Androgen-induced migration in NIH3T3 mouse fibroblasts: role of AR association with Filamin A.

Androgen stimulation triggers rapid activation of various signaling effectors in target cells (Migliaccio et al. 2000; Castoria et al. 2003). This occurs through a direct interaction of classical steroid receptors with proto-oncogenic tyrosine kinase (Src), the p85-regulatory subunit of Phosphatidylinositol 3-kinase (PI3K) and other signaling components. Activation of these pathways fosters cell cycle, prevents apoptosis and leads to cytoskeleton changes in reproductive as well as non reproductive cells. The role of signaling activation in steroid action has been corroborated by findings showing that mouse embryo NIH3T3 fibroblasts express very low amount of classical androgen receptor (AR) which does not activate gene transcription. This receptor, through rapid and extra nuclear action, triggers migration upon stimulation with physiological concentrations (10nM) of the non aromatizable androgen, R1881 (Castoria et al. 2003). By combining different approaches, it was observed that filamin A (FlnA) has an important role in androgen-stimulated migration. In NIH3T3 cells, 10 nM R1881 rapidly induces interaction of AR with filamin A at cytoskeleton. AR/FlnA complex recruits and activates integrin beta1. Androgen assembled AR/FlnA/integrin beta1 complex activates both Ras-related C3 botulinum toxin substrate 1 (Rac1) and focal adhesion kinase (FAK), which independently of each other coordinate cytoskeletal changes and cell motility of fibroblasts. Collected data indicate that AR/FlnA interaction in the extra-nuclear compartment of target cells plays a master role for the access of androgen to the signalling leading to cell migration. Such an interaction may impact human organ development as well as cancer progression and may offer new hints to gain a more tailored therapy of androgen-dependent human cancers

Androgen-Induced Cell Migration: Role of Androgen Receptor/Filamin A Association

Background: Androgen receptor (AR) controls male morphogenesis, gametogenesis and prostate growth as well as development of prostate cancer. These findings support a role for AR in cell migration and invasiveness. However, the molecular mechanism involved in AR-mediated cell migration still remains elusive. Methodology/Principal Findings: Mouse embryo NIH3T3 fibroblasts and highly metastatic human fibrosarcoma HT1080 cells harbor low levels of transcriptionally incompetent AR. We now report that, through extra nuclear action, AR triggers migration of both cell types upon stimulation with physiological concentrations of the androgen R1881. We analyzed the initial events leading to androgen-induced cell migration and observed that challenging NIH3T3 cells with 10 nM R1881 rapidly induces interaction of AR with filamin A (FlnA) at cytoskeleton. AR/FlnA complex recruits integrin beta 1, thus activating its dependent cascade. Silencing of AR, FlnA and integrin beta 1 shows that this ternary complex controls focal adhesion kinase (FAK), paxillin and Rac, thereby driving cell migration. FAK-null fibroblasts migrate poorly and Rac inhibition by EHT impairs motility of androgen-treated NIH3T3 cells. Interestingly, FAK and Rac activation by androgens are independent of each other. Findings in human fibrosarcoma HT1080 cells strengthen the role of Rac in androgen signaling. The Rac inhibitor significantly impairs androgen-induced migration in these cells. A mutant AR, deleted of the sequence interacting with FlnA, fails to mediate FAK activation and paxillin tyrosine phosphorylation in androgen-stimulated cells, further reinforcing the role of AR/FlnA interaction in androgen-mediated motility. Conclusions/Significance: The present report, for the first time, indicates that the extra nuclear AR/FlnA/integrin beta 1 complex is the key by which androgen activates signaling leading to cell migration. Assembly of this ternary complex may control organ development and prostate cancer metastasis

Nation-wide measure of variability in HCMV, EBV and BKV DNA quantification among centers involved in monitoring transplanted patients

BACKGROUND: Inter-laboratory variability in quantifying pathogens involved in viral disease following transplantation may have a great impact on patient care, especially when pre-emptive strategies are used for prevention. OBJECTIVES: The aim of this study was to analyze the variability in quantifying CMV, EBV and BKV DNA from 15 virology laboratories of the Italian Infections in Transplant Working Group (GLaIT) involved in monitoring transplanted patients. STUDY DESIGN: Panels from international Quality Control programs for Molecular Diagnostics (QCMD, year 2012), specific for the detection of CMV in plasma, CMV in whole blood (WB), EBV and BKV were used. Intra- and inter-laboratory variability, as well as, deviations from QCMD consensus values were measured. RESULTS: 100% specificity was obtained with all panels. A sensitivity of 100% was achieved for EBV and BKV evaluations. Three CMV samples, with concentrations below 3 log10 copies/ml, were not detected by a few centers. Mean intra-laboratory variability (% CV) was 1.6 for CMV plasma and 3.0 for CMV WB. Mean inter-laboratory variability (% CV) was below 15% for all of the tested panels. Inter-laboratory variability was higher for CMV in WB with respect to the CMV plasma panel (3.0 vs 1.6% CV). The percentiles 87.7%, 58.6%, 89.6% and 74.7% fell within±0.5 log10 difference of the consensus values for CMV plasma, CMV WB, EBV and BKV panels, respectively. CONCLUSIONS: An acceptable intra- and inter-laboratory variability, in comparison with international standards was observed in this study. However, further harmonization in viral genome quantification is a reasonable goal for the future

Long-term reduction of atrial tachyarrhythmia recurrences in patients paced for bradycardia-tachycardia syndrome

Background: Atrial tachyarrhythmias (AT) are considered progressive diseases. Several rhythm control therapies for treatment of AT have been proposed. Objectives: The Italian AT500 Registry was designed to prospectively study long-term AT evolution in patients paced for the brady-tachy form of sinus node disease (BT-SND). Methods: Three hundred forty-six BT-SND patients received an antitachycardia dual-chamber pace-maker and were followed-up for a minimum of 12 months (median 19 months). Prevention and antitachycardia pacing (ATP) features were enabled in all patients. Results: During the observation period, 224 (65%) patients were treated by antiarrhythmic drugs and 45 (13%) patients were cardioverted. Five patients suffered a stroke, 4 transient ischemic attack, 22 permanent AT, and 98 AT recurrences longer than 7 days. AT mean cycle length changed from 246 to 2 70 ms, and the percentage of patients with AT-related hospitalizations significantly decreased with an annual 28% relative reduction. AT burden and the percentage of patients with AT recurrences longer than 2 days remained constant with time in the overall population but decreased significantly in the subgroup of patients who did not develop permanent AT. High ATP efficacy was associated with an increasingly higher prevention of AT recurrences longer than 2 days. Conclusion: In a long-term observation of BT-SND patients, AT-related hospitalizations decreased significantly and mean AT cycle length increased significantly. The data suggest that rhythm control therapies induce inversion of AT progression. © 2005 Heart Rhythm Society. All rights reserved