DNA damage stress: Cui prodest?

DNA is an entity shielded by mechanisms that maintain genomic stability and are essential for living cells; however, DNA is constantly subject to assaults from the environment throughout the cellular life span, making the genome susceptible to mutation and irreparable damage. Cells are prepared to mend such events through cell death as an extrema ratio to solve those threats from a multicellular perspective. However, in cells under various stress conditions, checkpoint mechanisms are activated to allow cells to have enough time to repair the damaged DNA. In yeast, entry into the cell cycle when damage is not completely repaired represents an adaptive mechanism to cope with stressful conditions. In multicellular organisms, entry into cell cycle with damaged DNA is strictly forbidden. However, in cancer development, individual cells undergo checkpoint adaptation, in which most cells die, but some survive acquiring advantageous mutations and selfishly evolve a conflictual behavior. In this review, we focus on how, in cancer development, cells rely on checkpoint adaptation to escape DNA stress and ultimately to cell death

Recent advances on the mechanisms regulating cholangiocyte proliferation and the significance of the neuroendocrine regulation of cholangiocyte pathophysiology

Cholangiocytes are epithelial cells lining the biliary epithelium. Cholangiocytes play several key roles in the modification of ductal bile and are also the target cells in chronic cholestatic liver diseases (i.e., cholangiopathies) such as PSC, PBC, polycystic liver disease (PCLD) and cholangiocarcinoma (CCA). During these pathologies, cholangiocytes (which in normal condition are in a quiescent state) begin to proliferate acquiring phenotypes of neuroendocrine cells, and start secreting different cytokines, growth factors, neuropeptides, and hormones to modulate cholangiocytes proliferation and interaction with the surrounding environment, trying to reestablish the balance between proliferation/loss of cholangiocytes for the maintenance of biliary homeostasis. The purpose of this review is to summarize the recent findings on the mechanisms regulating cholangiocyte proliferation and the significance of the neuroendocrine regulation of cholangiocyte pathophysiology. To clarify the mechanisms of action of these factors we will provide new potential strategies for the management of chronic liver diseases

Bloom's syndrome protein is required for correct relocalization of RAD50/MRE11/NBS1 complex after replication fork arrest

Bloom's syndrome (BS) is a rare genetic disorder characterized by a broad range of symptoms and, most importantly, a predisposition to many types of cancers. Cells derived from patients with BS exhibit an elevated rate of somatic recombination and hypermutability, supporting a role for bleomycin (BLM) in the maintenance of genomic integrity. BLM is thought to participate in several DNA transactions, the failure of which could give raise to genomic instability, and to interact with many proteins involved in replication, recombination, and repair. In this study, we show that BLM function is specifically required to properly relocalize the RAD50/MRE11/NBS1 (RMN) complex at sites of replication arrest, but is not essential in the activation of BRCA1 either after stalled replication forks or γ-rays. We also provide evidence that BLM is phosphorylated after replication arrest in an Ataxia and RAD3-related protein (ATR)-dependent manner and that phosphorylation is not required for subnuclear relocalization. Therefore, in ATR dominant negative mutant cells, the assembly of the RMN complex in nuclear foci after replication blockage is almost completely abolished. Together, these results suggest a relationship between BLM, ATR, and the RMN complex in the response to replication arrest, proposing a role for BLM protein and RMN complex in the resolution of stalled replication forks

Contribution of resident stem cells to liver and biliary tree regeneration in human diseases

Two distinct stem/progenitor cell populations of biliary origin have been identified in the adult liver and biliary tree. Hepatic Stem/progenitor Cells (HpSCs) are bipotent progenitor cells located within the canals of Hering and can be differentiated into mature hepatocytes and cholangiocytes; Biliary Tree Stem/progenitor Cells (BTSCs) are multipotent stem cells located within the peribiliary glands of large intrahepatic and extrahepatic bile ducts and able to differentiate into hepatic and pancreatic lineages. HpSCs and BTSCs are endowed in a specialized niche constituted by supporting cells and extracellular matrix compounds. The actual contribution of these stem cell niches to liver and biliary tree homeostatic regeneration is marginal; this is due to the high replicative capabilities and plasticity of mature parenchymal cells (i.e., hepatocytes and cholangiocytes). However, the study of human liver and biliary diseases disclosed how these stem cell niches are involved in the regenerative response after extensive and/or chronic injuries, with the activation of specific signaling pathways. The present review summarizes the contribution of stem/progenitor cell niches in human liver diseases, underlining mechanisms of activation and clinical implications, including fibrogenesis and disease progression

How to Manage Self-Poisoning With Baclofen in Alcohol Use Disorder? Current Updates

Specialists in addiction medicine continue to debate whether baclofen is still indicated to treat alcohol use disorders in view of conflicting results as to its efficacy. This review summarizes current knowledge on self-poisoning with baclofen focusing of alcohol-use disorder in order to provide an overview of the reliable scientific knowledge on management of such an intoxication. Moreover, as alcohol-dependent patients experience many psychiatric co-morbidities, the risk in suicide attempt using baclofen seems real. Numerous studies have suggested that patients given daily-doses of baclofen higher than 80 mg/day are more likely to attempt suicides than others. Following an ingestion of a large amount of baclofen, central nervous system depression is usually observed. Seizures require the patient to be admitted in intensive care unit and should be treated like other toxicological seizures. Cardiac complications include prolonged QTc interval, degree heart block, premature atrial contractions, and supraventricular tachycardia, hypotension and bradycardia. In cases of intoxication, the elimination half-life of baclofen may last between 12 and 36 h post-overdose and renal failure is known to delay its clearance. Rarely measured in clinical practice, the toxic level of baclofen blood level ranges from 1.1 to 3.5 mg/l, and coma or fatal intoxication are observed from 6 to 9.6 mg/l. Baclofen withdrawal has been observed but making the diagnosis of withdrawal in case of suspected self-poisoning is difficult as baclofen intoxication and baclofen withdrawal share many clinical signs. Admission to hospital to manage of suicide attempt with baclofen is mandatory and should not be limited to baclofen alone. It needs to include other aspects of the overall care of patients with alcohol disorders (psychological and psychosocial interventions, management of comorbid mental conditions and physical complications)

Vasopressin regulates the growth of the biliary epithelium in polycystic liver disease

The neurohypophysial hormone arginine vasopressin (AVP) acts by three distinct receptor subtypes: V1a, V1b, and V2. In the liver, AVP is involved in ureogenesis, glycogenolysis, neoglucogenesis and regeneration. No data exist about the presence of AVP in the biliary epithelium. Cholangiocytes are the target cells in a number of animal models of cholestasis, including bile duct ligation (BDL), and in several human pathologies, such as polycystic liver disease characterized by the presence of cysts that bud from the biliary epithelium. In vivo, liver fragments from normal and BDL mice and rats as well as liver samples from normal and ADPKD patients were collected to evaluate: (i) intrahepatic bile duct mass by immunohistochemistry for cytokeratin-19; and (ii) expression of V1a, V1b and V2 by immunohistochemistry, immunofluorescence and real-time PCR. In vitro, small and large mouse cholangiocytes, H69 (non-malignant human cholangiocytes) and LCDE (human cholangiocytes from the cystic epithelium) were stimulated with vasopressin in the absence/presence of AVP antagonists such as OPC-31260 and Tolvaptan, before assessing cellular growth by MTT assay and cAMP levels. Cholangiocytes express V2 receptor that was upregulated following BDL and in ADPKD liver samples. Administration of AVP increased proliferation and cAMP levels of small cholangiocytes and LCDE cells. We found no effect in the proliferation of large mouse cholangiocytes and H69 cells. Increases were blocked by preincubation with the AVP antagonists. These results showed that AVP and its receptors may be important in the modulation of the proliferation rate of the biliary epithelium

Phosphorylation by CK2 regulates MUS81/EME1 in mitosis and after replication stress

The MUS81 complex is crucial for preserving genome stability through the resolution of branched DNA intermediates in mitosis. However, untimely activation of the MUS81 complex in S-phase is dangerous. Little is known about the regulation of the human MUS81 complex and how deregulated activation affects chromosome integrity. Here, we show that the CK2 kinase phosphorylates MUS81 at Serine 87 in late-G2/mitosis, and upon mild replication stress. Phosphorylated MUS81 interacts with SLX4, and this association promotes the function of the MUS81 complex. In line with a role in mitosis, phosphorylation at Serine 87 is suppressed in S-phase and is mainly detected in the MUS81 molecules associated with EME1. Loss of CK2-dependent MUS81 phosphorylation contributes modestly to chromosome integrity, however, expression of the phosphomimic form induces DSBs accumulation in S-phase, because of unscheduled targeting of HJ-like DNA intermediates, and generates a wide chromosome instability phenotype. Collectively, our findings describe a novel regulatory mechanism controlling the MUS81 complex function in human cells. Furthermore, they indicate that, genome stability depends mainly on the ability of cells to counteract targeting of branched intermediates by the MUS81/EME1 complex in S-phase, rather than on a correct MUS81 function in mitosis

H3 histamine receptor-mediated activation of protein kinase calpha inhibits the growth of cholangiocarcinoma in vitro and in vivo

Histamine regulates functions via four receptors (HRH1, HRH2, HRH3, and HRH4). The D-myo-inositol 1,4,5-trisphosphate (IP(3))/Ca(2+)/protein kinase C (PKC)/mitogen-activated protein kinase pathway regulates cholangiocarcinoma growth. We evaluated the role of HRH3 in the regulation of cholangiocarcinoma growth. Expression of HRH3 in intrahepatic and extrahepatic cell lines, normal cholangiocytes, and human tissue arrays was measured. In Mz-ChA-1 cells stimulated with (R)-(alpha)-(-)-methylhistamine dihydrobromide (RAMH), we measured (a) cell growth, (b) IP(3) and cyclic AMP levels, and (c) phosphorylation of PKC and mitogen-activated protein kinase isoforms. Localization of PKC alpha was visualized by immunofluorescence in cell smears and immunoblotting for PKC alpha in cytosol and membrane fractions. Following knockdown of PKC alpha, Mz-ChA-1 cells were stimulated with RAMH before evaluating cell growth and extracellular signal-regulated kinase (ERK)-1/2 phosphorylation. In vivo experiments were done in BALB/c nude mice. Mice were treated with saline or RAMH for 44 days and tumor volume was measured. Tumors were excised and evaluated for proliferation, apoptosis, and expression of PKC alpha, vascular endothelial growth factor (VEGF)-A, VEGF-C, VEGF receptor 2, and VEGF receptor 3. HRH3 expression was found in all cells. RAMH inhibited the growth of cholangiocarcinoma cells. RAMH increased IP(3) levels and PKC alpha phosphorylation and decreased ERK1/2 phosphorylation. RAMH induced a shift in the localization of PKC alpha expression from the cytosolic domain into the membrane region of Mz-ChA-1 cells. Silencing of PKC alpha prevented RAMH inhibition of Mz-ChA-1 cell growth and ablated RAMH effects on ERK1/2 phosphorylation. In vivo, RAMH decreased tumor growth and expression of VEGF and its receptors; PKC alpha expression was increased. RAMH inhibits cholangiocarcinoma growth by PKC alpha-dependent ERK1/2 dephosphorylation. Modulation of PKC alpha by histamine receptors may be important in regulating cholangiocarcinoma growth. (Mol Cancer Res 2009;7(10):1704-13