Rudolf Mihalik

Staurosporine Induces Necroptotic Cell Death under Caspase-Compromised Conditions in U937 Cells

For a long time necrosis was thought to be an uncontrolled process but evidences recently have revealed that necrosis can also occur in a regulated manner. Necroptosis, a type of programmed necrosis is defined as a death receptor-initiated process under caspase-compromised conditions. The process requires the kinase activity of receptor-interacting protein kinase 1 and 3 (RIPK1 and RIPK3) and mixed lineage kinase domain-like protein (MLKL), as a substrate of RIPK3. The further downstream events remain elusive. We applied known inhibitors to characterize the contributing enzymes in necroptosis and their effect on cell viability and different cellular functions were detected mainly by flow cytometry. Here we report that staurosporine, the classical inducer of intrinsic apoptotic pathway can induce necroptosis under caspase-compromised conditions in U937 cell line. This process could be hampered at least partially by the RIPK1 inhibitor necrotstin-1 and by the heat shock protein 90 kDa inhibitor geldanamycin. Moreover both the staurosporine-triggered and the classical death ligand-induced necroptotic pathway can be effectively arrested by a lysosomal enzyme inhibitor CA-074-OMe and the recently discovered MLKL inhibitor necrosulfonamide. We also confirmed that the enzymatic role of poly(ADP-ribose)polymerase (PARP) is dispensable in necroptosis but it contributes to membrane disruption in secondary necrosis. In conclusion, we identified a novel way of necroptosis induction that can facilitate our understanding of the molecular mechanisms of necroptosis. Our results shed light on alternative application of staurosporine, as a possible anticancer therapeutic agent. Furthermore, we showed that the CA-074-OMe has a target in the signaling pathway leading to necroptosis. Finally, we could differentiate necroptotic and secondary necrotic processes based on participation of PARP enzyme.

Syndecan-1 (CD138) expression in human non-Hodgkin lymphomas

Syndecan‐1, an important transmembrane heparan sulphate proteoglycan, is expressed in distinct stages of differentiation of normal lymphoid cells :  in pre‐B cells and Ig‐producing plasma cells; however, its normal function, or presence in lymphoid malignancies, is still largely unknown. The expression of syndecan‐1 (CD138) was studied in 57 human non‐Hodgkin lymphomas using immunocytochemistry and immunohistochemistry. Positive expression of syndecan‐1 was found in the plasma cells in chronic lymphoblastic leukaemia (B‐CLL) cases, in different plasmocytoid lymphomas as well as in myeloma. All normal and malignant T cells, or CD5+ cells other than B‐CLL proved to be negative. These results strongly suggest that syndecan‐1 expression is a characteristic phenotypic marker for B‐CLL and lymphoplasmocytoid lymphomas and could be used for diagnostic purposes.

Necroptosis: Biochemical, Physiological and Pathological Aspects

Programmed cell death is a key component of tissue homeostasis, normal development and wide variety of diseases. Conventional view refers to programmed cell death form as caspase-mediated apoptosis while necrosis is considered as an accidental and unwanted cell demise, carried out in a non-regulated manner and caused by extreme conditions. However, accumulating evidences indicate that necrotic cell death can also be a regulated process. The term necroptosis has been introduced to describe a cell death receptor-induced, caspase-independent, highly regulated type of programmed cell death process with morphological resemblance of necrosis. Necroptosis recently has been found to contribute to a wide range of pathologic cell death forms including ischemic brain injury, neurodegenerative diseases and viral infection, therefore a better understanding of the necroptotic signaling machinery has clinical relevance.

ROR1 expression is not a unique marker of CLL.

Recent studies have identified receptor tyrosine kinase‐like orphan receptor 1 (ROR1) on the surface of chronic lymphoid leukaemia (CLL) cells. In order to determine whether ROR1 expression is a suitable surrogate marker for the diagnosis of CLL we analysed the mRNA level of ROR1 in different types of non‐Hodgkin lymphomas (NHL), and detected elevated levels of ROR1 compared to control peripheral mononuclear cells in several entities (CLL ≥ mantle cell lymphoma (MCL) > marginal zone lymphoma (MZL) >> diffuse large B‐cell lymphoma > follicular lymphoma). ROR1 protein was expressed intensely on the cell surface of lymphoma cells with leukaemic blood count detected by three colour immunofluorescence. Our results indicate that ROR1 expression is not limited to CLL cases, but it is more prevalent in NHLs, mainly in MCL where it is expressed intensely and MZL where it is expressed moderately, suggesting a general role of ROR1 in lymphoma genesis and/or maintenance. Copyright

Proteasome inhibitors sensitize colon carcinoma cells to TRAIL-induced apoptosis via enhanced release of Smac/DIABLO from the mitochondria

The synergistic interaction between proteasome inhibitors and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a promising approach to induce cell death in tumor cells. However, the molecular and biochemical mechanisms of this synergism have been proven to be cell type specific. We therefore focused our investigation on TRAIL-resistant colon carcinoma cells in this study. DNA fragmentation, mitochondrial membrane depolarization and increased caspase-3-like enzyme activity was exclusively induced only by combined treatment with proteasome inhibitors (epoxomicin, MG132, bortezomib/PS-341) and TRAIL. The expression level of anti-apoptotic proteins (XIAP, survivin, Bcl-2, Bcl-Xl), regulated by NF-κB transcription factor, was not effected by any of these treatments. TRAIL alone induced only partial activation of caspase-3 (p20), while the combination of TRAIL and proteasome inhibition led to the full proteolytic activation of caspase-3 (p17). Only the combination treatment induced marked membrane depolarization and the release of cytochrome c, HtrA2/Omi and Smac/DIABLO. Apoptosis-inducing factor (AIF) was not released in any of these conditions. These results are consistent with a model where the full activation of caspase-3 by caspase-8 is dependent on the release of Smac/DIABLO in response to the combined treatment. This molecular mechanism, independent of the inhibition NF-kB activity, may provide rationale for the combination treatment of colon carcinomas with proteasome inhibitors and recombinant TRAIL or agonistic antibody of TRAIL receptors. (Pathology Oncology Research Vol 12, No 3, 133–142)

Detection of drug-induced apoptosis by flow cytometry after alkaline extraction of ethanol fixed cells

A new flow cytometric method was developed to detect apoptotic cells with fragmented DNA and to determine cell cycle distribution of viable cells, in the same sample, by propidium iodide staining. Apoptosis, in HT58 human B lymphoma cells, was induced by etoposide and/or by staurosporine. Using appropriate alkaline solutions (between 1-10 mN NaOH in 150 mM saline) followed by neutralization with buffer solution, the fragmented DNA can be extracted quantitatively from ethanol fixed cells. Further, good resolution of the cell cycle distribution can be obtained in unimpaired cells without RNase treatment. Furthermore, unlike the widely used hypotonic-detergent extraction of unfixed cells, the suggested extraction method can prevent drug-induced disintegration of dead cells when karyorrhexis occurs.

APOPTOTIC AND NON-APOPTOTIC MODES OF PROGRAMMED CELL DEATH IN MCF-7 HUMAN BREAST CARCINOMA CELLS

Apoptosis is a specific mode of programmed cell death (PCD), recognized by characteristic morphological and molecular changes. Here we present evidence for a non‐apoptotic type of PCD in human MCF‐7 breast carcinoma cells. We used TNF‐α and tyrphostin AG213 to induce apoptotic and non‐apoptotic cell death respectively in vitro. Microscopic and immunohistochemical studies, together with DNA analysis and flow cytometric analysis of p53 and bcl‐2 oncogene expression, revealed some novel characteristics of non‐apoptotic cell death. We show here for the first time some of the biochemical features of an experimentally induced non‐apoptotic PCD and emphasize the distinct biochemical events leading to apoptotic and non‐apoptotic PCD.

Syndecan-1-Dependent Homotypic Cell Adhesion in HT58 Lymphoma Cells

Objectives: Many cellular functions are controlled by cell-cell and cell-matrix interactions. It has recently been found that syndecans, transmembrane heparan sulphate (HS) proteoglycans, can act as receptors or co-receptors and modulate cell adhesion. Our aim was to study the role of syndecan-1 in the aggregation of human lymphoma cells, and to investigate its effect on cell survival. Methods: Immunocytochemistry, confocal laser scanning microscopy, flow cytometry and aggregation/reaggregation bio-assays were used on HT58, BL41/95 and Raji lymphoma cell lines. Results: Bio-assays showed that the aggregation of HT58 cells was inhibited by heparin, HS, removal of the HS chain and binding of the anti-syndecan-1 monoclonal antibody. In the search for a counter-receptor of syndecan-1, several adhesion molecules were tested, but none of them proved to be the adhesion partner. In the case of heparitinase/trypsin digestion with long-term inhibition of HS synthesis (sodium chlorate treatment), the inhibited aggregation was accompanied by cell cycle arrest and the induction of apoptosis. Conclusions: The results obtained showed that surface syndecan-1 expression contributes to homotypic adhesion. In addition, HS chains, including those on syndecan-1, take part in the regulation of cell proliferation and active cell death in HT58 lymphoma cells.

Bone marrow derived mesenchymal stem/stromal cells transduced with full length human TRAIL repress the growth of rhabdomyosarcoma cells in vitro

Rhabdomyosarcoma (RMS) is the most frequent juvenal cancer originating from skeletal muscle and patient survival is poor in case of metastatic disease. New targeted therapeutics are critically needed. In a recent Hematologica paper Kuci et al. [1][1] reported that in vitro cultured cytokine-induced

Possible involvement of protein kinase C-ϵ in phorbol ester-induced growth inhibition of human lymphoblastic cells

Abstract Sustained activation of members of the protein kinase C (PKC) family is known to influence the growth and differentiation of various cell types, however, the specific roles for individual isoforms mediating these cellular events have yet to be elucidated. Activation of PKC by phorbol esters leads to growth inhibition in certain cell lines. The HT58 human B lymphoblastic cell may serve as a cellular model system to investigate the participation of individual isoforms in the initial events of growth arrest induced by phorbol ester. Determination of cell cycle and investigation of apoptosis were performed by flow cytometric measurements. Phorbol ester-induced translocation and down-regulation of the conventional α, β and the novel ϵ isoforms of PKC were demonstrated by Western blot analysis. At lower concentrations (0.5 ng/ml) phorbol myristate acetate (PMA) stimulated a G1 arrest with retention of viability in the human HT58 B lymphoblastic cell. The protein kinase inhibitor staurosporine at a concentration of 25 nM did not significantly alter HT58 cell viability. However, staurosporine (25 nM) induced apoptosis in cells preincubated for 4 hr with 0.5–1.0 ng/ml PMA. The translocation of PKC-ϵ was observed within 30 min exposure to 0.5 ng/ml PMA. After a 4 hr treatment, evidence for down-regulation and an altered phosphorylation state of PKC-ϵ was seen. In contrast, the conventional α and β isoforms were practically uneffected by this PMA treatment. At higher PMA concentrations (50 ng/ml) the α and β isoforms showed a significant down-regulation. The preferential alterations in PKC-ϵ observed under the conditions required for PMA to influence the growth and survival of HT58 cells suggest a role for the Ca 2+ -independent ϵ isoform in mediating the initial events of the phorbol ester stimulated cellular responses.