Julien Defoiche

Mechanisms of leukemogenesis induced by bovine leukemia virus: prospects for novel anti-retroviral therapies in human

In 1871, the observation of yellowish nodules in the enlarged spleen of a cow was considered to be the first reported case of bovine leukemia. The etiological agent of this lymphoproliferative disease, bovine leukemia virus (BLV), belongs to the deltaretrovirus genus which also includes the related human T-lymphotropic virus type 1 (HTLV-1). This review summarizes current knowledge of this viral system, which is important as a model for leukemogenesis. Recently, the BLV model has also cast light onto novel prospects for therapies of HTLV induced diseases, for which no satisfactory treatment exists so far.

Valproate synergizes with purine nucleoside analogues to induce apoptosis of B-chronic lymphocytic leukaemia cells

Resistance to chemotherapy and drug toxicity are two major concerns of chronic lymphocytic leukaemia (B‐CLL) treatment by purine nucleoside analogues (PNA, i.e. fludarabine and cladribine). We hypothesized that targeting epigenetic changes might address these issues and evaluated the effect of the histone deacetylase inhibitor valproate (VPA) at a clinically relevant concentration. VPA acted in a highly synergistic/additive manner with fludarabine and cladribine to induce apoptosis of B‐CLL cells. Importantly, VPA also restored sensitivity to fludarabine in B cells from poor prognosis CLL patients who became resistant to chemotherapy. Mechanism of apoptosis induced by VPA alone or combined with fludarabine or to cladribine was caspase‐dependent and involved the extrinsic pathway. VPA, but neither fludarabine nor cladribine, enhanced the production of reactive oxygen species (ROS) and inhibition of ROS with N‐acetylcysteine decreases apoptosis of CLL cells. VPA stimulates hyperphosphorylation of p42/p44 ERK, cytochrome c release and overexpression of Bax and Fas. Together, our data indicate that VPA may ameliorate the outcome of PNA‐based therapeutic protocols and provide a potential alternative treatment in both the relapsed and front‐line resistant patients and in patients with high risk features.

Reduction of B cell turnover in chronic lymphocytic leukaemia.

Whether chronic lymphocytic leukaemia (CLL) is a latent or a proliferating disease has been intensively debated. Whilst the dogma that CLL results from accumulation of dormant lymphocytes is supported by the unresponsiveness of leukaemic cells to antigens and polyclonal activators, recent in vivo kinetic measurements indicate that B lymphocytes do divide at significant rates in CLL. However, an important and still unanswered question is whether CLL cells proliferate faster or slower compared with their normal counterparts. This report addressed directly this point and compared B‐cell kinetics in CLL subjects and healthy controls, using a pulse‐chase approach based on incorporation of deuterium from 6,6‐2H2‐glucose into DNA. We confirmed that B cells proliferated at significant levels in CLL but found that the proliferation rates were reduced compared with healthy subjects (mean 0·47 vs. 1·31%/d respectively, P = 0·007), equivalent to an extended doubling time of circulating B cells (147 d vs. 53 d). In conclusion, CLL B cells proliferate at reduced levels compared with healthy controls. CLL is thus characterized by an aberrant B‐cell kinetics with a decrease in cell turnover, an observation that may impact on elaboration of efficient therapeutic strategies.

Measurement of proliferation and disappearance of rapid turnover cell populations in human studies using deuterium-labeled glucose

Cell proliferation may be measured in vivo by quantifying DNA synthesis with isotopically labeled deoxyribonucleotide precursors. Deuterium-labeled glucose is one such precursor which, because it achieves high levels of enrichment for a short period, is well suited to the study of rapidly dividing cells, in contrast to the longer term labeling achieved with heavy water (2H2O). As deuterium is non-radioactive and glucose can be readily administered, this approach is suitable for clinical studies. It has been widely applied to investigate human lymphocyte proliferation, but solid tissue samples may also be analyzed. Rate, duration and route (intravenous or oral) of [6,6-2H2]-glucose administration should be adapted to the target cell of interest. For lymphocytes, cell separation is best achieved by fluorescence activated cell sorting (FACS), although magnetic bead separation is an alternative. DNA is then extracted, hydrolyzed enzymatically and analyzed by gas chromatography mass spectrometry (GC/MS). Appropriate mathematical modeling is critical to interpretation. Typical time requirements are as follows: labeling, 10–24 h; sampling, ∼3 weeks; DNA extraction/derivatization, 2–3 d; and GC/MS analysis, ∼2 d.

Emphasis on cell turnover in two hosts infected by bovine leukemia virus: a rationale for host susceptibility to disease.

Bovine leukemia virus (BLV) is a deltaretrovirus that infects and induces accumulation of B-lymphocytes in the peripheral blood and lymphoid tissues of cattle, leading to leukemia/lymphoma. BLV can also be experimentally transmitted to sheep, in which disease appears earlier and at higher frequencies. Abnormal accumulation of leukemic B-lymphocytes results from an alteration of different parameters that include cell proliferation and death as well as migration to lymphoid tissues. Interestingly, B lymphocyte turnover is increased in BLV-infected sheep but reduced in cattle, revealing a potential relationship between cell kinetics and disease progression.

Measurement of ribosomal RNA turnover in vivo by use of deuterium-labeled glucose.

BACKGROUND Most methods for estimation of rates of RNA production are not applicable in human in vivo clinical studies. We describe here an approach for measuring ribosomal RNA turnover in vivo using [6,6-(2)H(2)]-glucose as a precursor for de novo RNA synthesis. Because this method involves neither radioactivity nor toxic metabolites, it is suitable for human studies. METHODS For method development in vitro, a lymphocyte cell line (PM1) was cultured in the presence of [6,6-(2)H(2)]-glucose. RNA was extracted, hydrolyzed enzymatically to ribonucleosides, and derivatized to either the aldonitrile tetra-acetate or the pentafluoro triacetate derivative of the pentose before GC-MS. We identified optimum derivatization and analysis conditions and demonstrated quantitative incorporation of deuterium from glucose into RNA of dividing cells. RESULTS Pilot clinical studies demonstrated the applicability of this approach to blood leukocytes and solid tissues. A patient with chronic lymphocytic leukemia received [6,6-(2)H(2)]-glucose (1 g/kg) orally in aliquots administered every 30 min for a period of 10 h. When we analyzed CD3(-) B cells that had been purified by gradient centrifugation and magnetic-bead adhesion, we observed deuterium enrichment, a finding consistent with a ribosomal RNA production rate of about 7%/day, despite the slow division rates observed in concurrent DNA-labeling analysis. Similarly, in 2 patients with malignant infiltration of lymph nodes, administration of [6,6-(2)H(2)]-glucose (by intravenous infusion for 24 h) before excision biopsy allowed estimation of DNA and RNA turnover in lymph node samples. CONCLUSIONS Our study results demonstrate the proof-of-principle that deuterium-labeled glucose may be used to analyze RNA turnover, in addition to DNA production/cell proliferation, in clinical samples.

In vivo ribosomal RNA turnover is down-regulated in leukaemic cells in chronic lymphocytic leukaemia.

Human B-cell chronic lymphocytic leukaemia (B-CLL) is characterized by the progressive and massive accumulation of CD5 B-cells in blood. The long average lifespan of B-CLL cells, 6 months (Messmer et al, 2005; Defoiche et al, 2008), exceeds that of healthy B cells, 2 months (Defoiche et al, 2008). Mechanisms by which B-CLL cells escape normal celldeath pathways include upregulation of anti-apoptotic genes, e.g. BCL2, MCL1 and XIAP, and down-regulation of cell cycle genes; hence most B-CLL cells are arrested in G0/G1 (Zenz et al, 2010). We hypothesized that down-regulation of metabolic activity may represent a further mechanism by which B-CLL cells avoid programmed cell-death. Several lines of evidence support this paradigm. Firstly, gene expression studies reveal consistently reduced activity of genes regulating intermediary metabolism (aldehyde dehydrogenase, mitochondrial isocitrate dehydrogenase, adenosine deaminase and glucose transporter type-3 genes) (Zheng et al, 2002). Secondly, progressive B-CLL is associated with reduced cellular RNA content (Ricciardi et al, 2001), indicative of reduced ribosomal mass and reduced protein synthetic activity. Thirdly, much earlier direct (albeit ex vivo) measurements of ribosomal assembly using radio-labelled uridine or methylmethionine showed impaired assembly of new ribosomes in B-CLL (Rubin, 1971). In order to test whether RNA metabolism is downregulated in B-CLL, we used deuterium-labelled glucose as a safe, non-radioactive tracer to measure in vivo rRNA synthesis (Defoiche et al, 2009). This approach exploits the fact that deuterium is incorporated from labelled glucose into the pentose moiety of newly synthesized RNA in proportion to its rate of production. Selectivity for ribosomal RNA (rRNA) is achieved by sampling after the predicted

Reduced expression of cell cycle‐associated genes in B lymphocytes purified from the peripheral blood of early‐stage B chronic lymphocytic leukaemia patients – response to di Iasio et al

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