Inactivation of the putative ubiquitin-E3 ligase PDLIM2 in classical Hodgkin and anaplastic large cell lymphoma.

Apart from its unique histopathological appearance with rare tumor cells embedded in an inflammatory background of bystander cells, classical Hodgkin lymphoma (cHL) is characterized by an unusual activation of a broad range of signaling pathways involved in cellular activation. This includes constitutive high-level activity of nuclear factor-κB (NF-κB), Janus kinase/signal transducer and activator of transcription (JAK/STAT), activator protein-1 (AP-1) and interferon regulatory factor (IRF) transcription factors (TFs) that are physiologically only transiently activated. Here, we demonstrate that inactivation of the putative ubiquitin E3-ligase PDLIM2 contributes to this TF activation. PDLIM2 expression is lost at the mRNA and protein levels in the majority of cHL cell lines and Hodgkin and Reed-Sternberg (HRS) cells of nearly all cHL primary samples. This loss is associated with PDLIM2 genomic alterations, promoter methylation and altered splicing. Reconstitution of PDLIM2 in HRS cell lines inhibits proliferation, blocks NF-κB transcriptional activity and contributes to cHL-specific gene expression. In non-Hodgkin B-cell lines, small interfering RNA-mediated PDLIM2 knockdown results in superactivation of TFs NF-κB and AP-1 following phorbol 12-myristate 13-acetate (PMA) stimulation. Furthermore, expression of PDLIM2 is lost in anaplastic large cell lymphoma (ALCL) that shares key biological aspects with cHL. We conclude that inactivation of PDLIM2 is a recurrent finding in cHL and ALCL, promotes activation of inflammatory signaling pathways and thereby contributes to their pathogenesis.

The tumour suppressor p53 is frequently nonfunctional in Sézary syndrome.

BACKGROUND: Primary cutaneous T-cell lymphomas (CTCLs) are a heterogeneous group with Sézary syndrome (SS) as one of the most aggressive variants. Recently, we identified a loss of E2A as a recurrent event in SS, which enhanced proliferation via upregulation of the proto-oncogene MYC. MYC-induced transformation usually requires deleterious alterations of key apoptotic genes including p53; however, p53 functionality and mutation status in SS are unclear. OBJECTIVES: We investigated functionality of p53 signalling by pharmacological treatment with the MDM2 antagonist nutlin-3, which might result in p53 activation. Furthermore, we analysed the TP53 mutation status in CTCL cell lines and highly purified tumour cells from patients with SS by mRNA and DNA sequencing. METHODS: We analysed the apoptosis induction due to nutlin-3 treatment in various SS cell lines and primary patient samples by annexin V/propidium iodide staining. Induction of p53 target genes was analysed by immunoblotting, and TP53 was sequenced at the mRNA and DNA level. RESULTS: We identified various TP53 mutations and an impaired p53 signalling in the vast majority of the investigated cell lines and primary SS cells. CONCLUSIONS: In accordance with the importance of MYC deregulation in SS, p53 signalling is frequently nonfunctional in SS. However, although most likely ineffective as exclusive treatment in SS, it remains possible that pharmacological p53 activation could be beneficial in combination with other approaches including classical chemotherapeutics.

Serum/glucocorticoid-regulated kinase 1 (SGK1) is a prominent target gene of the transcriptional response to cytokines in multiple myeloma and supports the growth of myeloma cells.

Multiple myeloma (MM) is a paradigm for a malignant disease that exploits external stimuli of the microenvironment for growth and survival. A thorough understanding of the complex interactions between malignant plasma cells and their surrounding requires a detailed analysis of the transcriptional response of myeloma cells to environmental signals. We determined the changes in gene expression induced by interleukin (IL)-6, tumor necrosis factor-α, IL-21 or co-culture with bone marrow stromal cells in myeloma cell lines. Among a limited set of genes that were consistently activated in response to growth factors, a prominent transcriptional target of cytokine-induced signaling in myeloma cells was the gene encoding the serine/threonine kinase serum/glucocorticoid-regulated kinase 1 (SGK1), which is a down-stream effector of PI3-kinase. We could demonstrate a rapid, strong and sustained induction of SGK1 in the cell lines INA-6, ANBL-6, IH-1, OH-2 and MM.1S as well as in primary myeloma cells. Pharmacologic inhibition of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway abolished STAT3 phosphorylation and SGK1 induction. In addition, small hairpin RNA (shRNA)-mediated knock-down of STAT3 reduced basal and induced SGK1 levels. Furthermore, downregulation of SGK1 by shRNAs resulted in decreased proliferation of myeloma cell lines and reduced cell numbers. On the molecular level, this was reflected by the induction of cell cycle inhibitory genes, for example, CDKNA1/p21, whereas positively acting factors such as CDK6 and RBL2/p130 were downregulated. Our results indicate that SGK1 is a highly cytokine-responsive gene in myeloma cells promoting their malignant growth.

High-level expression of Mastermind-like 2 contributes to aberrant activation of the NOTCH signaling pathway in human lymphomas.

Inappropriate activation of the NOTCH signaling pathway, for example, by activating mutations, contributes to the pathogenesis of various human malignancies. Here, we demonstrate that aberrant expression of an essential NOTCH coactivator of the Mastermind-like (MAML) family provides an alternative mechanism to activate NOTCH signaling in human lymphoma cells. We detected high-level MAML2 expression in several B cell-derived lymphoma types, including classical Hodgkin lymphoma (cHL) cells, relative to normal B cells. Inhibition of MAML-protein activity by a dominant negative form of MAML or by small hairpin RNAs targeting MAML2 in cHL cells resulted in downregulation of the NOTCH target genes HES7 and HEY1, which we identified as overexpressed in cHL cells, and in reduced proliferation. Furthermore, a NOTCH gene-expression signature in cHL cells confirmed their cell-autonomous NOTCH activity. Finally, in line with the essential role of MAML proteins for assembly and activity of the NOTCH transcriptional complex (NTC), we show that MAML-derived small-peptide constructs block NOTCH activity and disrupt NTC formation in vitro. These data strongly suggest direct targeting of the NTC as treatment strategy for NOTCH-dependent malignancies.

[The molecular pathogenesis of classical Hodgkin lymphoma]. / Die molekulare Pathogenese des klassischen Hodgkin-Lymphoms.

Despite the fact that classical Hodgkin lymphoma (HL) has been described more than 170 years ago, only over the last 15 years significant advances regarding its molecular pathogenesis have been achieved. The lack of a specific lineage profile in combination with the low number of the malignant mononuclear Hodgkin- and multinucleated Reed-Sternberg- (HRS-) cells in the affected lymph nodes prevented for a long time both the identification of its cell of origin and of genomic and molecular defects. The development of methods for the analysis of micromanipulated single cells made it possible to demonstrate a B cell origin of HRS cells. However, it has become clear that the normal B cell-specific gene expression program in HRS cells is disrupted by various molecular lesions. Furthermore, molecular and genomic defects of various signaling pathways could be identified in HRS cells, including the NF-kappaB, JAK/STAT and MAPK-AP-1 signaling pathways, which protect HRS cells from apoptotic cell death. Despite significant advances in the treatment of HL, the considerable long term toxicity of conventional therapies requires the development of new non-genotoxic therapeutic strategies. Therefore, it will be a central aim to develop new treatment strategies based on these insights into HL pathogenesis.

Pharmacologic activation of p53-dependent and p53-independent apoptotic pathways in Hodgkin/Reed-Sternberg cells.

The status of the p53 pathway in classical Hodgkin lymphoma (cHL) remains unclear, and a lack of proven TP53 mutations contrasts with often high expression levels of p53 protein. In this study, we demonstrate that pharmacologic activation of the p53 pathway with the murine double minute 2 (MDM2) antagonist nutlin-3 in Hodgkin lymphoma-derived cell lines leads to effective apoptosis induction and sensitizes the cells to other anticancer drugs. Cells with mutant p53 are resistant to nutlin-3, but sensitive to geldanamycin, a pharmacologic inhibitor of heat shock 90 kDa protein (HSP90), indicating that HSP90 inhibition can induce apoptosis in a p53-independent manner. Conversely, cells with defects in the HSP90/nuclear factor-kappa B pathway expressing wild-type p53 are more resistant to geldanamycin, but still sensitive to nutlin-3. Our results suggest that selective activation of p53 by MDM2 antagonists as a single agent or in combination with conventional chemotherapeutics and/or inhibitors of p53-independent survival pathways may offer effective treatment options for patients with cHL. Importantly, because nutlins and HSP90 inhibitors are non-genotoxic agents, their use might offer a means to reduce the genotoxic burden of current chemotherapeutic regimens.

Loss of the tissue-specific proapoptotic BH3-only protein Nbk/Bik is a unifying feature of renal cell carcinoma.

We report for the first time inactivation of a tissue-specific Bcl-2 homology domain 3 (BH3)-only protein as a common aspect in human cancer. In detail, we show that loss of the BH3-only protein natural born killer (Nbk)/Bcl-2-interacting killer (Bik) is a common feature of clear-cell renal cell carcinoma (RCC). While strong Nbk expression is found in the renal tubuli and the epithelial lining of the glomerula, a consistent loss of Nbk expression was observed in primary RCC tissue and RCC cell lines. Mutation of Nbk is, however, rare, whereas deletion of the Nbk gene at 22q13.2 is frequent. In addition to loss of heterozygosity (LOH), DNA methylation mediates transcriptional silencing of the Nbk gene. The conditional restoration of Nbk/Bik expression led to apoptotic death of RCC but not of nonmalignant renal epithelia. A broader expression analysis of RCC cell lines for BH3-only proteins revealed that loss of Nbk coincides with failure to express Bim, whereas Puma, Bid and BNIP3 are readily detectable and, in case of Puma, inducible by p53. These data delineate a role for defects in BH3-only proteins as tumor suppressors in RCC and may explain at the same time the impressive clinical apoptosis resistance of RCC.

[Quality of life in chronic venous insufficiency (CVI). Results of a study with the newly developed Tübingen Questionnaire for measuring quality of life of patients with chronic venous insufficiency]. / Lebensqualität bei chronisch venöser Insuffizienz (CVI). Ergebnisse einer Untersuchung mit dem neu entwickelten Tübinger Fragebogen zur Messung der Lebensqualität von CVI-Patienten (TLQ-CVI).

Across the boundaries of the medical specialties we have realized how important the concepts of quality of life and disease-related coping behavior are to understand the patients' subjective perception of the medical condition and its treatment. Although standardized instruments are already available for different medical indications and even in the related fields of peripheral vascular and cardiac diseases, phlebology still lacks standardized concepts for evaluating quality of life and/or disease-related coping behavior in patients with chronic venous insufficiency (CVI). We report on a newly developed instrument specifically designed for recording quality of life in patients with CVI. It not only meets the requirements of psychometric standards, but has also proven its applicability in clinical use. The "Tübingen Questionnaire for measuring Quality of Life in patients with CVI (TLQ-CVI)" and the results of a study on quality of life in 142 patients with various stages of chronic venous insufficiency are presented. It was possible to distinguish between Stage I/II and Stage III CVI patients with respect to parameters such as "leg complaints" and "day-to-day fears and worries". These convey clinically relevant insights into the patient's subjective perception of the disease and how they cope. The information gathered provides a set of reasonable target scores for clinical studies presently being carried out at various study centers in Germany incorporating the TLQ-CVI.

Interphase cytogenetic analysis of distinct X-chromosomal translocation breakpoints in synovial sarcoma.

Synovial sarcomas show a specific translocation involving chromosomes X and 18, t(X;18)(p11.2;q11.2). Two distinct X-chromosomal breakpoints occur in different synovial sarcoma tumour samples. These breakpoints are located within two related genomic regions containing ornithine aminotransferase-like sequences, termed OATL1 and OATL2. Preliminary observations indicated the potential correlation of OATL1-associated breakpoints with biphasic tumours and OATL2-associated breakpoints with monophasic fibrous tumours. The present study uses interphase cytogenetics to investigate the nature of chromosomal aberrations in frozen synovial sarcoma tissue samples. Two-colour fluorescence in situ hybridization (FISH) was performed using probes specific for the centromeres of chromosome X or 18, along with yeast artificial chromosome probes corresponding to the distinct breakpoint regions on Xp. One monophasic epithelial and two monophasic fibrous synovial sarcomas showed an OATL2-associated breakpoint, while a biphasic tumour revealed a hybridization pattern indicating a breakpoint within the OATL1 region. These results confirm our previous suggestion of a relationship between alternative breakpoints in Xp11.2 and different histological phenotypes observed in synovial sarcomas. They also demonstrate the utility of the two-colour hybridization approach for the identification of chromosomal changes in interphase nuclei isolated from frozen tissues.

Refined mapping of the human Ets-related gene Elk-1 to Xp11.2-p11.4, distal to the OATL1 region.

The gene for human Elk-1, an Ets-related transcription factor, has previously been localized to a region that lies on the short arm of chromosome X and that is involved in specific chromosomal translocations associated with synovial sarcoma and renal adenocarcinomas. We have used fluorescence in situ hybridization and a panel of tumor-derived somatic cell hybrids to refine the localization of Elk-1, in particular with regard to the rearrangements in these tumors. Elk-1 has been assigned to Xp11.2-p11.4, distal to the OATL1 region.

Molecular cloning of the synovial sarcoma-specific translocation (X;18)(p11.2;q11.2) breakpoint.

The chromosomal translocation (X;18)(p11.2;q11.2) represents the cytogenetic hallmark of human synovial sarcomas. Two related but distinct breakpoints within band Xp11.2 were reported previously by us and others using breakpoint-spanning YACs in conjunction with FISH. Interestingly, we found that the occurrence of these alternative breakpoints corresponds to the presence of different histologic characteristics of the tumors involved. Here we report the isolation, via subcloning of one of our YAC-derived cosmids, of probes which specifically hybridize to altered restriction fragments in tumor DNAs as compared to normal controls. By using a synovial sarcoma-derived der(X) containing somatic cell hybrid, which exhibits the more distal breakpoint, one of these aberrantly hybridizing fragments could be isolated via preparative gel electrophoresis. This fragment appears to contain chromosome X- and 18-derived sequences, as revealed by both FISH on normal metaphase spreads and Southern blot analysis of X- and 18-only somatic cell hybrids. We conclude that this genomic fragment is chimaeric in nature and contains the translocation breakpoint region. In addition, our results indicate that, in contrast to our findings on the X chromosome, a single locus on chromosome 18 may be involved in the development of different (sub)types of synovial sarcoma.

Perception of knowledge what administrators and assistants know.

1. Nurse administrators are responsible for the delivery of health care in long-term care settings, and it is nursing assistants who provide the majority of care meeting the basic needs of elderly in these settings. 2. A difference in knowledge perception exists between nurse assistants and nurse administrators on information needed by nurse assistants in the home care and nursing home settings. 3. Nurse assistants would benefit from expanded nurse assistant training programs that would include not only the physical, but also the psychological and legal needs of the elderly. 4. Improving the knowledge base and the competencies of nurse assistants would improve quality of care in long-term care settings.

On call is not enough: social workers in ED.

A study indicates that ED staff would refer to hospital social workers the increasing numbers of patients who need their services if, and only if, they are on duty in the ED itself.