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Doxorubicin induces senescence or apoptosis in rat neonatal cardiomyocytes by regulating the expression levels of the telomere binding factors 1 and 2

Low or high doses of doxorubicin induce either senescence or apoptosis, respectively, in cardiomyocytes. The mechanism by which different doses of doxorubicin may induce different stress-response cellular programs is not well understood. A recent study showed that the level of telomere dysfunction may induce senescence or apoptosis. We investigated the pathways to both apoptosis and senescence in neonatal rat cardiomyocytes and in H9c2 cells exposed to a single pulsed incubation with low or high doses of doxorubicin. High-dose doxorubicin strongly reduces TRF2 expression while enhancing TRF1 expression, and it determines early apoptosis. Low-dose doxorubicin induces downregulation of both TRF2 and TRF1, and it also increases the senescence-associated-beta-galactosidase activity, downregulates the checkpoint kinase Chk2, induces chromosomal abnormalities, and alters the cell cycle. The involvement of TRF1 and TRF2 with apoptosis and senescence was assessed by short interfering RNA interference. The cells maintain telomere dysfunction and a senescent phenotype over time and undergo late death. The increase in the phase>4N and the presence of micronuclei and anaphase bridges indicate that cells die by mitotic catastrophe. p38 modulates TRF2 expression, whereas JNK and cytoplasmic p53 regulate TRF1. Pretreatment with specific inhibitors of MAPKs and p53 may either attenuate the damage induced by doxorubicin or shift the cellular response to stress from senescence to apoptosis. In conclusion, various doses of doxorubicin induce differential regulation of TRF1 and TRF2 through p53 and MAPK, which is responsible for inducing either early apoptosis or senescence and late death due to mitotic catastrophe.

p38 MAPK and JNK Antagonistically Control Senescence and Cytoplasmic p16INK4A Expression in Doxorubicin-Treated Endothelial Progenitor Cells

Patients treated with low-dose anthracyclines often show late onset cardiotoxicity. Recent studies suggest that this form of cardiotoxicity is the result of a progenitor cell disease. In this study we demonstrate that Cord Blood Endothelial Progenitor Cells (EPCs) exposed to low, sub-apoptotic doses of doxorubicin show a senescence phenotype characterized by increased SA-b-gal activity, decreased TRF2 and chromosomal abnormalities, enlarged cell shape, and disarrangement of F-actin stress fibers accompanied by impaired migratory ability. P16 INK4A localizes in the cytoplasm of doxorubicin-induced senescent EPCs and not in the nucleus as is the case in EPCs rendered senescent by different stimuli. This localization together with the presence of an arrest in G2, and not at the G1 phase boundary, which is what usually occurs in response to the cell cycle regulatory activity of p16INK4A, suggests that doxorubicin-induced p16 INK4A does not regulate the cell cycle, even though its increase is closely associated with senescence. The effects of doxorubicin are the result of the activation of MAPKs p38 and JNK which act antagonistically. JNK attenuates the senescence, p16 INK4A expression and cytoskeleton remodeling that are induced by activated p38. We also found that conditioned medium from doxorubicin-induced senescent cardiomyocytes does not attract untreated EPCs, unlike conditioned medium from apoptotic cardiomyocytes which has a strong chemoattractant capacity. In conclusion, this study provides a better understanding of the senescence of doxorubicin-treated EPCs, which may be helpful in preventing and treating late onset cardiotoxicity.

Wolfram syndrome: new mutations, different phenotype.

Background Wolfram Syndrome (WS) is an autosomal recessive neurodegenerative disorder characterized by Diabetes Insipidus, Diabetes Mellitus, Optic Atrophy, and Deafness identified by the acronym “DIDMOAD”. The WS gene, WFS1, encodes a transmembrane protein called Wolframin, which recent evidence suggests may serve as a novel endoplasmic reticulum calcium channel in pancreatic β-cells and neurons. WS is a rare disease, with an estimated prevalence of 1/550.000 children, with a carrier frequency of 1/354. The aim of our study was to determine the genotype of WS patients in order to establish a genotype/phenotype correlation. Methodology/Principal Findings We clinically evaluated 9 young patients from 9 unrelated families (6 males, 3 females). Basic criteria for WS clinical diagnosis were coexistence of insulin-treated diabetes mellitus and optic atrophy occurring before 15 years of age. Genetic analysis for WFS1 was performed by direct sequencing. Molecular sequencing revealed 5 heterozygous compound and 3 homozygous mutations. All of them were located in exon 8, except one in exon 4. In one proband only an heterozygous mutation (A684V) was found. Two new variants c.2663 C>A and c.1381 A>C were detected. Conclusions/Significance Our study increases the spectrum of WFS1 mutations with two novel variants. The male patient carrying the compound mutation [c.1060_1062delTTC]+[c.2663 C>A] showed the most severe phenotype: diabetes mellitus, optic atrophy (visual acuity 5/10), deafness with deep auditory bilaterally 8000 Hz, diabetes insipidus associated to reduced volume of posterior pituitary and pons. He died in bed at the age of 13 years. The other patient carrying the compound mutation [c.409_424dup16]+[c.1381 A>C] showed a less severe phenotype (DM, OA).

Sublethal Doses of an Anti-erbB2 Antibody Leads to Death by Apoptosis in Cardiomyocytes Sensitized by Low Prosenescent Doses of Epirubicin: The Protective Role of Dexrazoxane

The cardiotoxic synergism resulting from the sequential treatment with anthracyclines and trastuzumab has been attributed to the trastuzumab-induced loss of the erbB2-related functions that serve as a salvage pathway against the damaging effects of anthracyclines. Cellular senescence is a novel mechanism of cardiotoxicity induced by subapoptotic doses of anthracyclines. After having identified prosenescent and proapoptotic doses of epirubicin and rat MAb c-erbB2/Her-2/neu Ab-9 clone B10 (B10), an anti-erbB2 monoclonal antibody, we investigated the effects of the sequential treatment with prosenescent doses of both drugs on H9c2 cells and neonatal rat cardiomyocytes pretreated with or without the cardioprotective agent dexrazoxane. Cells were analyzed by senescence-associated β-galactosidase, single-stranded DNA, annexin/propidium double staining, F-actin, and mitochondrial transmembrane potential. ErbB2 expression levels, AKT activation, and the effects of the inhibition of nicotinamide adenine dinucleotide phosphate oxidase [NAD(P)H oxidase] and phosphoinositide-3-OH kinase (PI3K) were also assessed. Data demonstrate that 1) the toxic effects of epirubicin mainly occur through NAD(P)H oxidase activation; 2) the erbB2 overexpression induced by epirubicin is a redox-sensitive mechanism largely dependent on NAD(P)H oxidase; 3) the loss of erbB2-related functions caused by B10 determines marginal cellular changes in untreated cells, but causes massive death by apoptosis in cells previously exposed to a prosenescent dose of epirubicin, 4) dexrazoxane promotes survival pathways, as demonstrated by the activation of Akt and the PI3K-dependent erbB2 overexpression; and 5) it also prevents epirubicin-induced senescence and renders epirubicin-treated cells more resistant to treatment with B10. Data underline the importance of NAD(P)H oxidase in epirubicin-induced cardiotoxicity and shed new light on the protective mechanisms of dexrazoxane.

Mother and daughter carrying the same KCNJ11 mutation but with a different response to switching from insulin to sulfonylurea

KCNJ11 gene mutations are related to permanent neonatal diabetes mellitus (PNDM). Glycemic stability minimizes the risk of complications. Sulfonylureas (SU) are the proven best therapeutic option. We report a 18-month follow-up of switching from insulin to SU in a mother and her daughter with PNDM due to KCNJ11 mutation.

Estimation of genetic risk for Type 1 diabetes mellitus in newborns on dried blood spot

Background: The main contribution to genetic susceptibility for Type 1 Diabetes Mellitus (T1 DM) is conferred by the Human Leukocyte Antigens (HLA). Aim: We evaluated the feasibility of large scale screening on Dried Blood Spot (DBS) to estimate the genetic risk for T1 DM in newborns. Subjects and methods: Peripheral blood DBS samples from 256 newborns, were genotyped for HLA DRB1 and DQB1 alleles identification by a commercially available assay based on a dissociation enhancer lanthanide fluorescence system available in many newborn screening laboratories. Results were compared with those obtained in two wide multicentric studies on cord blood (DIABFIN and PREVEFIN). Results: Genotyping on DBS revealed 6 subjects at high risk for T1DM, 99 at moderate risk for T1DM and the remaining at low risk for T1DM. We found 100% concordance between both techniques for HLA-DQB1 and DRB1 determination, confirming the feasibility of large scale screening on DBS. Conclusions: DBSs represent a resource for future studies about new genetics markers. This assay for estimate the genetic risk of T1DM on DBS showed an excellent sensitivity, specificity and accuracy compared with conventional techniques. Moreover, this assay resulted less expensive, and it could be easily performed on material already collected for newborn screening programs.

Cell-cell bond modulates vascular smooth muscle cell responsiveness to Angiotensin II.

Cell attachment is provided by cell-matrix and cell-cell bonds, and acts as a regulator of vascular smooth muscle cell (VSMC) survival, activity and homeostasis, as well as of VSMCs response to pathogenic stimuli. In this work we elicited an exclusive cell-cell contact by culturing A7r5 VSMCs on agarose-coated wells to form floating cell clusters, and we demonstrated that a steady state with a reduced response to the vasoactive peptide Angiotensin II (ATII) was induced. We found that clustered VSMCs showed subcortical stabilization of beta-catenin and Caveolin 1 (Cav1), unlike adherent confluent counterparts. We demonstrated that beta-catenin and Cav1 stabilization at the membrane level hampers the molecular cross-talk induced by ATII-activated AT1 receptor (AT1R), thereby impeding the phosphorylation of Cav1 and IGF1R, the NADPH oxidase activity, and counteracting ATII-dependent hypertrophy. Thus, elective cell-cell bond might modulate the proatherogenic activity of ATII, reducing the adverse vascular remodelling associated with AT1R activation.

Neonatal Diabetes Caused by Pancreatic Agenesia: Which other genes should be used for diagnosis?

Permanent neonatal diabetes (PND) is an extremely rare condition. Few cases of PND caused by pancreas-development failure have been described, and only a few genetic causes have been identified so far (1). We describe a boy with PND caused by pancreas agenesis, currently age 7 years. He is the first child of healthy, unrelated parents. He was born at 35 weeks of gestation, with severe intrauterine growth restriction (weight 1,620 g; length 45 cm). In the first day of life he developed severe hyperglycemia, and neonatal diabetes was diagnosed and treated with insulin, initially intravenously and then subcutaneously. Atrial septal defect was diagnosed in absence of …