Daniel Jung

Adenovirus 5 and chimeric adenovirus 5/F35 employ distinct B-lymphocyte intracellular trafficking routes that are independent of their cognate cell surface receptor

Gene transfer applications with adenovirus (Ad) type 5 are limited by its native tropism, hampering their use in several cell types. To address this limitation, several Ad vectors bearing chimeric fiber have been produced to take advantage of the different cellular receptors used by other subgroups of Ads. In this study, we have compared the transduction efficiency of Ad5 and the chimeric Ad5/F35 in primary human B lymphocytes and B-cell lines as a function of the developmental stage. We found that transduction efficiencies of the two Ads differ independently of their targeted cellular receptor but are related to the intracellular localization of the virus. In efficiently transduced cells, Ads were localized in early endosomes or cytosol, whereas in poorly transduced cells they were localized within late endosomes/lysosomes. Finally, we demonstrate that treatment of cells with phosphatase inhibitors known to redirect endocytosis towards caveolae, increased Ad5/F35 transduction efficiency.

Iron specific growth inhibition of Burkitt's lymphoma cells in vitro, associated with a decrease in translocated c-myc expression

The cellular proto‐oncogene c‐myc is an important transcription factor that plays a role in several cellular activities such as proliferation, differentiation, and apoptosis. It follows that regulation of c‐myc expression is crucial for maintaining cell integrity. Amplification or translocation can convert this proto‐oncogene into an activated oncogene, thereby deregulating c‐myc expression. Changes in the expression of c‐myc leading to malignant cell growth and tumor progression can also be triggered by extrinsic factors. It has been reported that iron can increase cell proliferation, mainly by stimulating DNA synthesis as well as by enhancing c‐myc expression. Here, we studied the effect of iron on cells in which c‐myc expression is deregulated by either chromosomal translocation or gene amplification. When added to Burkitts lymphoma cell lines, iron markedly inhibits cell proliferation. This effect is mediated by a cell cycle arrest in G2/M, followed by an important decrease in c‐myc expression, whereas no effect could be observed in a cell line harboring amplified c‐myc. Moreover, the down‐regulation of c‐myc expression, which is independent from cell cycle blockade, leads to cell death by apoptosis. Collectively, our results suggest the existence of a novel iron‐dependent cell cycle regulatory mechanism involving modulation of translocated c‐myc gene expression.

c‐Src tyrosine kinase co‐associates with and phosphorylates signal transducer and activator of transcription 5b which mediates the proliferation of normal human B lymphocytes

c‐Src is the normal human cellular protein homologue of the viral oncogene v‐src. c‐Src activity was reported recently to increase in CD40‐activated human B lymphocytes, suggesting its involvement in proliferation. To elucidate the exact role of c‐Src in this process, we investigated the effects of c‐Src over‐expression on normal B lymphocyte growth. B lymphocytes purified from human peripheral blood were infected with Ad5/F35 vector encoding either a constitutively active c‐Src (c‐Src/dominant‐positive) or a dominant‐negative c‐Src (c‐Src/DN). Little variation of B lymphocytes expansion could be observed between control enhanced yellow fluorescent protein and c‐Src/dominant‐positive‐infected cells. In contrast, over‐expression of c‐Src/DN results in a 40% inhibition of B lymphocyte expansion. These results suggest that DN c‐Src may compete with endogenous c‐Src, resulting in partial inhibition of a transcriptional pathway involved in B lymphocyte proliferation. We demonstrate further that c‐Src can phosphorylate signal transducer and activator of transcription 5b (STAT5b) on tyrosine 699 and that c‐Src and STAT5b co‐associate during B lymphocyte proliferation. These results confirm an important role for c‐Src in the expansion of normal human B lymphocytes in vitro, in which c‐Src may regulate STAT5b in the intracellular signalling pathway important for the proliferation of normal human B lymphocytes.

Free radicals act as effectors in the growth inhibition and apoptosis of iron-treated Burkitt's lymphoma cells

The addition of ferric citrate to Burkitts lymphoma (BL) cell lines inhibits growth, leads to the accumulation of cells in the phase G2/M of the cell cycle and to the modulation of translocated c-myc expression. The increase in the labile iron pool (LIP) of iron-treated BL cells leads to cytotoxicity. Indeed, intracellular free iron catalyzes the formation of highly reactive compounds such as hydroxyl radicals and nitric oxide (NO) that damages macromolecular components of cells, eventually resulting in apoptosis. In this report, we have investigated the possible involvement of free radicals in the response of Ramos cells to iron. When added to Ramos cells, iron increased the intracellular levels of peroxide/peroxynitrite and NO. Moreover, the addition of free radicals scavengers (TROLOX® and Carboxy-PTIO) neutralized the effects of iron on Ramos cells while addition of an NO donor or hydrogen peroxide (H2O2) to cells generated effects which partially mimicked those induced by iron addition. Collectively, our results suggest the involvement of free radicals as effectors in the iron specific growth inhibition of BL cells observed in vitro.

Suppression of protein phosphatase 2A activity enhances Ad5/F35 adenovirus transduction efficiency in normal human B lymphocytes and in Raji cells

Investigation of the molecular processes which control the development and function of lymphocytes is essential for our understanding of humoral immunity, as well as lymphocyte associated pathogenesis. Adenovirus-mediated gene transfer provided a powerful tool to investigate these processes. We have previously demonstrated that adenoviral vector Ad5/F35 transduces plasma cell lines at a higher efficiency than primary B cells, owing to differences in intracellular trafficking. Given that phosphatases are effectors of intracellular trafficking, here we have analyzed the effects of a panel of phosphatase inhibitors on Ad5/F35 transduction efficiency in B lymphocytes in the present study. FACS analysis was conducted to determine Ad5/F35-EYFP transduction efficiency in lymphoid cells, including human primary B cells, following serine/threonine phosphatase (PSP) inhibitor treatment. We further used confocal microscopy to analyze intracellular trafficking and fate of CY3 labeled Ad5/F35 vectors, in PSP treated lymphoid cell. Finally, we analyzed the MAPK pathway by Western blot in PSP treated cells. Adenoviral transduction efficiency was unresponsive to inhibition of PP1 whereas inhibition of PP2A by cantharidic acid, or PP1 and PP2A by okadaic acid, substantially increased transduction efficiency. Importantly, confocal microscopy analyses revealed that inhibition of PP2A shut down adenovirus recycling. Moreover, inhibition of PP2A resulted in increased phosphorylation of AKT, ERK1/2 and MEK1/2. Taken together, these results suggest that Ad5/F35 is more efficiently transduced in cells following PP2A inhibition. Our results are in agreement with reports indicating that PP2A is involved in the formation of recycling vesicles and might be of interest for gene therapy applications.

2-Methoxyestradiol induce the conversion of human peripheral blood memory B lymphocytes into plasma cells.

2-Methoxyestradiol (2ME), an end-metabolite of 17beta-estradiol, is an antiproliferative agent that is currently being tested in clinical trials for cancer treatment. We hereby report that sub-cytotoxic concentrations of 2ME influence the in vitro proliferation of human peripheral blood B lymphocytes. More surprisingly, we have observed that 2ME induces the conversion of CD138(-) B lymphocytes into CD138(+) cells of phenotype similar to immunoglobulin (Ig)-secreting plasma cells. Normal human B lymphocytes expressing CD138 increased in response to 2ME in a dose-dependent fashion, from 2% at baseline up to 31% in cells cultured in the presence of 0.75 microM 2ME. Moreover, most of the converted cells were also CD27(+) and secreted high levels of IgG (151 microg/10(6)cells/24h). IEF studies revealed that conversion occurred in a polyclonal manner. We then exploited this effect of 2ME to gain further insights into the molecular mechanisms that govern changes in transcription factors involved in plasma cells differentiation. Plasma cells generated by 2ME treatment of normal human B lymphocytes expressed elevated levels of IRF4 and reduced levels of Pax5 and Bcl-6. Similarly, levels of XBP-1 and Blimp-1 transcripts were increased. Our results suggest that the differentiation of peripheral blood B lymphocytes into plasma cells requires a similar modulation of transcription factors expression that for tonsil and bone marrow B lymphocytes.