-
[show abstract]
[hide abstract]
ABSTRACT: CD95 (APO-1/Fas) is a member of the death receptor (DR) family. Stimulation of CD95 leads to induction of apoptotic and non-apoptotic signaling pathways. The formation of the CD95 death-inducing signaling complex (DISC) is the initial step of CD95 signaling. Activation of procaspase-8 at the DISC leads to the induction of DR-mediated apoptosis. The activation of procaspase-8 is blocked by cellular FLICE-inhibitory proteins (c-FLIP). This review is focused on the role in the CD95-mediated signaling of the death effector domain-containing proteins procaspase-8 and c-FLIP. We discuss how dynamic cross-talk between procaspase-8 and c-FLIP at the DISC regulates life/death decisions at CD95.
Cell death and differentiation 11/2011; 19(1):36-41. · 8.24 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The human T-cell leukemia virus type-1 (HTLV-1)-associated adult T-cell leukemia/lymphoma (ATL) is incurable by currently known therapies. ATL samples and cell lines derived from ATL patients show restricted sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and CD95 ligand (CD95L). We have recently shown that HTLV-1-infected cells express elevated levels of cellular caspase-8 FLICE-inhibitory protein (c-FLIP) conferring resistance to receptor-mediated apoptosis. This finding underscores the demand to develop new strategies for treatment of ATL. In this study, we show that the naturally occurring herbal compound Rocaglamide (Roc) sensitizes CD95L- and TRAIL-induced apoptosis in HTLV-1-infected cells by downregulation of c-FLIP expression. Investigation of the molecular mechanism of Roc-mediated downregulation of c-FLIP revealed that it inhibits phosphorylation of the translation initiation factor 4E (eIF4E), a key factor that controls the rate-limiting step of translation, through inhibition of the MEK-ERK-MNK1 signaling pathway. This event prevents de novo synthesis of short-lived proteins such as c-FLIP in HTLV-1-infected cells. Our data suggest that Roc may serve as an adjuvant for TRAIL-based anticancer therapy.
Cell death and differentiation 02/2011; 18(2):362-70. · 8.24 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Stimulation of CD95/Fas/APO-1 results in the induction of both apoptotic and non-apoptotic signaling pathways. The processes regulating these two opposing pathways have not been thoroughly elucidated to date. In this study, using quantitative immunoblots, imaging, and mathematical modeling, we addressed the dynamics of the DED proteins of the death-inducing signaling complex (DISC), procaspase-8, and cellular FLICE inhibitory proteins (c-FLIPs) to the onset of CD95-mediated ERK1/2 and p38 mitogen-activated protein kinase (MAPK) activation. We found that CD95 DISC-induced caspase-8 activity is important for the initiation of ERK1/2 and p38 MAPK activation. The long c-FLIP isoform, c-FLIP(L), and the short c-FLIP isoform, c-FLIP(R), inhibited MAPK induction by blocking caspase-8 processing at the DISC. Furthermore, we built a mathematical model describing CD95 DISC-mediated MAPK activation and apoptosis. The model quantitatively defined the dynamics of DED proteins, procaspase-8, and c-FLIP, which lead to caspase-8 activation and induction of apoptotic and non-apoptotic signaling pathways. In conclusion, the combination of biochemical analysis with mathematical modeling provides evidence for an important role of caspase-8 in CD95-mediated activation of MAPKs, while c-FLIP exerts a regulatory function in this process.
Cell Death & Disease 01/2011; 2:e212. · 5.33 Impact Factor
-
G Polier,
J Ding,
B V Konkimalla,
D Eick,
N Ribeiro,
R Köhler,
M Giaisi,
T Efferth,
L Desaubry, P H Krammer,
M Li-Weber
[show abstract]
[hide abstract]
ABSTRACT: The wogonin-containing herb Scutellaria baicalensis has successfully been used for curing various diseases in traditional Chinese medicine. Wogonin has been shown to induce apoptosis in different cancer cells and to suppress growth of human cancer xenografts in vivo. However, its direct targets remain unknown. In this study, we demonstrate for the first time that wogonin and structurally related natural flavones, for example, apigenin, chrysin and luteolin, are inhibitors of cyclin-dependent kinase 9 (CDK9) and block phosphorylation of the carboxy-terminal domain of RNA polymerase II at Ser(2). This effect leads to reduced RNA synthesis and subsequently rapid downregulation of the short-lived anti-apoptotic protein myeloid cell leukemia 1 (Mcl-1) resulting in apoptosis induction in cancer cells. We show that genetic inhibition of Mcl-1 or CDK9 expression by siRNA is sufficient to mimic flavone-induced apoptosis. Pull-down and in silico docking studies demonstrate that wogonin directly binds to CDK9, presumably to the ATP-binding pocket. In contrast, wogonin does not inhibit CDK2, CDK4 and CDK6 at doses that inhibit CDK9 activity. Furthermore, we show that wogonin preferentially inhibits CDK9 in malignant compared with normal lymphocytes. Thus, our study reveals a new mechanism of anti-cancer action of natural flavones and supports CDK9 as a therapeutic target in oncology.
Cell Death & Disease 01/2011; 2:e182. · 5.33 Impact Factor
-
E Antiga,
C C Kretz,
R Klembt,
D Massi,
V Ruland,
C Stumpf,
G Baroni,
M Hartmann,
W Hartschuh,
W Volpi,
E Del Bianco,
A Enk,
P Fabbri, P H Krammer,
M Caproni,
A Kuhn
[show abstract]
[hide abstract]
ABSTRACT: The purpose of this study was to characterize regulatory T cells (T(reg)) in skin lesions and peripheral blood from patients with dermatomyositis (DM) and to determine the serum levels of regulatory cytokines in the disease. In skin biopsy specimens from patients with DM, immunohistochemistry was performed for CD4(+), CD25(+), forkhead/winged helix transcription factor (FoxP3)(+), transforming growth factor (TGF)-β(+) and interleukin (IL)-10(+) cells. Additionally, we defined the number of T(reg) subpopulations in peripheral blood by flow cytometry using monoclonal antibodies against CD4, CD25, FoxP3, CD45RO, CD95, CCR4 and CLA. The levels of TGF-β and IL-10 were also determined in serum samples from patients with DM by enzyme-linked immunosorbent assays. Controls included patients with cutaneous lupus erythematosus, psoriasis and atopic dermatitis (AD) as well as healthy donors. The frequency of FoxP3(+) cells was significantly reduced in skin lesions from patients with DM (p < 0.001) compared to psoriasis and AD. Moreover, the number of cells positive for TGF-β was lower in DM than in psoriasis and AD, while IL-10(+) cells were significantly reduced only compared to psoriasis. The number of CD4(+)CD25(++)FoxP3(+) T(reg) in the peripheral blood of patients with DM was significantly reduced compared to healthy controls (p < 0.05), whereas other cell populations showed no significant differences. Finally, TGF-β and IL-10 serum levels were significantly lower in patients with DM compared to healthy controls (p < 0.05). These data suggest that the depletion of T(reg) and their main effector cytokines in the skin and the serum of patients with DM may be an important factor in the pathogenesis of the disease.
Journal of Autoimmunity 12/2010; 35(4):342-50. · 7.37 Impact Factor
-
S Klein,
C C Kretz,
V Ruland,
C Stumpf,
M Haust,
W Hartschuh,
M Hartmann,
A Enk,
E Suri-Payer,
N Oberle, P H Krammer,
A Kuhn
[show abstract]
[hide abstract]
ABSTRACT: To determine the frequency and suppressive capacity of regulatory T cells (T(reg)) and their association with clinical parameters in patients with systemic scleroderma (SSc).
Peripheral blood from 25 patients with SSc, 15 patients with localised scleroderma (LS) and 29 healthy controls (HC) was studied. Analysis of CD4(+) forkhead box P3 (Foxp3)(+) and CD4(+)CD25(++)Foxp3(+) T(reg) subpopulations was carried out by flow cytometry and cell proliferation was quantified by (3)H-thymidine incorporation. Quantitative analysis of T(reg) was further performed in skin biopsies from 17 patients with SSc and 21 patients with LS using anti-CD4 and anti-Foxp3 monoclonal antibodies for immunohistochemistry.
The frequency of CD4(+)Foxp3(+) and CD4(+)CD25(++)Foxp3(+) T(reg) in peripheral blood from patients with SSc was not significantly different from that of patients with LS or HC. The suppressive capacity of CD4(+)CD25(++) T(reg) in SSc was also found to be similar to that of HC. Phenotypic and functional data revealed no significant difference between the limited or diffuse form of SSc. Moreover, therapy with bosentan showed no significant effect on the frequency of T(reg) during the course of the disease. However, the frequency of T(reg) in skin lesions from patients with SSc or LS, determined as the percentage of CD4(+) cells expressing Foxp3 in the inflammatory infiltrate, was significantly reduced compared with other inflammatory skin diseases.
These results indicate that although the authors found no defect in the frequency or function of peripheral T(reg) subpopulations, the reduction of CD4(+)Foxp3(+) T(reg) in the skin of patients with SSc may be important in the pathogenesis of the disease.
Annals of the rheumatic diseases 11/2010; 70(8):1475-81. · 8.11 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Drugs with tumor selectivity may have an important benefit in chemotherapies. We have previously shown that Rocaglamide(s), derived from the medicinal plant Aglaia, kills various leukemic cells through the mitochondrial apoptosis pathway with only minor toxicities to normal lymphocytes. Here, we show further that Rocaglamide preferentially promotes activation-induced cell death in malignant T cells by differential regulation of c-FLIP and CD95L expression. Rocaglamide enhances and also prolongs activation-induced JNK activation in malignant T cells leading to downregulation of c-FLIP but upregulation of CD95L expression. We also show that malignant T cells express a significantly higher amount of Bid - the molecular linker that bridges the receptor-mediated to the mitochondria-mediated apoptosis pathway. Conversely, a substantially lower amount of c-FLIP in response to T-cell stimulation compared to normal T cells is observed. This difference may provide a therapeutic window for cancer treatment. The effect of Rocaglamide on sensitization of activation-induced cell death in malignant T cells was further demonstrated in vivo in a mouse model. Our study demonstrates that Rocaglamide may be a potential anticancer drug that simultaneously targets both c-FLIP and CD95L expressions in tumor cells. This study may also provide a new clue to design a more efficient chemotherapy by using a combination of stimuli that engage the receptor-mediated and the mitochondria-mediated death pathway.
Cell death and differentiation 05/2009; 16(9):1289-99. · 8.24 Impact Factor
-
B Franz,
B Fritzsching,
A Riehl,
N Oberle,
C-D Klemke,
J Sykora,
S Quick,
C Stumpf,
M Hartmann,
A Enk,
T Ruzicka, P H Krammer,
E Suri-Payer,
A Kuhn
[show abstract]
[hide abstract]
ABSTRACT: To define the phenotype and function of CD4+,CD25+ regulatory T cells (Treg) in patients with cutaneous lupus erythematosus (CLE), a heterogeneous autoimmune disease characterized primarily by inflammatory skin lesions.
The number of Treg in skin specimens obtained from patients with various subtypes of CLE was investigated by immunohistochemical analysis, using anti-Foxp3 and anti-CD4 monoclonal antibodies. Furthermore, characterization of peripheral blood CD4+,CD25+ Treg from normal healthy donors and patients with CLE was carried out by flow cytometry, analyzing the expression of Foxp3 and Treg subpopulations. We also purified CD4+,CD25(high) Treg obtained from patients with CLE and tested the sensitivity of these cells to CD95L-mediated apoptosis.
Quantitative analysis of CD4+ T cells in skin lesions from patients with CLE revealed that the number was similar to that in lesions from patients with other chronic inflammatory diseases, but the number of Foxp3+ Treg in CLE was significantly reduced. There was no correlation between disease subtype and the frequency of Foxp3+ Treg in the skin of patients with CLE. In peripheral blood, no significant differences were observed in the number and phenotype of CD4+,CD25+ Treg or in the sensitivity to apoptosis of CD4+,CD25(high) Treg derived from patients with CLE and those derived from normal healthy donors.
These data suggest that an organ-specific abnormality of Treg in the skin underscores the importance of analyzing Treg in the affected tissue. Such a local process might give insight into the pathogenic mechanisms of CLE and differs from a global peripheral dysfunction as reported for patients with a systemic manifestation of the disease.
Arthritis & Rheumatism 07/2007; 56(6):1910-20. · 7.87 Impact Factor
-
Cell Death and Differentiation 04/2007; 14(3):634-6. · 8.85 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in many transformed cells, suggesting TRAIL as an ideal candidate for cancer gene therapy. A main obstacle in cancer therapy is intrinsic or acquired therapy resistance of malignant cells. To study induction of resistance against TRAIL, we generated lentiviral vectors allowing efficient TRAIL expression and apoptosis induction in a variety of human cancer cell lines. Within days upon TRAIL overexpression, cells became resistant towards TRAIL, but not to CD95 ligation or DNA damage by cisplatin. Cell surface expression of TRAIL receptors 1 and 2 was completely abrogated in resistant cells due to intracellular retention of the receptors by TRAIL. SiRNA directed against TRAIL resensitized the resistant cells by restoring cell surface expression of TRAIL receptors. These findings represent a novel resistance mechanism towards TRAIL, specifically caused by TRAIL overexpression, and question the use of TRAIL expression in tumor-cell targeting gene therapy.
Cell Death and Differentiation 10/2006; 13(10):1740-51. · 8.85 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The function of Fc receptors (FcR) on mouse spleen cells as detected by a sensitive resetting system using antibody-labeled bovine erythrocytes as indicator cells was shown to be inhibited by anti-Ia antiserum. Furthermore, the la specificities of each I-region sublocus (I-1A, I-1B I-C) seem to be associated with FcR, since FcR function was also inhibited when the blocking anti-Ia antiserum recognized only restricted Ia specificities. Antiserum directed against antigen coded for by the K-end of the major histocompatibility complex did not inhibit FcR function. The function of C3 receptors and Fc receptors on macrophages and Fc-receptor-positive cells in the fetal liver was not inhibited by anti-Ia antiserum. These findings arc discussed in view of a possible arrangement of Fc receptors and Ia antigens on the cell membrane.
Scandinavian Journal of Immunology 06/2006; 5(3):205 - 212. · 2.23 Impact Factor
-
C-D Klemke,
B Fritzsching,
B Franz,
E V Kleinmann,
N Oberle,
N Poenitz,
J Sykora,
A H Banham,
G Roncador,
A Kuhn,
S Goerdt, P H Krammer,
E Suri-Payer
[show abstract]
[hide abstract]
ABSTRACT: Cutaneous T-cell lymphomas (CTCL) are mainly comprised of two variants: mycosis fungoides (MF) with CD4(+) tumor cells confined to the skin and the leukemic Sézary syndrome with tumor cell spread to the blood. In this study, we investigated cutaneous expression of the regulatory T-cell (T(reg)) marker FOXP3 in 30 CTCL patients. Immunohistochemical analysis revealed significantly lower numbers of CD4(+)FOXP3(+) cells within the dermal lymphomononuclear infiltrate of Sézary patients (16% FOXP3(+) cells of CD4(+) cells) in contrast to MF (43% FOXP3(+) cells (P<0.05)) and rare types of CTCL (45% FOXP3(+) cells). Furthermore, CD4(+)FOXP3(+) T cells were also markedly reduced in the CD4(+) population within the peripheral blood of Sézary patients compared to controls as determined by fluorescence-activated cell sorter, quantitative PCR and functional analyses. The data support the conclusion that the neoplastic cells in CTCL do not express the T(reg) marker FOXP3. Our data also identify Sézary syndrome as, to our knowledge, the first reported neoplastic disease with a clear reduction in T(reg) numbers within the CD4(+) population. This lack of T(reg) might account for the more aggressive nature of Sézary syndrome compared with other CTCL.
Leukemia 06/2006; 20(6):1123-9. · 9.56 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Formation of the CD95 (APO-1/Fas) death inducing signaling complex (DISC) plays a central role in CD95 signaling. Previously, CD95 DISC composition was analyzed by two-dimensional gel electrophoresis and four major cytotoxicity-associated proteins (CAP1-4) were found. CAP1 and CAP2 were defined to be unmodified and phosphorylated FADD, respectively. CAP4 was identified as procaspase-8a. CAP3, however, has remained elusive. In this study, we demonstrate that CAP3 is an intermediate of procaspase-8 processing. CAP3 is generated within seconds of DISC formation and subsequently processed to the prodomain of procaspase-8a that is known as p26 (CAP5). These findings lead to new insights into the mechanism of procaspase-8 processing and apoptosis initiation.
Cell Death and Differentiation 04/2006; 13(3):489-98. · 8.85 Impact Factor
-
C Zuliani,
S Kleber,
S Klussmann,
T Wenger,
M Kenzelmann,
N Schreglmann,
A Martinez,
J A del Rio,
E Soriano,
P Vodrazka,
R Kuner,
H-J Groene,
I Herr, P H Krammer,
A Martin-Villalba
[show abstract]
[hide abstract]
ABSTRACT: The CD95 (Apo-1/Fas)/CD95 ligand (CD95L) system is best characterized as a trigger of apoptosis. Nevertheless, despite broad expression of CD95L and CD95 in the developing brain, absence of functional CD95 (lpr mice) or CD95L (gld mice) does not alter neuronal numbers. Here, we report that in embryonic hippocampal and cortical neurons in vivo and in vitro CD95L does not induce apoptosis. Triggering of CD95 in cultured immature neurons substantially increases neurite branches by promoting their formation. The branching increase occurs in a caspase-independent and death domain-dependent manner and is paralleled by an increase in the nonphosphorylated form of Tau. Most importantly, lpr and gld mutants exhibit a reduced number of dendritic branches in vivo at the time when synapse formation takes place. These data reveal a novel function for the CD95 system and add to the picture of guidance molecules in the developing brain.
Cell Death and Differentiation 02/2006; 13(1):31-40. · 8.85 Impact Factor
-
M Müller,
T Schilling,
A E Sayan,
A Kairat,
K Lorenz,
H Schulze-Bergkamen,
M Oren,
A Koch,
A Tannapfel,
W Stremmel,
G Melino, P H Krammer
[show abstract]
[hide abstract]
ABSTRACT: We investigated the mechanisms by which TAp73 beta and dominant-negative p73 (Delta Np73) regulate apoptosis. TAp73 beta transactivated the CD95 gene via the p53-binding site in the first intron. In addition, TAp73 beta induced expression of proapoptotic Bcl-2 family members and led to apoptosis via the mitochondrial pathway. Endogenous TAp73 was upregulated in response to DNA damage by chemotherapeutic drugs. On the contrary, DeltaNp73 conferred resistance to chemotherapy. Inhibition of CD95 gene transactivation was one mechanism by which DeltaNp73 functionally inactivated the tumor suppressor action of p53 and TAp73 beta. Concomitantly, DeltaNp73 inhibited apoptosis emanating from mitochondria. Thus, DeltaNp73 expression in tumors selects against both the death receptor and the mitochondrial apoptosis activity of TAp73 beta. The importance of these data is evidenced by our finding that upregulation of DeltaNp73 in hepatocellular carcinoma patients correlates with reduced survival. Our data indicate that Delta Np73 is an important gene in hepatocarcinogenesis and a relevant prognostic factor.
Cell Death and Differentiation 01/2006; 12(12):1564-77. · 8.85 Impact Factor
-
Cell Death and Differentiation 05/2005; 12(4):408-9. · 8.85 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Mathematical modeling is required for understanding the complex behavior of large signal transduction networks. Previous attempts to model signal transduction pathways were often limited to small systems or based on qualitative data only. Here, we developed a mathematical modeling framework for understanding the complex signaling behavior of CD95(APO-1/Fas)-mediated apoptosis. Defects in the regulation of apoptosis result in serious diseases such as cancer, autoimmunity, and neurodegeneration. During the last decade many of the molecular mechanisms of apoptosis signaling have been examined and elucidated. A systemic understanding of apoptosis is, however, still missing. To address the complexity of apoptotic signaling we subdivided this system into subsystems of different information qualities. A new approach for sensitivity analysis within the mathematical model was key for the identification of critical system parameters and two essential system properties: modularity and robustness. Our model describes the regulation of apoptosis on a systems level and resolves the important question of a threshold mechanism for the regulation of apoptosis.
The Journal of Cell Biology 10/2004; 166(6):839-51. · 10.26 Impact Factor
-
T M Ganten,
T L Haas,
J Sykora,
H Stahl,
M R Sprick,
S C Fas,
A Krueger,
M A Weigand,
A Grosse-Wilde,
W Stremmel, P H Krammer,
H Walczak
[show abstract]
[hide abstract]
ABSTRACT: Tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) exhibits potent antitumour activity upon systemic administration in mice without showing the deleterious side effects observed with other apoptosis-inducing members of the TNF family such as TNF and CD95L. TRAIL may, thus, have great potential in the treatment of human cancer. However, about 60% of tumour cell lines are not sensitive to TRAIL. To evaluate the mechanisms of tumour resistance to TRAIL, we investigated hepatocellular carcinoma (HCC) cell lines that exhibit differential sensitivity to TRAIL. Pretreatment with chemotherapeutic drugs, for example, 5-fluorouracil (5-FU), rendered the TRAIL-resistant HCC cell lines sensitive to TRAIL-induced apoptosis. Analysis of the TRAIL death-inducing signalling complex (DISC) revealed upregulation of TRAIL-R2. Caspase-8 recruitment to and its activation at the DISC were substantially increased after 5-FU sensitisation, while FADD recruitment remained essentially unchanged. 5-FU pretreatment downregulated cellular FLICE-inhibitory protein (cFLIP) and specific cFLIP downregulation by small interfering RNA was sufficient to sensitise TRAIL-resistant HCC cell lines for TRAIL-induced apoptosis. Thus, a potential mechanism for TRAIL sensitisation by 5-FU is the increased effectiveness of caspase-8 recruitment to and activation at the DISC facilitated by the downregulation of cFLIP and the consequent shift in the ratio of caspase-8 to cFLIP at the DISC.
Cell Death and Differentiation 08/2004; 11 Suppl 1:S86-96. · 8.85 Impact Factor
-
Cell Death and Differentiation 02/2003; 10(1):144-5. · 8.85 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Apoptosis is a morphologically distinct form of cell death involved in many physiological and pathological processes. The death receptor CD95 (APO-1/Fas) and its ligand (L) CD95L are critically involved in activation-induced-cell-death (AICD) of activated T-cells. Here we show that the anti-inflammatory sesquiterpene lactone parthenolide derived from the European traditional herb-medicine feverfew and many Mexican India medicinal plants suppresses expression of the CD95L and CD95 at the mRNA levels, thus, preventing T-cells from AICD. We demonstrate that parthenolide blocks NF-kappaB binding to the two NF-kappa binding sites of the CD95L promoter and suppresses promoter activity upon T-cell activation. Aberrant expression of CD95 and, particularly CD95L is dangerous and may lead to severe diseases. Our study indicates that parthenolide supports T-cell survival by down-regulating the CD95 system, at least in part, and, therefore, may have therapeutic potential as a new anti-apoptotic substance against AICD in T-cells.
Cell Death and Differentiation 12/2002; 9(11):1256-65. · 8.85 Impact Factor
