Primary effusion lymphomas (PELs) associated with infection by the Kaposi's sarcoma–associated herpesvirus (KSHV/HHV-8) have constitutive nuclear factor (NF)–κB activity that is essential for their survival, but the source of this activity is unknown. We report that viral FADD-like interleukin-1-β–converting enzyme [FLICE/caspase 8]-inhibitory protein (FLIP) activates NF-κB more potently than cellular FLIP in B cells and that it is largely responsible for NF-κB activation in latently infected PEL cells. Elimination of vFLIP production in PEL cells by RNA interference results in significantly decreased NF-κB activity, down-regulation of essential NF-κB–regulated cellular prosurvival factors, induction of apoptosis, and enhanced sensitivity to external apoptotic stimuli. vFLIP is the first virally encoded gene shown to be essential for the survival of naturally infected tumor cells.

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IntroductionChronic lymphocytic leukemia (CLL) is characterized by the abnormal expansion of cells in a CD5 ϩ (B-1) cell clone. 1,2 In this distinctive form of leukemia, most of the malignant cells are in the G 0 /G 1 phase of the cell cycle. 3,4 Disease results from the progressive accumulation of tumor cells which do not proliferate rapidly, but fail to undergo death. 5 In this regard, CLL provides a paradigm for studies of apoptosis inhibition in lymphocytes. In contrast to other B-cell neoplasms such as follicular lymphoma, Burkitt lymphoma, and mantle cell lymphoma that are characterized by specific cytogenetic abnormalities and dysregulated oncogenes, CLL is not distinguished by a common genetic defect. 6 Rather, the diagnosis of CLL is determined by the morphologic and phenotypic properties of the slowly dividing B cells.Despite their prolonged survival in vivo, most CLL cells do not survive for more than a few days in vitro. A longstanding puzzle for investigators is the propensity of CLL B cells to undergo spontaneous apoptosis in culture. Several lines of evidence support that nonmalignant cells and other host elements extrinsic to CLL cells provide survival signals to the tumor cells in vivo. These factors would include bone marrow stromal cells, 7 immune complexes in the context of accessory leukocytes, 8 "nurse-like" cells, 9 and soluble cytokines such as interleukin 4 (IL-4) 10 and tumor necrosis factor alpha (TNF␣). 11,12 On the basis of observations that CD40 ligation in CLL B cells is a powerful stimulus to induction of NF-B 13,14 and survival, [13][14][15][16] we have proposed that CLL tumor growth in vivo would be promoted by inflammatory elements such as CD40 ligand (CD154), cytokines, and antigens to which the tumor cells bind. 4,17 Like nonmalignant B-1 cells, CLL cells usually express surface IgM (sIgM) with the capacity to bind multiple infectious and autologous structures. [18][19][20][21] In the murine system it is established that the presence of autologous antigens for which the B-cell receptor (BCR) has affinity can positively influence nonmalignant CD5 ϩ B-cell fate and differentiation. 22 An emerging body of literature indicates CLL tumor cells often bear mutated Ig gene sequences, [23][24][25] suggesting that the cells have interacted with antigens in vivo. However, the intracellular signaling pathways by which antigens mediate survival in murine and human B-1 cells, and in leukemic B cells, are largely unknown.The purpose of this study was to determine how sIgM engagement affects CLL cell survival, and to identify key molecular components of an antigen-receptor-driven survival program in the leukemic cells. Here, using freshly isolated cells from more than 20 patients with CLL, we determined that BCR crosslinking favors survival in a process characterized by caspase inhibition, induction of NF-B, and expression of antiapoptotic molecules including bcl-2, mcl-1, and bfl-1. The antiapoptotic effects of sIgM engagement in CLL cells were magnified when CD40 was also engaged, and were...

Kaposi sarcoma–associated herpesvirus (KSHV), or human herpervirus 8 (HHV-8), is a γ-herpesvirus that infects human lymphocytes and is associated with primary effusion lymphoma (PEL). Currently, the role of viral infection in the transformation of PEL cells is unknown. One possibility is that KSHV, like the lymphotropic viruses Epstein-Barr virus (EBV) and human T-cell leukemia virus I (HTLV-I), activates the transcription factor NF-κB to promote survival and proliferation of infected lymphocytes. To examine this possibility, we assessed NF-κB activity in KSHV-infected PEL cell lines and primary tumor specimens by electrophoretic mobility shift assay (EMSA). We observed that NF-κB is constitutively activated in all KSHV-infected lymphomas, and consists of 2 predominant complexes, p65/p50 heterodimers and p50/p50 homodimers. Inhibition experiments demonstrated that Bay 11-7082, an irreversible inhibitor of IκBα phosphorylation, completely and specifically abrogated the NF-κB/DNA binding in PEL cells. PEL cells treated with Bay 11 demonstrated down-regulation of the NF-κB inducible cytokine interleukin 6 (IL-6), and apoptosis. These results suggest that NF-κB activity is necessary for survival of KSHV-infected lymphoma cells, and that pharmacologic inhibition of NF-κB may be an effective treatment for PEL.

Primary effusion lymphoma (PEL) is a distinct type of B-cell non-Hodgkin lymphoma characterized by the presence of Kaposi's sarcoma-associated herpesvirus (KSHV/human herpesvirus 8). Despite having a genotype and gene expression signature of highly differentiated B cells, PEL does not usually express surface or cytoplasmic immunoglobulin (Ig). We show the lack of Oct-2 and OCA-B transcription factors to be responsible, at least in part, for this defect in Ig production. Like Ig genes, ORF50, the key regulator of the switch from latency to lytic reactivation, contains an octamer motif within its promoter. We therefore examined the impact of Oct-2 and OCA-B on ORF50 activation. The binding of Oct-1 to the ORF50 promoter has been shown to significantly enhance ORF50 transactivation. We found that Oct-2, on the other hand, inhibited ORF50 expression and consequently lytic reactivation by competing with Oct-1 for the octamer motif in the ORF50 promoter. Our data suggest that Oct-2 downregulation in infected cells would be favorable to KSHV in allowing for efficient viral reactivation.

Kaposi sarcoma–associated herpesvirus (KSHV), or human herpervirus 8 (HHV-8), is a γ-herpesvirus that infects human lymphocytes and is associated with primary effusion lymphoma (PEL). Currently, the role of viral infection in the transformation of PEL cells is unknown. One possibility is that KSHV, like the lymphotropic viruses Epstein-Barr virus (EBV) and human T-cell leukemia virus I (HTLV-I), activates the transcription factor NF-κB to promote survival and proliferation of infected lymphocytes. To examine this possibility, we assessed NF-κB activity in KSHV-infected PEL cell lines and primary tumor specimens by electrophoretic mobility shift assay (EMSA). We observed that NF-κB is constitutively activated in all KSHV-infected lymphomas, and consists of 2 predominant complexes, p65/p50 heterodimers and p50/p50 homodimers. Inhibition experiments demonstrated that Bay 11-7082, an irreversible inhibitor of IκBα phosphorylation, completely and specifically abrogated the NF-κB/DNA binding in PEL cells. PEL cells treated with Bay 11 demonstrated down-regulation of the NF-κB inducible cytokine interleukin 6 (IL-6), and apoptosis. These results suggest that NF-κB activity is necessary for survival of KSHV-infected lymphoma cells, and that pharmacologic inhibition of NF-κB may be an effective treatment for PEL.

. (1997). Fundam. Appl. Toxicol 38,[75][76][77][78][79][80][81][82][83][84][85][86][87][88] The extent to which cardiorespiratory infirmity and other sublethal effects of saxitoxin (STX) and tetrodotoxin (TTX) can be reversed by 4-aminopyridine (4-AP) was investigated in guinea pigs chronically instrumented for the concurrent electrophysiological recordings of electrocorticogram (ECoG), diaphragmatic electromyogram (DEMG), Lead II electrocardiogram, and neck skeletal muscle electromyogram. Animals were intoxicated with either STX or TTX (2 and 3 /xg/kg, im) to produce a state of progressive cardiorespiratory depression (depicted by decreasing DEMG amplitude, bradypnea, and bradycardia). At the point where cardiorespiratory performance was most seriously compromised («30 min posttoxin), 4-AP (1 or 2 mg/kg, im) was administered. The therapeutic effect of 4-AP was striking in that, within minutes, the toxin-induced diaphragmatic blockade, bradypnea, bradycardia, and depressed cortical activity were all restored to a level either comparable to, or surpassing, that of control. The optimal 4-AP dose level was determined to be 2 mg/kg (im) based on analyses of cardiorespiratory activity profiles throughout the course of intoxication and 4-AP treatment. At the dose levels (either 1 or 2 mg/ kg) used to restore ventilatory function and cardiovascular performance, 4-AP produced no sign of seizures and convulsions. Although less serious secondary effects such as cortical excitant/arousal effect (indicated by ECoG power spectral analysis) and transient periods of skeletal muscle fasciculation were observed, these events were of minor concern particularly in view of the remarkable therapeutic effects of 4-AP. © i»»7 society of Toxicology.Saxitoxin (STX) and tetrodotoxin (TTX) are among the deadliest nonprotein neurotoxins known (lethal dose in guinea pigs «*5 ^g/kg, im; unpublished observations).

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