Paola Porcedda

DNA Vaccination Against Rat Her-2/Neu p185 More Effectively Inhibits Carcinogenesis Than Transplantable Carcinomas in Transgenic BALB/c Mice

The ability of vaccination with plasmids coding for the extracellular and the transmembrane domain of the product of transforming rat Her-2/neu oncogene (r-p185) to protect against r-p185+ transplantable carcinoma (TUBO) cells and mammary carcinogenesis was evaluated. In normal BALB/c mice, DNA vaccination elicits anti-r-p185 Ab, but only a marginal CTL reactivity, and protects against a TUBO cell challenge. Massive reactive infiltration is associated with TUBO cell rejection. In BALB/c mice transgenic for the rat Her-2/neu gene (BALB-neuT), DNA vaccination elicits a lower anti-r-p185 Ab response, no CTL activity and only incompletely protects against TUBO cells, but markedly hampers the progression of carcinogenesis. At 33 wk of age, when control BALB-neuT mice display palpable tumors in all mammary glands, about 60% of immunized mice are tumor free, and tumor multiplicity is markedly reduced. Tumor-free mammary glands still display the atypical hyperplasia of the early stages of carcinogenesis, and a marked down-modulation of r-p185, along with a massive reactive infiltrate. However, BALB-neuT mice protected against mammary carcinogenesis fail to efficiently reject a TUBO cell challenge. This suggests that the mechanisms required for the rejection of transplantable tumors may not coincide with those that inhibit the slow progression of carcinogenesis.

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.

Insertion of the DNA for the 163-171 peptide of IL1beta enables a DNA vaccine encoding p185(neu) to inhibit mammary carcinogenesis in Her-2/neu transgenic BALB/c mice.

An assessment was made of the effectiveness of DNA vaccination in prevention of the mammary adenocarcinomas of BALB/c female mice transgenic for the activated rat Her-2/neu oncogene. Atypical hyperplasia is evident in their mammary glands when they are 6 weeks old and in situ carcinoma by the 13th week. Palpable invasive carcinomas appear around the 17th week and are always evident in all 10 glands by the 33rd week. Intramuscular vaccinations with 100 μg plasmid DNA encoding the extracellular domain of the Her-2/neu p185 (ECD) performed at the 6th, 12th, 18th and 24th week provided no significant protection, whereas those ECD plasmids in which the DNA coding for the immunomodulatory 163–171 (VQGEESNDK) nonapeptide of human IL1β (ECD-IL1βp) had been inserted both delayed carcinogenesis and reduced tumor multiplicity. This reduction was associated with a marked immune-inflammatory reaction and a conspicuous leukocyte infiltrate located in the stroma surrounding the hyperplastic mammary ductul-alveolar structures. It was also directly correlated with a high anti-p185neu antibody production and an immunoglobulin switch to IgG2a and IgA. No anti-p185neu cytotoxic response was found. No significant protection was obtained when the DNA coding for the non-active peptide 189–197 of IL1β was inserted.

High Basal γH2AX Levels Sustain Self‐Renewal of Mouse Embryonic and Induced Pluripotent Stem Cells

Phosphorylation of histone H2AX (γH2AX) is known to be the earliest indicator of DNA double‐strand breaks. Recently, it has been shown that mouse embryonic stem cells (mESCs) have very high basal levels of γH2AX, even when they have not been exposed to genotoxic agents. As the specialized role of high basal γH2AX levels in pluripotent stem cells is still debated, we investigated whether H2AX phosphorylation is important in maintaining self‐renewal of these cells. Here, we report that not only mESCs but also mouse‐induced pluripotent stem cells (miPSCs), have high basal levels of γH2AX. We show that basal γH2AX levels decrease upon ESC and iPSC differentiation and increase when the cells are treated with self‐renewal‐enhancing small molecules. We observe that self‐renewal activity is highly compromised in H2AX−/− cells and that it can be restored in these cells through reconstitution with a wild‐type, but not a phospho‐mutated, H2AX construct. Taken together, our findings suggest a novel function of H2AX that expands the knowledge of this histone variant beyond its role in DNA damage and into a new specialized biological function in mouse pluripotent stem cells. STEM CELLS2012;30:1414–1423

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.

Analysis of Secondary V(D)J Rearrangements in Mature, Peripheral T Cells of Ataxia-Telangiectasia Heterozygotes

Ataxia-telangiectasia (AT) is a rare recessive disease with pleiotropic involvement of the nervous and lymphoid systems. AT heterozygotes have a population frequency of about 1%, and although not manifesting any overt clinical symptoms, they have an increased mortality, mainly because of cancer and ischemic heart disease. We and others have described a mature T lymphocyte population with an altered T cell receptor surface expression (“TCR variant”) that reactivates the recombination activating genes (RAG) and is expanded in the blood of patients with AT. In view of the known role of V(D)J recombination in the onset of tumorigenic translocations, we proposed that the increased RAG activity was responsible for the predisposition of AT homozygotes to develop mature-type T leukemia/lymphoma. In the present report, we used cytofluorimetry to quantify the TCR variant population and the memory/naïve T-cell compartments in the blood of AT heterozygotes compared with AT patients and controls. We assessed the expression of different recombinase genes through RT-PCR/oligotyping and cytofluorometric analysis and searched for rearrangement intermediates by ligase-mediated PCR in T-cell lines from four heterozygous carriers. We found the TCR variant population was increased on average 2 × in AT heterozygotes (vs 10 × in homozygotes) compared with controls, and naïve CD4+ T lymphocytes were reduced on average 0.5 × (vs 0.1 × in homozygotes). We were able to demonstrate recombinase gene expression in all four heterozygous T-cell lines, and rearrangement intermediates, indicative of ongoing V(D)J recombination, in two. These rearrangements were compatible with V-gene replacement, a mechanism of receptor editing described for Ig and TCRα genes, to our knowledge not previously documented for TCRβ. In conclusion, we found that RAG reactivation and secondary V(D)J rearrangements, potential risk factors of mature-type leukemia in AT homozygotes, also take place in AT heterozygous carriers and might place this large population fraction at an increased risk of leukemia/lymphoma.

Unsuitability of lymphoblastoid cell lines as surrogate of cryopreserved isolated lymphocytes for the analysis of DNA double-strand break repair activity.

As first task of a comprehensive investigation on DNA repair genotype-phenotype correlations, the suitability of Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs) as surrogate of cryopreserved peripheral blood mononuclear cells (PBMCs) in DNA repair phenotypic assays was evaluated. To this aim the amount of DNA damage induced by gamma-rays and DNA repair capacity were evaluated in unstimulated (G(0)) and mitogen-simulated (G(2)) PBMC from 20 healthy subjects and in EBV-transformed LCL obtained from the same individuals. Phosphorylation of histone H2AX, micronuclei and chromosomal aberrations were the end-points investigated. The results obtained show higher basal frequencies of binucleated cells bearing micronuclei and nucleoplasmic bridge (NPB) in LCL with respect to PBMC, suggesting that EBV transformation may be associated with chromosomal instability. After irradiation, higher levels of micronuclei were induced in G(0)-treated PBMC compared to cycling LCL; conversely, NPB were more frequent in LCL than in PBMC. Moreover, higher levels of chromosomal aberrations were observed in G(2)-treated PBMC compared to LCL. Concerning gamma-H2AX measurements, phosphorylation levels 1h after treatment and dephosphorylation kinetics were basically similar in LCL and in PBMC. However, while Spearmans test showed a strong correlation between the results obtained in replicated experiments with PBMC, high inter-experimental variability and poor reproducibility was observed in the experiments performed with LCL, possibly due to the intrinsic instability of LCL. In summary, both the analysis of gamma-H2AX and the evaluation of chromosome damage highlighted a larger inter-experimental variability in the results obtained with LCL compared to PBMC. Noteworthy, the two set of results proved to lack any significant correlation at the individual level. These results indicate that LCL may be unsuitable for investigating genotype-phenotype correlations with phenotypic DNA repair assays, especially when low impact functional genetic variants are involved.

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.

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.