G-quadruplex ligand RHPS4 radiosensitizes glioblastoma xenograft in vivo through a differential targeting of bulky differentiated- and stem-cancer cells.

BACKGROUND: Glioblastoma is the most aggressive and most lethal primary brain tumor in the adulthood. Current standard therapies are not curative and novel therapeutic options are urgently required. Present knowledge suggests that the continued glioblastoma growth and recurrence is determined by glioblastoma stem-like cells (GSCs), which display self-renewal, tumorigenic potential, and increased radio- and chemo-resistance. The G-quadruplex ligand RHPS4 displays in vitro radiosensitizing effect in GBM radioresistant cells through the targeting and dysfunctionalization of telomeres but RHPS4 and Ionizing Radiation (IR) combined treatment efficacy in vivo has not been explored so far. METHODS: RHPS4 and IR combined effects were tested in vivo in a heterotopic mice xenograft model and in vitro in stem-like cells derived from U251MG and from four GBM patients. Cell growth assays, cytogenetic analysis, immunoblotting, gene expression and cytofluorimetric analysis were performed in order to characterize the response of differentiated and stem-like cells to RHPS4 and IR in single and combined treatments. RESULTS: RHPS4 administration and IR exposure is very effective in blocking tumor growth in vivo up to 65 days. The tumor volume reduction and the long-term tumor control suggested the targeting of the stem cell compartment. Interestingly, RHPS4 treatment was able to strongly reduce cell proliferation in GSCs but, unexpectedly, did not synergize with IR. Lack of radiosensitization was supported by the GSCs telomeric-resistance observed as the total absence of telomere-involving chromosomal aberrations. Remarkably, RHPS4 treatment determined a strong reduction of CHK1 and RAD51 proteins and transcript levels suggesting that the inhibition of GSCs growth is determined by the impairment of the replication stress (RS) response and DNA repair. CONCLUSIONS: We propose that the potent antiproliferative effect of RHPS4 in GSCs is not determined by telomeric dysfunction but is achieved by the induction of RS and by the concomitant depletion of CHK1 and RAD51, leading to DNA damage and cell death. These data open to novel therapeutic options for the targeting of GSCs, indicating that the combined inhibition of cell-cycle checkpoints and DNA repair proteins provides the most effective means to overcome resistance of GSC to genotoxic insults.

Targeting anandamide metabolism rescues core and associated autistic-like symptoms in rats prenatally exposed to valproic acid.

Autism spectrum disorders (ASD) are characterized by altered sociability, compromised communication and stereotyped/repetitive behaviors, for which no specific treatments are currently available. Prenatal exposure to valproic acid (VPA) is a known, although still underestimated, environmental risk factor for ASD. Altered endocannabinoid activity has been observed in autistic patients, and endocannabinoids are known to modulate behavioral traits that are typically affected in ASD. On this basis, we tested the hypothesis that changes in the endocannabinoid tone contribute to the altered phenotype induced by prenatal VPA exposure in rats, with focus on behavioral features that resemble the core and associated symptoms of ASD. In the course of development, VPA-exposed rats showed early deficits in social communication and discrimination, compromised sociability and social play behavior, stereotypies and increased anxiety, thus providing preclinical proof of the long-lasting deleterious effects induced by prenatal VPA exposure. At the neurochemical level, VPA-exposed rats displayed altered phosphorylation of CB1 cannabinoid receptors in different brain areas, associated with changes in anandamide metabolism from infancy to adulthood. Interestingly, enhancing anandamide signaling through inhibition of its degradation rescued the behavioral deficits displayed by VPA-exposed rats at infancy, adolescence and adulthood. This study therefore shows that abnormalities in anandamide activity may underlie the deleterious impact of environmental risk factors on ASD-relevant behaviors and that the endocannabinoid system may represent a therapeutic target for the core and associated symptoms displayed by autistic patients.

PML nuclear body disruption impairs DNA double-strand break sensing and repair in APL.

Proteins involved in DNA double-strand break (DSB) repair localize within the promyelocytic leukemia nuclear bodies (PML-NBs), whose disruption is at the root of the acute promyelocytic leukemia (APL) pathogenesis. All-trans-retinoic acid (RA) treatment induces PML-RARα degradation, restores PML-NB functions, and causes terminal cell differentiation of APL blasts. However, the precise role of the APL-associated PML-RARα oncoprotein and PML-NB integrity in the DSB response in APL leukemogenesis and tumor suppression is still lacking. Primary leukemia blasts isolated from APL patients showed high phosphorylation levels of H2AX (γ-H2AX), an initial DSBs sensor. By addressing the consequences of ionizing radiation (IR)-induced DSB response in primary APL blasts and RA-responsive and -resistant myeloid cell lines carrying endogenous or ectopically expressed PML-RARα, before and after treatment with RA, we found that the disruption of PML-NBs is associated with delayed DSB response, as revealed by the impaired kinetic of disappearance of γ-H2AX and 53BP1 foci and activation of ATM and of its substrates H2AX, NBN, and CHK2. The disruption of PML-NB integrity by PML-RARα also affects the IR-induced DSB response in a preleukemic mouse model of APL in vivo. We propose the oncoprotein-dependent PML-NB disruption and DDR impairment as relevant early events in APL tumorigenesis.

Heme-based catalytic properties of human serum albumin.

Human serum albumin (HSA): (i) controls the plasma oncotic pressure, (ii) modulates fluid distribution between the body compartments, (iii) represents the depot and carrier of endogenous and exogenous compounds, (iv) increases the apparent solubility and lifetime of hydrophobic compounds, (v) affects pharmacokinetics of many drugs, (vi) inactivates toxic compounds, (vii) induces chemical modifications of some ligands, (viii) displays antioxidant properties, and (ix) shows enzymatic properties. Under physiological and pathological conditions, HSA has a pivotal role in heme scavenging transferring the metal-macrocycle from high- and low-density lipoproteins to hemopexin, thus acquiring globin-like reactivity. Here, the heme-based catalytic properties of HSA are reviewed and the structural bases of drug-dependent allosteric regulation are highlighted.

Characterization of HuH6, Hep3B, HepG2 and HLE liver cancer cell lines by WNT/ß - catenin pathway, microRNA expression and protein expression profile.

Somatic mutations in the genes members of WNT/ß-catenin pathway, especially in CTNNB1 codifying for ß-catenin, have been found to play an important role in hepatocarcinogenesis. The purpose of this work is to characterize alterations of the WNT/ß-catenin signalling pathway, and to study the expression pattern of a panel of microRNAs and proteins potentially involved in the pathogenesis of liver cancer. In this respect, the molecular characterization of the most used liver cancer cell lines HuH6, Hep3B, HepG2, and HLE, could represent a useful tool to identify novel molecular markers for hepatic tumour. A significant modulation of FZD7, NLK, RHOU, SOX17, TCF7L2, TLE1, SLC9A3R1 and WNT10A transcripts was observed in all the four liver cancer cell lines. The analysis of selected microRNAs showed that miR-122a, miR-125a and miR-150 could be suitable candidates to discriminate tumoural versus normal human primary hepatocytes. Finally, Grb-2 protein expression resulted to be increased more than two-fold in liver cancer cell lines in comparison to normal human primary hepatocytes. These advances in the knowledge of molecular mechanisms involved in the pathogenesis of liver cancer may provide new potential biomarkers and molecular targets for the diagnosis and therapy.

A case report of a patient with microcephaly, facial dysmorphism, chromosomal radiosensitivity and telomere length alterations closely resembling "Nijmegen breakage syndrome" phenotype.

Genetic heterogeneity in Nijmegen breakage syndrome (NBS) is highlighted by patients showing clinical and cellular features of NBS but with no mutations in NBS1 and normal levels of nibrin. NBS is an autosomal recessive disorder, whose clinical cellular signs include growth and developmental defects, dysmorphic facies, immunodeficiency, cancer predisposition, chromosomal instability and radiosensitivity. NBS is caused by mutations in the NBS1 gene, whose product is part of the MRE11/RAD50/NBS1 complex involved in the DNA double-strand break (DSB) response pathway. Since the identification of the NBS1 gene, patients with NBS clinical signs, particularly severe congenital microcephaly, are screened for mutations in the NBS1 gene. Further analyses include X-ray-induced chromosome aberrations, telomere analysis, kinetics of DSBs repair, levels of a panel of proteins involved in the maintenance of genetic stability, radiation-induced phosphorylation of various substrates and cell cycle analysis. We describe a patient with a NBS clinical phenotype, chromosomal sensitivity to X-rays but without mutations in the whole NBS1 or in the Cernunnos gene. Enhanced response to irradiation was mediated neither by DSBs rejoining defects nor by the NBS/AT-dependent DNA-damage response pathway. Notably, we found that primary fibroblasts from this patient displayed telomere length alterations. Cross-talk between pathways controlling response to DSBs and those involved in maintaining telomeres has been shown in the present patient. Dissecting the cellular phenotype of radiosensitive NBS-like patients represents a useful tool for the research of new genes involved in the cellular response to DSBs.

Genetic heterogeneity for a Nijmegen breakage-like syndrome.

Nijmegen breakage syndrome (NBS) is a rare, autosomal-recessive chromosome instability disorder characterized by growth and developmental defects, immunodeficiency, high susceptibility to lymphoid malignancies, hypersensitivity to ionizing radiation and aberrant cell-cycle checkpoint control. The disease is caused by mutations in the NBS1 gene, which encodes nibrin, a component of the hMre11-Rad50-p95 complex involved in cellular response to DNA double-strand breaks. Genetic heterogeneity has been suggested in at least two patients with the NBS phenotype, but no mutation in the NBS1 gene; recently, mutations in the gene encoding the enzyme ligase IV have been identified in patients with signs of NBS. We describe a boy with an NBS clinical phenotype but no mutation in either the NBS1 or the LIG4 genes. The analysis of his cellular phenotype reveals chromosome instability and radiosensitivity, but normal cell-cycle checkpoint control. In addition, a literature review was carried out to summarize and compare data of all NBS-like patients reported to date. This case confirms genetic heterogeneity for NBS. We believe that dissecting the clinical and cellular phenotypes of this and other NBS-like patients will provide useful information for the research of new genes involved in cellular response to DNA damage and the assessment of cancer risk in NBS-like syndrome.

G2-phase radiation response in lymphoblastoid cell lines from Nijmegen breakage syndrome.

The relationship between G2-phase checkpoint activation, cytoplasmic cyclin-B1 accumulation and nuclear phosphorylation of p34CDC2 was studied in Nijmegen breakage syndrome cells treated with DNA damaging agents. Experiments were performed on lymphoblastoid cell lines from four Nijmegen breakage syndrome patients with different mutations, as well as on cells from an ataxia telangiectasia patient. Lymphoblastoid cell lines were irradiated with 0.50-2 Gy X-rays and the percentage of G2-phase accumulated cells was evaluated by means of flow cytometry in samples that were harvested 24 h later. The G2-checkpoint activation was analysed by scoring the mitotic index at 2 and 4 h after treatment with 0.5 and 1 Gy X-rays and treatment with the DNA double-strand break inducer calicheamicin-gamma1. Cytoplasmic accumulation of cyclin-B1 was evaluated by means of fluorescence immunostaining or Western blotting, in cells harvested shortly after irradiation with 1 and 2 Gy. The extent of tyrosine 15-phosphorylated p34CDC2 was assessed in the nuclear fractions. Nijmegen breakage syndrome cells showed suboptimal G2-phase checkpoint activation respect to normal cells and were greatly different from ataxia telangiectasia cells. Increased cytoplasmic cyclin-B1 accumulation was detected by both immunofluorescence and immunoblot in normal as well as in Nijmegen breakage syndrome cells. Furthermore, nuclear p34CDC2. phosphorylation was detected at a higher level in Nijmegen breakage syndrome than in ataxia telangiectasia cells. In conclusion, our data do not suggest that failure to activate checkpoints plays a major role in the radiosensitivity of Nijmegen breakage syndrome cells.

[Changes in left ventricular filling after acute increase of afterload in essential arterial hypertension with or without left ventricular hypertrophy]. / Variazioni del riempimento ventricolare sinistro dopo aumento acuto del postcarico nella ipertensione arteriosa essenziale con e senza ipertrofia ventricolare sinistra.

It is debatable whether a direct causal link exists between left ventricular hypertrophy (LVH) and abnormal left ventricular filling (LVF) in arterial hypertension (AH). To assess if LVH is responsible for peculiar patterns of LVF, we examined 30 selected subjects, similar in age and body surface area, by Doppler echocardiography: 10 hypertensives (SBP: 158 +/- 15; DBP: 105 +/- 7 mmHg) with LVH (IM: 154 +/- 19 mg/m2) (Group 1); 10 hypertensives (SBP: 157 +/- 9; DBP: 101 +/- 5 mmHg) without LVH (IM: 105 +/- 17 mg/m2; Group 2); 10 normotensives without familiar history of AH (Group 3). LVF was analyzed in baseline conditions and during transient afterload increase, obtained by isometric exercise (handgrip, HG, for 5 min at 30% of maximal effort). At rest: 1) A wave peak velocity resulted significantly higher (p less than 0.01) in Group 1 (61 +/- 3 cm/s) versus Group 3 (52 +/- 7 cm/s), and E wave peak velocity was significantly lower in Group 1 (58 +/- 13 cm/s) versus Group 3 (74 +/- 10 cm/s); 2) Group 2 values (E wave: 66 +/- 12; A wave: 58 +/- 15 cm/s) were intermediate between those of groups 1 and 3, and with no significant differences; 3) E/A ratio resulted significantly lower both in Group 1 (1.04 +/- 0.2) and Group 2 (1.21 +/- 0.2) versus Group 3 (1.6 +/- 0.4).(ABSTRACT TRUNCATED AT 250 WORDS)