Melek Ergin

Analysis of expression of nuclear factor κB (NF-κB) in multiple myeloma: Downregulation of NF-κB induces apoptosis

Nuclear factor‐κB (NF‐κB) is an important transcription factor that regulates survival in many cells. Activated NF‐κB has been shown to protect some haematopoietic neoplastic cells from apoptosis. In the present study, we analysed NF‐κB status in 13 primary samples from patients with multiple myeloma (MM) and in four myeloma cell lines including U266, RPMI 8226, HS‐Sultan and K620. Constitutive activation of NF‐κB was evaluated by either immunohistochemistry or immunofluorescence using a monoclonal mouse anti‐human p65 (Rel A) antibody, which recognizes the unbound, active form of p65 (Rel A). Constitutively active NF‐κB was present in all MM patient samples as well as in all four myeloma cell lines. Inhibition of constitutively active NF‐κB, by either proteasome inhibitors (MG132, gliotoxin) or inhibitors of IκB phosphorylation (Bay117082, and Bay117085), induced apoptosis as demonstrated by both flow cytometric analysis and light microscopic morphological evaluation. This chemically induced apoptosis was associated with decreased DNA binding of nuclear NF‐κB as determined by the electrophoretic mobility shift assay. In addition, adenovirus vector with dominant negative IκBα (Ad5IκB) was used for inhibition of NF‐κB in the U266 cell line. Compared with wild‐type, super‐repressor‐treated cells showed an increased level of apoptosis. These results suggest that constitutive expression of NF‐κB plays an important role in plasma cell survival in MM.

Characterization of NF-κB expression in Hodgkin's disease: Inhibition of constitutively expressed NF-κB results in spontaneous caspase-independent apoptosis in Hodgkin and Reed-Sternberg cells

Although the neoplastic cells of classical Hodgkin’s disease (CHD) demonstrate high levels of constitutively active nuclear NF-κB, the precise physiologic and clinical significance of NF-κB expression is currently undefined. Expression of active NF-κB p65(Rel A) was evaluated in patient samples of CHD and nodular lymphocyte predominance Hodgkin’s disease. The action of the chemical NF-κB inhibitors gliotoxin and MG132 and the effect of NF-κB inhibition utilizing an adenovirus vector carrying a dominant-negative IκBα mutant (Ad5IκB) were then demonstrated in CHD cell lines (L428, KMH2, and HS445). Hodgkin and Reed-Sternberg (HRS) cells from all patient and cell line specimens showed strong immunopositivity for active p65(Rel A). Expression was also seen in lymphocytic/histiocytic cells from all cases of nodular lymphocyte predominance Hodgkin’s disease. After chemical NF-κB inhibition, p65(Rel A) was significantly reduced in nuclear extracts from cultured HRS cells as revealed by electrophoretic mobility shift assays. Furthermore, chemical NF-κB inhibition resulted in time- and concentration-dependent apoptosis in HRS cells. With the exception of MG132-induced apoptosis in HS445, apoptosis by chemical NF-κB inhibition was not significantly altered by preincubation with various caspase inhibitors (z-DQMD-FMK, z-DEVD-FMK, z-VAD-FMK, z-VEID-FMK, and z-IETD-FMK). Regardless of the chemical inhibitor used, no significant change in caspase-3 functional activity was found in CHD cell lines. HRS cells infected with Ad5IκB also showed a marked increase in spontaneous apoptosis compared with wild type adenovirus-infected and control cells. Overall, the inhibition of active NF-κB in HRS cells resulting in spontaneous caspase-independent apoptosis demonstrates a critical role for NF-κB in HRS cell survival and resistance to apoptosis.

Inhibition of tyrosine kinase activity induces caspase-dependent apoptosis in anaplastic large cell lymphoma with NPM-ALK (p80) fusion protein

OBJECTIVE The t(2;5)(p23;q35) translocation creates a fusion gene NPM-ALK (p80) that encodes a product with tyrosine kinase activity believed to play an important role in development of anaplastic large cell lymphoma (ALCL). Our study was aimed to analyze tyrosine kinase activity and phosphotyrosine in ALCLs. We were also interested in determining the effect of tyrosine kinase inhibitors on survival of ALCL. METHODS Eleven cases of ALCL and three ALCL cell lines with t(2;5)(Karpas-299, SUPM2, SU-DHL-1) and 10 Hodgkins disease (HD) samples were stained with anti-phosphotyrosine antibody. The tyrosine kinase activity, p80 phosphorylation, and the apoptotic effects of two tyrosine kinase inhibitors, herbimycin A and STI-571, were determined on ALCL cell lines. RESULTS Herbimycin A had showed both a time- and dose-dependent apoptotic effect on all three cell lines, while STI-571 demonstrated a minimal effect. Following herbimycin A treatment, a decrease in tyrosine kinase activity in the ALCL cell lines and inhibition in NPM-ALK (p80) autophosphorylation was demonstrated by immunoprecipitation and Western blotting. Herbimycin A-induced apoptosis was accompanied by caspase-3 activation. Furthermore, apoptosis induced by herbimycin A was blocked by both z-VAD-FMK and z-DEVD-FMK, suggesting a critical role of caspases. CONCLUSIONS These findings indicate that tyrosine kinase activity is a common characteristic of ALCLs and necessary for ALCL cell survival. These findings further suggest that therapies targeting tyrosine kinases, including p80, may have clinical utility.

Protein kinase C‐delta is commonly expressed in multiple myeloma cells and its downregulation by rottlerin causes apoptosis

Summary. The growth and proliferation of multiple myeloma (MM) cells are influenced by various cytokines produced by bone marrow stromal cells. As cytokine interaction between malignant plasma cells and neighbouring stromal cells is important in the pathogenesis of MM, the understanding of intracellular signalling events elicited by this interaction is of central importance. Recent reports have shown that protein kinase C (PKC) is directly involved in modulating apoptosis in different cells types, including those of haematopoietic neoplasms. In the present study, we analysed the expression patterns of PKC isoforms in the myeloma cell lines U266, RPMI‐8226 and K620. This analysis demonstrated common expression of PKC‐δ, PKC‐ι, PKC‐µ and PKC‐ζ in all three myeloma cell lines. PKC‐δ expression in plasma cells from 11 patients with MM was also shown by immunohistochemistry, utilizing a monoclonal mouse anti‐human PKC‐δ antibody. U266 cells treated with the broad PKC inhibitor safingol (l‐threo‐dihydrosphingosine) or the PKC‐δ‐specific inhibitor rottlerin (3′‐[(8‐Cinnamoyl‐5,7‐dihydroxy‐2,2‐dimethyl‐2H‐1‐benzopyran‐6‐yl)methyl]‐2′,4′,6′‐trihydroxy‐5′‐methylacetophenone) showed decreased PKC‐δ in the particulate fraction and resulted in significant apoptosis. Primary myeloma cells also showed apoptosis after treatment with the PKC inhibitors, as detected by both flow cytometric and morphological evaluation. Our results indicate that PKC‐δ is commonly expressed in myeloma cells and plays an important role in plasma cell survival.

Expression of the Tumor Necrosis Factor Receptor-Associated Factors (TRAFs) 1 and 2 is a Characteristic Feature of Hodgkin and Reed-Sternberg Cells

Tumor necrosis factor receptor–associated factors (TRAFs) are a recently established group of proteins involved in the intracellular signal transduction of several members of the tumor necrosis factor receptor (TNFR) superfamily. Recently, specific members of the TRAF family have been implicated in promoting cell survival as well as activation of the transcription factor NF-κB. We investigated the constitutive expression of TRAF1 and TRAF2 in Hodgkin and Reed–Sternberg (HRS) cells from archived paraffin-embedded tissues obtained from 21 patients diagnosed with classical Hodgkins disease (HD). In a selective portion of cases, examination of HRS cells for Epstein-Barr virus (EBV)–encoded RNA was performed by in situ hybridization, and the results were compared with the magnitude of TRAF1 and TRAF2 staining. We also determined the TRAF profile in the classical HD cell lines L428, KMH2, and HS445 by Western blotting using a series of antibodies that specifically recognize the six individual TRAF family proteins (TRAF1–TRAF6). Moderate to high constitutive expression of TRAF1 and TRAF2 were found in 19 of 21 and 20 of 21 cases of classical HD, respectively. Of the remaining cases, one case showed weak expression of TRAF1, and another case showed weak expression of both proteins. No relationship was found between the staining intensity of the TRAF proteins and EBV expression in HRS cells. Strong constitutive expression of TRAF1 was also identified in the HD cell line L428, compared with the relatively weak expression observed in KMH2 and HS445. All three HD cell lines showed strong expression of TRAF2 protein and moderate, comparatively equal expression of TRAF4 and TRAF6. In contrast, TRAF3 was not expressed in the HD cell lines. Although KMH2 showed weak expression, the remaining HD cell lines also lacked TRAF5 protein. These data demonstrate that constitutive expression of TRAF1 and TRAF2 is a characteristic feature of HRS cells from both patient and cell line specimens. Furthermore, with the exception of TRAF1 expression, HRS cells from the three HD cell lines showed similar TRAF protein expression patterns. Overall, these findings demonstrate the expression of several TRAF proteins in HD. Significantly, the altered regulation of selective TRAF proteins may reflect HRS cell response to stimulation from the microenvironment and potentially contribute both to apoptosis resistance and cell maintenance of HRS cells.

Characterization of apoptosis induced by protein kinase C inhibitors and its modulation by the caspase pathway in acute promyelocytic leukaemia

Acute promyelocytic leukaemia (APL;M3) is a unique form of acute myelogenous leukaemia characterized by t(15;17) translocation. The induction of apoptosis via inhibiting protein kinase C (PKC) has been recently viewed as a promising tool for the eradication of several malignant disorders. In the present study, we investigated the effect of two different protein kinase C inhibitors, Gö6976 and safingol, on the induction of apoptosis in the APL cell line NB4 and its all trans retinoic acid (ATRA)‐resistant variant NB4.306. The effect of the PKC inhibitors on leukaemic cells obtained from three APL patients was also studied. We also evaluated the possible involvement of the caspases in apoptosis induced by PKC inhibitors. Significant time‐ and concentration‐dependent apoptotic changes were demonstrated using Gö6976 and safingol. In addition, our results demonstrated that the caspases were involved in the apoptosis induced by the PKC inhibitors. In conclusion, our study illustrates that the PKC inhibitors Gö6976 and safingol induce apoptosis in APL and hence could be potential therapeutic agents for the treatment of this disease.

Detection of T-cell receptor-γ gene rearrangement in lymphoproliferative disorders by temperature gradient gel electrophoresis

OBJECTIVE Polymerase chain reaction amplification of DNA for T-cell receptor (TCR) gene rearrangement analysis is helpful in the evaluation of T-cell lymphoproliferative disorders. Detection of polymerase chain reaction products is limited by the poor resolution of bands analyzed by agarose or polyacrylamide gel electrophoresis. To improve the detection of a clonal T-cell population, we used temperature gradient gel electrophoresis (TGGE) as an alternative method for analysis of TCR gene rearrangement. DESIGN One hundred eighteen archival DNA samples were randomly selected based on previous Southern blot analysis results. Samples included 58 T-cell neoplasms with positivity for TCR beta gene rearrangement, 22 cases of reactive hyperplasia with germline pattern for both TCR beta and J(H), and 38 patients with B-cell lymphoma. MOLT-16, a T-cell lymphoblastic cell line, was used for the sensitivity assay. Polymerase chain reaction was performed using GC-clamped multiplex primers to amplify the TCR gamma locus and was analyzed by TGGE. The range of temperature gradients was empirically determined for optimal resolution of bands. RESULTS The sensitivity of TGGE was 0.1% when DNA from the MOLT-16 cell line was serially diluted with DNA from reactive lymphoid tissue. Fifty-four (93%) of 58 T-cell neoplasms with TCR beta gene rearrangements showed rearrangement patterns by TCR gamma TGGE, and only 1 of 60 samples (reactive or B-cell lymphomas) showed evidence of gene rearrangement by TGGE. Patients with T-cell neoplasm and involvement of multiple sites showed an identical migration pattern by TGGE analysis. CONCLUSION We demonstrate that TGGE is an effective method for analysis of TCR gene rearrangement in the evaluation of nodal and extranodal lymphoid lesions.

Transfection of caspase-3 in the caspase-3-deficient Hodgkin's disease cell line, KMH2, results in enhanced sensitivity to CD95-, TRAIL-, and ARA-C-induced apoptosis

BACKGROUND CD95(Fas/apo-1) is a cell surface protein member of the tumor necrosis factor receptor family that serves an important role in the induction of apoptosis in several cell types. Although expression of CD95 has been detected on Hodgkin/Reed Sternberg (HRS) cells in situ, our understanding of the biological significance of this molecule in Hodgkins disease (HD) is limited. DESIGN We analyzed both CD95-related apoptotic signaling and its effects on the expression of several factors involved in the regulation of apoptotic mechanisms including: caspase-3, caspase-8, bcl-2, bcl-x, and Bax in HD cell lines (L-428, L-540, HDLM-2, HS-445, and KM-H2). RESULTS HD cell lines showed similar expression levels of CD95 and all but KM-H2 demonstrated variable increases in apoptosis after CD95 stimulation by the agonistic monoclonal antibody, CH11. There was no significant correlation between CD95 sensitivity and constitutive expression levels of caspase-8, bcl-2, bcl-x, and Bax. Caspase-3 transcript was demonstrated by reverse transcriptase polymerase chain reaction (RT-PCR) in all cell lines but protein was at low to nearly undetectable levels in KM-H2 cells. Transfection of KM-H2 cells with pro-caspase-3 resulted in a markedly enhanced apoptotic response to CD95 stimulation that was blocked by pretreatment with the caspase-3 inhibitor, DEVD-FMK. In addition, pro-caspase-3-transfected KM-H2 cells showed significantly increased sensitivity to other caspase-3-dependent apoptotic stimuli, including the death-inducing ligand, TRAIL, and the chemotherapeutic agent, Ara-C. CONCLUSION These data demonstrate that caspase-3 expression plays an important role in CD95-mediated apoptosis in HD cell lines. Furthermore, lack of or decreased expression of caspase-3 in HD cells impairs their apoptotic response not only to CD95 but also to other caspase-3-dependent apoptotic stimuli.