Cyclin D1 inhibits mitochondrial activity in B cells.

Cyclin D1 is a cell cycle regulatory protein that acts at the G1-S transition, following its binding to and activation by the cyclin-dependent kinases 4 or 6. Cyclin D1 is absent from the entire B-cell lineage but is present in a large percentage of 2 types of malignant B-cell hemopathy--mantle cell lymphoma and multiple myeloma--suggesting a major role of this protein in the malignancy process. We show here, in an experimental model of cyclin D1 fusion protein transduction in mature B cells, that, cyclin D1 inhibits total mitochondrial activity. Cyclin D1 is localized at the outer mitochondrial membrane, bound to a voltage-dependent anion channel through its central domain, and it competes with hexokinase 2 for binding to this channel. The bound cyclin D1 decreases the supply of ADP, ATP, and metabolites, thereby reducing energy production. This function of cyclin D1 was also reported by others in normal and transformed mammary epithelial cells, suggesting that it may be ubiquitous.

ZAP-70 intron1 DNA methylation status: determination by pyrosequencing in B chronic lymphocytic leukemia.

ZAP-70 expression is a strong prognostic indicator in chronic lymphocytic leukemia. However, ZAP-70 quantification by flow cytometry lacks sufficient standardization. Based upon the correlation between ZAP-70 expression and its gene methylation status, we have developed a quantitative pyrosequencing assay for the determination of ZAP-70 methylation adapted for routine use. Methylation in four CpG pairs (C-223, C-243, C-254, and C-267) in the first intron of ZAP-70 is associated with repression of ZAP-70. Moreover, it correlates with CD38 expression (n=111, p<.0001), IgHv mutation status (n=106, p<.0001), time to treatment (p<.0001), and overall survival (p=.0014). Pyrosequencing of ZAP-70 provides a good alternative to flow cytometry.

Relevance of cyclin D1b expression and CCND1 polymorphism in the pathogenesis of multiple myeloma and mantle cell lymphoma.

BACKGROUND: The CCND1 gene generates two mRNAs (cyclin D1a and D1b) through an alternative splicing at the site of a common A/G polymorphism. Cyclin D1a and b proteins differ in their C-terminus, a region involved in protein degradation and sub-cellular localization. Recent data have suggested that cyclin D1b could be a nuclear oncogene. The presence of cyclin D1b mRNA and protein has been studied in two hemopathies in which cyclin D1 could be present: multiple myeloma (MM) and mantle cell lymphoma (MCL). The A/G polymorphism of CCND1 has also been verified in a series of patients. METHODS: The expression of cyclin D1 mRNA isoforms has been studied by real-time quantitative PCR; protein isoforms expression, localization and degradation by western blotting. The CCND1 polymorphism was analyzed after sequencing genomic DNA. RESULTS: Cyclin D1 mRNA isoforms a and b were expressed in mantle cell lymphoma (MCL) and multiple myeloma (MM). Cyclin D1b proteins were present in MCL, rarely in MM. Importantly, both protein isoforms localized the nuclear and cytoplasmic compartments. They displayed the same short half-life. Thus, the two properties of cyclin D1b recognized as necessary for its transforming activity are missing in MCL. Moreover, CCND1 polymorphism at the exon/intron boundary had no influence on splicing regulation in MCL cells. CONCLUSION: Our results support the notion that cyclin D1b is not crucial for the pathogenesis of MCL and MM.

High-purity selection and maintenance of gene expression in human neuroblastoma cells stably over-expressing GFP fusion protein. Application for opioid receptors desensitization studies.

Chronic use of opiates such as morphine is associated with drug tolerance, which is correlated with the desensitization of opioid receptors. This latter process involves phosphorylation of opioid receptors by G protein-coupled receptors kinases (GRKs) and subsequent uncoupling by beta-arrestins. To explore these molecular mechanisms, neuronal cell lines, endogenously expressing the opioid receptors, provide an ideal cellular model. Unfortunately, there are two major drawbacks: (1) these cells are refractory to cDNA introduction, resulting in low transfection efficiency; (2) continuous culturing of transfected cells invariably leads to phenotypic drift of the cultures even after an antibiotic selection. So, these cells were dropped in favor of heterologous expression systems, which are easier to transfect but whose relevance as adequate cellular model for studying opioid receptor regulation should be questioned, as recently demonstrated by [Haberstock-Debic, H., Kim, K.A.,Yu, Y.J., von Zastrow, M., 2005. Morphine promotes rapid, arrestin-dependent endocytosis of mu-opioid receptors in striatal neurons. J. Neurosci. 25, 7847-7857]. In this work, we describe a method, based on fluorescence-activated cell sorting (FACS), to select and maintain a high proportion of transfected SK-N-BE cells (a neuronal cell line endogenously expressing human Delta-Opioid Receptor (hDOR)), expressing the beta-arrestin1 fused to green fluorescent protein (GFP). While in functional experiments, we were not able to observe a major effect in non-sorted SK-N-BE cells expressing beta-arrestin1-GFP, the enrichment by 18-fold with FACS resulted in a robust increase of beta-arrestin1-GFP expression associated with strong hDOR desensitization. Moreover, this method also allows to counteract the phenotypic drift and to maintain a high-purity selection of SK-N-BE cells expressing beta-arrestin1-GFP. Thus, this approach provides a valuable tool for exploring opioid receptors desensitization in neuronal cells.