Salim Abdisalaam

Human Ventricular Unloading Induces Cardiomyocyte Proliferation

BACKGROUND The adult mammalian heart is incapable of meaningful regeneration after substantial cardiomyocyte loss, primarily due to the inability of adult cardiomyocytes to divide. Our group recently showed that mitochondria-mediated oxidative DNA damage is an important regulator of postnatal cardiomyocyte cell cycle arrest. However, it is not known whether mechanical load also plays a role in this process. We reasoned that the postnatal physiological increase in mechanical load contributes to the increase in mitochondrial content, with subsequent activation of DNA damage response (DDR) and permanent cell cycle arrest of cardiomyocytes. OBJECTIVES The purpose of this study was to test the effect of mechanical unloading on mitochondrial mass, DDR, and cardiomyocyte proliferation. METHODS We examined the effect of human ventricular unloading after implantation of left ventricular assist devices (LVADs) on mitochondrial content, DDR, and cardiomyocyte proliferation in 10 matched left ventricular samples collected at the time of LVAD implantation (pre-LVAD) and at the time of explantation (post-LVAD). RESULTS We found that post-LVAD hearts showed up to a 60% decrease in mitochondrial content and up to a 45% decrease in cardiomyocyte size compared with pre-LVAD hearts. Moreover, we quantified cardiomyocyte nuclear foci of phosphorylated ataxia telangiectasia mutated protein, an upstream regulator of the DDR pathway, and we found a significant decrease in the number of nuclear phosphorylated ataxia telangiectasia mutated foci in the post-LVAD hearts. Finally, we examined cardiomyocyte mitosis and cytokinesis and found a statistically significant increase in both phosphorylated histone H3-positive, and Aurora B-positive cardiomyocytes in the post-LVAD hearts. Importantly, these results were driven by statistical significance in hearts exposed to longer durations of mechanical unloading. CONCLUSIONS Prolonged mechanical unloading induces adult human cardiomyocyte proliferation, possibly through prevention of mitochondria-mediated activation of DDR.

Scanning fluorescence correlation spectroscopy techniques to quantify the kinetics of DNA double strand break repair proteins after γ-irradiation and bleomycin treatment

A common feature of DNA repair proteins is their mobilization in response to DNA damage. The ability to visualizing and quantifying the kinetics of proteins localizing/dissociating from DNA double strand breaks (DSBs) via immunofluorescence or live cell fluorescence microscopy have been powerful tools in allowing insight into the DNA damage response, but these tools have some limitations. For example, a number of well-established DSB repair factors, in particular those required for non-homologous end joining (NHEJ), do not form discrete foci in response to DSBs induced by ionizing radiation (IR) or radiomimetic drugs, including bleomycin, in living cells. In this report, we show that time-dependent kinetics of the NHEJ factors Ku80 and DNA-dependent protein kinase catalytic subunits (DNA–PKcs) in response to IR and bleomycin can be quantified by Number and Brightness analysis and Raster-scan Image Correlation Spectroscopy. Fluorescent-tagged Ku80 and DNA–PKcs quickly mobilized in response to IR and bleomycin treatments consistent with prior reports using laser-generated DSBs. The response was linearly dependent on IR dose, and blocking NHEJ enhanced immobilization of both Ku80 and DNA–PKcs after DNA damage. These findings support the idea of using Number and Brightness and Raster-scan Image Correlation Spectroscopy as methods to monitor kinetics of DSB repair proteins in living cells under conditions mimicking radiation and chemotherapy treatments.

RAD51 interconnects between DNA replication, DNA repair and immunity

Abstract RAD51, a multifunctional protein, plays a central role in DNA replication and homologous recombination repair, and is known to be involved in cancer development. We identified a novel role for RAD51 in innate immune response signaling. Defects in RAD51 lead to the accumulation of self-DNA in the cytoplasm, triggering a STING-mediated innate immune response after replication stress and DNA damage. In the absence of RAD51, the unprotected newly replicated genome is degraded by the exonuclease activity of MRE11, and the fragmented nascent DNA accumulates in the cytosol, initiating an innate immune response. Our data suggest that in addition to playing roles in homologous recombination-mediated DNA double-strand break repair and replication fork processing, RAD51 is also implicated in the suppression of innate immunity. Thus, our study reveals a previously uncharacterized role of RAD51 in initiating immune signaling, placing it at the hub of new interconnections between DNA replication, DNA repair, and immunity.

Cytoskeletal Regulation of CD44 Membrane Organization and Interactions with E-selectin

Background: CD44 on neutrophils is a ligand for E-selectin on endothelial cells. Results: CD44 forms actin-dependent clusters by binding to ezrin/radixin/moesin (ERM) proteins and ankyrin. Conclusion: Cytoskeletal interactions regulate CD44 clustering, mobility, and function. Significance: CD44 organizes at nanometer scale on cell surfaces. Interactions of CD44 on neutrophils with E-selectin on activated endothelial cells mediate rolling under flow, a prerequisite for neutrophil arrest and migration into perivascular tissues. How CD44 functions as a rolling ligand despite its weak affinity for E-selectin is unknown. We examined the nanometer scale organization of CD44 on intact cells. CD44 on leukocytes and transfected K562 cells was cross-linked within a 1.14-nm spacer. Depolymerizing actin with latrunculin B reduced cross-linking. Fluorescence resonance energy transfer (FRET) revealed tight co-clustering between CD44 fused to yellow fluorescent protein (YFP) and CD44 fused to cyan fluorescent protein on K562 cells. Latrunculin B reduced FRET-reported co-clustering. Number and brightness analysis confirmed actin-dependent CD44-YFP clusters on living cells. CD44 lacking binding sites for ankyrin and for ezrin/radixin/moesin (ERM) proteins on its cytoplasmic domain (ΔANKΔERM) did not cluster. Unexpectedly, CD44 lacking only the ankyrin-binding site (ΔANK) formed larger but looser clusters. Fluorescence recovery after photobleaching demonstrated increased CD44 mobility by latrunculin B treatment or by deleting the cytoplasmic domain. ΔANKΔERM mobility increased only modestly, suggesting that the cytoplasmic domain engages the cytoskeleton by an additional mechanism. Ex vivo differentiated CD44-deficient neutrophils expressing exogenous CD44 rolled on E-selectin and activated Src kinases after binding anti-CD44 antibody. In contrast, differentiated neutrophils expressing ΔANK had impaired rolling and kinase activation. These data demonstrate that spectrin and actin networks regulate CD44 clustering and suggest that ankyrin enhances CD44-mediated neutrophil rolling and signaling.

Tumor suppressor protein DAB2IP participates in chromosomal stability maintenance through activating spindle assembly checkpoint and stabilizing kinetochore-microtubule attachments

Defects in kinetochore-microtubule (KT-MT) attachment and the spindle assembly checkpoint (SAC) during cell division are strongly associated with chromosomal instability (CIN). CIN has been linked to carcinogenesis, metastasis, poor prognosis and resistance to cancer therapy. We previously reported that the DAB2IP is a tumor suppressor, and that loss of DAB2IP is often detected in advanced prostate cancer (PCa) and is indicative of poor prognosis. Here, we report that the loss of DAB2IP results in impaired KT-MT attachment, compromised SAC and aberrant chromosomal segregation. We discovered that DAB2IP directly interacts with Plk1 and its loss inhibits Plk1 kinase activity, thereby impairing Plk1-mediated BubR1 phosphorylation. Loss of DAB2IP decreases the localization of BubR1 at the kinetochore during mitosis progression. In addition, the reconstitution of DAB2IP enhances the sensitivity of PCa cells to microtubule stabilizing drugs (paclitaxel, docetaxel) and Plk1 inhibitor (BI2536). Our findings demonstrate a novel function of DAB2IP in the maintenance of KT-MT structure and SAC regulation during mitosis which is essential for chromosomal stability.

Replication stress induced site-specific phosphorylation targets WRN to the ubiquitin-proteasome pathway

Faithful and complete genome replication in human cells is essential for preventing the accumulation of cancer-promoting mutations. WRN, the protein defective in Werner syndrome, plays critical roles in preventing replication stress, chromosome instability, and tumorigenesis. Herein, we report that ATR-mediated WRN phosphorylation is needed for DNA replication and repair upon replication stress. A serine residue, S1141, in WRN is phosphorylated in vivo by the ATR kinase in response to replication stress. ATR-mediated WRN S1141 phosphorylation leads to ubiquitination of WRN, facilitating the reversible interaction of WRN with perturbed replication forks and subsequent degradation of WRN. The dynamic interaction between WRN and DNA is required for the suppression of new origin firing and Rad51-dependent double-stranded DNA break repair. Significantly, ATR-mediated WRN phosphorylation is critical for the suppression of chromosome breakage during replication stress. These findings reveal a unique role for WRN as a modulator of DNA repair, replication, and recombination, and link ATR-WRN signaling to the maintenance of genome stability.

COMT val158met polymorphism and molecular alterations in the human dorsolateral prefrontal cortex: Differences in controls and in schizophrenia

The single nucleotide val158met polymorphism in catechol o-methyltransferase (COMT) influences prefrontal cortex function. Working memory, dependent on the dorsolateral prefrontal cortex (DLPFC), has been repeatedly shown to be influenced by this COMT polymorphism. The high activity COMT val isoform is associated with lower synaptic dopamine levels. Altered synaptic dopamine levels are expected to lead to molecular adaptations within the synapse and within DLPFC neural circuitry. In this human post mortem study using high quality DLPFC tissue, we first examined the influence of the COMT val158met polymorphism on markers of dopamine neurotransmission, N-methyl-d-aspartate (NMDA) receptor subunits and glutamatic acid decarboxylase 67 (GAD67), all known to be critical to DLPFC circuitry and function. Next, we compared target gene expression profiles in a cohort of control and schizophrenia cases, each characterized by COMT genotype. We find that the COMT val allele in control subjects is associated with significant upregulation of GluN2A and GAD67 mRNA levels compared to met carriers. Comparisons between control and schizophrenia groups reveal that GluN2A, GAD67 and DRD2 are differentially regulated between diagnostic groups in a genotype specific manner. Chronic antipsychotic treatment in rodents did not explain these differences. These data demonstrate an association between COMTval158met genotype and gene expression profile in the DLPFC of controls, possibly adaptations to maintain DLPFC function. In schizophrenia val homozygotes, these adaptations are not seen and could reflect pathophysiologic mechanisms related to the known poorer performance of these subjects on DLPFC-dependent tasks.

Abstract 2490: Rad51 suppresses innate immune response by blocking MRE11-mediated degradation of newly replicated genome

Purpose of the study: Eukaryotic cells accrue DNA damage as a result of endogenous metabolic activities such as DNA replication, recombination errors or environmental exposures such as ionizing radiation, ultra-violet light and chemical mutagens. Unrepaired DNA damage leads to tumorigenesis. Rad51 is a multifunctional protein that plays a central role in DNA replication and homologous recombination repair. It is known that defects in Rad51 function can cause cancer. The goal of this study is to identify a novel role for Rad51 outside of its known functions in DSB repair and replication fork processing. Methods: Since Rad51 knockout is lethal to cells, we generated an inducible system in which we can down-regulate Rad51 expression in HT1080 cells after Doxycycline treatment. To determine the effect of Rad51-knockdown in global gene expression pattern, we carried out unbiased microarray gene expression analysis and after induction of DNA damage and replication stress by radiation. ssDNA and dsDNA in the cytosolic fractions were quantified using Quant-iT OliGreen and PicoGreen Assay Kits. For cytoplasmic BrdU detection, exponentially grown cells were labeled with BrdU for 18-20 h and then immunostained with anti-BrdU antibody. Additionally, we measured the expression and post-translational modification of proteins involved in innate immune signaling by western blotting. We also employed DNA fiber assay to determine the role of Rad51 in replication fork processing. Results: We found that defects in Rad51 lead to the accumulation of self-DNA in the cytoplasm, triggering a STING-mediated innate immune response after replication stress and DNA damage. Mechanistically, the unprotected newly replicated genome in the absence of Rad51 is degraded by the exonuclease activity of Mre11, and the fragmented nascent DNA accumulates in the cytosol, initiating an innate immune response. Our data revealed that in addition to playing roles in homologous recombination-mediated DNA double-strand break repair and replication fork processing, Rad51 is also implicated in the suppression of innate immunity. Conclusion: Rad51 plays a novel role in immunity outside its known functions in DSB repair and replication fork processing. We discovered that the lack of Rad51 leads to the upregulation of innate immune response pathway genes upon DNA damage and replication induced by irradiation. We found that in the absence of Rad51 the newly replicated genome is degraded by the exonuclease activity of Mre11. We also showed that these degraded nascent DNA fragments are exported to the cytoplasm, triggering innate immune response signaling. Our study reveals a previously unidentified role for Rad51 in triggering an innate immune response, and places Rad51 at the hub of new interconnections between DNA replication, DNA repair, and immunity. Funding: This work was supported by NIH R01AG053341 grants. Citation Format: Kalayarasan Srinivasan, Souparno Bhattacharya, Salim Abdisalaam, Shibani Mukherjee, Asaithamby Aroumougame. Rad51 suppresses innate immune response by blocking MRE11-mediated degradation of newly replicated genome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2490. doi:10.1158/1538-7445.AM2017-2490

Number and brightness analysis to quantify the kinetics of EGFR and its mutants in living cells before and after γ-irradiation

The epidermal growth factor receptor (EGFR), which is over-expressed in tumors cells of epithelial origin is one of the determinants of tumor responses to ionizing radiation. Recently, it has been shown that higher EGFR expression levels lead to higher tumor resistance to radiation therapy through the activation of cell proliferation and survival pathways. In this study, a raster-scan imaging technique known as Number and Brightness (N&B) analysis has been employed to demonstrate the nuclear translocation of EGFR in living cells under a variety of experimental conditions. About 80% of wild type (WT) EGFR translocated to the nucleus after γ-irradiation while the L858R and ▵E746-E750 mutant EGFR did not. Subsequently, the effects of γ-irradiation together with an EGFR-blocking antibody (cetuximab) were monitored simultaneously in the same cell lines expressing EGFR and its mutants. In the combined radiation and cetuximab treatment, about 26 % of WT were translocated to the nucleus, while the L858R and ▵E746-E750 mutant EGFR did not. These results are consistent with findings attained by standard molecular techniques and support the hypothesis that a cytosolic pool of EGFR exists that cannot be accessed by cetuximab and can therefore contribute to treatment resistance.

In vitro binding kinetics of DNA double strand break repair proteins Ku70/80 and DNA-PKcs quantified by fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy

DNA double-strand breaks (DSBs) are one of the most lethal types of DNA damage that occurs in eukaryotic cells. There are two distinct pathways of repairing DSBs, homologous recombination (HR) and non-homologous end joining (NHEJ). In the NHEJ repairing pathway, DSB recognition and repair initiation is directed by the interaction of DNAbinding subunit Ku70/80 heterodimer with the DNA-PK protein catalytic subunit (DNA-PKcs). Mutations in these proteins result in repair stalling and eventual DNA misrepair that may lead to genomic instability. Studying the binding kinetics of these repair proteins is therefore important for understanding the conditions under which DSB repair stalls. Currently open questions are, what is the minimum DNA length that this complex needs to get a foothold onto a DSB and how tightly does DNA-PKcs bind onto the DNA-Ku70/80 complex. Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Cross-Correlation Spectroscopy (FCCS) techniques have the potential to give information about the binding kinetics of DNA-protein and protein-protein interactions at the single-molecule level. In this work, FCS/FCCS measurements were performed to explore the minimum DNA base-pair (bp) length that Ku70/80 needed as a foothold to bind effectively onto the tips of different lengths of double-stranded DNA (dsDNA) fragments that mimic DSBs. 25 bp, 33 bp and 50 bp of dsDNA were used for these experiments and binding was studied as a function of salt concentration in solution. It was found that the 25 bp binding was weak even at physiological salt concentrations while the dissociation constant (Kd) remained constant for 33 and 50 bp dsDNA strand lengths. These studies indicated that the minimum binding length for the Ku70/8 is in the vicinity of 25 bp. The specificity of binding of Ku70/80 was proven by competitive binding FCCS experiments between Cy5-labeled DNA, GFP-Ku70/80 and titrations of unlabeled Ku70/80. Finally, using FCCS it was possible to estimate the apparent Kd for DNA-PKcs binding to the DNA-Ku70/80 complex and the induced dissociation of DNA-PKcs from that complex by phosphorylation was observed in real time.