journal Articles

  • Clinical interpretation and implications of whole-genome sequencing. JAMA : the journal of the American Medical Association Dewey, F. E., Grove, M. E., Pan, C., Goldstein, B. A., Bernstein, J. A., Chaib, H., Merker, J. D., Goldfeder, R. L., Enns, G. M., David, S. P., Pakdaman, N., Ormond, K. E., Caleshu, C., Kingham, K., Klein, T. E., Whirl-Carrillo, M., Sakamoto, K., Wheeler, M. T., Butte, A. J., Ford, J. M., Boxer, L., Ioannidis, J. P., Yeung, A. C., Altman, R. B., Assimes, T. L., Snyder, M., Ashley, E. A., Quertermous, T. 2014; 311 (10): 1035-1045


    Whole-genome sequencing (WGS) is increasingly applied in clinical medicine and is expected to uncover clinically significant findings regardless of sequencing indication.To examine coverage and concordance of clinically relevant genetic variation provided by WGS technologies; to quantitate inherited disease risk and pharmacogenomic findings in WGS data and resources required for their discovery and interpretation; and to evaluate clinical action prompted by WGS findings.An exploratory study of 12 adult participants recruited at Stanford University Medical Center who underwent WGS between November 2011 and March 2012. A multidisciplinary team reviewed all potentially reportable genetic findings. Five physicians proposed initial clinical follow-up based on the genetic findings.Genome coverage and sequencing platform concordance in different categories of genetic disease risk, person-hours spent curating candidate disease-risk variants, interpretation agreement between trained curators and disease genetics databases, burden of inherited disease risk and pharmacogenomic findings, and burden and interrater agreement of proposed clinical follow-up.Depending on sequencing platform, 10% to 19% of inherited disease genes were not covered to accepted standards for single nucleotide variant discovery. Genotype concordance was high for previously described single nucleotide genetic variants (99%-100%) but low for small insertion/deletion variants (53%-59%). Curation of 90 to 127 genetic variants in each participant required a median of 54 minutes (range, 5-223 minutes) per genetic variant, resulted in moderate classification agreement between professionals (Gross κ, 0.52; 95% CI, 0.40-0.64), and reclassified 69% of genetic variants cataloged as disease causing in mutation databases to variants of uncertain or lesser significance. Two to 6 personal disease-risk findings were discovered in each participant, including 1 frameshift deletion in the BRCA1 gene implicated in hereditary breast and ovarian cancer. Physician review of sequencing findings prompted consideration of a median of 1 to 3 initial diagnostic tests and referrals per participant, with fair interrater agreement about the suitability of WGS findings for clinical follow-up (Fleiss κ, 0.24; P < 001).In this exploratory study of 12 volunteer adults, the use of WGS was associated with incomplete coverage of inherited disease genes, low reproducibility of detection of genetic variation with the highest potential clinical effects, and uncertainty about clinically reportable findings. In certain cases, WGS will identify clinically actionable genetic variants warranting early medical intervention. These issues should be considered when determining the role of WGS in clinical medicine.

    View details for DOI 10.1001/jama.2014.1717

    View details for PubMedID 24618965

  • The immunoglobulin heavy chain gene 3 ' enhancers induce Bcl2 deregulation and lymphomagenesis in murine B cells LEUKEMIA Xiang, H., Noonan, E. J., Wang, J., Duan, H., Ma, L., Michie, S., Boxer, L. M. 2011; 25 (9): 1484-1493


    Human follicular B-cell lymphoma is associated with the t(14;18) chromosomal translocation that juxtaposes the Bcl2 proto-oncogene with the immunoglobulin heavy chain (Igh) locus, resulting in the deregulated expression of Bcl2. Our previous studies have shown that the Igh 3' enhancers deregulate the Bcl2 expression in vitro. However, the effects of the Igh 3' enhancer elements on Bcl2 expression in vivo are not known. To investigate the role of the Igh 3' enhancers in Bcl2 deregulation, we used gene targeting to generate knock-in mice in which four DNase I-hypersensitive regions from the murine Igh 3' region were integrated 3' of the Bcl2 locus. Increased levels of Bcl2 mRNA and protein were observed in the B cells of Igh-3'E-bcl2 mice. B cells from Igh-3'E-bcl2 mice showed an extended survival in vitro compared with B cells from wild-type (Wt) mice. The Bcl2 promoter shift from P1 (the 5' promoter) to P2 (the 3' promoter) was observed in B cells from Igh-3'E-bcl2 mice, similar to human t(14;18) lymphomas. The IgH-3'E-bcl2 mice developed monoclonal B-cell follicular lymphomas, which were slowly progressive. These studies show that the Igh 3' enhancers have an important role in the deregulation of Bcl2 and B-cell lymphomagenesis in vivo.

    View details for DOI 10.1038/leu.2011.115

    View details for Web of Science ID 000294665400014

    View details for PubMedID 21606958

  • Functional long-range interactions of the IgH 3 ' enhancers with the bcl-2 promoter region in t(14;18) lymphoma cells ONCOGENE Duan, H., Xiang, H., Ma, L., Boxer, L. M. 2008; 27 (53): 6720-6728


    To better understand the mechanisms underlying the role of the immunoglobulin heavy-chain gene (IgH) 3' enhancers on bcl-2 transcriptional deregulation in t(14;18) lymphoma, we characterized the physical interactions of the IgH 3' enhancer region with the bcl-2 promoters. Using the chromosome conformation capture technique, we found that the IgH 3' enhancers physically interact with the bcl-2 promoter region over a 350 kb genomic region in t(14;18) lymphoma cells. No interactions of the bcl-2 promoter region with sequences distant to the IgH enhancers were observed. The physical interactions of the IgH enhancers with the bcl-2 5' region are functionally involved in the transcriptional control of bcl-2. The histone deacetylase inhibitor, trichostatin A, repressed bcl-2 transcription and decreased the IgH enhancer-bcl-2 promoter region interactions. We showed by chromatin immunoprecipitation assay and small interference RNA transfection studies that the POU2 family transcription factor Oct-2 and its cofactor Bob-1 have an important function in mediating the IgH enhancer-bcl-2 promoter region interactions. This study reveals a new aspect of the regulatory role of the IgH 3' enhancers on bcl-2 transcription in t(14;18) lymphomas.

    View details for DOI 10.1038/onc.2008.286

    View details for Web of Science ID 000260866200002

    View details for PubMedID 18695675

  • Protein/DNA arrays identify nitric oxide-regulated cis-element and trans-factor activities some of which govern neuroblastoma cell viability NUCLEIC ACIDS RESEARCH Dhakshinamoorthy, S., Sridharan, S. R., Li, L., Ng, P. Y., Boxer, L. M., Porter, A. G. 2007; 35 (16): 5439-5451


    Toxic nitric oxide (NO) levels can regulate gene expression. Using a novel protein/DNA array, we show that toxic NO levels regulate the binding of trans-factors to various cis-elements in neuroblastoma cells, including CRE and those recognized by the transcription factors AP1, AP2, Brn-3a, EGR, E2F1 and SP1. Functionality of some of the cis-elements was confirmed by electro mobility shift and reporter assays. Interestingly, CREB, AP-1, Brn-3a, EGR and E2F1 can control mammalian cell viability. NO induced the anti-apoptotic Bcl-2 protein and its mRNA prior to the onset of death of 30-60% of the cells. Promoter analysis of the bcl-2 gene confirmed the involvement of a CRE in NO-dependent bcl-2 transcription. Neuroblastoma cells over-expressing bcl-2 became much more resistant to NO-induced apoptosis; conversely, Bcl-2 knockdown cells were rendered markedly more sensitive to NO. Together these results suggest that Bcl-2 counteracts NO-induced apoptosis in a fraction of the cell population. Thus, NO stimulates the binding of many trans-factors to their cognate cis-elements, some of which can regulate cell viability through transcriptional activation of target genes. Our results emphasize that a DNA/protein array approach can reveal novel, global transcription factor activities stimulated by cell death-regulating molecules.

    View details for DOI 10.1093/nar/gkm594

    View details for Web of Science ID 000250683300017

    View details for PubMedID 17702766

  • The immunoglobulin heavy-chain gene 3 ' enhancers deregulate bcl-2 promoter usage in t(14;18) lymphoma cells ONCOGENE Duan, H., Heckman, C. A., Boxer, L. M. 2007; 26 (18): 2635-2641


    In t(14;18) lymphomas, bcl-2 is juxtaposed to the immunoglobulin heavy-chain gene (IgH), resulting in increased bcl-2 transcription and resistance to apoptosis. Regulatory elements of both the bcl-2 promoter and the IgH enhancers are believed to play a role in the increased expression of bcl-2 in t(14;18) lymphoma cells. In addition, transcription of the translocated bcl-2 allele is deregulated with activation of the normally minor bcl-2 P2 promoter. The mechanisms involved in the promoter shift from P1 to P2 are not known. We found that the murine IgH 3' enhancers increased bcl-2 P2 promoter activity in an episomal model of the translocation, and IgH enhancer region HS12 had the greatest effect. Quantitative chromatin immunoprecipitation (ChIP) assays revealed that localized histone H3 hyperacetylation of the P2 promoter was observed on the translocated allele in t(14;18) DHL-4 cells and also on the stably transfected bcl-2 promoter-IgH enhancer episomal construct. Analysis of the HS12 enhancer region revealed that a previously identified nuclear factor-kappaB (NF-kappaB) site and a previously uncharacterized downstream Cdx site, both of which are conserved in the human and murine IgH enhancers, were important for its enhancer activity and promoter activation. ChIP assays showed that C/EBPbeta bound to the HS12 Cdx site in vivo, and mutation of this site abrogated the binding of C/EBPbeta. Reduced expression of C/EBPbeta by transfection of small interfering RNA or interference with NF-kappaB activity decreased transcription from the bcl-2 promoters. These results demonstrate that the IgH 3' enhancers, particularly HS12, are important for the deregulation of bcl-2 promoter usage in t(14;18) lymphomas.

    View details for DOI 10.1038/sj.onc.1210061

    View details for Web of Science ID 000245831200010

    View details for PubMedID 17043638

  • Activation of the c-myc p1 promoter in Burkitt's lymphoma by the hs3 immunoglobulin heavy-chain gene enhancer LEUKEMIA Hu, H., Kanda, K., Zhang, L., Boxer, L. M. 2007; 21 (4): 747-753


    The expression of c-myc is deregulated in Burkitt's lymphoma by the translocation t(8;14). Most of the increased c-myc expression is from the P1 promoter, which is normally a minor promoter. How the P1 promoter is activated by the immunoglobulin heavy chain gene enhancers is not understood. We identified a YY1 site in the immunoglobulin heavy-chain gene HS3 enhancer, which increased c-myc P1 promoter activity, and a MARE site, which decreased c-myc P1 activity. Small Maf proteins bound to the MARE site both in vitro and in vivo, recruited histone deacetylase 2, and resulted in deacetylation of histones H3 and H4 at the c-myc promoter region. In contrast, YY1 recruited CBP and increased histone acetylation at the c-myc promoter. Rb interacts with YY1 to prevent DNA binding in normal B cells, but no significant interaction with YY1 was detected in Burkitt's cells, and binding of YY1 to the HS3 enhancer was observed by chromatin immunoprecipitaton. Increased expression of MafK and/or decreased expression of YY1 by silencing RNA downregulated endogenous c-myc mRNA levels and increased the sensitivity of the cells to doxorubicin. Mutation of the major active sites (nuclear factor-kappa B and YY1) in the enhancers prevented c-myc activation.

    View details for DOI 10.1038/sj.leu.2404583

    View details for Web of Science ID 000245117600022

    View details for PubMedID 17287852

  • Artificial zinc(II) complexes regulate cell cycle and apoptosis-related genes in tumor cell lines CHEMBIOCHEM Gao, J., Liu, Y., Zhou, Y., Boxer, L. M., Woolley, F. R., Zingaro, R. A. 2007; 8 (3): 332-340


    Various proteins involved in transcriptional regulation possess highly selective DNA-binding domains, known as zinc fingers. However, little is known about small-molecule zinc(II) complexes in the regulation of gene expression and programmed cell death. A new family of zinc(II) complexes is reported, which might be useful against human cancer cells. By using template synthesis and in vitro cell-line screening, a set of zinc(II) complexes has been found to induce apoptosis of cancer cells and display single-reagent in vitro cytotoxicity. The method used to synthesize the molecules resulted in "built-in" luminescent behavior. Confocal optical imaging clearly demonstrated penetration through the cell membrane by these metal complexes. We have discovered that C3, the meso-zinc(II) complex is an extremely efficient regulator of the cell cycle and anti-apoptosis genes bcl-2 and bcl-xL. This study provides a new insight into the development of zinc(II) complexes as potential drugs.

    View details for DOI 10.1002/cbic.200600299

    View details for Web of Science ID 000244440100010

    View details for PubMedID 17203500

  • Role of the cyclic AMP response element in the bcl-2 promoter in the regulation of endogenous Bcl-2 expression and apoptosis in murine B cells MOLECULAR AND CELLULAR BIOLOGY Xiang, H., Wang, J., Boxer, L. M. 2006; 26 (22): 8599-8606


    We have previously shown for B-cell lines that the cyclic AMP response element (CRE) is a major positive regulatory site in the bcl-2 promoter. However, the role of the CRE in the regulation of endogenous bcl-2 expression in vivo has not been characterized. We used gene targeting to generate knock-in mice in which a mutated CRE was introduced into the bcl-2 promoter region (mutCRE-bcl2 mice). Quantitative chromatin immunoprecipitation assays revealed that mutation of the CRE abolished the binding of CREB/ATF and CBP transcription factors to the bcl-2 promoter and greatly diminished the binding of NF-kappaB factors. The mutant CRE significantly reduced the expression of Bcl-2 in B cells and rendered them susceptible to surface immunoglobulin- and chemotherapeutic agent-induced apoptosis. The low levels of Bcl-2 were not changed with activation of the cells. The numbers of pre-B, immature B, and mature B cells in the bone marrow were decreased, as were the numbers of splenic B cells in mutCRE-bcl2 mice. Our findings indicate that the CRE in the bcl-2 promoter has an important functional role in the regulation of endogenous Bcl-2 expression and plays a critical role in the coordination of signals that regulate B-cell survival.

    View details for DOI 10.1128/MCB.01062-06

    View details for Web of Science ID 000241926100033

    View details for PubMedID 16982684

  • Transcription factor gata4 regulates cardiac BCL2 gene expression in vitro and in vivo. FASEB journal Kobayashi, S., Lackey, T., Huang, Y., Bisping, E., Pu, W. T., Boxer, L. M., Liang, Q. 2006; 20 (6): 800-802


    The transcription factor GATA-4 protects cardiomyocytes against doxorubicin-induced cardiotoxicity. Here, we report the identification of Bcl2 as a direct target gene of GATA4 that may mediate the prosurvival function of GATA4 in cardiomyocytes. Bcl2 transcript and protein levels were reduced by doxorubicin in neonatal rat ventricular cardiomyocytes (NRVC) and in mouse heart as determined by RT-PCR and Western blot analysis. The reduction in Bcl2 was prevented by overexpression of GATA4 in NRVC and in transgenic mouse heart. Also, expression of GATA4 increased baseline Bcl2 levels by 30% in NRVC and 2.7-fold in transgenic heart, indicating the sufficiency of GATA4 to up-regulate Bcl2 gene expression. GATA4 knockdown by siRNA reduced Bcl2 levels by 48% in NRVC, suggesting that GATA4 is required for Bcl2 constitutive gene expression. Transfection of HEK cells with GATA4 plasmids activated Bcl2 promoter and elevated Bcl2 protein levels. Deletion and mutagenesis analysis revealed that a consensus GATA motif at base -266 on the promoter conserved across multiple species is partially responsible for the promoter activity. Electrophoretic mobility shift and chromatin immunoprecipitation assays demonstrate that GATA4 directly bound to this GATA site. Together, these results indicate that GATA4 positively regulates cardiac Bcl2 gene expression in vitro and in vivo.

    View details for PubMedID 16469847

  • Transcription factor GATA4 regulates cardiac BCL2 gene expression in vitro and in vivo FASEB JOURNAL Kobayashi, S., Lackey, T., Huang, Y., Bisping, E., Pu, W. T., Boxer, L. M., Liang, Q. 2006; 20 (2): 800-?
  • Oct transcription factors mediate t(14;18) lymphoma cell survival by directly regulating bcl-2 expression ONCOGENE Heckman, C. A., Duan, H., Garcia, P. B., Boxer, L. M. 2006; 25 (6): 888-898


    Oct-1 and Oct-2 are members of the POU homeodomain family of transcriptional regulators and are critical for normal embryonic development. Gene-targeting studies showed that Oct-1 and Oct-2 are largely dispensable for B-cell development and immunoglobulin production, although both Oct-2 and Bob-1 are required for a proper immune response and germinal center formation. In these studies, we investigated the role of Oct factors in B-cell lymphomas. Recent investigations have shown increased expression of Oct-2 and Bob-1 in lymphomas, and we observed greatly increased levels of Oct-2 in lymphoma cells with the t(14;18) translocation. Decreased expression of Oct-1, Oct-2, or Bob-1 by RNA interference resulted in apoptosis and down-regulation of bcl-2 expression. Furthermore, Oct-2 induced bcl-2 promoter activity and mediated this effect through three regions in the bcl-2 P2 promoter. Although these regions did not contain canonical octamer motifs, we observed the direct interaction of Oct-2 with all three sites both in vitro by EMSA and in vivo by chromatin immunoprecipitation assay. Moreover, by mutation analysis we found that the ability of Oct-2 to activate bcl-2 required C/EBP, Cdx, and TATA-binding sites. Oct-2, therefore, acts as a cell survival factor in t(14;18) lymphoma cells by directly activating the antiapoptotic gene bcl-2.

    View details for DOI 10.1038/sj.onc.1209127

    View details for Web of Science ID 000235212700008

    View details for PubMedID 16186795

  • CCAAT/enhancer binding protein alpha (C/EBP alpha) and C/EBP alpha myeloid oncoproteins induce Bcl-2 via interaction of their basic regions with nuclear factor-kappa B p50 MOLECULAR CANCER RESEARCH Paz-Priel, I., Cai, D. H., Wang, D. H., Kowalski, J., Blackford, A., Liu, H. T., Heckman, C. A., Gombart, A. F., Koeffler, H. P., Boxer, L. M., Friedman, A. D. 2005; 3 (10): 585-596


    The CEBPA gene is mutated in 10% of acute myeloid leukemia (AML) cases. We find that CEBPA and Bcl-2 RNA levels correlate highly in low-risk human AMLs, suggesting that inhibition of apoptosis via induction of bcl-2 by CCAAT/enhancer binding protein alpha (C/EBPalpha) or its mutant variants contributes to transformation. C/EBPalphap30, lacking a NH2-terminal transactivation domain, or C/EBPalphaLZ, carrying in-frame mutations in the leucine zipper that prevent DNA binding, induced bcl-2 in hematopoietic cell lines, and C/EBPalpha induced bcl-2 in normal murine myeloid progenitors and in the splenocytes of H2K-C/EBPalpha-Emu transgenic mice. C/EBPalpha protected Ba/F3 cells from apoptosis on interleukin-3 withdrawal but not if bcl-2 was knocked down. Remarkably, C/EBPalphaLZ oncoproteins activated the bcl-2 P2 promoter despite lack of DNA binding, and C/EBPalphap30 also activated the promoter. C/EBPalpha and the C/EBPalpha oncoproteins cooperated with nuclear factor-kappaB (NF-kappaB) p50, but not p65, to induce bcl-2 transcription. Endogenous C/EBPalpha preferentially coimmunoprecipitated with p50 versus p65 in myeloid cell extracts. Mutation of residues 297 to 302 in the C/EBPalpha basic region prevented induction of endogenous bcl-2 or the bcl-2 promoter and interaction with p50 but not p65. These findings suggest that C/EBPalpha or its mutant variants tether to a subset of NF-kappaB target genes, including Bcl-2, via p50 to facilitate gene activation and offer an explanation for preferential in-frame rather than out-of-frame mutation of the leucine zipper with sparing of the basic region in C/EBPalphaLZ oncoproteins. Targeting interaction between C/EBPalpha basic region and NF-kappaB p50 may contribute to the therapy of AML and other malignancies expressing C/EBPs.

    View details for DOI 10.1158/1541-7786.MCR-05-0111

    View details for Web of Science ID 000233027400006

    View details for PubMedID 16254192

  • Regulatory elements in the immunoglobulin heavy chain gene 3 '-enhancers induce c-myc deregulation and lymphomagenesis in murine B cells JOURNAL OF BIOLOGICAL CHEMISTRY Wang, J. H., Boxer, L. M. 2005; 280 (13): 12766-12773


    Burkitt's lymphoma is invariably associated with chromosomal translocations that juxtapose the c-myc proto-oncogene with regulatory elements of the immunoglobulin heavy (IgH) or light chain loci resulting in the deregulation of c-myc expression. However, the enhancer elements mediating c-myc deregulation in vivo remain largely unidentified. To investigate the role of the IgH 3'-enhancers in c-myc deregulation, we used gene targeting to generate knock-in mice in which four DNase I hypersensitive regions from the murine IgH 3'-region were integrated into the 5'-region of the c-myc locus. The IgH 3'-enhancers induced the up-regulation of c-myc expression specifically in B cells of IgH-3'-E-myc mice. After approximately 10 months, the mice developed a Burkitt-like B cell lymphoma with the phenotype of B220+, IgM+, and IgD(low). Analysis of immunoglobulin gene rearrangements indicated that the lymphoma cells were of clonal origin. The presence of a rapidly expanding population of B cells in the spleen and bone marrow of young knock-in mice at 2-4 months of age was observed. Premalignant splenic B cells of knock-in mice showed higher spontaneous and induced apoptosis; however, malignant B cells were more resistant to apoptosis. The p53-ARF-Mdm2 pathway was disabled in half of the lymphomas examined, in most cases through Mdm2 overexpression. Although c-myc expression was increased in premalignant B cells, the promoter shift from P2 to P1 was observed only in malignant B cells. Our studies demonstrate that the IgH 3'-enhancers play an important role in c-myc deregulation and B cell lymphomagenesis in vivo.

    View details for DOI 10.1074/jbc/M412446200

    View details for Web of Science ID 000227922000084

    View details for PubMedID 15687498

  • Histone deacetylase inhibitors down-regulate bcl-2 expression and induce apoptosis in t(14;18) lymphomas MOLECULAR AND CELLULAR BIOLOGY Duan, H., Heckman, C. A., Boxer, L. M. 2005; 25 (5): 1608-1619


    Histone deacetylase (HDAC) inhibitors are promising antitumor agents, but they have not been extensively explored in B-cell lymphomas. Many of these lymphomas have the t(14;18) translocation, which results in increased bcl-2 expression and resistance to apoptosis. In this study, we examined the effects of two structurally different HDAC inhibitors, trichostatin A (TSA) and sodium butyrate (NaB), on the cell cycle, apoptosis, and bcl-2 expression in t(14;18) lymphoma cells. We found that in addition to potent cell cycle arrest, TSA and NaB also dramatically induced apoptosis and down-regulated bcl-2 expression, and overexpression of bcl-2 inhibited TSA-induced apoptosis. The repression of bcl-2 by TSA occurred at the transcriptional level. Western blot analysis and quantitative chromatin immunoprecipitation (ChIP) assay showed that even though HDAC inhibitors increased overall acetylation of histones, localized histone H3 deacetylation occurred at both bcl-2 promoters. TSA treatment increased the acetylation of the transcription factors Sp1 and C/EBPalpha and decreased their binding as well as the binding of CBP and HDAC2 to the bcl-2 promoters. Mutation of Sp1 and C/EBPalpha binding sites reduced the TSA-induced repression of bcl-2 promoter activity. This study provides a mechanistic rationale for the use of HDAC inhibitors in the treatment of human t(14;18) lymphomas.

    View details for DOI 10.1128/MCB.25.5.1608-1619.2005

    View details for Web of Science ID 000227085700004

    View details for PubMedID 15713621

  • A genome-wide view of the in vitro response to L-asparaginase in acute lymphoblastic leukemia CANCER RESEARCH Fine, B. M., Kaspers, G. J., Ho, M., Loonen, A. H., Boxer, L. M. 2005; 65 (1): 291-299


    To investigate the effect of l-asparaginase on acute lymphoblastic leukemia (ALL), we used cDNA microarrays to obtain a genome-wide view of gene expression both at baseline and after in vitro exposure to l-asparaginase in cell lines and pediatric ALL samples. In 16 cell lines, a baseline gene expression pattern distinguished l-asparaginase sensitivity from resistance. However, for 28 pediatric ALL samples, no consistent baseline expression pattern was associated with sensitivity to l-asparaginase. In particular, baseline expression of asparagine synthetase (ASNS) was not predictive of response to l-asparaginase. After exposure to l-asparaginase, 5 cell lines and 10 clinical samples exhibited very similar changes in the expression of a large number of genes. However, the gene expression changes occurred more slowly in the clinical samples. These changes included a consistent increase in expression of tRNA synthetases and solute transporters and activating transcription factor and CCAAT/enhancer binding protein family members, a response similar to that observed with amino acid starvation. There was also a consistent decrease in many genes associated with proliferation. Taken together, the changes seem to reflect a consistent coordinated response to asparagine starvation in both cell lines and clinical samples. Importantly, in the clinical samples, increased expression of ASNS after l-asparaginase exposure was not associated with in vitro resistance to l-asparaginase, indicating that ASNS-independent mechanisms of in vitro l-asparaginase resistance are common in ALL. These results suggest that targeting particular genes involved in the response to amino acid starvation in ALL cells may provide a novel way to overcome l-asparaginase resistance.

    View details for Web of Science ID 000226080200036

    View details for PubMedID 15665306

  • Neurotrophin-3 and a CREB-mediated signaling pathway regulate Bcl-2 expression in oligodendrocyte progenitor cells JOURNAL OF NEUROCHEMISTRY Saini, H. S., Gorse, K. M., Boxer, L. M., Sato-Bigbee, C. 2004; 89 (4): 951-961


    Our previous results suggested that the transcription factor CREB mediates the actions of neuroligands and growth factor signals that coupled to different signaling pathways may play different roles along oligodendrocyte (OLG) development. We showed before that CREB phosphorylation in OLG progenitors is up-regulated by neurotrophin-3 (NT-3); and moreover CREB is required for NT-3 to stimulate the proliferation of these cells. We now show that treatment of OLG progenitors with NT-3 is also accompanied by an increase in the levels of the anti-apoptotic protein Bcl-2. Interestingly, the presence of a putative CREB binding site (CRE) in the Bcl-2 gene raised the possibility that CREB could also be involved in regulating Bcl-2 expression in the OLGs. Supporting this hypothesis, the NT-3 dependent increase in Bcl-2 levels is abolished by inhibition of CREB expression. In addition, transient transfection experiments using various regions of the Bcl-2 promoter and mutation of the CRE site indicate a direct role of CREB in regulating Bcl-2 gene activity in response to NT-3. Furthermore, protein-DNA binding assays show that the CREB protein from freshly isolated OLGs indeed binds to the Bcl-2 promoter CRE. Together with our previous results, these observations suggest that CREB may play an important role in linking proliferation and survival pathways in the OLG progenitors.

    View details for DOI 10.1111/j.1471-4159.2004.02365.x

    View details for Web of Science ID 000221544100016

    View details for PubMedID 15140194

  • Gene expression patterns associated with recurrent chromosomal translocations in acute lymphoblastic leukemia BLOOD Fine, B. M., Stanulla, M., Schrappe, M., Ho, M., Viehmann, S., HARBOTT, J., Boxer, L. M. 2004; 103 (3): 1043-1049


    We obtained a global view of gene expression in both cell lines and pediatric acute lymphoblastic leukemia (ALL) samples that harbor one of several selected chromosomal abnormalities. When the cell lines were studied alone, we found that these chromosomal abnormalities were associated with the predominant variation in transcriptional programs across the set of cell lines studied. When cell lines and clinical samples were studied together, we found that each chromosomal abnormality (TEL/AML1, BCR/ABL, or MLL abnormalities) was associated with a characteristic gene expression signature that was shared by both cell lines and clinical samples. However, BCR/ABL was associated with a much more heterogeneous pattern of expression than were TEL/AML1 and MLL abnormalities. This observation has important implications for the study of BCR/ABL ALL. In addition, we systematically identified genes whose expression was associated with TEL/AML1, BCR/ABL, or MLL abnormalities in both clinical samples and cell lines. Although some of these genes have previously been described, many have not previously been reported to be associated with one of these chromosomal abnormalities. Notably, we found that the erythropoietin receptor (EPOR) is consistently highly expressed in TEL/AML1 ALL compared with BCR/ABL or MLL.

    View details for DOI 10.1182/blood-2003-05-1518

    View details for Web of Science ID 000188393800051

    View details for PubMedID 14525776

  • Critical elements of the immunoglobulin heavy chain gene enhancers for deregulated expression of bcl-2 CANCER RESEARCH Heckman, C. A., Cao, T., Somsouk, L., Duan, H., Mehew, J. W., Zhang, C. Y., Boxer, L. M. 2003; 63 (20): 6666-6673


    Translocation of the bcl-2 gene to the immunoglobulin heavy chain gene is the most common alteration in follicular lymphoma. The result is the deregulated expression of bcl-2 and increased resistance to cell death. Regulation of the immunoglobulin heavy chain gene is controlled in part by four DNase I-hypersensitive regions located 3' of the gene. Here, we show that these four enhancer regions also contribute to bcl-2 up-regulation in t(14;18) cells. The enhancers are able to individually or in combination activate bcl-2 promoter activity. The HS4 enhancer region was found to impart the largest positive effect on the bcl-2 promoter, activating it by 6-fold, whereas addition of the HS1,2 region with HS4 increased promoter activity by approximately 9-fold. Nuclear factor kappaB binding sites were shown to be primarily responsible for the positive activity contributed by the HS1,2 and HS4 regions, and we observed the in vivo interaction of these factors with the human immunoglobulin heavy chain gene enhancer regions in t(14;18) cells. In addition, two Sp1 binding sites in HS4 were also found to positively influence bcl-2 activity, and Sp1 was observed to interact with the human HS4 enhancer in vivo. These results suggest that the interactions of the nuclear factor kappaB and Sp1 transcription factors with the immunoglobulin heavy chain enhancer region are important for bcl-2 deregulation in t(14;18) cells.

    View details for Web of Science ID 000186213800014

    View details for PubMedID 14583460

  • Regulation of Bcl-2 expression by C/EBP in t(14;18) lymphoma cells ONCOGENE Heckman, C. A., Wheeler, M. A., Boxer, L. M. 2003; 22 (39): 7891-7899


    In follicular lymphomas with the t(14;18) translocation, there is increased expression of the bcl-2 gene, which is dependent upon regulatory elements within the bcl-2 5' flanking region and the immunoglobulin heavy-chain gene enhancers. We found that t(14;18) lymphomas expressed C/EBPalpha, which is not normally expressed in B lymphocytes. Expression of C/EBPalpha increased bcl-2 expression, and two regions of the bcl-2 P2 promoter that mediated this effect were identified. C/EBPbeta was also able to increase bcl-2 promoter activity through these sites. The 5' site was GC-rich and did not contain a C/EBP consensus sequence; however, C/EBP was observed to interact with this site both in vitro by EMSA and in vivo by chromatin immunoprecipitation assay. The 3' region contained the Cdx site, which mediates the effect of A-Myb on the bcl-2 promoter. In vivo binding studies revealed that C/EBP interacted with this region of the bcl-2 promoter as well. Decreased expression of C/EBP factors due to targeting of their transcripts by siRNA molecules resulted in downregulation of Bcl-2 protein. We conclude that C/EBPalpha and C/EBPbeta contribute to the deregulated expression of Bcl-2 in t(14;18) lymphoma cells.

    View details for DOI 10.1038/sj.onc.1206639

    View details for Web of Science ID 000185388200003

    View details for PubMedID 12970736

  • Cytokine-mediated down-regulation of the transcription factor cAMP-response element-binding protein in pancreatic beta-cells JOURNAL OF BIOLOGICAL CHEMISTRY Jambal, P., Masterson, S., Nesterova, A., Bouchard, R., Bergman, B., Hutton, J. C., Boxer, L. M., Reusch, J. E., Pugazhenthi, S. 2003; 278 (25): 23055-23065


    Cytokines are known to induce apoptosis of pancreatic beta-cells. Impaired expression of the anti-apoptotic gene bcl-2 is one of the mechanisms involved. In this study, we identified a defect involving transcription factor cAMP-response element-binding protein (CREB) in the expression of bcl-2. Exposure of mouse pancreatic beta-cell line, MIN6 cells, to cytokines (interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma) led to a significant (p < 0.01) decrease in Bcl-2 protein and mRNA levels. Cytokines decreased (56%) the activity of the bcl-2 promoter that contains a cAMP-response element (CRE) site. Similar decreases were seen with a luciferase reporter gene driven by tandem repeats of CRE and a CREB-specific Gal4-luciferase reporter, suggesting a defect at the level of CREB. The active phospho form (serine 133) of CREB diminished significantly (p < 0.01) in cells exposed to cytokines. Examination of signaling pathways upstream of CREB revealed a reduction in the active form of Akt. Cytokine-induced decrease of bcl-2 promoter activity was partially restored when cells were cotransfected with a constitutively active form of Akt. Several end points of cytokine action including decreases in phospho-CREB, phospho-Akt, and BCl-2 levels and activation of caspase-9 were observed in isolated mouse islets. Overexpression of wild-type CREB in MIN6 cells by plasmid transfection and adenoviral infection led to protection against cytokine-induced apoptosis. Adenoviral transfer of dominant-negative forms of CREB, on the other hand, resulted in activation of caspase-9 and exaggeration of cytokine-induced beta-cell apoptosis. Together, these results point to CREB as a novel target for strategies aimed at improving the survival of beta-cells.

    View details for DOI 10.1074/jbc.M212450200

    View details for Web of Science ID 000183503900113

    View details for PubMedID 12679364

  • Oxidative stress-mediated down-regulation of bcl-2 promoter in hippocampal neurons JOURNAL OF NEUROCHEMISTRY Pugazhenthi, S., Nesterova, A., Jambal, P., Audesirk, G., Kern, M., Cabell, L., Eves, E., Rosner, M. R., Boxer, L. M., Reusch, J. E. 2003; 84 (5): 982-996


    Generation of oxidative stress/reactive oxygen species (ROS) is one of the causes of neuronal apoptosis. We have examined the effects of ROS at the transcriptional level in an immortalized hippocampal neuronal cell line (H19-7) and in rat primary hippocampal neurons. Treatment of H19-7 cells with hydrogen peroxide (150 micro m) resulted in a 40% decrease in Bcl-2 protein and a parallel decrease in bcl-2 mRNA levels. H19-7 cells overexpressing bcl-2 were found to be resistant to ROS-induced apoptosis. We had previously shown that bcl-2 promoter activity is positively regulated by the transcription factor cyclic AMP response element binding protein (CREB) in neurons. In the present study, we demonstrate that ROS decreases the activity of luciferase reporter gene driven by a cyclic AMP response element site containing bcl-2 promoter. Exposure of neurons to ROS for 6 h resulted in basal and fibroblast growth factor-2-stimulated phosphorylation/activation of CREB. Chronic 24 h treatment with ROS led to a significant (p < 0.01) decrease in CREB protein and CREB mRNA levels. Adenoviral overexpression of wild type CREB in H19-7 cells resulted in significant (p < 0.01) protection against ROS-induced apoptosis through up-regulation of Bcl-2 expression whereas dominant negative CREB exaggerated the injury. These findings demonstrate that loss of CREB function contributes to oxidative stress-induced neuronal dysfunction.

    View details for DOI 10.1046/j.1471-4159.2003.01606.x

    View details for Web of Science ID 000181055900009

    View details for PubMedID 12603823

  • Impaired proliferation and survival of activated B cells in transgenic mice that express a dominant-negative cAMP-response element-binding protein transcription factor in B cells JOURNAL OF BIOLOGICAL CHEMISTRY Zhang, C. Y., Wu, Y. L., Boxer, L. M. 2002; 277 (50): 48359-48365


    The cAMP-response element-binding protein (CREB) is activated by phosphorylation on serine 133 and mediates the proliferative response to a number of different signals. A mutant CREB with a serine to alanine substitution at position 133 (CREBM1) functions as a dominant-negative inhibitor. Transgenic mice that express the dominant-negative CREB protein in B lymphocytes were developed as a means to study the effects of the inhibition of CREB function on B-cell proliferation and survival. We have shown previously that CREB up-regulates Bcl-2 expression in B cells in response to activation signals. B cells from CREBM1 transgenic mice expressed lower levels of Bcl-2 with and without stimulation. Proliferation of B cells from the transgenic mice was impaired in part by lack of induction of activator protein 1 (AP1) transcription factors. B cells from the transgenic mice were more susceptible to induction of apoptosis with several different agents, consistent with the decreased expression of Bcl-2. These studies demonstrate that B-cell activation requires phosphorylation of CREB for the proliferative response and to protect against activation-induced apoptosis.

    View details for DOI 10.1074/jbc.M209329200

    View details for Web of Science ID 000179789600052

    View details for PubMedID 12374787

  • NF-kappa B activates Bcl-2 expression in t(14;18) lymphoma cells ONCOGENE Heckman, C. A., Mehew, J. W., Boxer, L. M. 2002; 21 (24): 3898-3908


    The t(14;18) translocation, which is characteristic of follicular lymphoma, results in the overexpression of the bcl-2 gene dependent upon regulatory elements within the bcl-2 5' flanking region and the immunoglobulin heavy chain gene enhancers. Conflicting evidence exists on the effects of NF-kappaB expression on Bcl-2 levels in different cell types. Lymphoma cells with the t(14;18) translocation show high levels of nuclear NF-kappaB proteins. We observed decreased levels of endogenous Bcl-2 when the IkappaBalpha-super-repressor was expressed in a t(14;18) cell line. Deletion analysis of the bcl-2 promoter indicated that the repressive effect of the IkappaBalpha-super-repressor occurred through a region that contained no NF-kappaB consensus sequences. This highly active region contained a c-AMP response element (CRE) and several Sp1 binding sites. Chromatin immunoprecipitation assays with antibodies specific for the NF-kappaB and CREB/ATF family members, as well as Sp1, resulted in the isolation of this IkappaBalpha-super-repressor responsive region of the bcl-2 promoter. Mutation of the CRE and the two Sp1 sites in different combinations in bcl-2 reporter constructs resulted in the loss of bcl-2 promoter repression by the IkappaBalpha-super-repressor. We therefore conclude that the activation of bcl-2 by NF-kappaB in t(14;18) lymphoma cells is mediated through the CRE and Sp1 binding sites.

    View details for Web of Science ID 000175847200009

    View details for PubMedID 12032828

  • A myc-associated zinc finger protein-related factor binding site is required for the deregulation of c-myc expression by the immunoglobulin heavy chain gene enhancers in Burkitt's lymphoma JOURNAL OF BIOLOGICAL CHEMISTRY Hu, H. M., Arcinas, M., Boxer, L. M. 2002; 277 (12): 9819-9824


    The deregulation of expression of the c-myc gene in Burkitt's lymphoma results from the translocation that links one c-myc allele to one of the immunoglobulin genes. This physical linkage promotes interactions between c-myc and immunoglobulin gene regulatory elements that affect c-myc transcription initiation and elongation. We have located a region in the c-myc promoter that is required for the complete activation by the immunoglobulin heavy chain gene enhancer. This regulatory element contains a core sequence, GGGAGG, similar to the GA box recognized by the transcription factor Myc-associated zinc finger protein (MAZ). UV cross-link analysis indicated that the mass of this protein did not correspond to that of MAZ, suggesting that a protein related to but distinct from MAZ bound to this site. Mutation of this regulatory element resulted in a loss of promoter activity induced by the immunoglobulin heavy chain gene enhancer. This site was also required for the c-myc promoter shift from P2 to P1. In vivo footprinting revealed that this site was occupied on the translocated c-myc allele but not on the untranslocated allele. Taken together, these findings suggest that this regulatory element is required for the full activation of c-myc promoter activity by the immunoglobulin heavy chain gene enhancer.

    View details for DOI 10.1074/jbc.M111426200

    View details for Web of Science ID 000174549200031

    View details for PubMedID 11777933

  • Allele-specific analysis of transcription factors binding to promoter regions METHODS Heckman, C. A., Boxer, L. M. 2002; 26 (1): 19-26


    In vivo footprinting techniques are useful for the identification of regulatory elements mediating transcriptional control of a gene. However, regulation of a gene can differ between the two alleles, and further steps must be taken to distinguish between the regulatory elements occupied on one allele and those used on the second allele. Many hematologic malignancies result from chromosomal translocations, which, in some cases, relocate a gene to a transcriptionally active region leading to the deregulated expression of that gene. This situation provides an example of differential expression between two alleles. In studying the t(14; 18) and t(8; 14) translocations, which involve the bcl-2 and c-myc proto-oncogenes, respectively, we have been able to identify regulatory elements important in mediating the activation of the translocated alleles and the silencing of the normal alleles. Following in vivo methylation and isolation of genomic DNA, we were able to separate the translocated and normal alleles by electrophoresis. Using the ligation-mediated polymerase chain reaction (LMPCR) technique, we could then assess protein interactions on the two different alleles. A detailed description of this methodology with examples from our studies are provided with a discussion of how these techniques may be applied to the study of other genes.

    View details for Web of Science ID 000175572600004

    View details for PubMedID 12054901

  • The critical role of the PE21 element in oncostatin M-mediated transcriptional repression of the p53 tumor suppressor gene in breast cancer cells ONCOGENE Li, C., Ahlborn, T. E., Tokita, K., Boxer, L. M., Noda, A., Liu, J. W. 2001; 20 (57): 8193-8202


    Cytokine oncostatin M (OM) exerts growth-inhibitory and differentiative effects on breast cancer cells. Previously we showed that the transcription from the p53 gene in breast cancer cells was down regulated by OM. To elucidate the molecular mechanisms underlying the OM effect on p53 transcription, in this study, we dissected the p53 promoter region and analysed the p53 promoter activity in breast tumor cells. We showed that treatment of MCF-7 cells with OM induced a dose- and time-dependent suppression of p53 promoter activity. The p53 promoter activity was decreased to 35% of control at 24 h and further decreased to 20% at 48 h by OM at concentrations of 5 ng/ml and higher. Deletion of the 5'-flanking region of the p53 promoter from -426 to -97 did not affect the OM effect. However, further deletion to -40 completely abolished the repressive effect of OM. The p53 promoter region -96 to -41 contains NF-kappaB and c-myc binding sites, and a newly identified UV-inducible element PE21. Mutations to disrupt NF-kappaB binding or c-myc binding to the p53 promoter decreased the basal promoter activity without affecting the OM-mediated suppression, whereas mutation at the PE21 motif totally abolished the OM effect. We further demonstrated that insertion of PE21 element upstream of the thymidine kinase minimal promoter generated an OM response analogous to that of the p53 promoter. Finally, we detected the specific binding of a nuclear protein with a molecular mass of 87 kDa to the PE21 motif. Taken together, we demonstrate that OM inhibits the transcription of the p53 gene through the PE21 element. Thus, the PE21 element is functionally involved in p53 transcription regulated by UV-induction and OM suppression.

    View details for Web of Science ID 000172507800004

    View details for PubMedID 11781835

  • Translocations involving c-myc and c-myc function ONCOGENE Boxer, L. M., DANG, C. V. 2001; 20 (40): 5595-5610


    c-MYC is the prototype for oncogene activation by chromosomal translocation. In contrast to the tightly regulated expression of c-myc in normal cells, c-myc is frequently deregulated in human cancers. Herein, aspects of c-myc gene activation and the function of the c-Myc protein are reviewed. The c-myc gene produces an oncogenic transcription factor that affects diverse cellular processes involved in cell growth, cell proliferation, apoptosis and cellular metabolism. Complete removal of c-myc results in slowed cell growth and proliferation, suggesting that while c-myc is not required for cell proliferation, it acts as an integrator and accelerator of cellular metabolism and proliferation.

    View details for Web of Science ID 000170887500003

    View details for PubMedID 11607812

  • The cyclic AMP response element in the Bcl-2 promoter confers inducibility by hypoxia in neuronal cells MOLECULAR BRAIN RESEARCH Freeland, K., Boxer, L. M., Latchman, D. S. 2001; 92 (1-2): 98-106


    In neuronal cells, expression of the anti-apoptotic Bcl-2 gene is induced by hypoxia and produces a protective effect. We show here that this effect is dependent upon the cyclic AMP response element (CRE) in the Bcl-2 promoter since mutation of this element abolishes the response and the isolated CRE can confer the response on a heterologous promoter. Interestingly however, the CRE in the Bcl-2 promoter does not render the promoter responsive to cyclic AMP and is not essential for its response to nerve growth factor. Despite the lack of cyclic AMP responsiveness, activation of the Bcl-2 promoter via the CRE in response to hypoxia requires the CREB transcription factor and is associated with the enhanced phosphorylation of CREB on serine 133 and enhanced transcriptional activation by the CREB-binding protein, CBP, in response to hypoxia. This finding establishes the importance of the CRE in the induction of Bcl-2 gene expression by hypoxia, allowing the Bcl-2 protein to protect neuronal cells against this damaging stimulus.

    View details for Web of Science ID 000170808300011

    View details for PubMedID 11483246

  • Molecular mechanisms of transcriptional control of bcl-2 and c-myc in follicular and transformed lymphoma CANCER RESEARCH Arcinas, M., Heckman, C. A., Mehew, J. W., Boxer, L. M. 2001; 61 (13): 5202-5206


    A synergistic interaction of Bcl-2 and c-Myc plays a role in lymphomagenesis in mice and in some patients as well. Progression of follicular lymphoma to a more aggressive lymphoma is seen in the majority of patients, and approximately 10% of the transformed lymphomas have a translocation of c-myc in addition to the translocation of bcl-2 found in the original follicular lymphoma. We investigated whether transcriptional deregulation of bcl-2 and c-myc could be examined in primary lymphoma cells by in vivo footprinting and in vitro protein-DNA binding studies. A matched pair of follicular and transformed lymphoma samples was examined. The transformed lymphoma had acquired a translocation of c-myc into the immunoglobulin heavy chain locus. High levels of bcl-2 expression were observed in both the follicular and transformed lymphomas, whereas the expression of c-myc was low in the follicular lymphoma and increased in the transformed lymphoma. In vivo footprint analysis revealed that a CRE site and a Cdx site in the bcl-2 promoter were occupied on the translocated alleles but not on the normal alleles in both the follicular and transformed lymphomas. Two nuclear factor kappaB sites were occupied on the translocated c-myc allele in the transformed lymphoma. Gel shift analysis revealed that these proteins bound to their respective sites in the bcl-2 or c-myc promoter. There was no evidence that the presence of one of the translocations in the immunoglobulin heavy chain locus influenced the expression of the other translocated gene.

    View details for Web of Science ID 000169563300042

    View details for PubMedID 11431360

  • Suppression of apoptosis and granulocyte colony-stimulating factor-induced differentiation by an oncogenic form of Cbl EXPERIMENTAL HEMATOLOGY Sinha, S., Jancarik, J., Roginskaya, V., Rothermund, K., Boxer, L. M., Corey, S. J. 2001; 29 (6): 746-755


    The retroviral oncogene v-Cbl causes pre-B cell lymphomas and myeloid leukemias in mice, and its Drosophila homologue is oncogenic, causing enhanced receptor tyrosine kinase signaling. The human Cbl gene resides at 11q23. The aim of this study is to determine the effect of oncogenic Cbl on growth-regulating responses.The oncogenic mutant of Cbl (CblDelta1-357) was transfected into factor-dependent 32Dcl3 myeloid cells. Consequently, cell survival and differentiation were measured. Lyn, Syk, MAP kinase, and phosphatidylinositol 3'(PI3')-kinase activities, protein phosphorylation, Bcl-2 promoter activity, ubiquitination, and levels of Bcl-2, Bax, Bad, and Bcl-x(L) were determined. In addition, the effect of v-Cbl on TF-1 cell survival upon granulocyte-macrophage colony-stimulating factor withdrawal was studied.32Dcl3 and TF-1 cells expressing v-Cbl showed resistance to apoptosis upon growth factor withdrawal, and 32Dcl3 cells completely failed to respond to granulocyte colony-stimulating factor's induction of differentiation. Basal activities of Lyn, Syk, and PI3'-kinase were elevated in the v-Cbl line. There was neither enhanced tyrosine phosphorylation of cellular protein content, Cbl, or Jak2, nor serine phosphorylation of MAP kinase or Akt. After factor withdrawal, the level of Bcl-2 was greater in v-Cbl cells than in control cells.Neither increased Bcl-2 promoter activity nor decreased ubiquitination of Bcl-2 could account for increased Bcl-2 levels. v-Cbl-expressing 32Dcl3 cells were resistant to differentiation. v-Cbl suppresses apoptosis and differentiation, possibly through enhancement of Lyn, Syk, and PI3'-kinase activities and Bcl-2.

    View details for Web of Science ID 000169174900011

    View details for PubMedID 11378270

  • Negative regulation of bcl-2 expression by p53 in hematopoietic cells ONCOGENE Wu, Y. L., Mehew, J. W., Heckman, C. A., Arcinas, M., Boxer, L. M. 2001; 20 (2): 240-251


    The p53 protein activates promoters containing p53 binding sites, and it represses other promoters. We examined the effect of p53 on bcl-2 expression in both the DHL-4 B cell line and the K562 erythroleukemia line. Transient transfection analyses revealed that wild-type p53 repressed the bcl-2 full-length promoter. The region of the bcl-2 promoter that was responsive to p53 was mapped to the bcl-2 P2 minimal promoter region, and we showed that p53 and the TATA binding protein bound to the bcl-2 TATA sequence. The TATA binding protein, p53, histone deacetylase-1 and mSin3a could be co-immunoprecipitated from K562 cell nuclear extract. The TATA binding protein and mSin3a could be recovered in a complex at the bcl-2 promoter TATA sequence, however, the formation of this complex was not dependent on the presence of p53. Treatment of K562 cells with the histone deacetylase inhibitor, trichostatin A, resulted in an increase in bcl-2 promoter activity whether p53 was present or not. Therefore, we demonstrated that p53 and the histone deacetylases repress the bcl-2 promoter independently. Similar results were obtained when endogenous bcl-2 mRNA or protein levels were measured in response to either p53 or trichostatin A, and p53 expression resulted in enhanced apoptosis. RNase protection assays demonstrated that transcription from the endogenous 3' bcl-2 promoter was decreased by p53. The regions of p53 that were required for repression of the bcl-2 promoter were defined. We conclude that the TATA sequence in the bcl-2 P2 minimal promoter is the target for repression by p53, and that the interaction between p53 and TBP is most likely responsible for the repression. Mutation of p53 may play a role in the up-regulation of bcl-2 expression in some B cell lymphomas.

    View details for Web of Science ID 000166410900011

    View details for PubMedID 11313951

  • Nerve growth factor- and epidermal growth factor-regulated gene transcription in PC12 pheochromocytoma and INS-1 insulinoma cells EUROPEAN JOURNAL OF CELL BIOLOGY Groot, M., Boxer, L. M., Thiel, G. 2000; 79 (12): 924-935


    PC12 and INS-1 cells both express the nerve growth factor (NGF) receptors trkA and p75NTR and the epidermal growth factor receptor (EGF). In PC12 cells, NGF treatment initiates a signaling cascade that ultimately leads to a change of the genetic program of the cell. We have investigated the role of NGF in regulating gene transcription in PC12 and INS-1 cells, in order to define if there are NGF-regulated genes per se. Furthermore, to distinguish between growth factor stimulation via receptor tyrosine kinases in general and NGF-specific changes in gene transcription, we analyzed the effects of EGF on gene transcription. First, we tested the biological activities of fusion proteins consisting of the DNA-binding domain of the yeast transcription factor GAL4 and the phosphorylation-dependent activation domains of the transcription factors Elk1, CREB, ATF2 and c-jun in NGF- or EGF-treated PC12 cells. We found a striking increase in the transcriptional activity of the GAL4-Elk1 fusion protein that is a major substrate for the extracellular signal-regulated protein kinase (ERK). This effect was observed in NGF- as well as in EGF-treated PC12 cells. In INS-1 cells, however, the activity of the GAL4-Elk1 fusion protein was induced by NGF, but not by EGF. The effects of NGF and EGF on gene transcription were subsequently studied with plasmids containing reporter genes under control of the Egr-1, c-jun, HES-1 or Bc12 regulatory sequences. NGF stimulated Egr-1 promoter activities in PC12 and INS-1 cells, although the effect was much more pronounced in PC12 cells than in INS-1 cells. EGF also stimulated Egr-1 promoter activity in both PC12 and INS-1 cells. Stimulation of c-jun promoter activity by NGF was observed only in PC12 cells. Deletion mutagenesis demonstrated the importance of the 12-O-tetradecanoylphorbol-13-acetate response elements within the c-jun promoter for basal and NGF-mediated transcriptional induction. Likewise, NGF activated HES1 and Bcl2 P1 promoter activities in PC12 cells but not in INS-1 cells and EGF did not show any effects on these promoters. We conclude that in PC12 and INS-1 cells, NGF signaling leads to an activation of the ERK subtype of mitogen-activated protein kinases in the nucleus and a subsequent activation of Egr-1 gene transcription. The NGF-induced transcription of the c-jun, HES1 and Bc12 genes is, in contrast, cell type-specific, indicating that NGF can trigger different gene expression programs dependent on the signaling pathways present in a particular cell type. EGF is clearly able to activate gene transcription, suggesting that the differences in the biological activities of EGF and NGF cannot be explained by the inability of EGF to stimulate gene transcription.

    View details for Web of Science ID 000166061800006

    View details for PubMedID 11152283

  • NF-kappa B activity is required for the deregulation of c-myc expression by the immunoglobulin heavy chain enhancer JOURNAL OF BIOLOGICAL CHEMISTRY Kanda, K., Hu, H. M., Zhang, L., Grandchamps, J., Boxer, L. M. 2000; 275 (41): 32338-32346


    The c-myc gene is translocated to one of the immunoglobulin genes in Burkitt's lymphoma resulting in deregulated expression of c-myc. Several enhancers have been shown to be important for expression of the immunoglobulin heavy chain gene. Four enhancer regions (murine-hypersensitive sites (MHS) 1, 2, 3, and 4) located 3' of the murine immunoglobulin heavy chain gene play a role in activating expression of the translocated c-myc gene. The enhancer regions also result in a shift in transcriptional initiation from the P2 promoter to P1 that is characteristic of the translocated c-myc allele. We found that the most 3' enhancer region (MHS4) activated the c-myc promoter by 46-fold in the Raji Burkitt's lymphoma cell line, and it was the most active enhancer in these cells. The addition of enhancer regions MHS1,2 and 3 to MHS4 increased c-myc transcription by an additional 3-fold and resulted in the full promoter shift from P2 to P1. By deletion analysis of enhancer region MHS4, we located a region that was critical for the transcriptional activity of MHS4. Electrophoretic mobility shift assay analysis revealed that NF-kappaB/Rel family members bound to this region. Mutation of the NF-kappaB binding site abolished both the enhancer activity and the promoter shift activity of MHS4. An active NF-kappaB site was also identified in the human HS4 enhancer. Inhibition of c-myc promoter activity driven by the immunoglobulin enhancers was observed with expression of a super-repressor IkappaBalpha construct. These results indicate that the NF-kappaB/Rel transcription factors play an important role in the deregulation of the translocated c-myc gene in Burkitt's lymphoma and suggest that interference with NF-kappaB function may represent a new approach to the treatment of Burkitt's lymphoma.

    View details for Web of Science ID 000089858900107

    View details for PubMedID 10931834

  • Akt/protein kinase B up-regulates Bcl-2 expression through cAMP-response element-binding protein JOURNAL OF BIOLOGICAL CHEMISTRY Pugazhenthi, S., Nesterova, A., Sable, C., Heidenreich, K. A., Boxer, L. M., Heasley, L. E., Reusch, J. E. 2000; 275 (15): 10761-10766


    In our previous study we showed that insulin-like growth factor-I induces a cAMP-response element (CRE) site-containing Bcl-2 promoter through a novel signaling pathway involving mitogen-activated protein kinase kinase 6/p38beta mitogen-activated protein kinase/MAP kinase-activated protein kinase-3/cAMP-response element-binding protein (CREB) (Pugazhenthi, S., Miller, E., Sable, C., Young, P., Heidenreich, K. A., Boxer, L. M., and Reusch, J. E.-B. (1999) J. Biol. Chem. 274, 27529-27535). In the present investigation, we define a second pathway contributing to CREB-dependent up-regulation of Bcl-2 expression as a novel anti-apoptotic function of Akt signaling. To examine the role of Akt on Bcl-2 expression, a series of transient transfections using a luciferase reporter gene driven by the promoter region of Bcl-2 containing a CRE were carried out. Pharmacological inhibition of phosphatidylinositol (PI) 3-kinase, the upstream kinase of Akt, with LY294002 led to a 45% decrease in Bcl-2 promoter activity. The reporter activity was enhanced 2.3-fold by overexpression of active p110 subunit of PI 3-kinase and inhibited 44% by the dominant negative p85 subunit of PI 3-kinase. Cotransfection with 3-phosphoinositide-dependent kinase (PDK1), which is required for the full activation of Akt, resulted in enhanced luciferase activity. Insulin-like growth factor-I-mediated induction of Bcl-2 promoter activity was decreased significantly (p < 0.01) by the dominant negative forms of p85 subunit of PI 3-kinase, PDK1, and Akt. These data indicate that regulation of Bcl-2 expression by IGF-I involves a signaling cascade mediated by PI 3-kinase/PDK1/Akt/CREB. Furthermore, we measured the Bcl-2 mRNA in PC12 cells overexpressing Akt by real-time quantitative reverse transcription-polymerase chain reaction using the TaqMan(TM) fluorogenic probe system. We observed a 2.1-fold increase in Bcl-2 mRNA levels in the Akt cell line compared with control PC12 cells, supporting the observation that enhanced CREB activity by Akt signaling leads to increased Bcl-2 promoter activity and cell survival.

    View details for Web of Science ID 000086466600009

    View details for PubMedID 10753867

  • A-Myb up-regulates bcl-2 through a Cdx binding site in t(14;18) lymphoma cells JOURNAL OF BIOLOGICAL CHEMISTRY Heckman, C. A., Mehew, J. W., Ying, G. G., Introna, M., Golay, J., Boxer, L. M. 2000; 275 (9): 6499-6508


    In follicular lymphoma, bcl-2 is translocated to the immunoglobulin heavy chain locus leading to deregulation of bcl-2 expression. We examined the role of Myb proteins in the regulation of bcl-2 expression in lymphoma cells. We showed that A-Myb up-regulates bcl-2 promoter activity. Northern and Western analyses demonstrated that A-Myb was expressed in the DHL-4 t(14;18) cell line. In t(14;18) cells and mature B cells, A-Myb up-regulated bcl-2 expression, whereas B- and c-Myb had little effect on bcl-2 gene expression. Deletion analysis of the bcl-2 5'-region identified a region responsive to A-Myb in t(14;18) cells. A potential binding site for the Cdx homeodomain proteins was located in this sequence. Analysis of the A-Myb-responsive region by UV cross-linking experiments revealed that a 32-kDa protein formed a complex with this region, but direct binding by Myb proteins could not be demonstrated. A-Myb could be recovered along with Cdx2 when nuclear extracts were passed over the Cdx site. Mutagenesis of the Cdx binding site abolished binding by the 32-kDa protein and significantly reduced the ability of A-Myb to induce bcl-2 expression. A strong induction of bcl-2 P2 promoter activity was observed in cotransfection studies of DHL-4 cells with the A-Myb and Cdx2 expression vectors, and increased endogenous Bcl-2 protein expression was observed in B cells transfected with A-Myb and/or Cdx2 expression constructs.

    View details for Web of Science ID 000085654400066

    View details for PubMedID 10692454

  • The expression of p53 tumor suppressor gene in breast cancer cells is down-regulated by cytokine oncostatin M CELL GROWTH & DIFFERENTIATION Liu, J. W., Li, C., Ahlborn, T. E., Spence, M. J., Meng, L., Boxer, L. M. 1999; 10 (10): 677-683


    Previously (J. Liu, et al., Cell Growth Differ., 8: 667-676, 1997), we showed that oncostatin M (OM), a cytokine produced by activated T cells and macrophages, inhibited the proliferation of breast cancer cells derived from solid tumors and malignant effusions. OM-treated cells showed reduced growth rates and differentiated phenotypes. Because the p53 tumor suppressor protein plays an important role in cellular proliferation, we examined p53 protein expression in three OM-responsive breast cancer cell lines, MCF-7, MDA-MB231, and H3922. Western blot analysis showed that p53 protein levels in all three of the cell lines were decreased by OM treatment. Reduction of p53 protein was detected after 1 day of OM treatment and reached maximal suppression of 10-20% of control after 3 days in H3922 and 40% of control after 4 days in MCF-7 cells. A comparison of p53 mRNA in OM-treated cells versus untreated control cells showed that exposure to OM reduced the steady-state levels of p53 mRNA transcripts to an extent similar to that of the p53 protein levels. This observation suggests that the effect of OM on p53 protein expression does not occur at the posttranslational level. Nuclear run-on assays verified that OM decreased the number of actively transcribed p53 mRNAs, which suggests a transcriptional regulatory mechanism. The effect of OM on p53 expression seems to be mediated through the extracellular signal-regulated kinase (ERK) pathway, inasmuch as the inhibition of ERK activation with a specific inhibitor (PD98059) to the ERK upstream kinase mitogen/extracellular-regulated protein kinase kinase abrogated the OM inhibitory activity on p53 expression in a dose-dependent manner. In addition to OM, we showed that the p53 protein expression in MCF-7 cells was also decreased by phorbol 12-myristate 13-acetate treatment (PMA). Because both OM and PMA induce MCF-7 cells to differentiate, our data suggest that p53 expression in breast cancer cells is down-regulated during the differentiation process.

    View details for Web of Science ID 000083357500002

    View details for PubMedID 10547071

  • Insulin-like growth factor-I induces bcl-2 promoter through the transcription factor cAMP-response element-binding protein JOURNAL OF BIOLOGICAL CHEMISTRY Pugazhenthi, S., Millers, E., Sable, C., Young, P., Heidenreich, K. A., Boxer, L. M., Reusch, J. E. 1999; 274 (39): 27529-27535


    Insulin-like growth factor-I (IGF-I) is known to prevent apoptosis induced by diverse stimuli. The present study examined the effect of IGF-I on the promoter activity of bcl-2, a gene with antiapoptotic function. A luciferase reporter driven by the promoter region of bcl-2 from -1640 to -1287 base pairs upstream of the translation start site containing a cAMP-response element was used in transient transfection assays. Treatment of PC12 cells with IGF-I enhanced the bcl-2 promoter activity by 2.3-fold, which was inhibited significantly (p < 0.01) by SB203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK). Cotransfection of the bcl-2 promoter with MAPK kinase 6 and the beta isozyme of p38 MAPK resulted in 2-3-fold increase in the reporter activity. The dominant negative form of MAPKAP-K3, a downstream kinase activated by p38 MAPK, and the dominant negative form of cAMP-response element-binding protein, inhibited the reporter gene activation by IGF-I and p38beta MAPK significantly (p < 0.01). IGF-I increased the activity of p38beta MAPK introduced into the cells by adenoviral infection. Thus, we have characterized a novel signaling pathway (MAPK kinase 6/p38beta MAPK/MAPKAP-K3) that defines a transcriptional mechanism for the induction of the antiapoptotic protein Bcl-2 by IGF-I through the nuclear transcription factor cAMP-response element-binding protein in PC12 cells.

    View details for Web of Science ID 000082739100027

    View details for PubMedID 10488088

  • A zinc-finger transcription factor induced by TGF-beta promotes apoptotic cell death in epithelial Mv1Lu cells FEBS LETTERS Chalaux, E., Lopez-Rovira, T., Rosa, J. L., Pons, G., Boxer, L. M., Bartrons, R., Ventura, F. 1999; 457 (3): 478-482


    Transforming growth factor-beta (TGF-beta) superfamily members constitute a group of multifunctional factors that are able to stimulate apoptotic cell death in a variety of cells. In this report, we show that a zinc-finger transcription factor (TIEG) is an immediate early gene transcriptionally induced by TGF-beta in the epithelial Mv1Lu cell line. We also demonstrate that, mimicking TGF-beta effects, ectopic overexpression of TIEG is sufficient to trigger the apoptotic cell program in these cells, which is preceded by a decrease of Bcl-2 protein levels. Finally, apoptotic events elicited by TIEG overexpression can be effectively prevented by ectopic co-expression of Bcl-2. On the basis of these results we suggest that induction of TIEG expression has a role in the pro-apoptotic properties of TGF-beta.

    View details for Web of Science ID 000082454500039

    View details for PubMedID 10471833

  • p53 suppresses the activation of the Bcl-2 promoter by the Brn-3a POU family transcription factor JOURNAL OF BIOLOGICAL CHEMISTRY Budhram-Mahadeo, V., Morris, P. J., Smith, M. D., Midgley, C. A., Boxer, L. M., Latchman, D. S. 1999; 274 (21): 15237-15244


    The Brn-3a POU family transcription factor has been shown to strongly activate expression of the Bcl-2 proto-oncogene and thereby protect neuronal cells from programmed cell death (apoptosis). This activation of the Bcl-2 promoter by Brn-3a is strongly inhibited by the p53 anti-oncogene protein. This inhibitory effect of p53 on Brn-3a-mediated transactivation is observed with nonoverlapping gene fragments containing either the Bcl-2 p1 or p2 promoters but is not observed with other Brn-3a-activated promoters such as in the gene encoding alpha-internexin or with an isolated Brn-3a binding site from the Bcl-2 promoter linked to a heterologous promoter. In contrast, p53 mutants, which are incapable of binding to DNA, do not affect Brn-3a-mediated activation of the Bcl-2 p1 and p2 promoters. Moreover, Brn-3a and p53 have been shown to bind to adjacent sites in the p2 promoter and to directly interact with one another, both in vitro and in vivo, with this interaction being mediated by the POU domain of Brn-3a and the DNA binding domain of p53. The significance of these effects is discussed in terms of the antagonistic effects of Bcl-2 and p53 on the rate of apoptosis and the overexpression of Brn-3a in specific tumor cell types.

    View details for Web of Science ID 000081965200098

    View details for PubMedID 10329733

  • Activation of the Bcl-2 promoter by nerve growth factor is mediated by the p42/p44 MAPK cascade NUCLEIC ACIDS RESEARCH Liu, Y. Z., Boxer, L. M., Latchman, D. S. 1999; 27 (10): 2086-2090


    The Bcl-2 protein has an anti-apoptotic effect in neuronal and other cell types. We show for the first time that the Bcl-2 promoter is activated by the neuronal survival factor nerve growth factor (NGF) and that this effect is dependent on a region of the promoter from -1472 to -1414. This activation requires the Rap-1 G protein and the MEK-1 and p42/p44 MAPK enzymes but is independent of other NGF-activated signalling pathways involving protein kinase A or protein kinase C.

    View details for Web of Science ID 000080431800004

    View details for PubMedID 10219080

  • The N-terminal domain unique to the long form of the Brn-3a transcription factor is essential to protect neuronal cells from apoptosis and for the activation of Bcl-2 gene expression NUCLEIC ACIDS RESEARCH Smith, M. D., DAWSON, S. J., Boxer, L. M., Latchman, D. S. 1998; 26 (18): 4100-4107


    The ability of the POU family transcription factor Brn-3a to stimulate neurite outgrowth and the expression of the genes encoding neuronal proteins such as the neurofilaments and SNAP-25 has previously been shown to be dependent upon the C-terminal POU domain which can mediate both DNA binding and transcriptional activation. We show here, however, that the ability of Brn-3a to activate Bcl-2 expression and protect neuronal cells from apoptosis (programmed cell death) requires a distinct N-terminal activation domain. Bcl-2 gene activation and protection from apoptosis are thus produced only by the long form of Brn-3a which contains this domain and not by a naturally occurring short form lacking this domain or by the isolated POU domain, although all these forms of Brn-3a can stimulate neurite outgrowth. Hence Brn-3a is a multi-functional transcription factor with different regions of the factor mediating its different effects and two distinct forms with different properties being generated by alternative splicing.

    View details for Web of Science ID 000076004500002

    View details for PubMedID 9722627

  • Bcl-2 transcription from the proximal P2 promoter is activated in neuronal cells by the Brn-3a POU family transcription factor JOURNAL OF BIOLOGICAL CHEMISTRY Smith, M. D., Ensor, E. A., Coffin, R. S., Boxer, L. M., Latchman, D. S. 1998; 273 (27): 16715-16722


    The BCL-2 protein is able to protect neuronal and other cell types from apoptotic programmed cell death and plays a key role in regulating the rate of apoptosis during development of the nervous system. We have previously demonstrated that the Brn-3a POU domain transcription factor protects sensory neurons from apoptotic programmed cell death induced by nerve growth factor withdrawal. We report here that Bcl-2 transcription is predominantly initiated from the Bcl-2 P2 promoter in both the ND7 neuronal cell line and primary dorsal root ganglion neurons, in contrast to the predominant use of the Bcl-2 P1 promoter in other cell types. Moreover, Bcl-2 transcription initiated from the P2 region increases in ND7 cells stably overexpressing Brn-3a, resulting in enhanced BCL-2 protein levels. Similarly, the Bcl-2 P2 promoter is directly activated by Brn-3a in co-transfection assays in both ND7 cells and dorsal root ganglion neurons. Analysis of the Bcl-2 regulatory sequence revealed a binding site for Brn-3a that is required for maximal activation by Brn-3a both in transfected cells and during differentiation of ND7 cells. Together these data identify Brn-3a as the first transcription factor regulating Bcl-2 activity specifically in neuronal cells and indicate that the anti-apoptotic effect of Brn-3a is likely to be mediated, at least in part, via the up-regulation of Bcl-2 expression.

    View details for Web of Science ID 000074545200016

    View details for PubMedID 9642226

  • The WT1 protein is a negative regulator of the normal bcl-2 allele in t(14;18) lymphomas JOURNAL OF BIOLOGICAL CHEMISTRY Heckman, C., Mochon, E., Arcinas, M., Boxer, L. M. 1997; 272 (31): 19609-19614


    The translocated and normal bcl-2 alleles in the DHL-4 cell line with the t(14;18) translocation were separated by pulsed field electrophoresis. An in vivo footprint over a potential WT1 binding site in the bcl-2 5'-flanking sequence was identified on the normal silent allele. Electrophoretic mobility shift assays with the bcl-2 WT1 site demonstrated a single specific complex. UV cross-linking and Western analysis revealed that this gel shift complex contained WT1 protein. Deletion or mutation of the WT1 site resulted in an increase in activity of the bcl-2 promoter in DHL-4 cells. Cotransfection with a 3:1 ratio of a WT1 expression vector to the bcl-2 promoter construct led to a 3.0-fold repression of the bcl-2 promoter. Cotransfection with a WT1 expression vector and the bcl-2 promoter with the mutated WT1 site resulted in only 1.2-fold repression. We conclude that the WT1 site functions as a negative regulatory site for the normal silent bcl-2 allele in t(14;18) lymphomas. The WT1 site is not occupied on the translocated bcl-2 allele.

    View details for Web of Science ID A1997XP06300074

    View details for PubMedID 9235968

  • Identification of the major positive regulators of c-myb expression in hematopoietic cells of different lineages JOURNAL OF BIOLOGICAL CHEMISTRY Sullivan, J., Feeley, B., GUERRA, J., Boxer, L. M. 1997; 272 (3): 1943-1949


    The c-myb gene is primarily expressed in hematopoietic cells, and it is overexpressed in many leukemias. The regulation of its expression is of critical importance in hematopoietic cells. We identified the major positive regulatory sites in the 5'-flanking sequence of the human c-myb gene, and we found that the positive regulators differed in cells of different lineages. In the Molt-4 T-cell line, two Ets-like binding sites were required for the expression of c-myb. The 5' site played a minor role in the regulation of c-myb expression, and we demonstrated that a protein of 67 kDa bound to this site. Antibodies against Ets proteins showed no cross-reactivity with this protein. We showed that Ets-1 bound to the 3'-regulatory site in the c-myb promoter by electrophoretic mobility shift assay and antibody studies. Both of these Ets-like binding sites were nonfunctional in the DHL-9 B-cell line and the K562 myeloid cell line. We identified a novel transcription factor of 50.5 kDa that was required for expression of c-myb in these cell lines.

    View details for Web of Science ID A1997WD05800082

    View details for PubMedID 8999884

  • Induction of bcl-2 expression by phosphorylated CREB proteins during B-cell activation and rescue from apoptosis MOLECULAR AND CELLULAR BIOLOGY Wilson, B. E., Mochon, E., Boxer, L. M. 1996; 16 (10): 5546-5556


    Engagement of surface immunoglobulin on mature B cells leads to rescue from apoptosis and to proliferation. Levels of bcl-2 mRNA and protein increase with cross-linking of surface immunoglobulin. We have located the major positive regulatory region for control of bcl-2 expression in B cells in the 5'-flanking region. The positive region can be divided into an upstream and a downstream regulatory region. The downstream regulatory region contains a cyclic AMP-responsive element (CRE). We show by antibody supershift experiments and UV cross-linking followed by denaturing polyacrylamide gel electrophoresis that both CREB and ATF family members bind to this region in vitro. Mutations of the CRE site that result in loss of CREB binding also lead to loss of functional activity of the bcl-2 promoter in transient-transfection assays. The presence of an active CRE site in the bcl-2 promoter implies that the regulation of bcl-2 expression is linked to a signal transduction pathway in B cells. Treatment of the mature B-cell line BAL-17 with either anti-immunoglobulin M or phorbol 12-myristate 13-acetate leads to an increase in bcl-2 expression that is mediated by the CRE site. Treatment of the more immature B-cell line, Ramos, with phorbol esters rescues the cells from calcium-dependent apoptosis. bcl-2 expression is increased following phorbol ester treatment, and the increased expression is dependent on the CRE site. These stimuli result in phosphorylation of CREB at serine 133. The phosphorylation of CREB that results in activation is mediated by protein kinase C rather than by protein kinase A. Although the CRE site is necessary, optimal induction of bcl-2 expression requires participation of the upstream regulatory element, suggesting that phosphorylation of CREB alters its interaction with the upstream regulatory element. The CRE site in the bcl-2 promoter appears to play a major role in the induction of bcl-2 expression during the activation of mature B cells and during the rescue of immature B cells from apoptosis. It is possible that the CRE site is responsible for induction of bcl-2 expression in other cell types, particularly those in which protein kinase C is involved.

    View details for Web of Science ID A1996VH85200033

    View details for PubMedID 8816467

  • CREB proteins function as positive regulators of the translocated bcl-2 allele in t(14;18) lymphomas JOURNAL OF BIOLOGICAL CHEMISTRY Ji, L., Mochon, E., Arcinas, M., Boxer, L. M. 1996; 271 (37): 22687-22691


    The translocated and normal bcl-2 alleles in the DHL-4 cell line with the t(14;18) translocation were separated by pulsed field electrophoresis. An in vivo footprint over a cAMP response element (CRE) in the bcl-2 5'-flanking sequence was identified on the translocated allele. Electrophoretic mobility shift assays with the bcl-2 CRE demonstrated complexes with mobilities identical to those with a consensus CRE. UV cross-linking experiments revealed that proteins with molecular masses of 34, 43, and 67 kDa bound to the bcl-2 CRE site. Electrophoretic mobility shift assay with an antibody specific to the phosphorylated cAMP response-binding protein (CREB) demonstrated that phosphorylated CREB was present in DHL-4 cells. Treatment with phorbol 12-myristate 13-acetate (PMA) led to an increase in both the amount of phosphorylated CREB and the bcl-2 promoter activity. The response to PMA was dependent on an intact CRE site. The activity of the bcl-2 promoter was increased 20-fold in a construct with the immunoglobulin heavy chain enhancers, and mutation of the CRE site abolished most of the induction. The addition of PMA increased the activity of the bcl-2-immunoglobulin enhancer construct by 3.5-fold. Access to the CRE site is blocked in the silent normal bcl-2 allele, while CREB proteins bind to the site on the translocated allele. We conclude that the CRE site functions as a positive regulatory site for the translocated bcl-2 allele in t(14;18) lymphomas.

    View details for Web of Science ID A1996VG67200060

    View details for PubMedID 8798441

  • Identification of an inducible regulator of c-myb expression during T-cell activation MOLECULAR AND CELLULAR BIOLOGY Phan, S. C., Feeley, B., Withers, D., Boxer, L. M. 1996; 16 (5): 2387-2393


    Resting T cells express very low levels of c-Myb protein. During T-cell activation, c-myb expression is induced and much of the increase in expression occurs at the transcriptional level. We identified a region of the c-myb 5' flanking sequence that increased c-myb expression during T-cell activation. In vivo footprinting by ligation-mediated PCR was performed to correlate in vivo protein binding with functional activity. A protein footprint was visible over this region of the c-myb 5' flanking sequence in activated T cells but not in unactivated T cells. An electrophoretic mobility shift assay (EMSA) with nuclear extract from activated T cells and an oligonucleotide of this binding site demonstrated a new protein-DNA complex, referred to as CMAT for c-myb in activated T cells; this complex was not present in unactivated T cells. Because the binding site showed some sequence similarity with the nuclear factor of activated T cells (NFAT) binding site, we compared the kinetics of induction of the two binding complexes and the molecular masses of the two proteins. Studies of the kinetics of induction showed that the NFAT EMSA binding complex appeared earlier than the CMAT complex. The NFAT protein migrated more slowly in a sodium dodecyl sulfate-polyacrylamide gel than the CMAT protein did. In addition, an antibody against NFAT did not cross-react with the CMAT protein. The appearance of the CMAT binding complex was inhibited by both cyclosporin A and rapamycin. The CMAT protein appears to be a novel inducible protein involved in the regulation of c-myb expression during T-cell activation.

    View details for Web of Science ID A1996UG29900053

    View details for PubMedID 8628306

  • REPRESSION OF THE C-MYB GENE BY WT1 PROTEIN IN T-CELL AND B-CELL LINES JOURNAL OF BIOLOGICAL CHEMISTRY McCann, S., Sullivan, J., GUERRA, J., Arcinas, M., Boxer, L. M. 1995; 270 (40): 23785-23789


    The c-myb gene is primarily expressed in immature hematopoietic cells, and it is overexpressed in many leukemias. We have investigated the role of negative regulatory sites in the c-myb promoter in the Molt-4 T cell line and in the DHL-9 B cell line. A potential binding site for either the EGR-1 or WT1 protein was identified by in vivo footprinting in the 5'-flanking region of c-myb in a region of negative regulatory activity in T cells. We showed by electrophoretic mobility shift assay and electrophoretic mobility shift assay Western that WT1, EGR-1, and Sp1 bound to this site. A mutation of this site which prevented protein binding increased the activity of the c-myb promoter by 2.5-fold. In the DHL-9 B cell line, this site was nonfunctional; however, we found a potential EGF-1/WT1 site located more 3' in a region of negative regulatory activity. We showed that WT1, EGR-1, and Sp1 bound to this site, and that mutation of this site increased the activity of the c-myb promoter by 3.2-fold. Cotransfection of a WT1 expression vector repressed the activity of the c-myb promoter in both cell lines, and this repression was relieved when the EGR-1/WT1 sites were removed. Cotransfection of either an EGR-1 or Sp1 expression vector had no significant effect on the activity of the c-myb promoter. We conclude that WT1 is a negative regulator of c-myb expression in both T and B cell lines.

    View details for Web of Science ID A1995RY90900082

    View details for PubMedID 7559553



    In hematopoietic cell development, the c-myb transcription factor plays an important role. c-myb mRNA is expressed at high levels in immature proliferating cells and in leukemic cells. We have investigated the regulatory role of Myb protein binding to the human c-myb promoter. Three Myb binding sites have been described at approximately 600 bp upstream of the cap site. By transient transfection assays in hematopoietic cell lines, we found that deletion of the previously defined most 5' Myb binding site had no effect on activity, whereas deletion of the region containing the remaining two Myb binding sites resulted in an increase in activity in both a T-cell line and a myeloid cell line. To specifically test the importance of these two Myb binding sites, the activity of three-point mutation constructs was measured. Mutation of either Myb binding site resulted in an increase in activity compared with the wild-type promoter in T cells. Mutation of both sites produced even higher activity. Transfection of the Myb site mutants into the myeloid cell line resulted in no change in activity compared with the wild type construct. Results from gel shift analysis, UV cross-linking, and Western blots showed that both c-Myb and B-Myb bound to the Myb I and II sites. We conclude that the Myb family proteins negatively regulate c-myb expression in T-cell lines in contrast to the positive regulation via these sites, which has been shown in fibroblasts. In addition, in a myeloid cell line, the Myb binding sites are nonfunctional.

    View details for Web of Science ID A1995RT38600026

    View details for PubMedID 7655015



    The bcl-2 gene is differentially regulated during B-cell development, with low-level expression in pre-B cells and higher-level expression in mature B cells. These changes correlate with susceptibility to cell death by apoptosis and suggest that the Bcl-2 protein may play a role in the control of cell death during B-cell development. We have identified two negative regulatory regions in the human bcl-2 5' flanking and 5' untranslated regions in pre-B cells; these regions have no significant function in mature B cells. Further investigation of these regions revealed two pre-B-cell-specific enhancer elements (pi 1 sites) in the 5' negative regulatory region and one in the 3' negative regulatory region. Mutational analysis confirmed that these three sites functioned as negative regulators of the bcl-2 promoter in the pre-B-cell line Nalm-6. Electrophoretic mobility shift assays with each of the three sites demonstrated a complex of identical mobility to that formed with the immunoglobulin heavy-chain enhancer pi 1 site. UV cross-linking experiments revealed that a protein with a molecular mass of 58 kDa bound to the three bcl-2 sites and to the immunoglobulin enhancer site. This protein reacted with an antibody against Ets family proteins. Constructs with the isolated pi 1 sites linked to the simian virus 40 promoter were used in transient transfection experiments in the pre-B-cell line. The bcl-2 sites decreased expression of the simian virus 40 promoter, while the immunoglobulin enhancer site increased its expression. The pi 1 sites in the bcl-2 gene may play a role in the developmental regulation of bcl-2 expression during B-cell differentiation.

    View details for Web of Science ID A1995RE03000042

    View details for PubMedID 7791791



    We have identified an in vivo footprint over the PuF site on the translocated c-myc allele in Burkitt's lymphoma cells. The PuF site on the silent normal c-myc allele was unoccupied. We demonstrated by electrophoretic mobility shift assay, electrophoretic mobility shift assay with antibody, UV cross-linking followed by SDS-gel electrophoresis, and Western analysis that Nm23H2 in B cell nuclear extracts bound to the c-myc PuF site. Transfection experiments with c-myc promoter constructs in both DHL-9 and Raji cells revealed that the PuF site functioned as a positive regulatory element in B cells with a drop in activity with mutation of this site. Access to this site is blocked in the normal silent c-myc allele; these data suggest that the Nm23H2 protein is involved in deregulation of the translocated c-myc allele in Burkitt's lymphoma cells.

    View details for Web of Science ID A1995RB43900066

    View details for PubMedID 7768941



    An in vivo footprint over a potential NF-kappa B site in the first exon of the c-myc gene has been identified on the translocated allele in the Ramos Burkitt's lymphoma cell line. The potential NF-kappa B site in the 5' flanking sequence of c-myc was found to be occupied on the translocated allele in the Raji Burkitt's cell line. Electrophoretic mobility shift assays with each of these sequences demonstrated complexes with mobilities identical to those of the NF-kappa B site from the kappa light-chain gene. A supershift was obtained with anti-p50 antibody with the exon site. The upstream-site shift complex disappeared with the addition of anti-p50 antibody. Binding of NF-kappa B proteins to the c-myc exon and upstream sites was demonstrated by induction of binding upon differentiation of pre-B 70Z/3 cells to B cells. UV cross-linking experiments revealed that a protein with a molecular mass of 50 kDa bound to the exon and upstream sites. Transfection experiments with Raji cells demonstrated that both sites functioned as positive regulatory regions, with a drop in activity level when either site was mutated. Access to these sites is blocked in the silent normal c-myc allele in Burkitt's lymphoma cells, while Rel family proteins bind to these sites in the translocated allele. We conclude that the two NF-kappa B sites function as positive regulatory regions for the translocated c-myc gene in Burkitt's lymphoma.

    View details for Web of Science ID A1994PV67400026

    View details for PubMedID 7969136



    In vivo footprinting has been used to examine protein binding sites in the c-myc promoter during differentiation of HL60 and K562 cell lines. The c-myc gene is expressed in proliferating cells, but c-myc levels decrease dramatically during differentiation. A number of potential protein binding sites have been identified from in vitro studies of the c-myc promoter, but very little is known about occupancy of these sites in vivo. We have identified in vivo footprints at DNase hypersensitive sites II2 and III1 which disappear during differentiation, while a footprint at site IV is present only in differentiated cells. Footprints at DNase hypersensitive sites I and II1 do not change with differentiation. A protected region near DNase hypersensitive site III2 is present in both undifferentiated and differentiated cells, but it extends further 5' in undifferentiated cells. From the protected sequences we have been able to identify candidate transcription factors likely to be involved in the control of c-myc expression. By electrophoretic mobility shift assay we have demonstrated that a protein binds to the sequence at site IV. We have also examined the 3' end of the first exon and the 5' end of intron I and do not find any evidence for protein binding sites in this region that was thought to be important for the block to transcription elongation during differentiation.

    View details for Web of Science ID A1994PC05400031

    View details for PubMedID 8058334



    We have shown by transient transfection assays that c-Abl increases expression of a c-myc promoter construct although to a lesser extent than v-Abl. c-Abl has been reported to bind to a specific DNA sequence, the EP element, in the hepatitis B virus enhancer. A similar sequence exists in the promoter region of the c-myc gene, and we have identified a potential protein binding site in this region by in vivo footprinting. Gel shift analysis demonstrated that the hepatitis B virus enhancer and c-myc sites yield identical complexes. To determine whether c-Abl activates c-myc transcription by binding directly to the promoter region, several experiments were performed. Mutation of the EP site in the c-myc promoter had no effect on transcriptional activation of c-myc by c-Abl. Next, we demonstrated that the DNA binding domain of c-Abl was not required for the transcriptional activation of c-myc. Finally, by Western blot analysis, we have determined that c-Abl is not present in the gel shift complexes formed by either the hepatitis B virus enhancer or c-myc promoter. We conclude that c-Abl activates c-myc transcription indirectly with no requirement for DNA binding by c-Abl. We also find no evidence for the interaction of c-Abl with the EP sequence of either the hepatitis B virus enhancer or the c-myc promoter.

    View details for Web of Science ID A1994PK97300078

    View details for PubMedID 8063836



    Oncogenes are activated forms of cellular genes involved in normal cell growth and development. Some oncogenes play a role in human malignancies. In hematologic malignancies, researchers have identified many transcription factors as oncogenes based on one of the following criteria: their association with transforming retroviruses in animals, their translocation into either the immunoglobulin or T-cell receptor loci, or the production of fusion proteins resulting from chromosomal translocations. The molecular characterization of oncogenes in hematologic malignancies has led to the discovery of new methods for diagnosis and detection of minimal residual disease. In the future, researchers probably will develop novel treatment strategies to interfere with the function of these oncogenes.

    View details for Web of Science ID A1994NF16800001

    View details for PubMedID 8198369

Conference Proceedings

  • An NF-kappa B site in the IgH enhancer deregulates bcl-2 expression in t(14;18) lymphomas. Somsouk, L., Heckman, C. A., Mehew, J. W., Kanda, K., Boxer, L. M. FEDERATION AMER SOC EXP BIOL. 1999: A1465-A1465

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