Jozo Delic

Increased sensitivity of CLL-derived lymphocytes to apoptotic death activation by the proteasome-specific inhibitor lactacystin

Ubiquitin‐proteasome‐dependent protein processing appears to be an essential component in the control of radiation‐induced apoptosis in human lymphocytes. This control is altered in chronic lymphocytic leukaemia (CLL), compared to that of normal human lymphocytes which mainly showed high apoptotic values after irradiation, but in some cases no sensitivity was observed. Interestingly, lactacystin activated the apoptotic pathway in both radio‐resistant and sensitive CLL cells, at doses which had no effect in normal cells where significantly higher concentrations were required. Therefore the resistance of some CLL cells to apoptosis initiation by radiation does not correlate to observed increased sensitivity to lactacystin. The nuclear level of the transcription factor NF‐κB or the cytoplasmic level of IκBα remained unaltered upon irradiation or lactacystin CLL cells treatment, suggesting that the activity of the other factors involved in apoptotic death control were altered through proteasomal inhibition. These results strongly suggest an essential role of the ubiquitin system in apoptotic cell death control in CLL lymphocytes. The inhibition of proteasome‐ubiquitin‐dependent processing could be a discriminatory apoptotic stimulus between normal versus malignant lymphocytes and therefore might potentially be of use in this specific human pathology.

Ubiquitin-Proteasome System and Increased Sensitivity of B-CLL Lymphocytes to Apoptotic Death Activation

The ubiquitin-proteasome-dependent proteolytic system has been reported to regulate apoptotic cell death in many experimental cell models. We recently found that B-CLL (chronic lymphocytic leukemia) lymphocytes are hypersensitive to apoptotic death activation through specific inhibition of proteasome function by lactacystin. Lactacystin efficiently activates apoptotic death process in B-CLL lymphocytes at doses at which no apoptotic effect can be observed in normal human lymphocytes in which 10-fold higher doses of lactacystin are required to weakly induce apoptosis. This hypersensitivity of B-cell CLL may be a result of an altered ubiquitin pathway and proteasomal proteolysis in these malignant cells, and this alteration could be specific for this malignancy. Together with other published works, these results suggest that lactacystin, though not per se a discriminatory inhibitor of the ubiquitinated protein processing/degradation, can nonetheless be discriminatory in the apoptotic cell response between B-CLL and normal lymphocytes: the property that promises efficacy in clinical trials of B-cell CLL. This hypothesis is documented by the fact that lymphocytes from patients in complete remission become resistant to lactacystin-induced apoptosis as normal lymphocytes do.

High throughput toxicity screening and intracellular detection of nanomaterials

With the growing numbers of nanomaterials (NMs), there is a great demand for rapid and reliable ways of testing NM safety—preferably using in vitro approaches, to avoid the ethical dilemmas associated with animal research. Data are needed for developing intelligent testing strategies for risk assessment of NMs, based on grouping and read‐across approaches. The adoption of high throughput screening (HTS) and high content analysis (HCA) for NM toxicity testing allows the testing of numerous materials at different concentrations and on different types of cells, reduces the effect of inter‐experimental variation, and makes substantial savings in time and cost. HTS/HCA approaches facilitate the classification of key biological indicators of NM‐cell interactions. Validation of in vitro HTS tests is required, taking account of relevance to in vivo results. HTS/HCA approaches are needed to assess dose‐ and time‐dependent toxicity, allowing prediction of in vivo adverse effects. Several HTS/HCA methods are being validated and applied for NM testing in the FP7 project NANoREG, including Label‐free cellular screening of NM uptake, HCA, High throughput flow cytometry, Impedance‐based monitoring, Multiplex analysis of secreted products, and genotoxicity methods—namely High throughput comet assay, High throughput in vitro micronucleus assay, and γH2AX assay. There are several technical challenges with HTS/HCA for NM testing, as toxicity screening needs to be coupled with characterization of NMs in exposure medium prior to the test; possible interference of NMs with HTS/HCA techniques is another concern. Advantages and challenges of HTS/HCA approaches in NM safety are discussed. WIREs Nanomed Nanobiotechnol 2017, 9:e1413. doi: 10.1002/wnan.1413 For further resources related to this article, please visit the WIREs website.

Natural Phosphorylation of CD5 in Chronic Lymphocytic Leukemia B Cells and Analysis of CD5-Regulated Genes in a B Cell Line Suggest a Role for CD5 in Malignant Phenotype

Chronic lymphocytic leukemia (CLL) results in the accumulation of B cells, presumably reflecting the selection of malignant cell precursors with Ag combined with complex alterations in protein activity. Repeated BCR stimulation of normal B cells leads to anergy and CD5 expression, both of which are features of CLL. Because CD5 is phosphorylated on tyrosine following BCR engagement and negatively regulates BCR signaling in normal B cells, we investigated its phosphorylation status and found it to be naturally phosphorylated on tyrosine but not on serine residues in CLL samples. To analyze the role of CD5, we established a B cell line in which CD5 is phosphorylated. Gene profiling of vector vs CD5-transfected B cells pointed out gene groups whose expression was enhanced: Apoptosis inhibitors (BCL2), NF-κB (RELB, BCL3), Wnt, TGFβ, VEGF, MAPKs, Stats, cytokines, chemokines (IL-10, IL-10R, IL-2R, CCL-3, CCL-4, and CCR7), TLR-9, and the surface Ags CD52, CD54, CD70, and CD72. Most of these gene groups are strongly expressed in CLL B cells as compared with normal B cells. Unexpectedly, metabolic pathways, namely cholesterol synthesis and adipogenesis, are also enhanced by CD5. Conversely, CD5 inhibited genes involved in RNA splicing and processing, ribosome biogenesis, proteasome, and CD80 and CD86 Ags, whose expression is low in CLL. Comparison of CD5- vs tailless CD5-transfected cells further demonstrated the role of CD5 phosphorylation in the regulation of selected genes. These results support a model where CLL cells are chronically stimulated, leading to CD5 activation and cell survival. In addition to CD5 itself, we point to several CD5-induced genes as potential therapeutic targets.

New molecular markers in resistant B-CLL

B-chronic lymphocytic leukemia (B-CLL) is characterized by a highly variable clinical course which has long remained a stumbling block for clinicians. This variability appears to arise from complex molecular alterations identified in malignant cells from patient subsets. Recent studies have focused in particular on identifying new molecular markers to help predict the most effective and adapted treatments. In addition to the mutation status of immunoglobulin variable heavy-chain region (IgVH) genes, which is a well-established predictive factor in B-CLL, these new markers include defects of cell factors involved in the maintenance of genome stability, such as telomere function, DNA repair, ATM and p53. Other predictive factors, such as tyrosine kinase Zap-70 and soluble factors found in patient sera, may be associated with B-cell receptor signal transduction. Interestingly, an alteration of these factors fits closely, though not strikingly, with the absence of somatic mutations in IgVH genes, suggesting that the latter may be due either to epigenetic events leading to an unstable genome or to an inherited defect in the immune response of malignant B-cells. Recent lessons from Zap-70 expression/phosphorylation suggest that some of these markers may reflect the defective pathways in B-CLL cells rather than being markers of cell malignancy per se. Furthermore, specific subsets of markers are found in patient cells resistant to treatment. Current studies on gene expression profiling and proteomic analyses should soon lead to a better understanding of how these pathways are affected, especially in multi-drug resistant B-CLL.

Aberrant telomere structure is characteristic of resistant chronic lymphocytic leukaemia cells

Aberrant telomere structure is characteristic of resistant chronic lymphocytic leukaemia cells

Concomitant heterochromatinisation and down-regulation of gene expression unveils epigenetic silencing of RELB in an aggressive subset of chronic lymphocytic leukemia in males

BackgroundThe sensitivity of chronic lymphocytic leukemia (CLL) cells to current treatments, both in vitro and in vivo, relies on their ability to activate apoptotic death. CLL cells resistant to DNA damage-induced apoptosis display deregulation of a specific set of genes.MethodsMicroarray hybridization (Human GeneChip, Affymetrix), immunofluorescent in situ labeling coupled with video-microscopy recording/analyses, chromatin-immunoprecipitation (ChIP), polymerase chain reactions (PCR), real-time quantitative PCR (RT-QPCR) and bisulfite genome sequencing were the main methods applied. Statistical analyses were performed by applying GCRMA and SAM analysis (microarray data) and Students t-test or Mann & Whitneys U-test.ResultsHerein we show that, remarkably, in a resistant male CLL cells the vast majority of genes were down-regulated compared with sensitive cells, whereas this was not the case in cells derived from females. This gene down-regulation was found to be associated with an overall gain of heterochromatin as evidenced by immunofluorescent labeling of heterochromatin protein 1α (HP-1), trimethylated histone 3 lysine 9 (3metH3K9), and 5-methylcytidine (5metC). Notably, 17 genes were found to be commonly deregulated in resistant male and female cell samples. Among these, RELB was identified as a discriminatory candidate gene repressed in the male and upregulated in the female resistant cells.ConclusionThe molecular defects in the silencing of RELB involve an increase in H3K9- but not CpG-island methylation in the promoter regions. Increase in acetyl-H3 in resistant female but not male CLL samples as well as a decrease of total cellular level of RelB after an inhibition of histone deacetylase (HDAC) by trichostatin A (TSA), further emphasize the role of epigenetic modifications which could discriminate two CLL subsets. Together, these results highlighted the epigenetic RELB silencing as a new marker of the progressive disease in males.

In vitro biocompatibility of mesoporous metal (III; Fe, Al, Cr) trimesate MOF nanocarriers

The high porosity and versatile composition of the benchmarked mesoporous metal (Fe, Al, Cr) trimesate metal-organic frameworks (MIL-100(Fe, Al, Cr)) make them very promising solids in different strategic industrial and societal domains (separation, catalysis, biomedicine, etc.). In particular, MIL-100(Fe) nanoparticles (NPs) have been recently revealed to be one of the most promising and innovative next generation tools enabling multidrug delivery to overcome cancer resistance. Here, we analyzed the in vitro toxicity of the potential drug nanocarrier MIL-100(Fe) NPs and the effect of the constitutive cation by comparing its cytotoxicity with that one of its Cr and Al analogue NPs. Lung (A549 and Calu-3) and hepatic (HepG2 and Hep3B) cell lines were selected considering pulmonary, ingestion or intravenous exposure modes. First, the complete physicochemical characterization (structural, chemical and colloidal stability) of the MIL-100(Fe, Al, Cr) NPs was performed in the cell culture media. Then, their cytotoxicity was evaluated in the four selected cell lines using a combination of methods from cell impedance, cell survival/death and ROS generation to DNA damage for measuring genotoxicity. Thus, MIL-100(Fe, Al, Cr) NPs did not induce in vitro cell toxicity, even at high doses in the p53 wild type cell lines (A549 and calu-3 (lung) and HepG2 (liver)). The only toxic effect of MIL100-Fe was observed in the hepatocarcinoma cell line Hep3B, which is stress sensitive because it does not express TP53, the guardian of the genome.

Chromosomal DNA and p53 Stability, Ubiquitin System and Apoptosis in B-CLL Lymphocytes

The ubiquitin system regulates diverse biological processes such as DNA replication and repair, biogenesis of ribosome, peroxisome and nucleosome, cell cycle, stress response and signal transduction pathways. Thus, the reported role of the ubiquitin system in apoptotic death control as well the alteration of its control in carcinogenesis should come as no surprise. Indeed, we and other groups have reported that the ubiquitin system is involved in apoptotic cell death of normal human lymphocytes and that this control is altered in B lymphocytes derived from chronic lymphocytic leukemia patients (B-CLL), rendering these malignant cells hypersensitive to specific inhibition of protein degradation/processing through proteasomal function. This approach recently allowed us to demonstrate that the stability of the tumor suppressor and pro-apoptotic protein p53 is differentially regulated in B-CLL versus normal lymphocytes and that this difference might at least partly explain the impaired response of B-CLL lymphocytes to apoptotic death activation. These results strongly suggest an imbalance in p53 regulation in B-CLL cells that leads to a variable response to DNA damage and constitutively expressed chromosomal instability. The question we and others would like to address is whether this alteration, or more likely a subset of alterations of the ubiquitin-proteasome pathway, is specific to B-CLL malignancy or if it is a hallmark of cancer cells in general. In either case, a better understanding of the ubiquitin-dependent control of apoptosis should pave the way towards a methodological approach for in vitro development of discriminating treatments which may be of potential usefulness in clinical trials of B-CLL.

A new phosphorylated form of Ku70 identified in resistant leukemic cells confers fast but unfaithful DNA repair in cancer cell lines.

Ku70-dependent canonical nonhomologous end-joining (c-NHEJ) DNA repair system is fundamental to the genome maintenance and B-cell lineage. c-NHEJ is upregulated and error-prone in incurable forms of chronic lymphocytic leukemia which also displays telomere dysfunction, multiple chromosomal aberrations and the resistance to DNA damage-induced apoptosis. We identify in these cells a novel DNA damage inducible form of phospho-Ku70. In vitro in different cancer cell lines, Ku70 phosphorylation occurs in a heterodimer Ku70/Ku80 complex within minutes of genotoxic stress, necessitating its interaction with DNA damage-induced kinase pS2056-DNA-PKcs and/or pS1981-ATM. The mutagenic effects of phospho-Ku70 are documented by a defective S/G2 checkpoint, accelerated disappearance of γ-H2AX foci and kinetics of DNA repair resulting in an increased level of genotoxic stress-induced chromosomal aberrations. Together, these data unveil an involvement of phospho-Ku70 in fast but inaccurate DNA repair; a new paradigm linked to both the deregulation of c-NHEJ and the resistance of malignant cells.