Hypomethylation of CD30 CpG islands with aberrant JunB expression drives CD30 induction in Hodgkin lymphoma and anaplastic large cell lymphoma

High expression of CD30 and JunB is the hallmark of malignant cells in Hodgkin lymphoma (HL) and anaplastic large cell lymphoma (ALCL). Ligand-independent signaling by CD30 induces JunB, which activates the CD30 promoter, stabilizing CD30 expression and supporting the survival of Hodgkin-Reed-Sternberg (H-RS) and ALCL cells. Here we show for the first time CpG islands encompassing 60 CpG dinucleotides, located in the core promoter, exon 1 and intron 1 of CD30 gene. Analysis of the methylation status of CD30 CpG islands in H-RS, ALCL and unrelated cell lines reveals an inverse relationship between the extent of CD30 CpG methylation and CD30 expression. CD30 CpG islands of H-RS and ALCL cell lines are rarely methylated. Methylation of the CD30 promoter decreases CD30 induction and JunB action on the demethylated CD30 promoter enhances CD30 induction. CD30 and JunB are strongly expressed in H-RS and ALCL cells, whereas they are not expressed in nonmalignant lymphocytes in which CD30 CpG islands are rarely methylated. We conclude that constitutive action of aberrantly expressed JunB on hypomethylated CD30 CpG islands of lymphocytes triggers CD30 induction and initiates activation of the JunB-CD30-JunB loop, essential to the pathogenesis of HL and ALCL.     

Ets-1 activates overexpression of JunB and CD30 in Hodgkin's lymphoma and anaplastic large-cell lymphoma

Overexpression of CD30 and JunB is a hallmark of tumor cells in Hodgkin's lymphoma (HL) and anaplastic large-cell lymphoma (ALCL). We reported that CD30-extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinase (MAPK) signaling induces JunB, which maintains constitutive activation of the CD30 promoter. Herein, we localize a cis-acting enhancer in the JunB promoter that is regulated by Ets-1. We show that E26 transformation-specific-1 (Ets-1) (-146 to -137) enhances JunB promoter activation in a manner that is dependent on CD30 or the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK)-ERK1/2 MAPK pathway. Ets-1 knockdown reduces the expression of both JunB and CD30, and CD30 knockdown significantly reduces JunB expression in HL and ALCL cell lines. NPM-ALK knockdown also reduces JunB expression in ALCL cell lines expressing NPM-ALK. Collectively, these results indicate that CD30 and NPM-ALK cooperate to activate the ERK1/2 MAPK-Ets-1 pathway. Ets-1, constitutively activated by ERK1/2-MAPK, plays a central role in the overexpression of JunB and CD30, which are both involved in the pathogenesis of HL and ALCL.     

Cytoplasmic aggregation of TRAF2 and TRAF5 proteins in the Hodgkin-Reed-Sternberg cells

We previously reported that ligand-independent signaling by highly expressed CD30 in Hodgkin-Reed-Sternberg (H-RS) cells is responsible for constitutive activation of NF-kappa B. In the present study, we characterize the intracellular localization of tumor necrosis factor (TNF) receptor associated factor (TRAF) proteins in H-RS cells. Confocal immunofluorescence microscopy of cell lines derived from H-RS cells and HEK293 transformants highly expressing CD30 revealed aggregation of TRAF2 and TRAF5 in the cytoplasm as well as clustering near the cell membrane. In contrast, TRAF proteins were diffusely distributed in the cytoplasm in cell lines unrelated to Hodgkin's disease (HD) and control HEK293 cells. Furthermore, the same intracellular distribution of TRAF proteins was demonstrated in H-RS cells of lymph nodes of HD, but not in lymphoma cells in lymph nodes of non-Hodgkin's lymphoma. Dominant-negative TRAF2 and TRAF5 suppressed cytoplasmic aggregation along with constitutive NF-kappa B activation in H-RS cell lines. Confocal immunofluorescence microscopy also revealed co-localization of IKK alpha, NIK, and I kappa B alpha with aggregated TRAF proteins in H-RS cell lines. These results suggest involvement of TRAF protein aggregation in the signaling process of highly expressed CD30 and suggest they function as scaffolding proteins. Thus, cytoplasmic aggregation of TRAF proteins appears to reflect constitutive CD30 signaling which is characteristic of H-RS cells.     

Autocrine growth regulation of CD30 ligand in CD30-expressing Reed-Sternberg cells: distinction between Hodgkin's disease and anaplastic large cell lymphoma

Persistent expression of high levels of CD30 in Hodgkin's Reed-Sternberg (H-RS) cells and anaplastic large-cell lymphoma (ALCL) cells, but not in most T- or B-cell lymphomas, suggests the presence of an underlying mechanism leading to the abnormality and possible involvement of CD30 in the growth and survival of these two unique types of tumors. In this study, we examined the effect of CD30 ligand (CD30L) on CD30-positive H-RS and ALCL cells in long-term cultures or in primary cultures. CD30L induced various degrees of proliferation in three long-term cultured H-RS cell lines (L428, HDLM-2, and KM-H2) as well as in primary cultures of H-RS cells obtained directly from patients with Hodgkin's disease. In contrast, significant inhibition was observed in one of the ALCL cell lines (SU-DHL-1), but no growth inhibition or promotion in responding to exogenous CD30L was detected in three others (PB-1, JB-6, and McG-2), nor in primary cultures of ALCL cells. Expression of CD30L was determined by polymerase chain reaction in long-term cultured cells and by an immunohistochemical method in H-RS or ALCL cells de novo in tissue sections. None of the H-RS and ALCL cell lines was positive for CD30L. In tissue sections, we noticed that ALCL cells were generally CD30L-negative. In contrast, the anti-CD30L antibody reacted with a majority of H-RS cells with diffuse cytoplasmic staining. Most H-RS cells were CD30-positive, indicating co-expression of CD30 and CD30L in the majority of lymphoma cells. The persistent high levels of CD30 and CD30L expression in H-RS cells suggest that the autocrine CD30L-CD30 cytokine-receptor loop, in addition to having the paracrine activity previously thought to exist, could play important roles in the proliferation of H-RS cells. In contrast, the CD30L-mediated cytotoxicity may participate in the regression or slow progression of ALCL during the early phase of the disease in selected patients. However, when the disease progresses, the ALCL cells are likely to become resistant to exogenous CD30L.     

CD30 ligand expression in nonmalignant and Hodgkin's disease-involved lymphoid tissues.

The CD30 ligand (CD30L) is a type II transmembrane glycoprotein of the tumor necrosis factor ligand superfamily. Recent cloning of CD30L has enabled studies to explore its function and tissue distribution. For instance, recombinant CD30L has been shown to co-stimulate T cells and to act as mitogen for Hodgkin's disease (HD)-derived cell lines. The counter-receptor for CD30L, ie, CD30, is a type I cytokine receptor that is highly expressed by activated T cells, Hodgkin and Reed-Sternberg (H-RS) cells, and anaplastic large cell lymphoma cells. In the present study, recombinant membrane-bound and soluble human CD30L were instrumental to raise a monoclonal antibody (M80) recognizing membrane-bound CD30L on transfected and native cells. With this reagent, a panel of cultured lymphoma-derived cell lines as well as primary normal, reactive, and HD-involved lymphoid tissues were examined for expression of CD30L by immunostaining and flow cytometry. In reactive lymphnodes and tonsils, CD30L was expressed by a small subset of lymphoid cells, histiocytes, and granulocytes. Higher levels of CD30L expression were noted in HD lesions among bystander cells; ie, T cells and granulocytes that surrounded H-RS cells. Native CD30L displayed at the cell surface was functionally active as shown by the ability of fixed granulocytes to interact with CD30+ cell lines. Moreover, CD30L was detectable, although to a lower staining intensity, in primary H-RS cells of all HD tissues investigated regardless of the histological subtype and the phenotype of H-RS cells (ie, CD30+/CD40+ versus CD30-/CD40+). Co-expression of CD30 and CD30L that was seen on H-RS cells of all, except the CD30- nodular lymphocyte predominant, subtypes of HD may point to the use of this pair of molecules in paracrine and/or autocrine mitogenic cell interactions. Monoclonal antibody M80 may thus represent a useful tool for studying CD30L expression on cultured cell lines and primary cells from normal, reactive, and malignant tissues.     

IkappaBalpha independent induction of NF-kappaB and its inhibition by DHMEQ in Hodgkin/Reed-Sternberg cells

Constitutive nuclear factor kappaB (NF-kappaB) activation characterizes Hodgkin/Reed-Sternberg (H-RS) cells. Blocking constitutive NF-kappaB has been shown to be a potential strategy to treat Hodgkin lymphoma (HL). Here, for the first time we show that although constitutive NF-kappaB level of H-RS cell lines is very high, topoisomerase inhibitors further enhance NF-kappaB activation through IkappaB kinase activation in not only H-RS cell lines with wild-type IkappaBalpha, but also in those with IkappaBalpha mutations and lacking wild-type IkappaBalpha. Thus, both constitutive and inducible NF-kappaB are potential targets to treat HL. We also present the data that indicate the involvement of IkappaBbeta in NF-kappaB induction by topoisomerase inhibitors. A new NF-kappaB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ) inhibited constitutive NF-kappaB activity and induced apoptosis of H-RS cell lines. DHMEQ also inhibited the growth of H-RS cells without significant systemic toxicity in a NOD/SCID/gammac(null) (NOG) mice model. DHMEQ and topoisomerase inhibitors revealed enhancement of apoptosis of H-RS cells by blocking inducible NF-kappaB. Results of this study suggest that both constitutive and inducible NF-kappaB are molecular targets of DHMEQ in the treatment of HL. The results also indicate that IkappaBbeta is involved in NF-kappaB activation in H-RS cells and IkappaBbeta substitutes for IkappaBalpha in H-RS cells lacking wild-type IkappaBalpha.     

Aberrant expression of T cell and B cell markers in myelocyte/monocyte/histiocyte-derived lymphoma and leukemia cells. Is the infrequent expression of T/B cell markers sufficient to establish a lymphoid origin for Hodgkin's Reed-Sternberg cells?

Most Hodgkin's mononuclear cells and Reed-Sternberg (H-RS) cells are characterized by the expression of the antigen CD30, but not of T or B cell markers. A few H-RS cells, however, may express a limited number of T or B cell markers. Whether this expression is sufficient to allow the conclusion that H-RS cells are derived from T and/or B cells has been debated vigorously. The present study examined whether CD30 and aberrant T and B cell markers are expressed in cell lines that are well documented as being derived from the granulocyte/monocyte/histiocyte lineage. These cells included HL-60, KG-1, ML-1, THP-1, and U-937. Four other cell lines derived from patients with leukemias/lymphomas of monocytic or granulocytic origins also were studied. These cells included BV173, CML-Brown, CTV-2, and SU-DHL-1. If aberrant expression is detected, by analogy one may expect that rare T or B cell marker expression may occur in H-RS cells, because abundant evidence has indicated that H-RS cells may be related to cells in histiocyte lineage. In all nine of the cell lines studied, it was confirmed that numerous monocyte/granulocyte markers were expressed. The marker expression was enhanced after cells were induced to differentiate with phorbol ester (TPA) and tumor necrosis factor (TNF). It was noted that several T and B cell markers also were expressed by these cells. Unlike the expression of monocyte/granulocyte markers, the expression of T or B cell markers was not affected, or only minimally affected, by treatment of the cells with TPA or TNF. Five of the cell lines (BV173, CML-Brown, CTV-2, SU-DHL-1, and THP-1) were shown to be CD30-positive. In CTV-2 and BV173, the expression of CD30 was greatly increased after induction with phorbol ester or TNF. Based on these studies, the following conclusions were reached: 1) The expression of aberrant B or T cell markers is not an uncommon finding in granulocyte/monocyte/histiocyte-related neoplastic cells. 2) The expression of granulocyte/monocyte markers in these cells is related to the state of cell differentiation, whereas the expression of T or B cell markers is not. 3) CD30 is not necessarily a proliferation-related antigen, and its expression is not a sole property of T or B cells, but can be present in granulocyte/monocyte/histiocyte-related cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

CD95 ligand is expressed in Reed–Sternberg cells of Hodgkin's disease

Reed-Sternberg (RS) cells and their mononuclear variants, Hodgkin's (H) cells, are considered to be the neoplastic cells of Hodgkin's disease (HD). The cellular origin of H-RS cells remains the subject of considerable controversy, although most recent papers have claimed that H-RS cells are of B cell origin. Recently, however, it has been reported that some H-RS cells express granzyme B, as observed in cytotoxic T cells and/or natural killer cells, which also express CD95 ligand (FasL/APO-1L). In the present study, the expression of CD95L and granzyme B in H-RS cells of HD was investigated. CD95L was detected in H-RS cells in five of nine HD cases (one case of lymphocyte-rich classical HD, two of these cases of nodular sclerosis type, and two of four cases of mixed cellularity type). All three examined HD cell lines expressed CD95L in the cytoplasm, although cell surface expression was seen only in L428 cells. Three HD cases expressed both CD95L and granzyme B. It was concluded that CD95L is frequently expressed in H-RS cells, which is one of their notable characteristics; albeit it seems to be irrespective of cell lineage.     

Expression of p55 (Tac) interleukin-2 receptor (IL-2R), but not p75 IL-2R, in cultured H-RS cells and H-RS cells in tissues.

The authors studied the secretion of interleukin-2 (IL-2), the expression of interleukin-2 receptors (IL-2R; p55/Tac and p75), and the response to exogenous IL-2 by cultured Hodgkin's Reed-Sternberg cells (cell lines HDLM-1, HDLM-1d, and KM-H2) and T cells (H9, HuT78, HuT102, MOLT-4, and MT-2). All of these cells did not produce IL-2 or produced it in undetectable amounts, and their growth was not affected by the addition of anti-IL-2 or anti-IL-2R antibodies. This indicates that H-RS cells in long-term culture, as well as T cells, can grow independently of IL-2. The three H-RS cell lines, as well as two of the T-cell lines (HuT102 and MT-2), expressed Tac, whereas the other three T-cell lines were Tac negative. Expression of p75 was noted in the two Tac-positive T-cell lines, but not in cultured H-RS cells. The expression of Tac and p75 in HuT102 and MT-2 cells correlated well with their capacity to proliferate on treatment with exogenous IL-2. On IL-2 treatment, nucleic-acid uptake in Tac/p75-positive T cells increased approximately four- to sixfold, whereas the Tac/p75-negative T cells did not show increased proliferation. Unlike the T cells, the Tac-positive H-RS cells did not respond to IL-2. The lack of a proliferative response to IL-2 appears to be related to the absence of p75 in H-RS cells. A similar pattern (Tac positivity and p75 negativity) was noted in H-RS cells in lymph nodes involved by Hodgkin's disease. Thus the exogenous IL-2 released by surrounding T lymphocytes may not cause the proliferative activity of H-RS cells because of the lack of high-affinity IL-2 receptors in the latter cells. In contrast to H-RS cells in culture, H-RS cells in tissues were stained by a specific anti-IL-2 monoclonal antibody. This indicates that the expression of IL-2 or an IL-2-like substance by H-RS cells in tissues may be responsible, in part, for the great increase in the number of reactive T lymphocytes in tissues involved by Hodgkin's disease.     

Detection of Epstein-Barr virus in Hodgkin-Reed-Sternberg cells : no evidence for the persistence of integrated viral fragments inLatent membrane protein-1 (LMP-1)-negative classical Hodgkin's disease

Classical Hodgkin's disease (HD) is associated with Epstein-Barr virus (EBV) infection. Although in developing countries EBV can be demonstrated in Hodgkin-Reed-Sternberg (H-RS) cells in up to 95% of HD cases, in industrialized countries only about 50% of HD cases are associated with EBV. An open question remains whether EBV in the EBV-negative cases has escaped detection by standard screening procedures due to deletions in the viral genome associated with integration of viral fragments into the host cell genome. We, among others, recently described this phenomenon in Burkitt's lymphoma cells. To investigate whether H-RS cells in latent membrane protein-1 (LMP-1)-negative HD cases harbor fragments of the EBV genome, we combined fluorescence in situ hybridization (FISH) using a set of six overlapping DNA probes spanning the whole EBV genome with immunophenotyping of fresh frozen lymphoma sections. Results in the eight cases analyzed were as follows: in three LMP-1-positive cases, FISH analysis yielded specific signals for each EBV DNA probe in H-RS cells, which had been identified by morphology and CD30 staining. In contrast, none of the EBV DNA probes hybridized to the H-RS cells in the five LMP-1-negative cases. Thus, there is no evidence for the presence of fragments of the viral genome integrated into the host cell genome in the LMP-1-negative cases. Furthermore, in the LMP-1-positive cases analyzed, no large deletions in the viral genome were detected. These results show that, in classical HD, LMP-1-negative cases do not harbor EBV DNA within the H-RS cells. Whether, in these cases, a still unknown virus contributes to the transformation and maintenance of the malignant phenotype remains to be established.     

Lack of c-kit (CD117) expression in CD30+ lymphomas and lymphomatoid papulosis.

c-Kit receptor (CD117) is expressed by erythroid, megakaryocytic, and myeloid precursors and mature mast cells and has been reported to be expressed in CD30+ lymphomas such as Hodgkin's disease and anaplastic large-cell lymphoma. Imatinib mesylate, a well-established inhibitor of bcr-abl tyrosine kinase, and currently used for the treatment of patients with chronic myeloid leukemia, also inhibits c-kit receptor kinase activity. In view of the possible use of imatinib as experimental therapy for patients with c-kit-positive tumors, we assessed c-kit expression in CD30+ cell lines and lymphomas. The cell lines were assessed using multiple methods (RT-PCR, flow cytometry, and Western blot). c-Kit expression was also immunohistochemically assessed in 168 CD30+ lymphomas including 87 classical Hodgkin's disease, 63 anaplastic large-cell lymphoma, and 15 cutaneous anaplastic large-cell lymphoma. We also studied 18 cases of lymphomatoid papulosis, a CD30+ lesion closely related to cutaneous anaplastic large-cell lymphoma. Neither c-kit mRNA nor protein was detected in any of the cell lines assessed. Furthermore, treatment with imatinib did not inhibit proliferation of cell lines in vitro. Using immunohistochemistry, only one of 183 (0.5%) lesions was positive for c-kit, the positive case being an ALK-negative anaplastic large-cell lymphoma. Our data demonstrate that expression of c-kit receptor is exceedingly rare among CD30+ lymphomas and lymphomatoid papulosis, suggesting that c-kit receptor is unlikely to be an appropriate target for therapeutic options such as imatinib in patients with these tumors.     

Phenotypes and phorbol ester-induced differentiation of human histiocytic lymphoma cell lines (U-937 and SU-DHL-1) and Reed-Sternberg cells.

Hodgkin's mononuclear cells, Reed-Sternberg (H-RS) cells, and U-937 and SU-DHL-1 histocytic cell lines were induced to differentiate by phorbol ester in cultures. The phenotypes of cells were determined by a panel of antibodies specific for monocytes, histiocytes, and interdigitating reticulum cells. Before induction, SU-DHL-1 cells and H-RS cells expressed similar markers, such as HeFi-1, 2H9, 1A2, and 1E9. In addition, SU-DHL-1 cells were also stained by Tac and Leu M5. Other monocyte markers, including OK M1, Co Mo2, BRL Mo1, BRL Mo2, and Leu M3 were consistently negative in both types of cells. After induction, SU-DHL-1 cells conserved the same phenotype, but H-RS cells became negative for HeFi-1, 1A2, and 2H9. The U-937 cells expressed Leu M1 and Co Mo2 and became positive for Leu M5, OK M1, Co Mo2, BRL Mo2, 2H9, and 1E9 after phorbol ester induction. The U-937 cells did not express HeFi-1 or 1A2. The marker expression of H-RS cells, SU-DHL-1 cells, and U-937 cells were compared with those of histiocytes or interdigitating reticulum cells in lymphoid tissues and with neoplastic cells in true histiocytic lymphoma and malignant histiocytosis. It is concluded that SU-DHL-1, U-937, and H-RS cells are derived from or most closely related to fixed histiocytes, free histiocytes, and interdigitating reticulum cells, respectively. Our study further confirms the diagnosis of SU-DHL-1 as true histiocytic lymphoma but reveals that U-937 is a case of malignant histiocytosis rather than the previously diagnosed histiocytic lymphoma. The phenotypes and induction properties of SU-DHL-1 cells are quite different from those of U-937 cells, which suggests that true histiocytic lymphoma and malignant histiocytosis are two distinct disease entities.     

Lack of effect of colony-stimulating factors, interleukins, interferons, and tumor necrosis factor on the growth and differentiation of cultured Reed-Sternberg cells. Comparison with effects of phorbol ester and retinoic acid.

The neoplastic Hodgkin's Reed-Sternberg (H-RS) cells in Hodgkin's disease are surrounded in vivo by abundant reactive cells, the function of which may be attributed in part to their elaboration of various cytokines. Thus, a study of the interaction of H-RS cells with exogenous cytokines may provide information as to the mechanism of the clinical and histopathologic changes observed in Hodgkin's disease. This study examined the effect of various cytokines, and of phorbol ester (TPA) and retinoic acid, on the differentiation and proliferation of cultured H-RS cells (cell lines HDLM-1 and KM-H2). In addition, it was determined whether these cells were able to secrete cytokines after being treated with exogenous cytokines. The cytokines used included various types of interleukins (1, 2, and 3), colony-stimulating factors (GM, G, and M), interferons (alpha, beta, and gamma), and tumor necrosis factor (alpha). It was found that these cytokines, used alone or in combination, were not effective in modulating the proliferation and differentiation of cells, or the production of cytokines, in cultured H-RS cells. In contrast, this study revealed that retinoic acid can potentiate TPA-induced growth inhibition in cultured H-RS cells. Retinoic acid, when used alone, exhibited a minimal effect on cell differentiation. No synergistic effects of cytokines and retinoic acid on H-RS cells were observed. The failure of cultured H-RS cells to respond to exogenous cytokines suggests that, during the course of neoplastic transformation, of progression of disease, or of establishment of the cells in culture, H-RS cells lose their dependence on cytokines, although they retain the capacity to produce various cytokines.     

Cultured Reed-Sternberg cells HDLM-1 and KM-H2 can be induced to become histiocytelike cells. H-RS cells are not derived from lymphocytes.

Two Hodgkin's Reed-Sternberg cell (H-RS) lines, HDLM-1 and KM-H2, have phenotypes and functional properties very similar to those of H-RS cells in tissues. These two types of cells were induced to differentiate with a combination of phorbol ester, retinoic acid, and extracellular matrix. The induced cells displayed the morphology of histiocytes or histiocytelike cells, with a small, round or oval, eccentric nucleus and abundant cytoplasm. In ultrastructural studies, many cytoplasmic projections and rugae were observed. These induced cells exhibited abundant cytoplasmic lysosomal enzymes, such as esterase, acid phosphatase, alpha 1-antitrypsin, or lysozyme. The histiocytic nature of these induced cells was further confirmed by the increased expression of many monocyte/histiocyte markers, including CD11b, CD11c, CD13, CD14, CD15, CD33, CD68, Mac387, and 1E9. In functional tests, the induced cells were shown to produce interleukin-1, tumor necrosis factor, macrophage colony-stimulating factor, and/or prostaglandin E2. Phagocytosis was detected in less than 5% to 10% of the cells when Candida albicans was added to cultures. The results strongly suggest that H-RS cells are related to cells of histiocyte lineage.