Protein kinase c-ß-dependent activation of NF-κB in stromal cells is indispensable for the survival of chronic lymphocytic leukemia B cells in vivo.

Tumor cell survival critically depends on heterotypic communication with benign cells in the microenvironment. Here, we describe a survival signaling pathway activated in stromal cells by contact to B cells from patients with chronic lymphocytic leukemia (CLL). The expression of protein kinase C (PKC)-ßII and the subsequent activation of NF-κB in bone marrow stromal cells are prerequisites to support the survival of malignant B cells. PKC-ß knockout mice are insusceptible to CLL transplantations, underscoring the in vivo significance of the PKC-ßII-NF-κB signaling pathway in the tumor microenvironment. Upregulated stromal PKC-ßII in biopsies from patients with CLL, acute lymphoblastic leukemia, and mantle cell lymphoma suggests that this pathway may commonly be activated in a variety of hematological malignancies.

Immunotoxin BL22 induces apoptosis in mantle cell lymphoma (MCL) cells dependent on Bcl-2 expression.

Mantle cell lymphoma (MCL) is an incurable mature B cell proliferation, combining the unfavourable clinical features of aggressive and indolent lymphomas. The blastic variant of MCL has an even worse prognosis and new treatment options are clearly needed. We analysed the effects of BL22, an immunotoxin composed of the Fv portion of an anti- CD22 antibody fused to a 38-kDa Pseudomonas exotoxin-A fragment on four MCL cell lines as well as on primary cells of four MCL patients. Apoptosis induction by BL22 was much more pronounced in MCL cell lines with low Bcl-2 expression (NCEB-1, JeKo-1 and JVM-2) compared to Granta-519 cells with high Bcl-2 expression. While the expression of the antiapoptotic protein Mcl-1 declined (NCEB-1, Granta-519), Bcl-2 levels remained unchanged in Granta-519 cells. However transfection of BCL2 cDNA into NCEB-1, JeKo-1 and JVM-2 cells significantly reduced BL22-mediated toxicity. Accordingly we examined the effects of Bcl-2 inactivation in Granta-519 cells using siRNA. Indeed, apoptosis induction was strongly enhanced in Granta-519 cells with silenced Bcl-2. Our results were confirmed in freshly isolated MCL-cells from patients with leukaemic MCL. We conclude that Bcl-2 expression is important for mediating resistance against the immunotoxin BL22 in MCL cells.

Inhibition of cholesterol recycling impairs cellular PrP(Sc) propagation.

The infectious agent in prion diseases consists of an aberrantly folded isoform of the cellular prion protein (PrP(c)), termed PrP(Sc), which accumulates in brains of affected individuals. Studies on prion-infected cultured cells indicate that cellular cholesterol homeostasis influences PrP(Sc) propagation. Here, we demonstrate that the cellular PrP(Sc) content decreases upon accumulation of cholesterol in late endosomes, as induced by NPC-1 knock-down or treatment with U18666A. PrP(c) trafficking, lipid raft association, and membrane turnover are not significantly altered by such treatments. Cellular PrP(Sc) formation is not impaired, suggesting that PrP(Sc) degradation is increased by intracellular cholesterol accumulation. Interestingly, PrP(Sc) propagation in U18666A-treated cells was partially restored by overexpression of rab 9, which causes redistribution of cholesterol and possibly of PrP(Sc) to the trans-Golgi network. Surprisingly, rab 9 overexpression itself reduced cellular PrP(Sc) content, indicating that PrP(Sc) production is highly sensitive to alterations in dynamics of vesicle trafficking.

Prion infection influences murine endogenous retrovirus expression in neuronal cells.

Prions as causative agents of transmissible spongiform encephalopathies have been well investigated in experimental and modelling work. However, little is known about the molecular pathogenesis of prion-induced encephalopathies, the role of co-factors, and the interaction of prions with cellular components. We investigated the influence of prion infection on expression of murine endogenous retroviruses (ERVs), which compose approximately 10% of the mouse genome. Hypothalamic neuronal cells (GT1) and neuroblastoma cells (N2a) were examined. Both cell lines can be persistently infected with mouse adapted prion strains, i.e., RML. Using a mammalian retrovirus-specific DNA microarray and quantitative PCR methods, we compared the expression profiles of ERVs in prion-infected, uninfected, and anti-prion compound-treated murine neuronal cell lines, including clonal cell populations. The results suggest that prion infection influences ERV expression in neuronal cell lines, that this influence is cell line-specific, ERV-specific, and responsive to anti-prion compound treatment.

Cell line dependent RNA expression profiles of prion-infected mouse neuronal cells.

The overall impact of prion disease on gene expression is not well characterized. We have carried out a large-scale expression analysis of specific cell types commonly employed in studies of prion disease. Neuroblastoma cells (N2a) and hypothalamic neuronal cells (GT1) can be persistently infected with mouse-adapted scrapie prions, the latter demonstrating cytopathologic effects associated with prion neuropathology. Exploiting a mouse DNA microarray containing approximately 21,000 spotted cDNAs, we have identified several hundred differentially expressed sequences in the two cell lines when infected with prion strain RML. ScN2a and ScGT1 cells demonstrate unique changes in RNA profiles and both differ from the reported changes in human microglia and prion-infected brain studies albeit with some overlap. In addition, several of the identified changes are shared in common with other neurodegenerative diseases such as Alzheimer's disease. The results illustrate that prion infection differs in effect depending on cell type, which could be exploited for diagnostic or therapeutic intervention.