Nuclear localization and increased levels of transcription factor YB-1 in primary human breast cancers are associated with intrinsic MDR1 gene expression.

Breast cancers are either primarily resistant to chemotherapy (intrinsic resistance), or respond to chemotherapy but later recur with a multidrug-resistant phenotype because of overexpression of the multidrug transporter P-glycoprotein. The MDR1 gene encoding P-glycoprotein may be transcriptionally regulated by a Y-box transcription factor. We now report that, in multidrug-resistant MCF-7 breast cancer cells, nuclear localization of YB-1 is associated with MDR-1 gene expression. In drug-sensitive MCF-7 cells, however, YB-1 was localized to the cytoplasm. Regulated overexpression of YB-1 in drug-sensitive diploid breast epithelial cells induced MDR-1 gene expression and multidrug resistance. In 27 out of 27 untreated primary breast cancers, YB-1 protein was expressed in the cytoplasm although it was undetectable in normal breast tissue of these patients. In a subgroup of tumors (9/27), however, YB-1 was also localized to the nucleus and, in these cases, high levels of P-glycoprotein were present. These results show that in a subset of untreated primary breast cancers, nuclear localization of YB-1 protein is associated with intrinsic multidrug resistance. Our data show that YB-1 has an important role in controlling MDR1 gene transcription and this finding provides a basis for the analysis of molecular mechanisms responsible for intrinsic multidrug resistance in human breast cancer.

Blocking the transcription factor E2F/DP by dominant-negative mutants in a normal breast epithelial cell line efficiently inhibits apoptosis and induces tumor growth in SCID mice.

The transcription factor E2F is regulated during the cell cycle through interactions with the product of the retinoblastoma susceptibility gene and related proteins. It is thought that E2F-mediated gene regulation at the G1/S boundary and during S phase may be one of the rate-limiting steps in cell proliferation. It was reported that in vivo overexpression of E2F-1 in fibroblasts induces S phase entry and leads to apoptosis. This observation suggests that E2F plays a role in both cell cycle regulation and apoptosis. To further understand the role of E2F in cell cycle progression, cell death, and tumor development, we have blocked endogenous E2F activity in HBL-100 cells, derived from nonmalignant human breast epithelium, using dominant-negative mutants under the control of a tetracycline-dependent expression system. We have shown here that induction of dominant-negative mutants led to strong downregulation of transiently transfected E2F-dependent chloramphenicol acetyl transferase reporter constructs and of endogenous c-myc, which has been described as a target gene of the transcription factor E2F/DP. In addition, we have shown that blocking of E2F could efficiently protect from apoptosis induced by serum starvation within a period of 10 d, whereas control cells started to die after 24 h. Surprisingly, blocking of E2F did not alter the rate of proliferation or of DNA synthesis of these cells; this finding indicates that cell-cycle progression could be driven in an E2F-independent manner. In addition, we have been able to show that blocking of endogenous E2F in HBL-100 cells led to rapid induction of tumor growth in severe combined immunodeficiency mice. No tumor growth could be observed in mice that received mock-transfected clones or tetracycline to block expression of the E2F mutant constructs in vivo. Thus, it appears that E2F has a potential tumor-suppressive function under certain circumstances. Furthermore, we provide evidence that dysregulation of apoptosis may be an important step in tumorigenesis.

A post-docking role for synaptobrevin in synaptic vesicle fusion.

We have used the squid giant synapse to determine the role of synaptobrevin, integral membrane proteins of small synaptic vesicles, in neurotransmitter release. The sequence of squid synaptobrevin, deduced by cDNA cloning, is 65%-68% identical to mammalian isoforms and includes the conserved cleavage site for tetanus and botulinum B toxins. Injection of either toxin into squid nerve terminals caused a slow, irreversible inhibition of release without affecting the Ca2+ signal which triggers release. Microinjection of a recombinant protein corresponding to the cytoplasmic domain of synaptobrevin produced a more rapid and reversible inhibition of release, whereas two smaller peptide fragments were without effect. Electron microscopy of tetanus-injected terminals revealed an increased number of both docked and undocked synaptic vesicles. These data indicate that synaptobrevin participates in neurotransmitter release at a step between vesicle docking and fusion.

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

The pathogenesis and etiology of Hodgkin's 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 Hodgkin's lymphoma cells from undergoing apoptosis under stress conditions. Consistent with this dual function, Hodgkin's 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 Hodgkin's disease and for developing new therapeutic strategies against it.

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.

Anti-CD20- and B-cell receptor-mediated apoptosis: evidence for shared intracellular signaling pathways.

Clinical administration of the anti-CD20 antibody IDEC-C2B8 can induce remission of low-grade B-cell lymphoma. Whereas it has been suggested that the main mechanisms of action are complement-mediated and antibody-dependent cell-mediated cytotoxicity, we demonstrate that monoclonal antibody IDEC-C2B8 is a strong inducer of apoptosis in CD20-positive B-cell lymphoma cell lines reflecting different stages of lymphomagenesis. Thus, CD20-dependent apoptosis was inducible in human surface IgM-positive Burkitt's lymphoma cell lines as well as in more mature surface IgM-negative B-cell lymphoma cell lines carrying the t(14;18) translocation. Furthermore, in Burkitt's lymphoma cell lines, we observed a striking correlation between anti-CD20- and B-cell receptor-mediated apoptosis with regard to sensitivity toward the apoptotic stimuli and the execution of the apoptotic pathway. Thus, induction of anti-CD20- or B-cell receptor-mediated apoptosis involved rapid up-regulation of the proapoptotic protein Bax. In addition, we show similar changes in the mRNA expression level of two early response genes, c-myc and Berg36, as well as activation of the mitogen-activated protein kinase family members p44 (extracellular signal-regulated kinase 1) and p42 (extracellular signal-regulated kinase 2) and activation of activator protein 1 (AP-1) DNA binding activity. These data support our hypothesis that both pathways are mediated in part by the same signal-transducing molecules. These results might help explain the resistance and regression of lymphomas to IDEC-C2B8 and give new insights in the signaling cascade after CD20 ligation.

Apoptosis-induced cleavage of keratin 15 and keratin 17 in a human breast epithelial cell line.

Keratin 15 (K15) and keratin 17 (K17) are intermediate filament (IF) type I proteins that are responsible for the mechanical integrity of epithelial cells. By analyzing the human breast epithelial cell line H184A1 before and after induction of apoptosis by high-resolution two-dimensional gel electrophoresis (2-DE) we identified the caspase-mediated cleavage of keratins 15 and 17. After induction of apoptosis three fragments of both K15 and K17 could be observed by 2 -DE. K15 and K17 proteolysis was observed during staurosporine-induced apoptosis and anoikis (anchorage-dependent apoptosis) as well and was shown to be caspase-dependent. By using mass spectrometry we could determine the caspase cleavage sites, one in K15 and two in K17. The sequence VEMD/A at the cleavage site located in the conserved linker region was found in K15 and K17. A further cleavage site was identified in the tail region of K17 with the recognition motif EVQD/G.

PI3-K/AKT/FKHR and MAPK signaling cascades are redundantly stimulated by a variety of cytokines and contribute independently to proliferation and survival of multiple myeloma cells.

IL-6 has been reported to play a central role in growth and survival of multiple myeloma (MM) cells. However, recently we have demonstrated that in the presence of bone marrow stromal cells, survival of MM cells becomes independent of the IL-6/gp130/STAT3 pathway questioning the singular role of IL-6 in MM. Therefore, it was the aim of this study to identify additional factors and signaling pathways that might contribute to the growth and survival of MM cells. We found that in addition to IL-6 a number of bone marrow derived cytokines such as LIF, VEGF, bFGF, MIP-1alpha, SDF-1alpha, IL-1beta, SCF and IL-3 activate the MAPK pathway and induce proliferation of MM.1S and RPMI-8226 MM cells. In addition, these cytokines independently phosphorylate the forkhead family member FKHR via PI3-K/AKT and support survival of primary human MM cells. Inhibition of these pathways induces apoptosis in MM cell lines and primary MM cells. Thus, we provide evidence that in addition to IL-6 a number of different factors trigger important growth-promoting pathways to support the proliferation and survival of MM cells. Therefore, blocking such pathways, rather than blocking a single factor, might be a promising approach for the development of novel treatment strategies in MM.

A novel recombinant bispecific single-chain antibody, bscWue-1 x CD3, induces T-cell-mediated cytotoxicity towards human multiple myeloma cells.

The development of antibody-based strategies for the treatment of multiple myeloma (MM) has been hampered so far by the fact that suitable plasma cell-specific surface antigens have been missing. However, recently a novel monoclonal antibody, designated Wue-1, has been generated that specifically recognizes normal and malignant human plasma cells. Therefore, Wue-1 is an interesting and promising candidate to develop novel immunotherapeutic strategies for the treatment of MM. One variant for an antibody-based strategy is the bispecific antibody approach. Recombinant bispecific single-chain (bsc) antibodies are especially interesting candidates because they show exceptional biological properties. We have generated a novel MM-directed recombinant bsc antibody, bscWue-1 x CD3, and analyzed the biological properties of this antibody using the MM cell line NCI-H929 and primary cells from the bone marrow of patients with MM. We were able to show that bscWue-1 x CD3 induces efficient and selective T-cell-mediated cell death of NCI-H929 cells and primary myeloma cells in nine out of 11 cases. The bscWue-1 x CD3 Ab is efficacious even at low E:T ratios, and with or without additional T-cell pre- or costimulation. Target cell lyses were specific for Wue-1 antigen-positive cells and could be blocked by the Wue-1 monoclonal antibody.

Efficient elimination of chronic lymphocytic leukaemia B cells by autologous T cells with a bispecific anti-CD19/anti-CD3 single-chain antibody construct.

Recently, we have shown that a novel recombinant bispecific single-chain antibody construct (bscCD19 x CD3), induces highly efficacious lymphoma-directed cytotoxicity mediated by unstimulated peripheral T lymphocytes. Functional analysis of bscCD19 x CD3 has so far been exclusively performed with human B lymphoma cell lines and T cells from healthy donors. Here we analysed the properties of bscCD19 x CD3 using primary B cells and autologous T cells from healthy volunteers or patients with B-cell chronic lymphocytic leukaemia (B-CLL). We show that bscCD19 x CD3 induces T-cell-mediated depletion of nonmalignant B cells in all four cases and depletion of primary lymphoma cells in 22 out of 25 cases. This effect could be observed at low effector-to-target (E:T) ratios and in the majority of cases without additional activation of autologous T cells by IL-2. Even in samples derived from patients heavily pretreated with different chemotherapy regimens, strong cytotoxic effects of bscCD19 x CD3 could be observed. The addition of bscCD19 x CD3 to patients' cells resulted in an upregulation of activation-specific cell surface antigens on autologous T cells and elevated levels of CD95 on lymphoma B cells. Although anti-CD95 antibody CH-11 failed to induce apoptosis in lymphoma cells, we provide evidence that B-CLL cell depletion by bscCD3 x CD3 is mediated at least in part by apoptosis via the caspase pathway.

The feed-forward loop between YB-1 and MYC is essential for multiple myeloma cell survival.

Y-box binding protein 1 (YB-1) functions as a translational regulator and has been suggested to elevate MYC mRNA translation via an internal ribosome entry segment (IRES) point mutation in multiple myeloma (MM). We show that YB-1-mediated translation of MYC mRNA occurs independently of the reported IRES mutation, as 87 MM patients (n=88) and all tested human MM cell lines (HMCLs) were negative for the mutation. We show for the first time that positive MYC staining predicts YB-1 co-expression in malignant plasma cells and YB-1/MYC co-expression increases from 30% in medullary to 70% in extramedullary MM. YB-1 knockdown in HMCLs reduced both MYC protein levels and MYC mRNA in the polysomal fraction, providing a mechanism by which YB-1 controls MYC translation. MYC transcription of YB-1 is demonstrated in HMCLs as MYC knockdown resulted in reduced YB-1 protein and mRNA levels. Furthermore, MYC activation in non-malignant mouse embryonic fibroblasts (MEFs) increased YB-1 mRNA, clearly indicating that MYC drives YB-1 transcription. Importantly, perturbation of the MYC/YB-1 oncogenic circuit leads to apoptosis in HMCLs. Here, we demonstrate that these two proteins co-regulate each other via combined transcriptional/translational activity establishing their pivotal role in MM cell survival. We therefore suggest that targeting the YB-1/mRNA interaction provides a new strategy for MM drug development.

[Heat shock protein 90 alpha und beta are overexpressed in multiple myeloma cells and critically contribute to survival]. / Die Hitzeschockproteine 90 alpha und beta sind im mutiplen Myelom überexprimiert und tragen zum Tumorzellüberleben bei.

HSP90's are overexpressed in different cancer types and they probably are required to sustain aberrant signalling in malignant cells. Recently, pharmacological inhibition of HSP90 was found to suppress growth of myeloma cell lines and in primary myeloma cells. Therefore, we wanted to investigate the role of HSP90alpha and HSP90beta in the pathogenesis of malignant myeloma (MM) in more detail. Immunohistochemistry was employed to examine the expression of HSP90alpha and HSP90beta in MM. The importance of HSP90 for survival of MM -cells was investigated by SiRNA-mediated knockdown of HSP90 and blockade of the IL-6R/STAT3 and the MAPK signaling pathways in vitro. HSP90alpha and HSP90beta were overexpressed in majority of investigated MM cases, but not in MGUS or in normal plasma cells. SiRNA-mediated knockdown of HSP90 or treatment with the novel HSP90 inhibitor 17-DMAG attenuated the levels of STAT3 and phospho-ERK and decreased the viability of MM cells. The knockdown of HSP90alpha was sufficient to induce apoptosis. This effect was strongly increased when both HSP90s were targeted, indicating a cooperation of both. HSP90 critically contributes to myeloma survival in the context of its microenvironment and therefore strengthen the potential value of HSP90 as a therapeutic target.

Prefractionation of protein samples for proteome analysis using reversed-phase high-performance liquid chromatography.

We describe an approach for fractionating complex protein samples prior to two-dimensional gel electrophoresis using reversed-phase high-performance liquid chromatography. Whole lysates of cells and tissue were prefractionated by reversed-phase chromatography and elution with a five-step gradient of increasing acetonitrile concentrations. The proteins obtained at each step were subsequently separated by high-resolution two-dimensional gel electrophoresis (2-DE). The reproducibility of this prefractionation technique proved to be optimal for comparing 2-DE gels from two different cell states. In addition, this method is suitable for enriching low-abundance proteins barely detectable by silver staining to amounts that can be detected by Coomassie blue and further analyzed by mass spectrometry.

Cleavage and shedding of E-cadherin after induction of apoptosis.

Apoptotic cell death induces dramatic molecular changes in cells, becoming apparent on the structural level as membrane blebbing, condensation of the cytoplasm and nucleus, and loss of cell-cell contacts. The activation of caspases is one of the fundamental steps during programmed cell death. Here we report a detailed analysis of the fate of the Ca(2+)-dependent cell adhesion molecule E-cadherin in apoptotic epithelial cells and show that during apoptosis fragments of E-cadherin with apparent molecular masses of 24, 29, and 84 kDa are generated by two distinct proteolytic activities. In addition to a caspase-3-mediated cleavage releasing the cytoplasmic domain of E-cadherin, a metalloproteinase sheds the extracellular domain from the cell surface during apoptosis. Immunofluorescence analysis confirmed that concomitant with the disappearance of E-cadherin staining at the cell surface, the E-cadherin cytoplasmic domain accumulates in the cytosol. In the presence of inhibitors of caspase-3 and/or metalloproteinases, cleavage of E-cadherin was almost completely blocked. The simultaneous cleavage of the intracellular and extracellular domains of E-cadherin may provide a highly efficient mechanism to disrupt cadherin-mediated cell-cell contacts in apoptotic cells, a prerequisite for cell rounding and exit from the epithelium.

Activation and activation-induced death of human tonsillar B cells and Burkitt lymphoma cells: lack of CD95 (Fas/APO-1) ligand expression and function.

Activation of T cells was shown to up-regulate the Fas ligand (FasL) which binds to the CD95 (APO-1/Fas) antigen and mediates activation-induced cell death (AICD) of activated T cells and T lymphoma cells. A recent report showed that mouse B cells express the FasL upon activation with lipopolysaccharide (LPS). We therefore asked whether activation of human B cells induces expression of FasL and whether AICD is mediated, as in T cells, through autocrine production of the FasL. We used human tonsillar B cells and Burkitt lymphoma cell lines which were activated by CD40 ligand, surface (s)IgM cross-linking, or LPS. Northern and Western blot analysis failed to detect FasL during B cell activation or AICD of both normal and malignant B cells. Low-level expression of FasL was detected by reverse transcriptase-polymerase chain reaction. Functional experiments, however, showed that FasL is not functionally expressed upon activation. IgM-mediated AICD in the tonsillar or Burkitt lymphoma B cells could not be inhibited by FasL blocking. Thus, our data show that, in contrast to T cells, activation of normal or malignant human B cells does not lead to functional FasL expression.

Overexpression of the death-promoting gene bax-alpha sensitizes human BL-41 Burkitt lymphoma cells for surface IgM-mediated apoptosis.

Major regulators of programmed cell death, or apoptosis, are the members of the bcl-2 gene family. Recently, we reported that surface(s) IgM triggering of the human B lymphoma cell line BL-41 led to strong induction of bax-alpha, a death-promoting member of the bcl-2 family, and subsequently to induction of apoptosis, suggesting a potential regulatory role of bax-alpha in sIgM-mediated cell death. In contrast, apoptosis-resistant subclones of this cell line showed only weak bax-alpha expression, which was not inducible by sIgM cross-linking. In this study, we were able to demonstrate the functional significance of this observation. We stably transfected bax-alpha into a BL-41 subline resistant against sIgM-mediated apoptosis. Several bax-alpha overexpressing clones could be selected, which all showed enhanced sensitivity for sIgM-mediated apoptosis. In contrast, no sensitive clone could be identified in a large number of mock controls. This clearly indicates that induction of bax-alpha is a critical regulatory step, which sensitizes B cells for sIgM-mediated apoptosis.

Preparative high-resolution two-dimensional electrophoresis enables the identification of RNA polymerase B transcription factor 3 as an apoptosis-associated protein in the human BL60-2 Burkitt lymphoma cell line.

Apoptosis or programmed cell death is essential in the process of controlling lymphocyte growth and selection. We identified RNA polymerase B transcription factor 3 (BTF3), which is associated with anti-IgM antibody-mediated apoptosis, using a subclone of the human Burkitt lymphoma cell line BL60. To identify the transcription factor BTF3, which is expressed only in minor amounts, we used preparative high-resolution two-dimensional gel electrophoresis (2DE) employing carrier ampholytes for isoelectric focusing. Comparison of the 2DE protein patterns from apoptotic and nonapoptotic cells showed BTF3 as a predominantly altered protein spot. The characterization of the differentially expressed transcription factor and 13 marker proteins described in this study were performed by internal Edman microsequencing and/or by peptide mass fingerprinting using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The proteome analysis was significantly improved by performing the newly developed preparative high-resolution two-dimensional gels employing high protein concentrations.

Involvement of NAK-1, the human nur77 homologue, in surface IgM-mediated apoptosis in Burkitt lymphoma cell line BL41.

The induction of apoptosis via surface IgM (sIgM) in immature B cells requires de novo transcription. To investigate the regulation of activation-induced cell death (AICD) in B cells we used a cell line model consisting of an Epstein-Barr virus-negative Burkitt lymphoma cell line (BL41), which is highly sensitive, and a subclone which is resistant to sIgM-mediated apoptosis (BL41/B5). Resistance in this cell line was not due to down-regulation of sIgM or functional impairment in signal transduction of the surface Ig complex. The zinc finger transcription factor nur77 has been implicated to play an important role in CD3-mediated apoptosis in murine T cells. We were able to demonstrate that surface IgM ligation and subsequent apoptosis in BL41 cells is associated with a concomitant induction of NAK-1, the human nur77 homologue. Induction of NAK-1 mRNA and DNA binding activity in the nucleus could be readily observed by means of Northern blot and electrophoretic mobility shift assay, respectively. In contrast, the resistant clone BL41/B5 did not show any NAK-1 expression upon stimulation. This suggests a role for NAK-1 in sIgM-mediated apoptosis of immature B cells.