Stimulation of the hypoxia pathway modulates chemotherapy resistance in Hodgkin's lymphoma cells.

Hodgkin's lymphoma (HL) is a malignant disease of the lymphatic system. The therapy has been improved during the last decades but there are still patients who cannot be cured, and the therapy is associated with several adverse late effects. Therefore, we asked which genes might be involved in the chemotherapy resistance of HL cells. We observed that HL cells became more resistant against cisplatin after treatment with cobalt chloride. Therefore, we analyzed which genes were differentially expressed between cells incubated in medium with or without cobalt chloride. We found several genes which were up- or downregulated in the presence of cobalt chloride and might be involved in the modulation of chemotherapy resistance. Cobalt chloride is a hypoxia-mimetic agent. Therefore, we tested chemo-resistance and gene expression of HL cells under hypoxic conditions and confirmed the results from the cobalt chloride experiments. Taken together, activation of the hypoxia pathway led to altered gene expression and drug resistance of HL cells. Differentially expressed genes might be interesting targets for the development of future treatment strategies against drug-resistant HL.

Preferentially Expressed Antigen in Melanoma (PRAME) and the PRAME Family of Leucine-Rich Repeat Proteins.

Preferentially expressed antigen in melanoma (PRAME) is the best characterized member of the PRAME family of leucine-rich repeat (LRR) proteins. Mammalian genomes contain multiple members of the PRAME family whereas in other vertebrate genomes only one PRAME-like LRR protein was identified. PRAME is a cancer/testis antigen that is expressed at very low levels in normal adult tissues except testis but at high levels in a variety of cancer cells. In contrast to most other cancer/testis antigens, PRAME is expressed not only in solid tumors but also in leukemia cells. Expression of PRAME and other members of the PRAME family is regulated epigenetically. PRAME interacts with varying pathways that might be directly involved in the malignant phenotype of cancer cells. For instance, PRAME is able to dominantly repress retinoic acid signaling in these cells. On the other hand, PRAME-derived peptides can be recognized as epitopes by cytotoxic T cells and PRAME represents an attractive target for immunological treatment strategies.

Prognostic Biomarkers for Hodgkin Lymphoma.

The prognosis of patients with classical Hodgkin lymphoma following chemo- and radiotherapy has been excellent during the last 4 decades. However, the development of secondary malignancies is of major concern. Therefore, the reduction of radiotherapy application is a major objective of ongoing clinical trials. De-escalation of treatment may increase the risk of relapses and thus may lead to reappearance of prognostic factors. Prognostic biomarkers might help to identify patients who are at increased risk of relapse. This review summarizes the current knowledge about potential prognostic biomarkers for patients with classical Hodgkin lymphoma.

Expression of dual-specificity phosphatase 5 pseudogene 1 (DUSP5P1) in tumor cells.

Sequencing of individual clones from a newly established cDNA library from the chemoresistant Hodgkin's lymphoma cell line L-1236 led to the isolation of a cDNA clone corresponding to a short sequence from chromosome 1. Reverse transcriptase-polymerase chain reaction indicated high expression of this sequence in Hodgkin's lymphoma derived cell lines but not in normal blood cells. Further characterization of this sequence and the surrounding genomic DNA revealed that this sequence is part of a human endogenous retrovirus locus. The sequence of this endogenous retrovirus is interrupted by a pseudogene of the dual specificity phosphatase 5 (DUSP5). Reverse transcriptase-polymerase chain reaction revealed high expression of this pseudogene (DUSP5P1) in HL cell lines but not in normal blood cells or Epstein-Barr virus-immortalized B cells. Cells from other tumor types (Burkitt's lymphoma, leukemia, neuroblastoma, Ewing sarcoma) also showed a higher DUSP5P1/DUSP5 ratio than normal cells. Furthermore, we observed that higher expression of DUSP5 in relation to DUSP5P1 correlated with the expression of the pro-apoptotic factor B cell leukemia/lymphoma 2-like 11 (BCL2L11) in peripheral blood cells and HL cells. Knock-down of DUSP5 in HL cells resulted in down-regulation of BCL2L11. Thus, the DUSP5/DUSP5P1 system could be responsible for regulation of BCL2L11 leading to inhibition of apoptosis in these tumor cells.

Impact of O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation and MGMT expression on dacarbazine resistance of Hodgkin's lymphoma cells.

We analyzed the methylation status of the O6-methylguanine-DNA methyltransferase (MGMT) promoter and mRNA expression in HL cells and assessed the response of these cells to dacarbazine. Expression of MGMT correlated with the presence of non-methylated promoters and cell lines with non-methylated promoters showed increased resistance against dacarbazine. KM-H2 cells expressed fusion transcripts between MGMT and proline-rich coiled-coil 2B (PRRC2B) but no wild type MGMT transcripts. Dacarbazine sensitivity suggested that fusion transcripts are translated into a protein with reduced functionality. MGMT promoter methylation predicts dacarbazine sensitivity of HL cells and it might be interesting to analyze this factor in HL patients.

Expression and Regulation of the Endogenous Retrovirus 3 in Hodgkin's Lymphoma Cells.

Human endogenous retroviruses (ERV) are an integral part of our genome. Expression of ERV is usually switched off but reactivation of ERV has been observed in varying human diseases including cancer. Recently, reactivation of ERV associated promoters in Hodgkin's lymphoma (HL) cells has been described. Despite relatively good prognosis, not all patients with HL can be cured with the established therapy and this therapy is associated with severe late side effects. Therefore, new targets are required for the development of future treatment strategies. Reactivated ERV might represent such target structures. Therefore, we asked which ERV loci are expressed in HL cells. Using DNA microarray analysis, we found no evidence for a general activation of ERV transcription in HL cells. In contrast, we observed down-regulation of ERV3, an ERV with potential tumor suppressor function, in HL cells in comparison to normal blood cells. Interestingly, ERV3 was also differentially expressed in published DNA microarray data from resting versus cycling B cells. Treatment of HL cells with the histone deacetylase inhibitor vorinostat strongly up-regulated ERV3 expression. In addition, we observed up-regulation in HL cells after treatment with hypoxia-mimetic cobalt(II) chloride. Like vorinostat, cobalt(II) chloride inhibited cell growth of HL cells. Our results suggest that cell cycle inhibition of HL cells is accompanied by up-regulation of ERV3.

Knock-down of PRAME increases retinoic acid signaling and cytotoxic drug sensitivity of Hodgkin lymphoma cells.

The prognosis for patients with Hodgkin lymphoma (HL) has improved in recent decades. On the other hand, not all patients can be cured with the currently established therapy regimes and this therapy is associated with several adverse late effects. Therefore it is necessary to develop new therapy strategies. After treatment of L-540 HL cells with 5'-azacytidine (5AC), we observed increased expression of the preferentially expressed antigen in melanoma (PRAME). In addition, we detected an increased resistance of 5AC-treated cells against cytotoxic drugs. We analyzed the influence of PRAME on cell survival of HL cells by knocking down PRAME in the chemotherapy resistant cell line L-428, a cell line that express PRAME at a high level. After knock-down of PRAME using vector based RNA interference we observed increased sensitivity for cisplatin, etoposide and retinoic acid. DNA microarray analysis of HL cells after PRAME knock-down indicated regulation of several genes including down-regulation of known anti-apoptotic factors. Increased retinoic acid signaling in these cells was revealed by increased expression of the retinoic acid metabolizing cytochrome P450 (CYP26B1), a transcriptional target of retinoic acid signaling. Our data suggest that PRAME inhibits retinoic acid signaling in HL cells and that the knock-down of PRAME might be an interesting option for the development of new therapy strategies for patients with chemo-resistant HL.

Curcuma Contra Cancer? Curcumin and Hodgkin's Lymphoma.

Curcumin, a phytochemical isolated from curcuma plants which are used as coloring ingredient for the preparation of curry powder, has several activities which suggest that it might be an interesting drug for the treatment or prevention of cancer. Curcumin targets different pathways which are involved in the malignant phenotype of tumor cells, including the nuclear factor kappa B (NFKB) pathway. This pathway is deregulated in multiple tumor entities, including Hodgkin's lymphoma (HL). Indeed, curcumin can inhibit growth of HL cell lines and increases the sensitivity of these cells for cisplatin. In this review we summarize curcumin activities with special focus on possible activities against HL cells.

Hodgkin's lymphoma RNA-transfected dendritic cells induce cancer/testis antigen-specific immune responses.

Cytotoxic T lymphocytes (CTL) can kill Hodgkin's lymphoma (HL) cells, and CTL have been used for the treatment of Epstein-Barr virus (EBV)-positive HL. For patients with EBV-negative HL, this strategy cannot be employed and alternative target structures have to be defined. In order to establish a system for the stimulation of HL-reactive T cells, we used dendritic cells (DC) as antigen-presenting cells for autologous T cells and transfected these DC with RNA from established HL cell lines. After stimulation of peripheral blood mononuclear cells (PBMC) with RNA-transfected DC, we analyzed the reactivity of primed PBMC by interferon gamma enzyme-linked immunospot. Our results suggest the presence of antigens with expression in HL cell lines and recognition of these antigens in combination with DC-derived human leukocyte antigen molecules. By the analysis of Gene Expression Omnibus microarray data sets from HL cell lines and primary HL samples in comparison with testis and other normal tissues, we identified HL-associated cancer testis antigens (CTA) including the preferentially expressed antigen in melanoma (PRAME). After stimulation of PBMC with RNA-transfected DC, we detected PRAME-reactive T cells. PRAME and other HL-associated CTA might be targets for HL-specific immune therapy or for the monitoring of HL-directed immune responses.

Histone deacetylase inhibition restores cisplatin sensitivity of Hodgkin's lymphoma cells.

Increasing evidence suggest that epigenetic mechanisms (e.g. histone modification by histone deacetylases) play a major role in the pathogenesis of Hodgkin's lymphoma (HL). We treated HL cell lines with the histone deacetylase inhibitor vorinostat and investigated the gene expression profile of these cells by using DNA microarrays. Vorinostat inhibited cell proliferation and induced chances in the gene expression profile of HL cells, including down regulation of interleukin-26 and CD30. Vorinostat also increased sensitivity for cisplatin. Our data suggest that the combination of vorinostat and chemotherapy might be an interesting option for patients with chemoresistant HL.