Serhan Alkan

Visualization of human herpesvirus type 8 in Kaposi's sarcoma by light and transmission electron microscopy.

Background:Human herpesvirus type 8 (HHV-8) has been associated with Kaposis sarcoma, body cavity-based lymphoma (BCBL), and multicentric Castlemans disease through DNA, in situ hybridization, and serologic studies. HHV-8 has been visualized only in HHV-8-positive/Epstein-Barr virus (EBV)-negative/cytomegalovirus (CMV)-negative BCBL cell lines, but not in HHV-8-positive/EBV-negative/CMV-negative Kaposis sarcoma lesions. Design:Kaposis sarcoma of the skin, lymph node, and spleen from three patients with AIDS were analysed for HHV-8, EBV and CMV DNA by polymerase chain reaction (PCR), for HHV-8 RNA (T1.1 riboprobe) by in situ hybridization (ISH), for viral inclusions by light microscopy, and for herpesviruses by transmission electron microscopy (TEM). Sections were also labeled with T1.1 counterstained with CD34, an endothelial cell marker. Results:The skin lesion was DNA PCR-positive for HHV-8 and CMV (nested, but not single PCR), the lymph node was positive for HHV-8 and EBV, and the spleen was positive for only HHV-8. TEM revealed infection by a virus displaying the typical morphology and cytopathicity of herpesviruses. Hexagonal nucleocapsids and mature enveloped virions were present in vasoformative spindle cells and mononuclear cells, often resembling lymphocytes. Extrapolating from TEM to standard light microscopy on hematoxylin and eosin-stained paraffin sections, eosinophilic, targetoid intranuclear inclusions were identified within spindle cells which often lined vascular lumina. The T1.1-riboprobe labeled CD34+ spindle cells containing intranuclear inclusions, as well as mononuclear cells within Kaposis sarcoma and residual lymphoid tissue. Conclusion:The herpesvirus visualized in Kaposis sarcoma lesions has morphologic and cytopathic features typical of human herpesviruses, productively infects vasoformative spindle cells and mononuclear cells, and is consistent with HHV-8. It can also form intranuclear inclusions that are identifiable by light microscopy in hematoxylin and eosin sections and by ISH.

Caspase activation and disruption of mitochondrial membrane potential during UV radiation-induced apoptosis of human keratinocytes requires activation of protein kinase C.

The induction of apoptosis in human keratinocytes by UV radiation involves caspase-mediated cleavage and activation of protein kinase C delta (PKCδ). Here we examined the role of PKC activation in caspase activation and disruption of mitochondria function by UV radiation. Inhibition of PKC partially blocked UV radiation-induced cleavage of PKCδ, pro-caspase-3, and pro-caspase-8, and the activation of these caspases. PKC inhibition also blocked the UV-induced loss of mitochondria membrane potential, but did not block the release of cytochrome c from mitochondria. Expression of the active catalytic domain of PKCδ was sufficient to induce apoptosis and disrupt mitochondrial membrane potential, however a kinase inactive PKCδ catalytic domain did not. Furthermore, the PKCδ catalytic fragment generated following UV radiation localized to the mitochondria fraction, as did ectopically expressed PKCδ catalytic domain. These results identify a functional role for PKC activation in potentiating caspase activation and disrupting mitochondrial function during UV-induced apoptosis.

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.

Analysis of histone deacetylase inhibitor, depsipeptide (FR901228), effect on multiple myeloma.

Multiple myeloma (MM) is a neoplastic proliferation of plasma cells and remains an incurable disease because of the development of drug resistance. Histone deacytylase (HDAC) inhibitors are a new class of chemotherapeutic reagents that cause growth arrest and apoptosis of neoplastic cells. Depsipeptide, a new member of the HDAC inhibitors, was found to be safe in humans and has been shown to induce apoptosis in various cancers. In order to evaluate the effects of depsipeptide, a MM cell line, U266 [interleukin (IL)‐6 dependent], was analysed for viability and apoptosis. The combined effect of depsipeptide with melphalan and changes in BCL‐2 family proteins (BCL‐2, BCL‐XL, BAX and MCL‐1) were also investigated. In addition, the RPMI 8226 cell line (IL‐6 independent), and primary patient myeloma cells were also analysed for apoptosis after depsipeptide treatment. Depsipeptide induced apoptosis in both U266 and RPMI 8226 cell lines in a time‐ and dose‐dependent fashion, and in primary patient myeloma cells. We also demonstrated that depsipeptide had an additive effect with melphalan (10 μmol/l). BCL‐2, BCL‐XL and MCL‐1 showed decreased expression in depsipeptide‐treated samples. Based on recent clinical trials demonstrating minimal clinical toxicity, our study supports the future clinical utilization of depsipeptide in the management of MM.

Histone deacetylase inhibitors induce caspase-dependent apoptosis and downregulation of daxx in acute promyelocytic leukaemia with t(15;17).

Histone deacetylase (HDAC) appears to play an important role in the pathogenesis of acute promyelocytic leukaemia (APL) as it is recruited by both PML–RARα and PLZF/RARα in leukaemic cells with t(15;17) and t(11;17) respectively. Recent studies have demonstrated that HDAC inhibitors can be therapeutically used in various neoplastic disorders including APL. Cell differentiation was considered the major mechanism of the anti‐leukaemic effects of HDAC inhibitors in APL. However, most of these studies either evaluated the effect of HDAC inhibitors in combination with all‐trans retinoic acid (ATRA) or focused on the less common form of APL with t(11;17). To investigate the cellular effects of HDAC inhibitors, including sodium butyrate, trichostatin A, and suberoylanilide hydroxamic acid (SAHA), we used two APL cell lines, NB4 and the ATRA‐resistant derivative NB4.306. Moreover, primary cells from five patients with cytogenetic evidence for t(15;17) were also studied. Our results demonstrated that HDAC inhibitors induce distinct caspase‐dependent apoptosis in APL, which showed both concentration‐and time‐dependence. In addition, changes in the apoptosis‐regulatory proteins, daxx, bcl‐2 and bax were analysed. HDAC inhibitors induced downregulation of daxx, but no significant changes were detected in bcl‐2 or bax. In conclusion, apoptosis induced by HDAC inhibitors in APL could provide an effective strategy for treatment of patients with t(15;17).

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.

Bruton tyrosine kinase is commonly overexpressed in mantle cell lymphoma and its attenuation by Ibrutinib induces apoptosis.

Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy that characteristically shows overexpression of cyclin-D1 due to an alteration in the t(11;14)(q13;q32) chromosomal region. Although there are some promising treatment modalities, great majority of patients with this disease remain incurable. The B-cell antigen receptor (BCR) signaling plays a crucial role in B-cell biology and lymphomagenesis. Bruton tyrosine kinase (BTK) has been identified as a key component of the BCR signaling pathway. Evidence suggests that the blockade of BTK activity by potent pharmacologic inhibitors attenuates BCR signaling and induces cell death. Notably, the expression levels and the role of BTK in MCL survival are still elusive. Here, we demonstrated a moderate to strong BTK expression in all MCL cases (n=19) compared to benign lymphoid tissues. Treatment of MCL cell lines (Mino or Jeko-1) with a potent BTK pharmacologic inhibitor, Ibrutinib, decreased phospho-BTK-Tyr(223) expression. Consistent with this observation, Ibrutinib inhibited the viability of both Mino and JeKo-1 cells in concentration- and time-dependent manners. Ibrutinib also induced a concentration-dependent apoptosis in both cell lines. Consistently, Ibrutinib treatment decreased the levels of anti-apoptotic Bcl-2, Bcl-xL, and Mcl-1 protein. These findings suggest that BTK signaling plays a critical role in MCL cell survival, and the targeting of BTK could represent a promising therapeutic modality for aggressive lymphoma.

Histone deacetylase inhibitor romidepsin has differential activity in core binding factor acute myeloid leukemia.

Purpose: Recruitment of histone deacetylases (HDAC) is a mechanism of transcriptional repression implicated in the differentiation block in acute myeloid leukemia (AML). We hypothesized that the HDAC inhibitor romidepsin could cause transcriptional derepression, up-regulation of specific target genes in AML, and differentiation of the leukemic clone. The primary objectives of the study were to evaluate the safety and efficacy of romidepsin in advanced AML. Experimental Design: Twenty patients were stratified into cohort A or B based on the absence or presence of chromosomal abnormalities known to recruit HDACs, including those involving core binding factor (CBF). Romidepsin was administered i.v. at 13 mg/m2/d on days 1, 8, and 15 of a 28-day cycle. Pharmacodynamic endpoints were evaluated at serial time points. Results: Common adverse effects noted were grade 1 to 2 nausea, anorexia, and fatigue. No objective evidence of antileukemic activity was seen in cohort A. In cohort B, although there were no clinical responses by standard criteria, antileukemic activity was observed in 5 of 7 patients. Two patients had clearance of bone marrow blasts and 3 patients had a >50% decrease in bone marrow blasts. Furthermore, in cohort B, at 24 h, there was a significant increase in MDR1 (P = 0.005), p15 (P = 0.01), and p14 (P < 0.0001) expression. In cohort A, although there was a trend toward up-regulation of MDR1, p15, and p14 expression, these changes were not statistically significant. Conclusion: Romidepsin has differential antileukemic and molecular activity in CBF AML. Development of this agent in CBF AML should focus on combinations that target related mechanisms of gene silencing such as DNA methylation.

IL-4 induces differentiation and expansion of Th2 cytokine-producing eosinophils.

Innate effector cells that produce Th2-type cytokines are critical in Th2 cell-mediated immune responses. However, it is not known how these cells acquire the ability to produce Th2 cytokines. IL-4 is a potent inducer that directs differentiation of naive CD4+ T cells into CD4+ Th2 effector cells. To determine whether IL-4 can induce differentiation and expansion of Th2 cytokine-producing innate cells, we used mice whose il-4 gene was replaced by a knock-in green fluorescence protein (gfp) gene. We found that, directly ex vivo, IL-4 increased the number of GFP+ cells in the airway and the lung tissue in an Ag-specific manner. The majority of GFP+ cells were eosinophils, suggesting that IL-4 plays a pivotal role in expanding IL-4-producing eosinophils in vivo. IL-4-producing eosinophils showed some unique features compared with IL-4-producing CD4+ T cells. They exhibited biallelic expression of the il-4 gene when stimulated and were more dominant IL-4- and IL-5-producing cells. Furthermore, we show that IL-4 drove bone marrow progenitor cells to differentiate into Th2 cytokine-producing eosinophils in vitro. These results strongly suggest IL-4 is a potent factor in directing bone marrow progenitor cells to differentiate into Th2 cytokine-producing eosinophils.

Analysis of expression of heat shock protein-90 (HSP90) and the effects of HSP90 inhibitor (17-AAG) in multiple myeloma.

Heat shock protein 90 (HSP90) is required for structural folding and maintenance of conformational integrity of various proteins, including several associated with cellular signaling. Recent studies utilizing 17-allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of HSP90, demonstrated an antitumor effect in solid tumors. To test whether HSP90 could be targeted in multiple myeloma (MM) patients, we first investigated expression of HSP90 by immunofluorescence and flow cytometric analysis in a myeloma cell line (U266) and primary myeloma cells. Following demonstration of HSP90 expression in myeloma cells, archival samples of 32 MM patients were analysed by immunoperoxidase staining. Myeloma cells in all patients showed strong cytoplasmic expression of HSP90 in all samples and 55% also demonstrated concurrent nuclear immunopositivity. Treatment of U266 and primary MM cells with 17AAG resulted in significantly increased apoptosis compared to untreated control cells. Analysis of anti-apoptotic BCL2 family proteins and akt in MM cells incubated with 17-AAG revealed down-regulation of BCL-2, BCL-XL, MCL-1 and akt. Furthermore, although a low concentration of bortezomib resulted in no cell death, a combination of 17AAG and bortezomib treatment revealed a synergistic apoptotic effect on the U266 cell line. These data suggest that targeted inhibition of HSP90 may prove to be a valid and innovative strategy for the development of future therapeutic options for MM patients.