Luca Orlando

A rapid flow cytometry test based on histone H2AX phosphorylation for the sensitive and specific diagnosis of ataxia telangiectasia

Ataxia telangiectasia (A‐T) is a progressive neurodegenerative disease with onset in early childhood, caused by mutations in the ATM (ataxia‐telangiectasia mutated) gene. Diagnosis relies on laboratory tests showing high levels of serum alphafetoprotein, cell sensitivity to ionizing radiation (IR) and absence or reduced levels of ATM protein. Many tests, however, are not sufficiently sensitive or specific for A‐T, have long turnaround times, or require large blood samples. This prompted us to develop a new flow cytometry method for the diagnosis of A‐T based on the measurement of histone H2AX phosphorylation. We established normal ranges of histone H2AX phosphorylation after 2 Gy IR by testing T‐cell lines, lymphoblastoid cell lines (LCLs) and/or peripheral blood mononuclear cells (PBMCs) or both from 20 genetically proven A‐T and 46 control donors. To further evaluate the specificity and sensitivity of the test, we analyzed cells from 19 patients suspected of having A‐T, and from one Friedreich Ataxia, one Ataxia with Oculomotor Apraxia type 2, and one Nijmegen Breakage Syndrome patients. Phosphorylated histone H2AX mean fluorescence intensity of irradiated A‐T cells was significantly lower than that of healthy donors. The intrastaining, intraassay, and interassay imprecisions were ≤13.22%. Sensitivity and specificity were virtually 100% when the test was performed on PBMCs. Screening of 19 consecutive new patients with suspected A‐T classified 15 patients as non‐A‐T and four as A‐T; diagnosis of the latter four was subsequently confirmed by DNA sequencing to identify ATM mutations. The Friedreich Ataxia patient, the Ataxia with Oculomotor Apraxia type 2 patient and the Nijmegen Breakage Syndrome patient were classified as non‐A‐T. This flow cytometry test is very sensitive, specific and rapid, and requires only 2 ml of blood. It may thus be proposed for the early differential diagnosis of A‐T as an alternative to methods requiring the production of LCLs.

Fluorescent Silica Nanoparticles Improve Optical Imaging of Stem Cells Allowing Direct Discrimination between Live and Early‐Stage Apoptotic Cells

Highly bright and photostable cyanine dye-doped silica nanoparticles, IRIS Dots, are developed, which can efficiently label human mesenchymal stem cells (hMSCs). The application procedure used to label hMSCs is fast (2 h), the concentration of IRIS Dots for efficient labeling is low (20 μg mL(-1) ), and the labeled cells can be visualized by flow cytometry, confocal microscopy, and transmission electron microscopy. Labeled hMSCs are unaffected in their viability and proliferation, as well as stemness surface marker expression and differentiation capability into osteocytes. Moreover, this is the first report that shows nonfunctionalized IRIS Dots can discriminate between live and early-stage apoptotic stem cells (both mesenchymal and embryonic) through a distinct external cell surface distribution. On the basis of biocompatibility, efficient labeling, and apoptotic discrimination potential, it is suggested that IRIS Dots can serve as a promising stem cell tracking agent.

Two-tier analysis of histone H2AX phosphorylation allows the identification of Ataxia Telangiectasia heterozygotes

BACKGROUND AND PURPOSE Ataxia Telangiectasia (A-T) heterozygotes constitute 0.36-1% of the general population. They have a higher risk of developing several types of cancer and may be more likely to suffer side-effects following radiotherapy than the general population. Their identification is both labor- and time-consuming and the sensitivity and specificity of the methods employed has not been evaluated. This paper describes a new approach to the identification of A-T heterozygotes based on a two-tier analysis of histone H2AX phosphorylation. MATERIALS AND METHODS We compared the T-cell phenotype after exposure to 2 Gy in nine obligate A-T heterozygotes and 17 normal donors. Examined end points were histone H2AX phosphorylation by flow cytometry 1 h after irradiation (kinase proficiency) and the residual gamma-H2AX foci by confocal microscopy 72 h after irradiation (DSB repair proficiency). RESULTS The sequential use of these two methods results in 100% positive predictive value (PPV), 67% negative predictive value (NPV), 78% sensitivity, and 100% specificity. The overall hit rate, i.e. the ratio between the true positives plus the true negatives and the total number of observations was 85%. CONCLUSIONS A-T heterozygotes can be identified by analysing irradiated T-cell H2AX phosphorylation level and residual gamma-H2AX foci.

Involvement of MRE11A and XPA gene polymorphisms in the modulation of DNA double-strand break repair activity: a genotype-phenotype correlation study.

DNA double-strand breaks (DSB) are the most lethal form of ionizing radiation-induced DNA damage, and failure to repair them results in cell death. In order to see if any associations exist between DNA repair gene polymorphisms and phenotypic profiles of DSB repair (DSBR) we performed a genotype-phenotype correlation study in 118 young healthy subjects (mean age 25.8±6.7years). Subjects were genotyped for 768 single nucleotide polymorphisms (SNPs) with a custom Illumina Golden Gate Assay, and an H2AX histone phosphorylation assay was done to test DSBR capacity. We found that H2AX phosphorylation at 1h was significantly lower in subjects heterozygous (no variant homozygotes were observed) for the XPA gene SNP rs3176683 (p-value=0.005), while dephosphorylation was significantly higher in subjects carrying the variant allele in three MRE11A gene SNPs: rs1014666, rs476137 and rs2508784 (p-value=0.003, 0.003 and 0.008, respectively). An additive effect of low-activity DNA repair alleles was associated with altered DSBR activity, as demonstrated by both H2AX phosphorylation at 1 h (p-trend <0.0001) and γH2AX dephosphorylation at 3h (p-trend <0.0001). Our study revealed that in addition to SNPs of genes that are well-established players in DSBR, non-DSBR genes, such as the XPA gene that is mainly involved in the nucleotide excision repair pathway, can also influence DSBR in healthy subjects. This suggests that successful DSBR may require both DSBR and non-DSBR mechanisms.

Expression of IFNγR2 mutated in a dileucine internalization motif reinstates IFNγ signaling and apoptosis in human T lymphocytes

In T lymphocytes, the internalization of the R2 chain of the IFN-γ receptor (IFN-γR2) prevents the switching-on of pro-apoptotic and anti-proliferative genes induced by the IFN-γ/STAT1 pathway. In fibroblasts, a critical role of controlling the IFN-γR2 internalization is played by the LI(255-256) intracellular motif. Here we show that, in human malignant T cells, the expression of a mutated IFN-γR2 chain in which the LI(255-256) internalization motif is replaced by two alanines (LI(255-256)AA) induces cell surface accumulation of the receptor and reinstates the cell sensitivity to IFN-γ. In comparison with T cells that expressed wild-type IFN-γR2, cells that expressed the mutated receptor displayed, in response to IFN-γ a sustained activation of STAT1. The activation of this signaling pathway leads to higher induction of MHC class I and FasL expression and triggered apoptosis. Malignant ST4 cells transduced with either wild-type or mutated receptor were able to grow in SCID mice, but only the proliferation of T cells expressing the mutated receptor was inhibited by IFN-γ. Finally, lentiviral-mediated transduction of the mutated receptor in T lymphoblasts from healthy donors reinstated their IFN-γ-dependent apoptosis. As a whole, these data indicate that perturbation of IFN-γR2 internalization by mutating the LI(255-256) motif induces a timely coordinated activation of IFN-γ/STAT1 signaling pathways that leads to the apoptosis of T cells.

A novel defect in mitochondrial p53 accumulation following DNA damage confers apoptosis resistance in Ataxia Telangiectasia and Nijmegen Breakage Syndrome T-cells

We have previously shown that whereas T-cells from normal individuals undergo accumulation of p53 and apoptosis when treated with the genotoxic agent Actinomycin D (ActD), those from Ataxia Telangiectasia (AT) and Nijmegen Breakage Syndrome (NBS) patients resist ActD-induced apoptosis [1]. We have now found similar resistance by the p53-null Jurkat T-cell line and by siRNA p53-knockdown normal T-cells. This evidence that ActD initiates a p53-dependent apoptotic responce prompted us to look for defective p53 accumulation by AT and NBS T-cells. Surprisingly the total p53 level was only slightly reduced compared to normal T cells but its intracellular localization was highly defective: p53 was poorly accumulated in the cytosol and nearly undetectable in mitochondria. In accordance with the dependence of ActD-induced apoptosis on a mitochondrial p53 function, in control T-cells specific inhibition of mitochondrial p53 translocation with μ pifithrin reduced apoptosis by 86%, whereas treatment with α pifithrin, which blocks p53-mediated transcription, had no effect. We also showed that nuclear export is not required for mitochondrial p53 translocation. Observation of an altered p53 ubiquitination pattern and Mdm2 accumulation in ActD-treated AT and NBS T-cells provided a mechanistic link to their defective extranuclear p53 localization. Our results disclose an undescribed defect in mitochondrial p53 accumulation in AT and NBS T-cells that makes them resistant to apoptosis following unrepairable DNA damage.

The cyclin-dependent kinase inhibitor 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole induces nongenotoxic, DNA replication-independent apoptosis of normal and leukemic cells, regardless of their p53 status

BackgroundCurrent chemotherapy of human cancers focuses on the DNA damage pathway to induce a p53-mediated cellular response leading to either G1 arrest or apoptosis. However, genotoxic treatments may induce mutations and translocations that result in secondary malignancies or recurrent disease. In addition, about 50% of human cancers are associated with mutations in the p53 gene. Nongenotoxic activation of apoptosis by targeting specific molecular pathways thus provides an attractive therapeutic approach.MethodsNormal and leukemic cells were evaluated for their sensitivity to 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) through cell viability and caspase activation tests. The apoptotic pathway induced by DRB was analysed by immunfluorescence and immunoblot analysis. H2AX phosphorylation and cell cycle analysis were performed to study the dependance of apoptosis on DNA damage and DNA replication, respectively. To investigate the role of p53 in DRB-induced apoptosis, specific p53 inhibitors were used. Statistical analysis on cell survival was performed with the test of independence.ResultsHere we report that DRB, an inhibitor of the transcriptional cyclin-dependent kinases (CDKs) 7 and 9, triggers DNA replication-independent apoptosis in normal and leukemic human cells regardless of their p53 status and without inducing DNA damage. Our data indicate that (i) in p53-competent cells, apoptosis induced by DRB relies on a cytosolic accumulation of p53 and subsequent Bax activation, (ii) in the absence of p53, it may rely on p73, and (iii) it is independent of ATM and NBS1 proteins. Notably, even apoptosis-resistant leukemic cells such as Raji were sensitive to DRB.ConclusionOur results indicate that DRB represents a potentially useful cancer chemotherapeutic strategy that employs both the p53-dependent and -independent apoptotic pathways without inducing genotoxic stress, thereby decreasing the risk of secondary malignancies.