Kurt Bommert

Constitutive nuclear factor-kappaB-RelA activation is required for proliferation and survival of Hodgkin's disease tumor cells.

The pathogenesis and etiology of Hodgkins disease, a common human malignant lymphoma, is still unresolved. As a unique characteristic, we have identified constitutive activation of the transcription factor nuclear factor (NF)-kappaB p50-RelA in Hodgkin/Reed-Sternberg (H/RS) cells, which discriminates these neoplastic cells from most cell types studied to date. In contrast to other lymphoid and nonlymphoid cell lines tested, proliferation of H/RS cells depended on activated NF-kappaB. Furthermore, constitutive NF-kappaB p50-RelA prevented Hodgkins lymphoma cells from undergoing apoptosis under stress conditions. Consistent with this dual function, Hodgkins lymphoma cells depleted of constitutive nuclear NF-kappaB revealed strongly impaired tumor growth in severe combined immunodeficient mice. Our findings identify NF-kappaB as an important component for understanding the pathogenesis of Hodgkins disease and for developing new therapeutic strategies against it.

Aberrantly expressed c‐Jun and JunB are a hallmark of Hodgkin lymphoma cells, stimulate proliferation and synergize with NF‐κB

AP‐1 family transcription factors have been implicated in the control of proliferation, apoptosis and malignant transformation. However, their role in oncogenesis is unclear and no recurrent alterations of AP‐1 activities have been described in human cancers. Here, we show that constitutively activated AP‐1 with robust c‐Jun and JunB overexpression is found in all tumor cells of patients with classical Hodgkins disease. A similar AP‐1 activation is present in anaplastic large cell lymphoma (ALCL), but is absent in other lymphoma types. Whereas c‐Jun is up‐regulated by an autoregulatory process, JunB is under control of NF‐κB. Activated AP‐1 supports proliferation of Hodgkin cells, while it suppresses apoptosis of ALCL cells. Furthermore, AP‐1 cooperates with NF‐κB and stimulates expression of the cell‐cycle regulator cyclin D2, proto‐oncogene c‐met and the lymphocyte homing receptor CCR7, which are all strongly expressed in primary HRS cells. Together, these data suggest an important role of AP‐1 in lymphoma pathogenesis.

c-FLIP Mediates Resistance of Hodgkin/Reed-Sternberg Cells to Death Receptor–induced Apoptosis

Resistance to death receptor–mediated apoptosis is supposed to be important for the deregulated growth of B cell lymphoma. Hodgkin/Reed-Sternberg (HRS) cells, the malignant cells of classical Hodgkins lymphoma (cHL), resist CD95-induced apoptosis. Therefore, we analyzed death receptor signaling, in particular the CD95 pathway, in these cells. High level CD95 expression allowed a rapid formation of the death-inducing signaling complex (DISC) containing Fas-associated death domain–containing protein (FADD), caspase-8, caspase-10, and most importantly, cellular FADD-like interleukin 1β–converting enzyme-inhibitory protein (c-FLIP). The immunohistochemical analysis of the DISC members revealed a strong expression of CD95 and c-FLIP overexpression in 55 out of 59 cases of cHL. FADD overexpression was detectable in several cases. Triggering of the CD95 pathway in HRS cells is indicated by the presence of CD95L in cells surrounding them as well as confocal microscopy showing c-FLIP predominantly localized at the cell membrane. Elevated c-FLIP expression in HRS cells depends on nuclear factor (NF)-κB. Despite expression of other NF-κB–dependent antiapoptotic proteins, the selective down-regulation of c-FLIP by small interfering RNA oligoribonucleotides was sufficient to sensitize HRS cells to CD95 and tumor necrosis factor–related apoptosis-inducing ligand–induced apoptosis. Therefore, c-FLIP is a key regulator of death receptor resistance in HRS cells.

Overexpression of the death-promoting gene bax-alpha which is downregulated in breast cancer restores sensitivity to different apoptotic stimuli and reduces tumor growth in SCID mice.

We have studied the expression of members of the bcl-2 family in human breast cancer. The expression pattern of these genes in breast cancer tissue samples was compared with the expression pattern in normal breast epithelium. No marked difference with regard to bcl-2 and bcl-xL expression was observed between normal breast epithelium and cancer tissue. In contrast, bax-alpha, a splice variant of bax, which promotes apoptosis, is expressed in high amounts in normal breast epithelium, whereas only weak or no expression could be detected in 39 out of 40 cancer tissue samples examined so far. Of interest, downregulation of bax-alpha was found in different histological subtypes. Furthermore, we transfected bax-alpha into breast cancer cell lines under the control of a tetracycline-dependent expression system. We were able to demonstrate for the first time that induction of bax expression in breast cancer cell lines restores sensitivity towards both serum starvation and APO-I/Fas-triggered apoptosis and significantly reduces tumor growth in SCID mice. Therefore, we propose that dysregulation of apoptosis might contribute to the pathogenesis of breast cancer at least in part due to an imbalance between members of the bcl-2 gene family.

Intrinsic inhibition of transcription factor E2A by HLH proteins ABF-1 and Id2 mediates reprogramming of neoplastic B cells in Hodgkin lymphoma

B cell differentiation is controlled by a complex network of lineage-restricted transcription factors. How perturbations to this network alter B cell fate remains poorly understood. Here we show that classical Hodgkin lymphoma tumor cells, which originate from mature B cells, have lost the B cell phenotype as a result of aberrant expression of transcriptional regulators. The B cell–specific transcription factor program was disrupted by overexpression of the helix-loop-helix proteins ABF-1 and Id2. Both factors antagonized the function of the B cell–determining transcription factor E2A. As a result, expression of genes specific to B cells was lost and expression of genes not normally associated with the B lineage was upregulated. These data demonstrate the plasticity of mature human lymphoid cells and offer an explanation for the unique classical Hodgkin lymphoma phenotype.* NOTE: In the version of this article initially published online, the directions to the panels for Figure 6e were incorrect in the legend and text. The legend for this panel should begin as follows: “Immunoblot (top), EMSA (bottom left) and RT-PCR (bottom right)….” The accompanying text should read as follows: “Transfection of L428 cells with a combination of these siRNAs efficiently reduced ABF-1 protein expression (Fig. 6e, top) and resulted in a substantial loss of E2A–ABF-1 DNA-binding activity (Fig. 6e, bottom left). After reduction of ABF-1 expression, we noted considerable downregulation of CSF1R and TCF7 expression and a moderate suppression of GATA3 expression (Fig. 6e, bottom right).” The error has been corrected for the HTML and print versions of the article.

Identification of Apoptosis-associated Proteins in a Human Burkitt Lymphoma Cell Line CLEAVAGE OF HETEROGENEOUS NUCLEAR RIBONUCLEOPROTEIN A1 BY CASPASE 3

Apoptosis or programmed cell death is essential in the process of controlling lymphocyte growth and selection. We identified proteins that are involved in anti-IgM antibody-mediated apoptosis using a subclone of the human Burkitt lymphoma cell line BL60. Apoptosis-associated proteins were detected by high resolution two-dimensional gel electrophoresis on a micropreparative scale. Comparison of the high resolution two-dimensional gel electrophoresis protein patterns from apoptotic and non-apoptotic cells showed differences in ∼80 spots including protein modifications. Analysis of the predominantly altered proteins was performed by internal Edman microsequencing and/or by peptide mass fingerprinting using matrix-assisted laser desorption/ionization mass spectrometry. Analysis was significantly improved by using new micropreparative high resolution two-dimensional gels employing high protein concentrations. The following 12 apoptosis-associated proteins were identified: heterogeneous nuclear ribonucleoprotein (hnRNP) A1, hnRNP C1/C2, FUSE-binding protein, dUTPase, lymphocyte-specific protein LSP1, UV excision repair protein RAD23 homologue B (HHR23B), 60 S acidic ribosomal protein P0 (L10E), heterochromatin protein 1 homologue α (HP1α), nucleolin, lamin, neutral calponin, and actin. Fragmentation of actin, hnRNP A1, hnRNP C1/C2, 60 S acidic ribosomal protein P0, lamin, and nucleolin could be inhibited by benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethyl ketone, a selective irreversible inhibitor of CPP32 (caspase 3).

Apoptosis-induced cleavage of beta-catenin by caspase-3 results in proteolytic fragments with reduced transactivation potential.

β-Catenin is a member of the Armadillo repeat protein family with a dual cellular function as a component of both the adherens junction complex and the Wnt/wingless signaling pathway. Here we show that β-catenin is proteolytically cleaved during anoikis and staurosporine-induced apoptosis. Cleavage of β-catenin was found to be caspase-dependent. Five cleavage products of β-catenin were identified in vivo and after in vitrocleavage by caspase-3. Amino acid sequencing and mass spectrometry analysis indicated two caspase-3 cleavage sites at the C terminus and three further sites at the N terminus, whereas the central Armadillo repeat region remained unaffected. All β-catenin cleavage products were still able to associate with E-cadherin and α-catenin and were found to be enriched in the cytoplasm. Functional analysis revealed that β-catenin deletion constructs resembling the observed proteolytic fragments show a strongly reduced transcription activation potential when analyzed in gene reporter assays. We therefore conclude that an important role of the β-catenin cleavage during apoptosis is the removal of its transcription activation domains to prevent its transcription activation potential.

Inactivating I kappa B epsilon mutations in Hodgkin/Reed-Sternberg cells.

The pathogenesis of Hodgkin lymphoma (HL) is still unclear. Previous investigations have demonstrated constitutive nuclear activity of the transcription factor NF kappa B (NF‐κB) in Hodgkin/Reed–Sternberg (HRS) cells as an important prerequisite in protecting these cells from apoptosis. As a molecular mechanism leading to constitutive NF‐κB activity in HRS cells, mutations of the NF‐κB inhibitor I kappa B alpha (IκBα) have recently been identified in classical (c) HL‐derived cell lines in a patient with cHL. In the present study, the NF‐κB inhibitor I kappa B epsilon (IκBε) has been analysed for somatic mutations in the same group of six patients already studied for IκBα mutations, as well as in cHL‐derived cell lines. In one cHL‐derived cell line (L428), a hemizygous frame‐shift mutation generating a pre‐terminal stop codon resulting in a severely truncated protein was found. Moreover, in the HRS cells of one patient, a hemizygous mutation affecting the 5′‐splicing site of intron 1 of the IκBε gene was found. These results, in combination with recently described IκBα mutations, indicate that defective NF‐κB inhibitors appear more frequent than previously thought and might explain the constitutive nuclear activity of NF‐κB in a significant proportion of cHL cases. Copyright

Induction of the death‐promoting gene bax‐α sensitizes cultured breast‐cancer cells to drug‐induced apoptosis

Resistance to apoptosis plays an important role in malignancies that are refractory to chemotherapy treatment. Recently we have shown that the expression of bax‐α, a death‐promoting member of the bcl‐2 family, is down‐regulated in breast cancer and have provided evidence that low bax expression might contribute to the pathogenesis of breast cancer. In this study we were able to demonstrate the role of this gene in chemotherapy‐induced apoptosis. We transfected bax‐α into the breast‐cancer cell lines R30C and MCF‐7 under the control of an inducible tetracycline‐dependent expression system. Induction of bax‐α expression did not affect viability by itself but strongly increased chemosensitivity to epirubicin. We were able to demonstrate that this sensitization is due to apoptosis. These data might explain the recently published observation that reduced expression of bax is associated with poor response rates to chemotherapy in breast cancer.

A role for β2 integrins (CD11/CD18) in the regulation of cytokine gene expression of polymorphonuclear neutrophils during the inflammatory response

Growing evidence supports the idea that adhesion via β2 integrins not only allows cellular targeting, but also induces intracellular signaling, which in turn activates functional responses of adherent cells. This study investigates whether β2 integrin‐mediated adhesion of human polymorphonuclear neutrophils (PMN) has a functional impact on cytokine production. Aggregation of the β2 integrin Mac‐1 (CD11b/CD18) by antibody cross‐linking was found to induce substantial de novo synthesis of IL‐8 mRNA as measured by semiquantitative RT‐PCR and Northern blotting technique, respectively. Induction of IL‐8 mRNA was also observed upon adhesion of PMN to immobilized fibrinogen, a functional equivalent of its clotting product fibrin that serves as a native ligand of Mac‐1. Results were confirmed using PMN derived from CD18‐deficient mice, which were unable to produce MIP‐2 mRNA, a homologue of human IL‐8, in the presence of immobilized fibrinogen. In contrast, a substantial increase of MIP‐2 mRNA was observed when wild‐type PMN were incubated on immobilized fibrinogen. In human PMN, ELISA technique showed that the gene activation that required tyrosine kinase activity resulted in a substantial production and secretion of biologically active IL‐8 and IL‐1β. In contrast, no TNF‐α or IL‐6 production was found, revealing that β2 integrins mediate differential expression of proinflammatory cytokines. The biological relevance of the present findings was confirmed in an in vivo model of acute inflammation. Altogether, the present findings provide evidence for a functional link between clotting and inflammatory responses that may contribute to the recruitment and/or activation of PMN and other cells at sites of lesion.—Walzog, B., Weinmann, P., Jeblonski, F., Scharffetter‐Kochanek, K., Bommert, K., Gaehtgens, P. A role for β2 integrins (CD11/CD18) in the regulation of cytokine gene expression of polymorphonuclear neutrophils during the inflammatory response. FASEB J. 13, 1855–1865 (1999)