AP-1 family transcription factors have been implicated in the control of proliferation, apoptosis and malignant transformation. However, their role in oncogenesis is unclear and no recurrent alterations of AP-1 activities have been described in human cancers. Here, we show that constitutively activated AP-1 with robust c-Jun and JunB overexpression is found in all tumor cells of patients with classical Hodgkin's disease. A similar AP-1 activation is present in anaplastic large cell lymphoma (ALCL), but is absent in other lymphoma types. Whereas c-Jun is up-regulated by an autoregulatory process, JunB is under control of NF-kappa B. Activated AP-1 supports proliferation of Hodgkin cells, while it suppresses apoptosis of ALCL cells. Furthermore, AP-1 cooperates with NF-kappa B and stimulates expression of the cell-cycle regulator cyclin D2, proto-oncogene c-met and the lymphocyte homing receptor CCR7, which are all strongly expressed in primary HRS cells. Together, these data suggest an important role of AP-1 in lymphoma pathogenesis.

B cell differentiation is controlled by a complex network of lineage-restricted transcription factors. How perturbations to this network alter B cell fate remains poorly understood. Here we show that classical Hodgkin lymphoma tumor cells, which originate from mature B cells, have lost the B cell phenotype as a result of aberrant expression of transcriptional regulators. The B cell-specific transcription factor program was disrupted by overexpression of the helix-loop-helix proteins ABF-1 and Id2. Both factors antagonized the function of the B cell-determining transcription factor E2A. As a result, expression of genes specific to B cells was lost and expression of genes not normally associated with the B lineage was upregulated. These data demonstrate the plasticity of mature human lymphoid cells and offer an explanation for the unique classical Hodgkin lymphoma phenotype.

Resistance to death receptor–mediated apoptosis is supposed to be important for the deregulated growth of B cell lymphoma. Hodgkin/Reed-Sternberg (HRS) cells, the malignant cells of classical Hodgkin's lymphoma (cHL), resist CD95-induced apoptosis. Therefore, we analyzed death receptor signaling, in particular the CD95 pathway, in these cells. High level CD95 expression allowed a rapid formation of the death-inducing signaling complex (DISC) containing Fas-associated death domain–containing protein (FADD), caspase-8, caspase-10, and most importantly, cellular FADD-like interleukin 1β–converting enzyme-inhibitory protein (c-FLIP). The immunohistochemical analysis of the DISC members revealed a strong expression of CD95 and c-FLIP overexpression in 55 out of 59 cases of cHL. FADD overexpression was detectable in several cases. Triggering of the CD95 pathway in HRS cells is indicated by the presence of CD95L in cells surrounding them as well as confocal microscopy showing c-FLIP predominantly localized at the cell membrane. Elevated c-FLIP expression in HRS cells depends on nuclear factor (NF)-κB. Despite expression of other NF-κB–dependent antiapoptotic proteins, the selective down-regulation of c-FLIP by small interfering RNA oligoribonucleotides was sufficient to sensitize HRS cells to CD95 and tumor necrosis factor–related apoptosis-inducing ligand–induced apoptosis. Therefore, c-FLIP is a key regulator of death receptor resistance in HRS cells.

IntroductionAlthough organic and inorganic arsenic-containing compounds are environmental toxins, they have been used for more than 100 years for the treatment of human diseases. 1,2 Salvarsan, active against syphilis, 3 or melarsoprol, still used for the treatment of African sleeping sickness, 4 are examples of organic arsenicals. Fowler solution, which was used for the treatment of chronic myeloid leukemia 5 (CML) or psoriasis, 6 is an example of inorganic arsenic. Most of these drugs have been replaced by others, as it became clear that arsenic causes diverse pathologies and increases the risk of cancer. 7,8 Since the discovery that arsenic trioxide (As 2 O 3 ) is an efficient drug for the treatment of acute promyelocytic leukemia 9 (APL), As 2 O 3 was reintroduced in current therapeutic concepts. Initial clinical studies focused on the treatment of hematopoietic malignancies. Currently, the action of arsenic on many other tumor entities is under investigation. 10,11 Depending on the cell type and the applied concentrations of arsenic, diverse cellular effects are observed (for a recent review, see Miller et al 12 ). Low concentrations can induce differentiation or cell cycle arrest, whereas high concentrations can induce apoptosis. Mechanisms leading to induction of apoptosis were extensively studied. An important role was attributed to arsenic-triggered degradation of specific proteins with prosurvival function. The strongest correlation was found with the degradation of the promyelocytic leukemia/retinoic acid receptor-␣ (PML/RAR-␣) protein in APL, 13 but also degradation of the viral Tax protein in human T-cell lymphotropic virus (HTLV)-induced T-cell leukemia has been reported. 14 However, these mechanisms cannot explain the induction of apoptosis in many other cell types. As a general finding, all 3 groups of mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK), p38, and Jun Nterminal kinase (JNK), are activated in response to arsenic. 15,16 This activation may be responsible for the carcinogenic effects of arsenic, but MAPK activation is not required for arsenic-induced apoptosis. 17 Furthermore, the generation of reactive oxygen species (ROS) has been implicated in arsenic-mediated apoptosis. 18 The biologic effects of arsenic may be attributed to structural and functional alterations of critical cellular proteins by its reactivity with sulfhydryl groups. 19 The resulting loss of function of specific enzymes, including kinases and phosphatases, functionally alters diverse signaling pathways. For example, arsenicmediated inhibition of MAPK phosphatases, which contain cysteines in their catalytic pocket, induces MAPK activation. 16 Arsenic exerts an opposite effect on the IB kinase (IKK) complex. 20 The IKK complex, which is composed of the 2 catalytic IKK␣ and IKK␤ subunits and the regulatory IKK␥/NEMO component, is the central mediator of nuclear factor-B (NF-B)-inducing stimuli. 21 Activation of the IKK complex results in phosphorylation of IBs, which subsequently ar...

The immunosuppressive macrolide rapamycin and its derivative everolimus (SDZ RAD, RAD) inhibit the mammalian target of rapamycin (mTOR) signaling pathway. In this study, we provide evidence that RAD has profound antiproliferative activity in vitro and in NOD/SCID mice in vivo against Hodgkin lymphoma (HL) and anaplastic large cell lymphoma (ALCL) cells. Moreover, we identified 2 molecular mechanisms that showed how RAD exerts antiproliferative effects in HL and ALCL cells. RAD down-regulated the truncated isoform of the transcription factor CCAAT enhancer binding protein ␤ (C/ EBP␤), which is known to disrupt terminal differentiation and induce a transformed phenotype. Furthermore, RAD inhibited constitutive nuclear factor B (NF-B) activity, which is a critical sur- IntroductionHodgkin lymphoma (HL) and anaplastic large cell lymphoma (ALCL) share morphologic and immunophenotypic markers in a subgroup of cases although they are biologically distinct entities. 1 Therefore, pathologic diagnosis is sometimes difficult to achieve and these cases are classified as "gray-zone lymphomas." 2 Moreover, in both entities novel therapeutic options are needed, as curative therapy of HL is compromised by a high risk of long-term complications, and anaplastic lymphoma kinase (ALK)-negative ALCL still has a very unfavorable prognosis with current treatment strategies. [3][4][5] The macrocyclic lactone everolimus (SDZ, RAD) RAD is a rapamycin derivative with potent immunosuppressive and antiproliferative properties. [6][7][8][9][10] It is further known to inhibit growth factor-driven cell proliferation of hematopoietic and nonhematopoietic cells. 6,10 In addition, RAD is a potent inhibitor of human Epstein-Barr virus (EBV)-transformed B lymphocytes in vitro and in vivo, arresting cell-cycle progression and increasing the apoptotic rate of EBV ϩ B cells. 10 Therefore, it has been suggested that RAD might be effective in the prevention and treatment of human posttransplant lymphoproliferative disorders. 10 Here, we investigated whether RAD inhibits tumor cell proliferation of HL and ALCL. We show that RAD significantly inhibits proliferation of HL and ALCL cells in vitro and arrests cell-cycle progression in G 0 /G 1 . Furthermore, we demonstrate that in vivo, RAD markedly suppresses tumor cell proliferation of HL and ALCL cells, xenotransplanted into NOD/SCID mice. Our data suggest that RAD might be used in combination chemotherapy for the treatment of HL and ALCL. Moreover, we studied the mechanisms of proliferation arrest mediated by the mammalian target of rapamycin (mTOR) inhibitor RAD to identify the molecular targets in HL and ALCL. The mTOR pathway controls the translation initiation machinery in response to nutrients and growth factors thereby coordinating cell growth with cell division. 11 A transcription factor that is a critical target of mTOR is the CCAAT enhancer binding protein (C/EBP)␤. 11-13 C/EBP␤ has previously been identified as an essential downstream target in tumors expressing activated cyclin D1. 14 Our dat...

IntroductionTranscription factor nuclear factor kappa B (NF-B) plays a key role in the regulation of immune and inflammatory responses, functions as a potent inhibitor of apoptosis, and is involved in malignant transformation of different cell types (for recent reviews see Rayet and Gelinas, 1 Karin and Ben-Neriah, 2 and Bonizzi and Karin 3 ). Depending on the stimulus, the duration of stimulation, and the cellular context, the NF-B family members p50, p52, p65 (RelA), RelB, and c-Rel form different homo-or heterodimers. p50 and p52 are derived from precursor molecules (p105 and p100), which can act as inhibitors of NF-B (IB) proteins. The activation of the NF-B pathway is controlled by IB proteins, which retain NF-B in the cytoplasm. Following activation, they are degraded by the proteasome while NF-B translocates into the nucleus where it activates transcription. 2 The IB kinase (IKK) complex plays a central role in this pathway by mediating the initial phosphorylation of IB proteins. 4 The Bcl-3 protein shares structural features with I〉 proteins. 5,6 It was initially discovered by investigation of the t(14;19)(q32.3; q13.2) translocation in B-cell chronic lymphocytic leukemia (B-CLL), which is associated with poor prognosis. 7 In line with an oncogenic function, E-Bcl-3 transgenic mice develop lymphoproliferative disorders, 8 Bcl-3 directly transforms cells, 9 and Bcl-3 excerts an antiapoptotic effect in B and T lymphocytes. 10,11 Furthermore, Bcl-3 overactivity has been suggested to be involved in the pathogenesis of breast cancer, 12 and Bcl-3 overexpression has been reported in a subgroup of anaplastic large-cell lymphomas (ALCLs). 13 Consistent with a noninhibitory function, Bcl-3 is located mainly in the nucleus, and it can function as a regulator at NF-B DNA binding sites. 5,6,[14][15][16][17] Bcl-3-mediated regulation of NF-B activity might occur indirectly by dissociation of the inhibitory NF-B (p50) 2 and (p52) 2 complexes from DNA 5 or, alternatively, by a function as transcriptional coactivator. [16][17][18] ALCLs are unique lymphomas originating from cytotoxic T cells (for recent review see Stein et al 19 ). They are defined by the proliferation of predominantly large lymphoid cells and expression of the tumor necrosis factor (TNF)-receptor family member CD30 and the cytotoxic molecules perforin and granzyme B. ALCLs express the anaplastic lymphoma kinase (ALK) in 50% to 70% of the cases, 19 most commonly in the context of the chromosomal translocation t(2;5)(p23;q35). The resulting nucleophosmin (NPM)-ALK fusion protein promotes cellular transformation. ALCLs share a number of molecular aberrations with classical Hodgkin lymphoma (cHL), which in the majority of cases derives from B cells. 20 Constitutive AP-1 (c-Jun and JunB) 21 or Notch-1 overactivity 22 is found in both of these lymphoma entities. Interestingly, the B-cell-derived neoplastic Hodgkin/Reed-Sternberg (HRS) cells of cHL have lost the B-cell-specific gene expression program, 23 whereas the T-cell-derived ALCLs have lost most of the...

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Summary Similar to classical Hodgkin lymphoma (HL) tumour cells, primary effusion lymphoma (PEL) originates from mature B cells but displays a non‐B cell phenotype, the mechanisms and consequences of which are not yet understood. This study showed that PEL lacked DNA binding activity of the B cell‐determining transcription factors E2A, EBF and Pax5. PEL overexpressed the E2A antagonists ABF‐1 and Id2, which have been described to block the B‐cell differentiation program in classical HL. However, in contrast to HL cells, B lineage‐inappropriate genes were not similarly upregulated in PEL, and reconstitution of B cell‐specific E2A homodimer activity in PEL induced apoptosis. These data demonstrate that lineage infidelity in PEL is not as pronounced as in HL, and that the loss of the B cell‐specific transcription factor E2A in PEL is implicated in apoptosis protection.