The molecular mechanisms underlying the pathogenesis of the malignant Hodgkin's/Reed-Sternberg (HRS) cells of Hodgkin's lymphoma (HL) are largely unknown. This study investigates the contribution of phosphatidyl-inositide 3 kinase (PI3-kinase) and demonstrates that Akt, a substrate of PI3-kinase, is constitutively activated in HL-derived cell lines. Several downstream effectors of Akt signalling, including glycogen synthase kinase 3 (GSK-3) alpha and beta and mTOR substrates 4E-BP1 and p70 S6 kinase, were also phosphorylated in HL cells. The mTOR inhibitor, rapamycin, inhibited phosphorylation of these proteins. Furthermore, LY294002 inhibited phosphorylation of p70 S6 kinase and 4E-BP1, suggesting that the phosphorylation of p70 S6 kinase and 4E-BP1 in HL cells is PI3-kinase dependent. Importantly, HRS cells of primary tumour samples not only expressed high levels of activated Akt but also displayed phosphorylation of downstream targets of Akt activation including GSK-3, 4E-BP1, and p70 S6 Kinase. Inhibition of PI3-kinase and mTOR showed only modest effects on cell survival at the lower serum concentrations. However, rapamycin and doxorubicin acted synergistically to reduce HL cell survival. A combination of rapamycin and chemotherapy should be investigated in the treatment of HL.

Fas ligand ͉ apoptosis ͉ small interfering RNA

IntroductionClassical Hodgkin lymphoma (HL) is derived from germinal center (GC) B cells and is characterized by malignant Hodgkin/ReedSternberg (HRS) cells in a background of nonmalignant "reactive" cells. 1 The Epstein-Barr virus (EBV) is present in HRS cells in approximately half of all patients with HL, in whom it expresses a restricted set of virus-latent genes; these include the major EBV oncogene latent membrane protein-1 (LMP1). 2 By mimicking a constitutively active CD40 receptor, LMP1 activates signaling pathways, such as NF-B, which enhance B-cell survival and are essential for EBV-induced transformation. 3,4 Polycomb group (PcG) genes are necessary for the maintenance and renewal of embryonic and adult stem cells, embryogenesis, and cell cycle regulation. 5,6 Two polycomb repressive complexes, PRC1 and PRC2, are required for the initiation and maintenance of gene silencing, respectively. 7-9 Bmi-1/PCGF4 (B lymphoma Mo-MLVinsertion region/ polycomb group ring finger 4) is a component of PRC1. 10,11 Bmi-1 induces lymphoid proliferation and the development of lymphomas in transgenic mice. 12-15 Bmi-1 is highly expressed in high-grade large B-cell lymphomas, mantle cell lymphoma, and nonlymphoid malignancies, such as colorectal cancer and non-small cell lung cancer. [16][17][18] Although Bmi-1 is highly expressed in HRS cells, 19-21 its regulation and contribution to the pathogenesis of HL are unknown. We show here that Bmi-1 is a transcriptional target of LMP1, that the expression of Bmi-1 promotes the survival of HL cells, and that Bmi-1 induces transcriptional changes in HL cells that include the down-regulation of the ataxia telangiectasia mutated (ATM) gene. Materials and methodsThe work undertaken in the study received ethical approval from the South Birmingham Research Ethics Committee (LREC no.0844). Cell lines and tissue samplesEBV-negative cell lines from mixed cellularity (MC) HL (KM-H2) and nodular sclerosis (NS) HL (L428) 22 were maintained in RPMI 1640 supplemented with 10% fetal calf serum, 2 mM L-glutamine, and 1% penicillin-streptomycin solution (Sigma-Aldrich, Poole, United Kingdom). An EBV-positive cell line from a patient with NS HL (L591) and an EBV-negative clone of this line (L591-SD3) were grown in the same way. 23 Paraffin-embedded HL biopsies were obtained from Queen Elizabeth Hospital, (Birmingham, United Kingdom), and their EBV status was determined by immunohistochemical staining for LMP1. 24 Transient transfection of HL linesTransfection of HL-derived cell lines was performed using the nucleofector unit supplied by Amaxa GmbH and described by Schakowski et al. 25 In brief, 2 ϫ 10 6 KM-H2 cells and 4 ϫ 10 6 other HL cell lines were pelleted at 1500g for 9 minutes. After resuspension in 100 L freshly prepared nucleofector solution kit T (catalog no. VCA-1002; Amaxa, Cologne, Germany), 2 g plasmid DNA was added to KM-H2 and 4 g was added to the other HL cells. Subsequently, KM-H2 cells were pulsed using program T-01, and the other HL cells were pulsed using program U-09; cells ...

Protection from death receptor (DR)-mediated apoptosis has been proposed as an important step in the development of malignancy, enabling tumour cells not only to survive and escape antitumour immune responses, but also to develop resistance to chemotherapy or other cancer treatments. An important regulator of DR-induced death is the cellular FADD-like IL-1beta-converting enzyme inhibitory protein (c-FLIP) which, when overexpressed, can protect tumour cells from apoptosis. This review focuses on the role of c-FLIP as a tumour progression factor, with particular emphasis on recent work from the authors' laboratory concerning the contribution of c-FLIP to the pathogenesis of Hodgkin's lymphoma. The possibility of targeting c-FLIP as an approach to the treatment of cancer and, in particular, Hodgkin's lymphoma is discussed.

We have used Townsend scores from postcode data to compare levels of material deprivation and Epstein–Barr virus (EBV)-positivity for 223 patients diagnosed with Hodgkin's disease (HD) in the period 1981–1997. The presence of EBV in HD tumours was determined using in situ hybridization to target the abundantly expressed EBV early RNAs. EBV was detected in the malignant Hodgkin and Reed–Sternberg cells in 47/223 HD cases (21%). There was found to be a tendency for higher Townsend scores (indicative of higher levels of material deprivation) in EBV-positive HD patients, but this association was not statistically significant. When various subgroups of patients from the study were examined separately the indication of higher Townsend scores in EBV-positive patients was found to be more marked for patients with mixed cellularity disease ( P = 0.09) and for females ( P = 0.03). The results of this study suggest that differences in the level of material deprivation are important in determining the likelihood of EBV-positive HD in the UK, particularly for certain subgroups of patients. It is not known what specific socioeconomic factors are responsible for these differences, although alterations in the timing or rate of primary EBV infection, or decline in the level of EBV-specific immunity, may be important. © 1999 Cancer Research Campaign

Hodgkin's lymphoma is one of the most common lymphoid cancers, particularly among young adults. Although there have been dramatic improvements in the treatment of Hodgkin's lymphoma, leading to high cure rates in some groups, current combination chemotherapy regimes are associated with significant secondary complications in long-term survivors. Furthermore, although a proportion of patients with Hodgkin's lymphoma will be cured, there still remains a significant rate of relapse and also a smaller proportion of poor responders who will go on to die of their disease. Therefore, developments in the treatment of Hodgkin's lymphoma must be directed at improving cure rates and reducing the burden of secondary complications. In recent years, the underlying pathogenesis of Hodgkin's lymphoma has become better understood. In particular, it is emerging that a key pathogenic event in Hodgkin's lymphoma is protection from Fas-induced cell death. Recent studies by the authors' group, and others, have demonstrated that this is, in part, due to the expression by Hodgkin/Reed-Sternberg cells of the cellular Fas-associated death domain-like IL-1 converting enzyme (FLICE)-like inhibitory protein molecule, a potent inhibitor of Fas-induced death. In this review, the role of cellular FLICE-like inhibitory protein in the pathogenesis of Hodgkin's lymphoma will be explored and also the possibility of targeting this molecule in order to provide an alternative and potentially safe approach to the treatment of Hodgkin's lymphoma will be investigated.

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