Research activities

· Longitudinal studies of sCD23 from patients with B-CLL

· Effect of Bisphosphonates on actin cytoskeleton and induction of apoptosis in tumor cells

· Deregulation of Notch2 signaling in B-CLL

· Regulation of hairy cell adhesion to bone marrow fibroblasts via VLA-4 / VCAM-1 by TGF-ß

· Mechanism of Action of Radon in Patients with Ankylosing Spondylitis (Morbus Bechterew):

Longitudinal studies of sCD23 from patients with B-CLL:

Soluble CD23 (sCD23) has been recognised 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 serum of 145 patients at diagnosis and serial determinations were carried out for eight years in 38 patients (24 patients in Rai 0-I and 14 patients in Rai II-IV stages). Results showed that, in patients with identical clinical stages at first presentation, the disease took different courses depending on initial sCD23 concentrations below or above specific threshold levels (group 1: 860 U/ml; group 2: 5900 U/ml). sCD23 higher than these thresholds was associated with faster progression into upper clinical stages. Serial determinations of sCD23 and calculation of sCD23-doubling time (sCD23-DT) demonstrated that patients with long sCD23-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 smouldering disease. As compared to lymphocyte doubling time (LTD), sCD23-DT showed a better predictive value for disease progression. However, sCD23 did not correlate with the in vitro sensitivity of B‑CLL cells to chemotherapy as demonstrated by proliferation assays (MTT). We conclude that B-CLL patients can be divided into different risk categories according to initial determinations of sCD23, and that sCD23-DT may serve as an additional important parameter in predicting disease progression.

Effect of Bisphosphonates on actin cytoskeleton and induction of apoptosis in tumor cells:

Increased bone resorption is a prominent feature in patients with cancer and might be attributed to the activation of the osteoclasts by soluble factors and cytokines released from the tumour cells. On the other hand, bisphosphonates are the most effective treatment for hypercalcemia of malignancy, they decrease the skeletal complications in patients with cancer and reduce bone metastasis. We addressed the question whether a direct effect of bisphosphonates on tmour cells may contribute to the mechanism leading to its beneficial effects.

In vitro effects of a potent member of bisphosphonate family (AHBuBP) on human lung adenocarcinoma cell line A549 were investigated. Results showed that AHBuBP (1-100 µM) inhibits cell proliferation, leads to striking morphological changes in the malignant cells (rounding and detachment) and induce tumour cell death by apoptosis. The nature of apoptotic cell death was assessed by microscopy, specific chromatin stain (Hoechst fluorochrome- 33258), annexin V and propidium iodide stains, DNA extraction and gel electrophoresis showing the typical DNA laddering. This effect was more prominent in actively proliferating than in serum deprived cells. Interestingly, the effect of AHBuBP on the tumour cells could be significantly reduced by mevalonate (1mM) or its metabolic intermediate geranylgeranyl pyrophosphates (20-60 µM) but not by farnesol, farnesol pyrophosphate or squalene. Studies on actin cytoskeleton showed that the bisphosphonate-induced morphological changes were associated with disruption of actin filaments in the tumour cells prior to death by apoptosis.

Results indicate that bisphosphonates may interfere with the mevalonate pathway in tumour cells thereby depriving the cells from isoprenid-intermediates that are essential for maintaining cytoskeletal structure and supporting cell growth and survival. This may also point to another aspect in the mechanisms of action of bisphosphonates that lead to inhibition of bone resorption associated with malignancy and reducing skeletal metastasis.

Deregulation of Notch2 signaling in B-CLL:

The overexpression of the transmembrane glycoprotein CD23 is one of the major characteristics of B-CLL cells. Besides the prognostic potential of its soluble cleavage product, sCD23, selective expression of the CD23a isoform is concurrent to a state of B-CLL cell survival, thereby providing a link between CD23a and the malfunction of apoptosis characteristic for this neoplastic B-cell type.

By electrophoretic mobility shift assays, we identified a transcription factor complex (C1) which binds sequence specific to one known and four newly identified putative CBF1 recognition sites in the CD23a core promoter region. The significance of this complex was underlined by the fact, that in B-cell samples the intensity of C1 correlated with their respective levels of CD23a transcription. Furthermore, using Epstein Barr virus (EBV) infected B-cells as a model for CBF1 mediated CD23a expression, C1 was found to be EBV inducible. Supershift assays revealed that the nuclear form of Notch2 is a component of C1 in B-CLL cells, supporting a model in which NotchIC activates transcription by binding to CBF1 tethered to DNA. Finally, RT-PCR analysis indicates that the Notch2 oncogene is overexpressed in B-CLL cells. These data suggest that deregulation of Notch2 signaling, which is known to inhibit differentiation and apoptosis, plays a pivotal role in the upregulation of the CD23 gene in B-CLL cells.

Regulation of hairy cell adhesion to bone marrow fibroblasts via VLA-4 / VCAM-1 by TGF-ß:

Bone marrow (BM) infiltration with the malignant hairy cells (HC) is a characteristic finding in patients with Hairy cell leukemia (HCL). In the BM, hairy cells are found in association with fibroblastic cells and surrounded with fine reticulin meshwork. Such selective tissue localization may be attributed to the adhesive and migratory properties of the HC and allows the cells to receive signals for survival and proliferation. We have recently reported that BM of HCL patients contain high levels of transforming growth factor-beta 1 (TGF-ß) as compared to healthy donors (HD). This cytokine plays an important role in regulating cell-cell and cell-matrix adhesion. Therefore, we investigated the in vitro interaction between the HC and BM fibroblasts (BMF) and the influence of TGF-ß on this process. Co-cultures of peripheral blood leukocytes or purified HC and BMF demonstrated a striking adhesive property of the HC to BMF. Within few minutes, HC were observed to adhere to and migrate beneath the fibroblasts. This was followed by formation of cell clusters and enhanced cell survival and proliferation. Under the same culture conditions, cells from HD remained in suspension. Adhesion assays revealed that TGF-ß further increases the adhesion of HC while neutralizing anti-TGF-ß antibodies inhibit it. In spite of the known inhibitory effect of TGF-ß on the proliferation of B cells, no such inhibitory effect was found on the HC. FACS and immunofluorescence studies revealed high expression of VLA-4 on the HC. Preincubation of the HC with antibodies against the a4 chain of VLA-4 integrin (CD49d) or preincubating the BMF with anti-VCAM-1 (CD106) Ab inhibited the adhesion of HC. This inhibition could be augmented by anti-TGF-ß Ab and reduced by TGF-ß. We conclude that the adhesion of the hairy cells to bone marrow fibroblasts through binding of VLA-4 integrin to its ligand VCAM-1 may represent a key step in marrow infiltration with the malignant cells and that TGF-ß may play an important role in this process.

Mechanism of Action of Radon in Patients with Ankylosing Spondylitis (Morbus Bechterew): Regualtion of TGF-ß Activities:

Morbus Bechterew/Ankylosing spondylitis (AS) is a chronic inflammatory disease of the axial joints. The disease is initiated by inflammatory reactions with lymphocytic infiltration at the attachment of ligaments to bone. Subsequent healing with fibrosis and calcification gives rise to bony ankylosis and ultimately leading to fusion of the whole spine. A promising beneficial effect for radon in therapy of AS has been demonstrated (A. Falkenbach, M. Herold 1998). However the mechanisms underlying this beneficial effects at cellular level are not clear.

Several cytokines might be involved in the pathogphysiology of AS and might be regulated by expopsure to radon. A potential candidate is transforming growth factor-beta (TGF-ß). This cytokine is known as a potent immunosuppressive agent and plays a major role in the healing processes, induction of fibrosis and bone formation (S. Wahl et al., 1992, W. Border et al, 1994, M. Centrella et al, 1994). This may suggest that it might be involved in different stages in the pathogenesis of AS. In accordance with this suggestion are the results of in situ hybridisation of biopsies from sacroiliac joints from patients with AS demonstrating the expression of TGF-ß mRNA particularly near the site of new bone formation (J. Braun et.al. 1995). Also studies on murine progressive ankylosis, which resembles human ankylosing spondylitis, showed the enhanced proliferative responsiveness of the spinal ligament fibroblasts to TGF-ß as compared to normal fibroblasts (H. Krug 1998).

TGF-ß is produced locally in a latent form that requires activation to induce a biological activity. However, the precise mechanism of TGF-ß activation in vivo is not clear. Recent investigations demonstrated that exposure of mice to radiation leads to in situ activation of latent TGF-ß (M. Barcellos-Hoff 1994) through a mechanism that involves the generation of reactive oxygen species (ROS) (M. Barcellos-Hoff 1996). H₂O₂ has been also shown to act as a mediator for TGF-ß-induced transcription of certain genes such as erg-1 (M. Ohba 1994). Interestingly, a-particles emitted from radon and radon daughters significantly increases the intracellular ROS (superoxide and hydrogen peroxide) in lung fibroblasts (P. Narayanan 1997). However, the effects of radon on TGF-ß expression and activation have not been investigated so far.

Our prelimenary results indicate that plasma level of TGF-ß1, particularly the active form, in patients with AS is increased after radon therapy in the heilstollen. Therefore, It is possible that intermittent exposure to radon, may lead to transient activation of TGF-ß which in turn, due to its potent anti-inflammatory effect, aborts the inflammatory reaction at the joints and then initiates a normal process of healing, ultimately leading to the therapeutic effect in patients with AS.