Martin Hilgarth

Reconstitution of PTEN activity by CK2 inhibitors and interference with the PI3-K/Akt cascade counteract the antiapoptotic effect of human stromal cells in chronic lymphocytic leukemia

Evidence suggests that tumor microenvironment is critically involved in supporting survival of chronic lymphocytic leukemia (CLL) cells. However, the molecular mechanisms of this effect and the clinical significance are not fully understood. We applied a microenvironment model to explore the interaction between CLL cells and stromal cells and to elucidate the role of phosphatidylinositol 3 kinase (PI3-K)/Akt/phosphatase and tensin homolog detected on chromosome 10 (PTEN) cascade in this process and its in vivo relevance. Primary human stromal cells from bone marrow, lymph nodes, and spleen significantly inhibited spontaneous apoptosis of CLL cells. Pan-PI3-K inhibitors (LY294002, wortmannin, PI-103), isotype-specific inhibitors of p110α, p110β, p110γ, and small interfering RNA against PI3-K and Akt1 counteracted the antiapoptotic effect of the stromal cells. Induction of apoptosis was associated with a decrease in phosphatidylinositol-3,4,5-triphosphate, PI3-K-p85, and dephosphorylation of phosphatidylinositol-dependent kinase-1 (PDK-1), Akt1, and PTEN. Freshly isolated peripheral blood mononuclear cells from patients with CLL (n = 44) showed significantly higher levels of phosphorylated Akt1, PDK-1, PTEN, and CK2 than healthy persons (n = 8). CK2 inhibitors (4,5,6,7-tetrabromo-1H-benzotriazole, apigenin, and 5,6-dichloro-1-β-D-ribofuranosylbenzimidazol) decreased phosphorylation of PTEN and Akt, induced apoptosis in CLL cells, and enhanced the response to fludarabine. In conclusion, bone marrow microenvironment modulates the PI3-K/Akt/PTEN cascade and prevents apoptosis of CLL cells. Combined inhibition of PI3-K/Akt and recovery of PTEN activity may represent a novel therapeutic concept for CLL.

Induction of apoptosis by proteasome inhibitors in B-CLL cells is associated with downregulation of CD23 and inactivation of Notch2.

Recently, proteasome inhibitors (PI) have attracted interest as novel anticancer agents in B-cell chronic lymphocytic leukemia (B-CLL). A prominent feature of B-CLL cells is the high expression of CD23, which is closely related to cell survival and is regulated by Notch2. Since several components of the Notch signaling cascade are tightly regulated by proteasomal degradation, we studied the effect of PI on Notch2 activity and CD23 expression. Exposure of B-CLL cells to PI led to induction of apoptosis, a time- and dose-dependent downregulation of CD23 expression and a decline in DNA binding of transcriptionally active Notch2. In contrast, the transcription factor NF-AT and its putative target gene CD5, which is highly expressed in B-CLL cells, were unaffected. When the late phase of PI-induced apoptosis was arrested by inhibition of caspase 3, the reduction of Notch2 activity was still observed, indicating that reduction of active Notch2 took place already during an earlier phase of apoptosis. Enforced expression of constitutively active Notch2 decreased PI-mediated apoptosis in a human B-cell line. These data indicate that downregulation of CD23 and loss of Notch2 activity are early steps in PI-induced apoptosis of B-CLL lymphocytes and may be part of the full apoptotic response.

TGF-β1 induces bone marrow reticulin fibrosis in hairy cell leukemia

The mechanisms that lead to reticulin fibrosis of bone marrow (BM) in hairy cell leukemia (HCL) are not fully understood. We therefore investigated the involvement of TGF-β1, a potent fibrogenic cytokine, in this process. Immunoassays revealed that TGF-β1 is present at higher concentrations in BM, serum, and plasma of HCL patients in comparison with healthy donors (P < 0.001). RT-PCR and immunofluorescence studies showed that TGF-β1 is overexpressed at the mRNA and protein levels in peripheral blood, spleen, and BM mononuclear cells and that hairy cells (HCs) are the main source of TGF-β1. Active TGF-β1 correlated significantly with grades of BM fibrosis, infiltration with HCs, and serum procollagen type III aminoterminal propeptide (PIIINP). Ex vivo studies demonstrated that TGF-β1 significantly enhances the production and deposition of reticulin and collagen fibers by BM fibroblasts. In addition, BM plasma of HCL patients increased the synthesis of type I and type III procollagens, the main components of reticulin fibers, at the mRNA and protein levels. This fibrogenic activity of BM plasma was abolished by neutralizing anti–TGF-β1 antibodies. These results show, for the first time to our knowledge, that TGF-β1 is highly expressed in HCs and is directly involved in the pathogenesis of BM reticulin fibrosis in HCL.

Effect of combined spa-exercise therapy on circulating TGF-beta1 levels in patients with ankylosing spondylitis.

ZusammenfassungZIEL: Um einen objektiven Indikator für das biologische Ansprechen von Patienten mit ankylosierender Spondylitis (AS, M. Bechterew) auf die Heilstollen-Behandlung in Badgastein zu finden, wurde die Rolle des anti-inflammatorischen Zytokins TGF-β1 (Transforming Growth Factor β 1) untersucht. METHODIK: 83 Patienten mit AS wurden 3–4 Wochen lang einer Therapie bestehend aus Hyperthermie, Exposition mit niedrigen Radon-Dosen und gymnastischen Übungen unterzogen. Das Ansprechen auf die Behandlung wurde durch das Ausmaß der Schmerzreduktion ermittelt. Als Kontrollen wurden 10 AS-Patienten, die eine konventionelle Therapie ohne Heilstollen-Exposition erhielten und 10 Patienten mit Rückenschmerzen ohne entzündliches Substrat (low back pain, LBP) in die Untersuchungen eingeschlossen. Vor und nach der Therapie wurden mittels Immunassays die Serumkonzentrationen von TGF-β1 bestimmt. RESULTATE: Nach Therapie wurde im Serum der ASPatienten ein signifikanter Anstieg von TGF-β1 (total und aktiviert) festgestellt. Die mittlere Konzentration des totalen TGF-β1 stieg von 28715 pg/ml auf 43136 pg/ml (p < 0,01) und die des aktivierten TGF-β1 von 77 auf 1096 pg/ml (p < 0.001). Bei Unterteilung der AS-Patienten hinsichtlich des Ausmaßes der Schmerzreduktion in zwei Gruppen, zeigte die Gruppe 1 (Schmerzreduktion: n = 46) einen 17-fachen Anstieg der Konzentration von aktiviertem TGF-β1 (96 bis 1654 pg/ml, p < 0.0001), während die Gruppe 2 (keine Schmerzreduktion: n = 37) nur einen 7-fachen Anstieg (53 bis 402 pg/ml, p < 0.01) aufwies. Bei LBP-Patienten wurde nur ein geringfügiger Anstieg des aktivierten TGF-β1 von 31 auf 42 pg/ml (p < 0.01) registriert und bei AS-Patienten, die lediglich einer konventionellen Therapie unterzogen wurden, fand sich keine signifikante Änderung der TGF-β1 Werte. SCHLUSSFOLGERUNG: Nach Heilstollen-Therapie in Badgastein fand sich im Blut von Patienten mit ankylosierender Spondylitis ein signifikanter Anstieg des zirkulierenden TGF-β1. Das Zytokin könnte auf Grund seiner antiinflammatorischen Eigenschaften auf den Heilungsprozess einwirken und somit zu einer Besserung der Schmerzen und der Mobilität der Patienten beitragen.SummaryBACKGROUND: Ankylosing spondylitis (AS) is a chronic inflammatory disease of the axial joints with no satisfactory therapy. Reduction of joint pain has been reported after a course of therapy at a spa, Gasteiner Heilstollen, in Badgastein in Austria. The mechanism underlying this beneficial effect is not clearly understood and objective evidence for the biological response to therapy is lacking. The aim of this study was to find evidence for a biological response to speleotherapy in patients with AS and to study the involvement of the antiinflammatory cytokine TGF-β1 in this response. PATIENTS AND METHODS: 83 patients with AS were treated in Badgastein for 3–4 weeks. Therapy included active exercises, hyperthermia and exposure to low doses of radon in a former mine. Response to therapy was assessed from measurement of morning pain and immunoassay of serum levels of TGF-β1 before and after therapy. Ten AS patients who received conventional therapy and 10 patients with low back pain (LBP) served as controls. RESULTS: A significant increase in TGF-β1 (total and active) was found in AS patients after spa therapy. Mean concentration of total TGF-β1 increased from 28,715 pg/ml to 43,136 pg/ml, (P < 0.01) and active TGF-β1 increased from 77 pg/ml to 1096 pg/ml (P < 0.001). When the AS patients were divided into two groups according to pain reduction, group 1 (decrease in morning pain, responders: n = 46) exhibited a 17-fold increase of active TGF-β1 levels (96 pg/ml to 1654 pg/ml, P < 0.0001) whereas group 2 (no change or an increase in morning pain: nonresponders: n = 37), showed only 7-fold increase (53 pg/ml to 402 pg/ml, P < 0.01). There was a moderate increase in active TGF-β1 from 31 pg/ml to 42 pg/ml (P < 0.05) in patients with LBP and no significant change was observed in the patients treated with conventional therapy. CONCLUSION: These results demonstrate a significant increase in circulating TGF-β1 in patients with AS after the combined spa-exercise therapy in Badgastein. The results also provide evidence for a biological response to speleotherapy and suggest that TGF-β, through its antiinflammatory function, may play a role in this response.

The role of soluble CD23 in distinguishing stable and progressive forms of B-chronic lymphocytic leukemia.

Soluble CD23 (sCD23) has been recognized as an important prognostic parameter in patients with chronic lymphocytic leukemia (B-CLL) at early clinical stages. There is, however, no clear information on its prognostic significance in advanced stages and on its role as an indicator for aggressive or indolent courses of disease. Therefore, sCD23 was measured in the serum of 145 patients at diagnosis and serial determinations were carried out for 8 years in 38 patients. The results indicate that in patients with identical clinical stages at first presentation the disease could take different courses depending on initial sCD23 concentrations below or above specific threshold levels (860 and 5900   U/ml). sCD23 higher than these thresholds was associated with faster progression into upper clinical stages. Furthermore, sCD23-doubling time (sCD23-DT) indicated that patients with long DT progressed slowly, while those with short DT had more aggressive disease. Particularly in patients with advanced disease stages, long sCD23-DT indicated development of smoldering disease. Since sCD23 levels reflect total tumor mass, determination of sCD23-DT has probably a better predictive value than lymphocyte doubling time. It appears that B-CLL patients can be divided into different risk categories according to initial determinations of sCD23 and that sCD23-DT is an additional important parameter in predicting disease progression.

Simultaneous occurrence of chronic myeloid leukemia and multiple myeloma: Evaluation by FISH analysis and in vitro expansion of bone marrow cells

Simultaneous occurrence of chronic myeloid leukemia and multiple myeloma: Evaluation by FISH analysis and in vitro expansion of bone marrow cells

Gliotoxin is a potent NOTCH2 transactivation inhibitor and efficiently induces apoptosis in chronic lymphocytic leukaemia (CLL) cells

Chronic lymphocytic leukaemia (CLL) cells express constitutively activated NOTCH2 in a protein kinase C (PKC)– dependent manner. The transcriptional activity of NOTCH2 correlates not only with the expression of its target gene FCER2 (CD23) but is also functionally linked with CLL cell viability. In the majority of CLL cases, DNA‐bound NOTCH2 complexes are less sensitive to the γ‐secretase inhibitor (GSI) DAPT. Therefore, we searched for compounds that interfere with NOTCH2 signalling at the transcription factor level. Using electrophoretic mobility shift assays (EMSA), we identified the Aspergillum‐derived secondary metabolite gliotoxin as a potent NOTCH2 transactivation inhibitor. Gliotoxin completely blocked the formation of DNA‐bound NOTCH2 complexes in CLL cells independent of their sensitivity to DAPT. The inhibition of NOTCH2 signalling by gliotoxin was associated with down regulation of CD23 (FCER) expression and induction of apoptosis. Short time exposure of CLL cells indicated that the early apoptotic effect of gliotoxin is independent of proteasome regulated nuclear factor κB activity, and is associated with up regulation of NOTCH3 and NR4A1 expression. Gliotoxin could overcome the supportive effect of primary bone marrow stromal cells in an ex vivo CLL microenvironment model. In conclusion, we identified gliotoxin as a potent NOTCH2 inhibitor with a promising therapeutic potential in CLL.

NOTCH2 links protein kinase C delta to the expression of CD23 in chronic lymphocytic leukaemia (CLL) cells

One characteristic of chronic lymphocytic leukaemia (CLL) lymphocytes is high expression of CD23, which has previously been identified as a downstream target for NOTCH2 signalling. The mechanisms regulating NOTCH2‐dependent CD23 expression, however, are largely unknown. This study showed that peripheral CLL cells overexpressed transcriptionally active NOTCH2 (N2IC), irrespective of their prognostic marker profile. When placed in culture, NOTCH2 activity was spontaneously decreased in 25 out of 31 CLL cases (81%) within 24 h. DNA‐bound N2IC complexes could be maintained by the protein kinase C (PKC) activator phorbol 12‐myristate 13‐acetate (PMA) or by γ‐interferon (IFN‐γ), two CLL characteristic inducers of CD23 expression. Inhibition of PKC‐δ by RNA interference or by rottlerin antagonised PMA‐induced NOTCH2 activation and also suppressed NOTCH2 activity in CLL cases with constitutively activated NOTCH2 signalling. In 23 out of 29 CLL cases tested (79%), DNA‐bound N2IC complexes were found to be resistant to the γ‐secretase inhibitor (GSI) DAPT, suggesting that GSIs will be only effective in a subset of CLL cases. These data suggest that deregulation of NOTCH2 signalling is critically involved in maintaining the malignant phenotype of CLL lymphocytes and point to a link between PKC‐δ and NOTCH2 signalling in the leukemic cells.

Gliotoxin Targets Nuclear NOTCH2 in Human Solid Tumor Derived Cell Lines In Vitro and Inhibits Melanoma Growth in Xenograft Mouse Model

Deregulation of NOTCH2 signaling is implicated in a wide variety of human neoplasias. The current concept of targeting NOTCH is based on using gamma secretase inhibitors (GSI) to regulate the release of the active NOTCH intracellular domain. However, the clinical outcome of GSI remains unsatisfactory. Therefore we analyzed human solid tumor derived cell lines for their nuclear NOTCH activity and evaluated the therapeutic potential of the NOTCH2 transactivation inhibitor gliotoxin in comparison to the representative GSI DAPT. Electrophoretic mobility shift assays (EMSA) were used as a surrogate method for the detection of NOTCH/CSL transcription factor complexes. The effect of gliotoxin on cell viability and its clinical relevance was evaluated in vitro and in a melanoma xenograft mouse model. Cell lines derived from melanoma (518A2), hepatocellular carcinoma (SNU398, HCC-3, Hep3B), and pancreas carcinoma (PANC1) express high amounts of nuclear NOTCH2. Gliotoxin efficiently induced apoptosis in these cell lines whereas the GSI DAPT was ineffective. The specificity of gliotoxin was demonstrated in the well differentiated nuclear NOTCH negative cell line Huh7, which was resistant to gliotoxin treatment in vitro. In xenotransplanted 518A2 melanomas, a single day dosing schedule of gliotoxin was well tolerated without any study limiting side effects. Gliotoxin significantly reduced the tumor volume in early (83 mm3 vs. 115 mm3, p = 0.008) as well as in late stage (218 mm3 vs. 576 mm3, p = 0.005) tumor models. In conclusion, NOTCH2 appears to be a key target of gliotoxin in human neoplasias and gliotoxin deserves further evaluation as a potential therapeutic agent in cancer management.

Critical assessment of the efficiency of CD34 and CD133 antibodies for enrichment of rabbit hematopoietic stem cells

Rabbits have many hereditary diseases common to humans and are therefore a valuable model for regenerative disease and hematopoietic stem cell (HSC) therapies. Currently, there is no substantial data on the isolation and/or enrichment of rabbit HSCs. This study was initiated to evaluate the efficiency of the commercially available anti‐CD34 and anti‐CD133 antibodies for the detection and potential enrichment of rabbit HSCs from peripheral blood. PBMCs from rabbit and human blood were labelled with different clones of anti‐human CD34 monoclonal antibodies (AC136, 581, and 8G12) and rabbit polyclonal CD34 antibody (pCD34) and anti‐human CD133 monoclonal antibodies (AC133 and 293C3). Flow cytometry showed a higher percentage of rabbit CD34+ cells labelled by AC136 in comparison to the clone 581 and pCD34 (P < 0.01). A higher percentage of rabbit CD133+ cells were also detected by 293C3 compared to the AC133 clone (P < 0.01). Therefore, AC136 clone was used for the indirect immunomagnetic enrichment of rabbit CD34+ cells using magnetic‐activated cell sorting (MACS). The enrichment of the rabbit CD34+ cells after sorting was low in comparison to human samples (2.4% vs. 39.6%). PCR analyses confirmed the efficient enrichment of human CD34+ cells and the low expression of CD34 mRNA in rabbit positive fraction. In conclusion, the tested antibodies might be suitable for detection, but not for sorting the rabbit CD34+ HSCs and new specific anti‐rabbit CD34 antibodies are needed for efficient enrichment of rabbit HSCs.