Ingo Ringshausen - Academia.edu (original) (raw)

Papers by Ingo Ringshausen

Cancer Cell, 2006

There is currently much interest in the idea of restoring p53 activity in tumor cells by inhibiti... more There is currently much interest in the idea of restoring p53 activity in tumor cells by inhibiting Hdm2/Mdm2. However, it has remained unclear whether this would also activate p53 in normal cells. Using a switchable endogenous p53 mouse model, which allows rapid and reversible toggling of p53 status between wild-type and null states, we show that p53 is spontaneously active in all tested tissues of mdm2-deficient mice, triggering fatal pathologies that include ablation of classically radiosensitive tissues. In apoptosis-resistant tissues, spontaneous unbuffered p53 activity triggers profound inhibition of cell proliferation. Such acute spontaneous p53 activity occurs in the absence of any detectable p53 posttranslational modification, DNA damage, or p19 ARF signaling and triggers rapid p53 degradation.

Cold Spring Harbor Symposia on Quantitative Biology, 2005

Antifungal Therapy with Itraconazole Impairs the Anti-Lymphoma Effects of Rituximab by Inhibiting Recruitment of CD20 to Cell Surface Lipid Rafts

Cancer Research, 2010

Immunotherapy with rituximab alone or in conjunction with chemotherapy has significantly improved... more Immunotherapy with rituximab alone or in conjunction with chemotherapy has significantly improved the treatment outcome of B-cell lymphoma patients. Nevertheless, a subpopulation of patients does not respond to rituximab. The reason for treatment failure as well as the exact mechanism of action is still uncertain. The function of rituximab has long been associated with the partitioning of CD20 molecules to membrane microdomains. Here, we show that concomitant antifungal treatment with itraconazole impairs the rituximab anti-lymphoma effect both in vitro and in vivo. At the molecular level, recruitment of CD20 to lipid rafts is inhibited in the presence of itraconazole. Furthermore, calcium influx, which is crucial for rituximab-mediated cell death, was nearly completely abolished by itraconazole treatment. In contrast, the antifungal drug caspofungin did not inhibit CD20 recruitment to lipid rafts, nor did it affect calcium influx or the cytotoxic effect of rituximab. The finding that itraconazole also abolished the cytotoxic effects of other therapeutic antibodies directed against lipid raft-associated molecules (i.e., CD20 and CD52) but not those against the non-raft-associated molecule CD33 further supported our proposed mechanism of action. Our results argue that concomitant medications must be adjusted carefully to achieve optimal antitumor effects with monoclonal antibodies.

Cell Cycle Inhibition in Malignant Lymphoma: Disease Control by Attacking the Cellular Proliferation Machinery

Current Drug Targets, 2006

Disruption of a proper regulation of cell proliferation can ultimately cause cancer. Most human B... more Disruption of a proper regulation of cell proliferation can ultimately cause cancer. Most human B cell malignancies are driven by chromosomal translocations or other genetic alterations which directly affect the function of critical cell cycle proteins, such as cyclins and cyclin-dependent kinases. In addition, the transformation of indolent lymphomas into aggressive malignancies is often accompanied by a loss of tumor suppressors controlling important cell cycle checkpoints. A better understanding of cell cycle deregulations in human tumors has promoted the introduction of a new class of antiproliferative drugs into cancer therapies. These drugs exert their function by specifically blocking important cell cycle proteins. In the present review we discuss how alterations in the cell cycle control contribute to the malignant transformation of B cells. Furthermore, we provide an overview of novel direct and indirect cell cycle inhibitors and their impact on the treatment of patients with B cell lymphomas.

British Journal of Haematology, 2003

Mantle cell lymphoma (MCL) is a distinctive non-Hodgkin's lymphoma subtype, characterized by over... more Mantle cell lymphoma (MCL) is a distinctive non-Hodgkin's lymphoma subtype, characterized by overexpression of cyclin D1 as a consequence of the chromosomal translocation t(11;14)(q13;q32). MCL remains an incurable disease, combining the unfavourable clinical features of aggressive and indolent lymphomas. The blastic variant of MCL, which is often associated with additional cytogenetic alterations, has an even worse prognosis and new treatment options are clearly needed. The present study investigated the effect of a specific proteasome inhibitor, lactacystin, on cell cycle progression and apoptosis in two lymphoma cell lines harbouring the t(11;14)(q13;q32) and additional cytogenetic alterations, including p53 mutation (NCEB) and p16 deletion (Granta 519). Granta cells were more susceptible to inhibition of the proteasome with respect to inhibition of proliferation and apoptosis induction.

Experimental Hematology, 2003

Methods. The effect of proteasome inhibition was analyzed using thymidine incorporation, annexin ... more Methods. The effect of proteasome inhibition was analyzed using thymidine incorporation, annexin V assays, and TUNEL staining. Immunoblots were performed to evaluate expression of proteins involved in cell cycle and apoptosis regulation.

In B-cell chronic lymphocytic leukemia (B-CLL), malignant cells seem to be arrested in the G 0 /e... more In B-cell chronic lymphocytic leukemia (B-CLL), malignant cells seem to be arrested in the G 0 /early G 1 phase of the cell cycle, and defective apoptosis might be involved in disease progression. However, increasing evidence exists that B-CLL is more than a disease consisting of slowly accumulating resting B cells: a proliferating pool of cells has been described in lymph nodes and bone marrow and might feed the accumulating pool in the blood. Rapamycin has been reported to inhibit cell cycle progression in a variety of cell types, including human B cells, and has shown activity against a broad range of human tumor cell lines. Therefore, we investigated the ability of rapamycin to block cell cycle progression in proliferating B-CLL cells. We have recently demonstrated that stimulation with CpG-oligonucleotides and interleukin-2 provides a valuable model for studying cell cycle regulation in malignant B cells. In our present study, we demonstrated that rapamycin induced cell cycle arrest in proliferating B-CLL cells and inhibited phosphorylation of p70s6 kinase (p70 s6k ).

Annals of Hematology, 2009

Although B-cell chronic lymphocytic leukemia (CLL) is treatable, it remains an incurable disease ... more Although B-cell chronic lymphocytic leukemia (CLL) is treatable, it remains an incurable disease and most patients inevitably suffer relapse. Many therapeutic options exist for those requiring therapy, including monoclonal antibodies and stem cell transplantation, but remissions tend to last shorter in the course of the disease. Targeting the cell cycle has recently been realized to be an attractive therapeutic approach in solid and hematological malignancies, and the proliferative nature of B-CLL is increasingly accepted. Here, we report data on a phase II pilot trial with the oral mammalian target of rapamycin (mTOR) inhibitor RAD001 5 mg/daily in patients with advanced B-CLL who had progressive disease after at least two lines of treatment. After treatment of seven patients, this trial was stopped because of toxicity concerns, although some degree of activity was observed (one partial remission, three patients with stable disease). Interestingly, cyclin E expression decreased in responding patients. Further strategies of mTOR inhibition by RAD001 in B-CLL should focus on different treatment schedules, adequate anti-infectious prophylaxis, or combinations with cytotoxic drugs.

Recruitment of PKC-βII to lipid rafts mediates apoptosis-resistance in chronic lymphocytic leukemia expressing ZAP70

Leukemia, 2010

ZAP-70 is a key signaling molecule in T cells. It couples the antigen-activated T-cell receptor t... more ZAP-70 is a key signaling molecule in T cells. It couples the antigen-activated T-cell receptor to downstream signaling pathways. Its expression in leukemic B-cells derived from a subgroup of patients with chronic lymphocytic leukemia (CLL) is associated with an aggressive course of the disease. However, its implication for the pathogenesis of aggressive CLL is still unclear. In this study, we show that the expression of ZAP-70 enhances the signals associated with the B-cell receptor, recruiting protein kinase C-betaII (PKC-betaII) into lipid raft domains. Subsequently, PKC-betaII is activated and shuttles from the plasma membrane to the mitochondria. We unravel that the antiapoptotic protein Bcl-2 and its antagonistic BH3-protein Bim(EL) are putative substrates for PKC-betaII. PKC-betaII-mediated phosphorylation of Bcl-2 augments its antiapoptotic function by increasing its ability to sequester more pro-apoptotic Bim(EL.) In addition, the phosphorylation of Bim(EL) by PKC-betaII leads to its proteasomal degradation. These changes confer leukemic cells to a more antiapoptotic state with aggressiveness of the disease. Most importantly, these molecular changes can be therapeutically targeted with the small molecule inhibitor Enzastaurin. We provide evidence that this compound is highly active in leukemic cells and augments the cytotoxic effects of standard chemotherapeutic drugs.

Annals of Hematology, 2006

The clinical course of chronic lymphocytic leukemia is variable. While some patients have indolen... more The clinical course of chronic lymphocytic leukemia is variable. While some patients have indolent disease, others require aggressive treatment within a short time after diagnosis. Differences in the expression of proteins regulating cell cycle and apoptosis may be responsible for the heterogeneous course of the disease. Recently, protein ZAP 70 [zeta-chain (T-cell receptor) associated protein kinase 70 kDa] has been found to be differentially expressed within two biologic subgroups, characterized by the presence or absence of somatic mutations in specific immunoglobulin heavy-chain variable region genes. In the present work, we analyzed highly purified B-CLL cells from 60 patients for ZAP 70 expression and the expression of cyclin E, bcl-2, bax, and mcl-1 as well as the ratios of bcl-2/bax and mcl-1/bax. The results indicate that cyclin E is expressed significantly higher in ZAP 70-positive as in ZAP 70-negative samples. We did not observe significant differences within the expression of Bcl-2 family member proteins. We conclude that higher cyclin E expression in samples of ZAP 70-positive patients may reflect a larger proliferating compartment in vivo compared to ZAP 70-negative patients and that cyclin E may add prognostic information in this context for patients with B-CLL.

British Journal of Haematology, 2010

Mantle cell lymphoma (MCL) is an incurable mature B cell proliferation, combining the unfavourabl... more Mantle cell lymphoma (MCL) is an incurable mature B cell proliferation, combining the unfavourable clinical features of aggressive and indolent lymphomas. The blastic variant of MCL has an even worse prognosis and new treatment options are clearly needed. We analysed the effects of BL22, an immunotoxin composed of the Fv portion of an anti- CD22 antibody fused to a 38-kDa Pseudomonas exotoxin-A fragment on four MCL cell lines as well as on primary cells of four MCL patients. Apoptosis induction by BL22 was much more pronounced in MCL cell lines with low Bcl-2 expression (NCEB-1, JeKo-1 and JVM-2) compared to Granta-519 cells with high Bcl-2 expression. While the expression of the antiapoptotic protein Mcl-1 declined (NCEB-1, Granta-519), Bcl-2 levels remained unchanged in Granta-519 cells. However transfection of BCL2 cDNA into NCEB-1, JeKo-1 and JVM-2 cells significantly reduced BL22-mediated toxicity. Accordingly we examined the effects of Bcl-2 inactivation in Granta-519 cells using siRNA. Indeed, apoptosis induction was strongly enhanced in Granta-519 cells with silenced Bcl-2. Our results were confirmed in freshly isolated MCL-cells from patients with leukaemic MCL. We conclude that Bcl-2 expression is important for mediating resistance against the immunotoxin BL22 in MCL cells.

Constitutive activation of the MAPkinase p38 is critical for MMP-9 production and survival of B-CLL cells on bone marrow stromal cells

Leukemia, 2004

In the present work we investigated the role and biological significance of mitogen activated pro... more In the present work we investigated the role and biological significance of mitogen activated protein kinases (MAPK) in B-cell chronic lymphocytic leukaemia (B-CLL). The MAPK p38 was constitutively activated in B-CLL, but not in normal peripheral B cells. In addition, we demonstrated that the upstream kinases of p38, MKK3/6 were also constitutively activated in B-CLL cells. Furthermore, we determined by EMSA that the p38 MAP kinase pathway was not linked to the constitutive high expression of NF-kappaB, a critical survival factor of B-CLL cells. Recently, it has been shown that serum levels of angiogenic factors like VEGF, bFGF and MMP-9 are elevated in the serum of CLL patients and correlate with an unfavorable prognosis. We showed that the constitutive expression of MMP-9 was dependent on p38-activity and inhibition of p38 strongly downregulated MMP-9 expression. Coculture of B-CLL cells and stromal cells can protect spontaneous apoptosis of leukemic B cells. To determine the role of permanently activated p38 and MMP-9 expression, we cocultured B-CLL cells with bone marrow stromal cells. Survival of B-CLL cells on stroma was severely impaired when p38 was inhibited. Furthermore, blockade of MMP-9 activity also antagonized the antiapoptotic effect of stromal cells.

In the present study we analyzed the role of phophatidylinositol-3 kinase (PI-3K) in B chronic ly... more In the present study we analyzed the role of phophatidylinositol-3 kinase (PI-3K) in B chronic lymphocytic leukemia (B-CLL) cells. PI-3K is activated by many stimuli and is linked to several different signaling pathways. We demonstrated that inhibition of PI-3K by a specific inhibitor, LY294002, induced apoptosis in B-CLL cells in vitro. This effect was specific for the inhibition of PI-3K because inhibition of other signaling pathways such as extracellular signaling-regulated kinase (ERK), p38, or p70S6 kinase did not affect spontaneous apoptosis. Furthermore, PI-3K was constitutively activated in freshly isolated B-CLL cells. Corresponding to enhanced apoptosis, LY294002 downregulated expression of the antiapoptotic proteins X-linked inhibitor of apoptosis protein (XIAP) and Mcl-1. Next, we investigated which factors downstream of PI-3K were activated in B-CLL cells. We demonstrated that protein kinase B/Akt is expressed in all tested CLL samples but no activation of Akt was detected. In contrast, we observed a constitutive activation of protein kinase C␦ (PKC␦) in freshly isolated B-CLL cells. PKC␦ is linked to PI-3K and is phosphorylated at Thr505 in response to PI-3K activation. We further demonstrated that tyrosine phosphorylation and activity of PKC␦ were dependent on PI-3K activity in B-CLL cells. Inhibition of PKC␦ by the specific inhibitor Rottlerin strikingly enhanced apoptosis. In contrast, peripheral blood B cells of healthy donors were resistant to inhibition of PI-3K or PKC␦. We conclude that activated PI-3K might be important in the pathogenesis of B-CLL, and survival signals might be mediated via PKC␦. Therefore, inhibition of PI-3K or PKC␦ may be an innovative approach to treat B-CLL. (Blood. 2002;100:3741-3748)

Annals of Hematology, 2010

Although B chronic lymphocytic leukemia (B-CLL) cells express the alpha chain of the interleukin-... more Although B chronic lymphocytic leukemia (B-CLL) cells express the alpha chain of the interleukin-2 (IL-2) receptor CD25, little is known about the effect of IL-2 on apoptosis in B-CLL cells. We have shown previously that stimulation of B-CLL cells with a CpG-oligonucleotide induces IL-2 high affinity receptors. In our current work, we analyzed the effect of IL-2 on apoptosis in resting B-CLL cells and in our model of activated B-CLL cells (CD25 high cells). IL-2 had modest antiapoptotic activity in resting B-CLL cells. In contrast, IL-2 was much more potent to prevent apoptosis in activated cells. Prevention of cell death was also associated with the maintenance of the mitochondrial membrane potential. While only limited regulation of apoptosis controlling proteins was observed in resting B-CLL cells, IL-2 had strong effects on MCL-1, Bcl-xl, and survivin expression and inhibited Bax cleavage in CD25 high cells. Interestingly, expression of Bcl-2 was reduced. Addition of IL-2 to activated B-CLL cells caused rapid phosphorylation of Akt, while IL-2 failed to significantly phosphorylate Akt in resting B-CLL cells. Pharmacological inhibition of Akt by LY294002 restored sensitivity of activated B-CLL cells to fludarabine. IL-2 might be an important survival factor in activated B-CLL cells and might contribute to disease progression by upregulation of several critical antiapoptotic proteins.

Leukemia, 2008

antibodies and immune complexes which accelerate platelet removal. 7 Based upon these preliminary... more antibodies and immune complexes which accelerate platelet removal. 7 Based upon these preliminary observations, we hypothesize that reducing exposure to platelet transfusions and stored platelet supernatant might yield better clinical outcomes in acute leukemia in adults. This hypothesis will need to be assessed in larger randomized trials. Washed, leukoreduced, ABO identical transfusions represent an inexpensive, technically simple and low-risk treatment strategy worth further investigation in patients with acute leukemia.

Leukemia, 2006

Constitutively activated signaling pathways contribute to the apoptosis-defect of B-CLL cells. Pr... more Constitutively activated signaling pathways contribute to the apoptosis-defect of B-CLL cells. Protein kinase C-d is a permanently activated kinase and a putative downstream target of phosphatidylinositol-3 kinase in B-CLL. Blockade of protein kinase C-d (PKC-d) by the highly specific inhibitor rottlerin induces apoptosis in chronic lymphocytic leukaemia (CLL) cells. By co-culturing bone marrow stromal and CLL cells, we determined that the proapoptotic effect of rottlerin is not abolished in the presence of survival factors, indicating that a targeted therapy against PKC-d might be a powerful approach for the treatment of CLL patients. The downstream events following rottlerin treatment engage mitochondrial and nonmitochondrial pathways and ultimately activate caspases that execute the apoptotic cell death. Herein we report that the inhibition of PKC-d decreases the expression of the important antiapoptotic proteins Mcl-1 and XIAP accompanied by a loss of the mitochondrial membrane potential Dw. In addition, we discovered that ZAP-70-expressing cells are significantly more susceptible to rottlerin-induced cell death than ZAP-70 negative cells. We finally observed that rottlerin can augment cell toxicity induced by standard chemotherapeutic drugs. Conclusively, PKC-d is a promising new target in the combat against CLL.

British Journal of Haematology, 2006

Diffuse large B-cell lymphoma (DLBCL) is a common lymphoma entity. Although a significant amount ... more Diffuse large B-cell lymphoma (DLBCL) is a common lymphoma entity. Although a significant amount of DLBCL patients can be cured with modern chemotherapeutic regimens, a substantial proportion of patients die because of progressive disease. Therefore, new therapeutic strategies are clearly needed. Inhibitors of mTOR [mammalian target of rapamycin (Rap)] represent a new class of antiproliferative drugs with applications as immunosuppressive and anticancer agents. Extensive safety data exist on the mTOR inhibitor RAD001, which is already approved as an immunosuppressant in organ transplant recipients. Rap and RAD001 inhibited cell cycle progression in DLBCL cells by inducing a G1 arrest without inducing apoptosis. Phosphorylation of the main targets of mTOR, p70 s6 kinase and 4-EBP-1 was reduced in cells cultured in the presence of RAD001. Cell cycle arrest was accompanied by reduced phosphorylation of the retinoblastoma protein (RB) as well as reduced expression of cyclin D3 and A in all cell lines. Although the effect of the chemotherapeutic agent vincristine (vin) was not enhanced by RAD001, rituximab-induced cytotoxicity was augmented in the rituximab-sensitive cell lines. mTOR inhibition is a promising therapeutic strategy in DLBCL by inducing a G1 arrest and augments rituximab-induced cytotoxicity. Therefore, combination of these drugs might be an interesting new therapeutic approach in DLBCL patients.