Modulation of Cellular Stemness for Enhanced Fat Grafting.

Low volumetric retention limits the utility of fat grafting. Although inclusion of stem cells and platelet-rich plasma have been proposed to enhance graft retention, accumulating evidence has failed to show a clear benefit. Here, we propose a strategy to pharmacologically enhance stemness of stem and progenitor cell populations in fat grafts to promote increased volume retention and tissue health. We also propose how to integrate stemness-promoting and differentiation-promoting therapies such as platelet-rich plasma, and viability promoting therapies within the common fat grafting workflow to achieve optimal fat grafting results.

Analysis of Major and Minor Pedicles in Flap Perfusion by Computational Fluid Dynamics.

Background: Type 2 muscle flaps are characterized by major and minor pedicles, such that the minor pedicle is unreliable, and the major pedicle is a requirement for the success of the flap. The role of the minor pedicle, beyond the decreased caliber and decreased vascular territory in comparison to the major pedicle, is poorly understood. We sought to model the fluid dynamics of a model flap containing a major and minor pedicle to understand differences between the pedicles and the implications on perfusion. Methods: We first generated a computer-assisted design model of a type 2 flap with a major and minor pedicle. We then performed computational fluid dynamics to analyze velocities and flow within the pedicles and flap. Results: In our investigation, we found that the flow velocity within the major pedicle was higher than the minor pedicle, indicative of decreased resistance to flow. Concomitantly, we found decreased pressures within the major pedicle, reflecting decreasing resistance to flow. Interestingly, we found increased kinematic viscosity in flap areas supplied by the minor pedicle, suggesting decreased flow rates and increased resistance. Conclusions: We identified that the major pedicle has increased flow velocity, decreased resistance, and decreased kinematic viscosity, suggesting its dominance in maintaining flap perfusion. Our study also identifies computational fluid dynamics as a powerful tool in studying flap perfusion dynamics.

LKB1 signaling and patient survival outcomes in hepatocellular carcinoma.

The liver is a major organ that is involved in essential biological functions such as digestion, nutrient storage, and detoxification. Furthermore, it is one of the most metabolically active organs with active roles in regulating carbohydrate, protein, and lipid metabolism. Hepatocellular carcinoma is a cancer of the liver that is associated in settings of chronic inflammation such as viral hepatitis, repeated toxin exposure, and fatty liver disease. Furthermore, liver cancer is the most common cause of death associated with cirrhosis and is the 3rd leading cause of global cancer deaths. LKB1 signaling has been demonstrated to play a role in regulating cellular metabolism under normal and nutrient deficient conditions. Furthermore, LKB1 signaling has been found to be involved in many cancers with most reports identifying LKB1 to have a tumor suppressive role. In this review, we use the KMPlotter database to correlate RNA levels of LKB1 signaling genes and hepatocellular carcinoma patient survival outcomes with the hopes of identifying potential biomarkers clinical usage. Based on our results STRADß, CAB39L, AMPKα, MARK2, SIK1, SIK2, BRSK1, BRSK2, and SNRK expression has a statistically significant impact on patient survival.

Unfolded protein response and angiogenesis in malignancies.

Cellular stress, arising from accumulation of unfolded proteins, occurs frequently in rapidly proliferating cancer cells. This cellular stress, in turn, activates the unfolded protein response (UPR), an interconnected set of signal transduction pathways that alleviate the proteostatic stress. The UPR is implicated in cancer cell survival and proliferation through upregulation of pro-tumorigenic pathways that ultimately promote malignant metabolism and neoangiogenesis. Here, we reviewed mechanisms of signaling crosstalk between the UPR and angiogenesis pathways, as well as transmissible ER stress and the role in tumor growth and development. To characterize differences in UPR and UPR-mediated angiogenesis in malignancy, we employed a data mining approach using patient tumor data from The Cancer Genome Atlas (TCGA). The analysis of TCGA revealed differences in UPR between malignant samples versus their non-malignant counterparts.

Targeting TRAF3IP2 inhibits angiogenesis in glioblastoma.

Increased vascularization, also known as neoangiogenesis, plays a major role in many cancers, including glioblastoma multiforme (GBM), by contributing to their aggressive growth and metastasis. Although anti-angiogenic therapies provide some clinical improvement, they fail to significantly improve the overall survival of GBM patients. Since various pro-angiogenic mediators drive GBM, we hypothesized that identifying targetable genes that broadly inhibit multiple pro-angiogenic mediators will significantly promote favorable outcomes. Here, we identified TRAF3IP2 (TRAF3-interacting protein 2) as a critical regulator of angiogenesis in GBM. We demonstrated that knockdown of TRAF3IP2 in an intracranial model of GBM significantly reduces vascularization. Targeting TRAF3IP2 significantly downregulated VEGF, IL6, ANGPT2, IL8, FZGF2, PGF, IL1ß, EGF, PDGFRB, and VEGFR2 expression in residual tumors. Our data also indicate that exogenous addition of VEGF partially restores angiogenesis by TRAF3IP2-silenced cells, suggesting that TRAF3IP2 promotes angiogenesis through VEGF- and non-VEGF-dependent mechanisms. These results indicate the anti-angiogenic and anti-tumorigenic potential of targeting TRAF3IP2 in GBM, a deadly cancer with limited treatment options.

Biallelic, Selectable, Knock-in Targeting of CCR5 via CRISPR-Cas9 Mediated Homology Directed Repair Inhibits HIV-1 Replication.

Transplanting HIV-1 positive patients with hematopoietic stem cells homozygous for a 32 bp deletion in the chemokine receptor type 5 (CCR5) gene resulted in a loss of detectable HIV-1, suggesting genetically disrupting CCR5 is a promising approach for HIV-1 cure. Targeting the CCR5-locus with CRISPR-Cas9 was shown to decrease the amount of CCR5 expression and HIV-1 susceptibility in vitro as well as in vivo. Still, only the individuals homozygous for the CCR5-Δ32 frameshift mutation confer complete resistance to HIV-1 infection. In this study we introduce a mechanism to target CCR5 and efficiently select for cells with biallelic frameshift insertion, using CRISPR-Cas9 mediated homology directed repair (HDR). We hypothesized that cells harboring two different selectable markers (double positive), each in one allele of the CCR5 locus, would carry a frameshift mutation in both alleles, lack CCR5 expression and resist HIV-1 infection. Inducing double-stranded breaks (DSB) via CRISPR-Cas9 leads to HDR and integration of a donor plasmid. Double-positive cells were selected via fluorescence-activated cell sorting (FACS), and CCR5 was analyzed genetically, phenotypically, and functionally. Targeted and selected populations showed a very high frequency of mutations and a drastic reduction in CCR5 surface expression. Most importantly, double-positive cells displayed potent inhibition to HIV-1 infection. Taken together, we show that targeting cells via CRISPR-Cas9 mediated HDR enables efficient selection of mutant cells that are deficient for CCR5 and highly resistant to HIV-1 infection.

Increased Efficiency for Biallelic Mutations of the CCR5 Gene by CRISPR-Cas9 Using Multiple Guide RNAs As a Novel Therapeutic Option for Human Immunodeficiency Virus.

CCR5 is a coreceptor of human immunodeficiency virus type 1 (HIV-1). Transplantation of hematopoietic stem cells homozygous for a 32-bp deletion in CCR5 resulted in a loss of detectable HIV-1 in two patients, suggesting that genetic strategies to knockout CCR5 expression would be a promising gene therapy approach for HIV-1-infected patients. In this study, we targeted CCR5 by CRISPR-Cas9 with a single-guide (sgRNA) and observed 35% indel frequency. When we expressed hCas9 and two gRNAs, the Surveyor assay showed that Cas9-mediated cleavage was increased by 10% with two sgRNAs. Genotype analysis on individual clones showed 11 of 13 carried biallelic mutations, where 4 clones had frameshift (FS) mutations. Taken together, these results indicate that the efficiency of biallelic FS mutations and the knockout of the CCR5 necessary to prevent viral replication were significantly increased with two sgRNAs. These studies demonstrate the knockout of CCR5 and the potential for translational development.

Anti-Cancer and Anti-Bacterial Effects of Terfezia boudieri-Derived Silver Nanoparticles.

BACKGROUND: The use of nanoparticles has markedly increased in biomedical sciences. The silver nanoparticles (AgNPs) have been investigated for their applicability to deliver chemotherapeutic/antibacterial agents to treat cancer or infections disease. However, the existing chemical and physical methods of synthesizing AgNPs are considered inefficient, expensive and toxic. METHODS: Natural products have emerged as viable candidates for nanoparticle production, including the use of Terfezia boudieri (T. boudieri), a member of the edible truffle family. Accordingly, our goal was to synthesize AgNPs using an aqueous extract of T. boudieri (green synthesized AgNPs). Since certain infectious agents are linked to cancer, we investigated their potential as anti-cancer and antibacterial agents. RESULTS: The synthesis of AgNPs was confirmed by the presence of an absorption peak at 450nm by spectroscopy. The physico-chemical properties of green synthesized AgNPs were analyzed by UV-Vis, FT-IR, XRD, SEM, and TEM. In addition, their potential to inhibit cancer cell (proliferation and the growth of infectious bacteria were investigated. CONCLUSION: The size of nanoparticles ranged between 20-30nm. They exerted significant cytotoxicity and bactericidal effects in a concentration and time-dependent manner compared to T. boudieri extract alone. Interestingly, the synthesis of smaller AgNPs was correlated with longer synthesis time and enhanced cytotoxic and bactericidal properties.

A Novel function of Nebivolol: Stimulation of Adipose-derived Stem Cell Proliferation and Inhibition of Differentiation.

Tissue engineering is limited by the time of culture expansion of cells needed for scaffold seeding. Thus, a simple means of accelerated stem cell proliferation could represent a significant advance. Here, Nebivolol was investigated for its effect on the replicative capacity of adipose-derived stem cells (ASCs). This study indicates that the number of ASCs with Nebivolol treatment showed a significant population increase of 51.5% compared to untreated cells (p<0.01). Cell cycle analysis showed a significant decrease in the percentage of ASCs in G1 phase with Nebivolol treatment compared to untreated cells (p<0.01), suggesting that Nebivolol shortens the G1 phase of ASCs, resulting in a faster proliferative rate. Furthermore, our results showed that Nebivolol significantly increased colony-forming units of ASCs (p<0.01). Despite increasing ASC proliferative potential, we showed that Nebivolol has an inhibitory effect on adipogenic and osteogenic differentiation potential as indicated by significantly reduced expression of CCAAT Enhancer Binding Protein alpha (P<0.01) and lipoprotein lipase (P<0.01) and inhibited activity of alkaline phosphatase (P<0.01), respectively. Taken together, these results showed that Nebivolol accelerated ASC proliferation through shortening G1 phase, while inhibiting both adipogenic and osteogenic potentials of ASCs. These data identify a novel and simple approach to accelerate stem cell expansion in vitro before cell differentiation.

Targeting TRAF3IP2, Compared to Rab27, is More Effective in Suppressing the Development and Metastasis of Breast Cancer.

Here we investigated the roles of Rab27a, a player in exosome release, and TRAF3IP2, an inflammatory mediator, in development and metastasis of breast cancer (BC) in vivo. Knockdown (KD) of Rab27a (MDAKDRab27a) or TRAF3IP2 (MDAKDTRAF3IP2) in triple negative MDA-MB231 cells reduced tumor growth by 70-97% compared to wild-type tumors (MDAw). While metastasis was detected in MDAw-injected animals, none was detected in MDAKDRab27a- or MDAKDTRAF3IP2-injected animals. Interestingly, micrometastasis was detected only in the MDAKDRab27a-injected group. In addition to inhibiting tumor growth and metastasis, silencing TRAF3IP2 disrupted inter-cellular inflammatory mediator-mediated communication with mesenchymal stem cells (MSCs) injected into contralateral mammary gland, evidenced by the lack of tumor growth at MSC-injected site. Of translational significance, treatment of pre-formed MDAw-tumors with a lentiviral-TRAF3IP2-shRNA not only regressed their size, but also prevented metastasis. These results demonstrate that while silencing Rab27a and TRAF3IP2 each inhibited tumor growth and metastasis, silencing TRAF3IP2 is more effective; targeting TRAF3IP2 inhibited tumor formation, regressed preformed tumors, and prevented both macro- and micrometastasis. Silencing TRAF3IP2 also blocked interaction between tumor cells and MSCs injected into the contralateral gland, as evidenced by the lack of tumor formation on MSCs injected site. These results identify TRAF3IP2 as a novel therapeutic target in BC.

Minocycline reverses IL-17A/TRAF3IP2-mediated p38 MAPK/NF-κB/iNOS/NO-dependent cardiomyocyte contractile depression and death.

Minocycline, an FDA-approved second-generation semisynthetic tetracycline, exerts antioxidant, anti-apoptotic and anti-inflammatory effects, independent of its antimicrobial properties. Interleukin (IL)-17A is an immune and inflammatory mediator, and its sustained induction is associated with various cardiovascular diseases. Here we investigated (i) whether IL-17A induces cardiomyocyte contractile depression and death, (ii) whether minocycline reverses IL-17A's negative inotropic effects and (iii) investigated the underlying molecular mechanisms. Indeed, treatment with recombinant mouse IL-17A impaired adult cardiomyocyte contractility as evidenced by a 34% inhibition in maximal velocity of shortening and relengthening after 4 h (P < .01). Contractile depression followed iNOS induction at 2 h (2.13-fold, P < .01) and NO generation at 3 h (3.71-fold, P <.01). Further mechanistic investigations revealed that IL-17A-dependent induction of iNOS occurred via TRAF3IP2, TRAF6, TAK1, NF-κB, and p38MAPK signaling. 1400 W, a highly specific iNOS inhibitor, suppressed IL-17A-induced NO generation and contractile depression, where as the NO donors SNAP and PAPA-NONOate both suppressed cardiomyocyte contractility. IL-17A also stimulated cardiomyocyte IL-1ß and TNF-α secretion, however, their neutralization failed to modulate IL-17A-mediated contractile depression or viability. Further increases of IL-17A concentration and the duration of exposure enhanced IL-1ß and TNF-α secreted levels, buthad no impact on adult cardiomyocyte viability. However, when combined with pathophysiological concentrations of IL-1ß or TNF-α, IL-17A promoted adult cardiomyocyte death. Importantly, minocycline blunted IL-17A-mediated deleterious effects, indicating its therapeutic potential in inflammatory cardiac diseases.

A New Humanized Mouse Model Mimics Humans in Lacking α-Gal Epitopes and Secreting Anti-Gal Antibodies.

Mice have been used as accepted tools for investigating complex human diseases and new drug therapies because of their shared genetics and anatomical characteristics with humans. However, the tissues in mice are different from humans in that human cells have a natural mutation in the α1,3 galactosyltransferase (α1,3GT) gene and lack α-Gal epitopes on glycosylated proteins, whereas mice and other nonprimate mammals express this epitope. The lack of α-Gal epitopes in humans results in the loss of immune tolerance to this epitope and production of abundant natural anti-Gal Abs. These natural anti-Gal Abs can be used as an adjuvant to enhance processing of vaccine epitopes to APCs. However, wild-type mice and all existing humanized mouse models cannot be used to test the efficacy of vaccines expressing α-Gal epitopes because they express α-Gal epitopes and lack anti-Gal Abs. Therefore, in an effort to bridge the gap between the mouse models and humans, we developed a new humanized mouse model that mimics humans in that it lacks α-Gal epitopes and secretes human anti-Gal Abs. The new humanized mouse model (Hu-NSG/α-Galnull) is designed to be used for preclinical evaluations of viral and tumor vaccines based on α-Gal epitopes, human-specific immune responses, xenotransplantation studies, and in vivo biomaterials evaluation. To our knowledge, our new Hu-NSG/α-Galnull is the first available humanized mouse model with such features.

RECK suppresses interleukin-17/TRAF3IP2-mediated MMP-13 activation and human aortic smooth muscle cell migration and proliferation.

Sustained inflammation and matrix metalloproteinase (MMP) activation contribute to vascular occlusive/proliferative disorders. Interleukin-17 (IL-17) is a proinflammatory cytokine that signals mainly via TRAF3 Interacting Protein 2 (TRAF3IP2), an upstream regulator of various critical transcription factors, including AP-1 and NF-κB. Reversion inducing cysteine rich protein with kazal motifs (RECK) is a membrane-anchored MMP inhibitor. Here we investigated whether IL-17A/TRAF3IP2 signaling promotes MMP-13-dependent human aortic smooth muscle cell (SMC) proliferation and migration, and determined whether RECK overexpression blunts these responses. Indeed, IL-17A treatment induced (a) JNK, p38 MAPK, AP-1, NF-κB, and CREB activation, (b) miR-21 induction, (c) miR-27b and miR-320 inhibition, (d) MMP-13 expression and activation, (e) RECK suppression, and (f) SMC migration and proliferation, all in a TRAF3IP2-dependent manner. In fact, gain of TRAG3IP2 function, by itself, induced MMP-13 expression and activation, and RECK suppression. Furthermore, treatment with recombinant MMP-13 stimulated SMC migration in part via ERK activation. Importantly, RECK gain-of-function attenuated MMP-13 activity without affecting its mRNA or protein levels, and inhibited IL-17A- and MMP-13-induced SMC migration. These results indicate that increased MMP-13 and decreased RECK contribute to IL-17A-induced TRAF3IP2-dependent SMC migration and proliferation, and suggest that TRAF3IP2 inhibitors or RECK inducers have the potential to block the progression of neointimal thickening in hyperplastic vascular diseases.

Breast Tumor Microenvironment Can Transform Naive Mesenchymal Stem Cells into Tumor-Forming Cells in Nude Mice.

How mesenchymal stem cells (MSCs) interact with tumor cells and promote tumor growth is not well understood. In this study, we demonstrate that when naive MSCs and malignant breast cancer cells (MDA-MB231) were injected into opposing mammary glands of an immunodeficient nude mouse, both cell types formed tumor-like masses within 8 weeks at the injected site. Surprisingly, MDA-MB231 cells were detected in the opposing mammary gland injected with the naive MSCs, indicating migration and crosstalk between naive MSCs and MDA-MB231 cells. Furthermore, when naive MSCs preexposed to MDA-MB231-derived conditioned medium (CM; MSCCM) or purified exosomes (Exo; MSCExo) were injected into mammary glands of nude mice, they too formed a tumor-like mass with stromal tissue within 14 weeks. Interestingly, cells dissociated from these primary explants also formed tumor-like masses. Finally, injecting MSCCM or MSCExo and naive MSCs into opposing mammary glands formed tumor-like masses on the naive MSC-injected side, suggesting migration and crosstalk between MSCCM or MSCExo with naive MSCs, similar to that observed between malignant MDA-MB231 cells and naive MSCs. Importantly, molecular analysis of MSCCM and MSCExo revealed DNA hypermethylation. These data demonstrate that MSCs and breast cancer cells communicate, resulting in the transformation of naive MSCs into cells capable of forming explants in nude mice. Our data also suggest that DNA hypermethylation might have contribute to their migration. Understanding the crosstalk between MSCs and tumor cells, and identifying the players involved in their interaction, will help us develop novel therapeutics for breast cancer regression and elimination.

The Anti-Angiogenic Effect of Atorvastatin in Glioblastoma Spheroids Tumor Cultured in Fibrin Gel: in 3D in Vitro Model

Purpose: Glioblastoma multiform (GBM) is the most aggressive glial neoplasm. Researchers have exploited the fact that GBMs are highly vascularized tumors. Anti-angiogenic strategies including those targeting VEGF pathway have been emerged for treatment of GBM. Previously, we reported the anti-inflammatory effect of atorvastatin on GBM cells. In this study, we investigated the anti-angiogenesis and apoptotic activity of atorvastatin on GBM cells. Methods: Different concentrations of atorvastatin (1, 5, 10µM) were used on engineered three-dimensional (3D) human tumor models using glioma spheroids and Human Umbilical Vein Endothelial cells (HUVECs) in fibrin gel as tumor models. To reach for these aims, angiogenesis as tube-like structures sprouting of HUVECs were observed after 24 hour treatment with different concentrations of atorvastatin into the 3-D fibrin matrix and we focused on it by angiogenesis antibody array. After 48 hours exposing with different concentrations of atorvastatin, cell migration of HUVECs were investigated. After 24 and 48 hours exposing with different concentrations of atorvastatin VEGF, CD31, caspase-3 and Bcl-2 genes expression by real time PCR were assayed. Results: The results showed that atorvastatin has potent anti-angiogenic effect and apoptosis inducing effect against glioma spheroids. Atorvastatin down-regulated the expression of VEGF, CD31 and Bcl-2, and induced the expression of caspase-3 especially at 10µM concentration. These effects are dose dependent. Conclusion: These results suggest that this biomimetic model with fibrin may provide a vastly applicable 3D culture system to study the effect of anti-cancer drugs such as atorvastatin on tumor malignancy in vitro and in vivo and atorvastatin could be used as agent for glioblastoma treatment.

TRAF3IP2, a novel therapeutic target in glioblastoma multiforme.

Glioblastoma multiforme (glioblastoma) remains one of the deadliest cancers. Pro-inflammatory and pro-tumorigenic mediators present in tumor microenvironment (TME) facilitate communication between tumor cells and adjacent non-malignant cells, resulting in glioblastoma growth. Since a majority of these mediators are products of NF-κB- and/or AP-1-responsive genes, and as TRAF3 Interacting Protein 2 (TRAF3IP2) is an upstream regulator of both transcription factors, we hypothesized that targeting TRAF3IP2 blunts tumor growth by inhibiting NF-κB and pro-inflammatory/pro-tumorigenic mediators. Our in vitro data demonstrate that similar to primary glioblastoma tumor tissues, malignant glioblastoma cell lines (U87 and U118) express high levels of TRAF3IP2. Silencing TRAF3IP2 expression inhibits basal and inducible NF-κB activation, induction of pro-inflammatory mediators, clusters of genes involved in cell cycle progression and angiogenesis, and formation of spheroids. Additionally, silencing TRAF3IP2 significantly increases apoptosis. In vivo studies indicate TRAF3IP2-silenced U87 cells formed smaller tumors. Additionally, treating existing tumors formed by wild type U87 cells with lentiviral TRAF3IP2 shRNA markedly regresses their size. Analysis of residual tumors revealed reduced expression of pro-inflammatory/pro-tumorigenic/pro-angiogenic mediators and kinesins. In contrast, the expression of IL-10, an anti-inflammatory cytokine, was increased. Together, these novel data indicate that TRAF3IP2 is a master regulator of malignant signaling in glioblastoma, and its targeting modulates the TME and inhibits tumor growth by suppressing the expression of mediators involved in inflammation, angiogenesis, growth, and malignant transformation. Our data identify TRAF3IP2 as a potential therapeutic target in glioblastoma growth and dissemination.

TRAF3IP2 mediates TWEAK/TWEAKR-induced pro-fibrotic responses in cultured cardiac fibroblasts and the heart.

Persistent inflammation promotes development and progression of heart failure (HF). TWEAK (TNF-Related WEAK Inducer Of Apoptosis), a NF-κB- and/or AP-1-responsive proinflammatory cytokine that signals via TWEAK receptor (TWEAKR), is expressed at high levels in human and preclinical models of HF. Since the adapter molecule TRAF3IP2 (TRAF3 Interacting Protein 2) is an upstream regulator of various proinflammatory pathways, including those activated by NF-κB and AP-1, we hypothesized that targeting TRAF3IP2 inhibits TWEAK-induced proinflammatory and pro-fibrotic responses in vitro and in vivo. Consistent with the hypothesis, forced expression of TRAF3IP2 upregulated TWEAK and its receptor expression in cultured adult mouse cardiac fibroblasts (CF). Further, exogenous TWEAK upregulated TRAF3IP2 expression in a time- and dose-dependent manner, suggesting a positive-feedback regulation of TRAF3IP2 and TWEAK. TWEAK also promoted TRAF3IP2 nuclear translocation. Confirming its critical role in TWEAK signaling, silencing TRAF3IP2 inhibited TWEAK autoregulation, TWEAKR upregulation, p38 MAPK, NF-κB and AP-1 activation, inflammatory cytokine expression, MMP and TIMP1 activation, collagen expression and secretion, and importantly, proliferation and migration. Recapitulating these in vitro results, continuous infusion of TWEAK for 7 days increased systolic blood pressure (SBP), upregulated TRAF3IP2 expression, activated p38 MAPK, NF-κB and AP-1, induced the expression of multiple proinflammatory and pro-fibrotic mediators, and interstitial fibrosis in hearts of wild type mice. These proinflammatory and pro-fibrotic changes occurred in conjunction with myocardial hypertrophy and contractile dysfunction. Importantly, genetic ablation of TRAF3IP2 inhibited these TWEAK-induced adverse cardiac changes independent of increases in SBP, indicating that TRAF3IP2 plays a causal role, and thus a therapeutic target, in chronic inflammatory and fibro-proliferative diseases.

A novel model to characterize structure and function of BRCA1.

BRCA1 plays a central role in DNA repair. Although N-terminal RING and C-terminal BRCT domains are studied well, the functions of the central region of BRCA1 are poorly characterized. Here, we report a structural and functional analysis of BRCA1 alleles and functional human BRCA1 in chicken B-lymphocyte cell line DT40. The combination of "homologous recombineering" and "RT-cassette" enables modifications of chicken BRCA1 gene in Escherichia coli. Mutant BRCA1 knock-in DT40 cell lines were generated using BRCA1 mutation constructs by homologous recombination with a targeting efficiency of up to 100%. Our study demonstrated that deletion of motifs 2-9 BRCA1Δ/Δ181-1415 (Caenorhabditis elegans BRCA1 mimic) or deletion of motif 1 BRCA1Δ/Δ126-136 decreased cell viability following cisplatin treatment. Furthermore, deletion of motifs 5 and 6 BRCA1Δ/Δ525-881 within DNA-binding region, even the conserved 7-amino acid deletion BRCA1Δ/Δ872-878 within motif 6, caused a decreased cell viability upon cisplatin treatment. Surprisingly, human BRCA1 is functional in DT40 cells as indicated by DNA damage-induced Rad 51 foci formation in human BRCA1 knock-in DT40 cells. These results demonstrate that those conserved motifs within the central region are essential for DNA repair functions of BRCA1. These findings provide a valuable tool for the development of new therapeutic modalities of breast cancer linked to BRCA1.

NPR3 protects cardiomyocytes from apoptosis through inhibition of cytosolic BRCA1 and TNF-α.

Natriuretic peptide receptor 3 (NPR3) is a clearance receptor by binding and internalizing natriuretic peptides (NPs) for ultimate degradation. Patients with cardiac failure show elevated NPs. NPs are linked to poor long-term survival because of their apoptotic effects. However, the underling mechanisms have not been identified yet. Here we report the role of NPR3 in anti-apoptosis via the breast cancer type 1 susceptibility protein (BRCA1) and tumor necrosis factor α (TNF-α ). To demonstrate a role for NPR3 in apoptosis, stable H9C2 cardiomyocyte cell lines using shRNA to knockdown NPR3 were generated. The activities of caspase-3, 8, and 9 were significantly increased in NPR3 knockdown H9C2 cardiomyocytes. Knockdown of NPR3 increased the expression of BRCA1. Also NPR3 knockdown remarkably increased the activity of cAMP response element-binding protein (CREB), a positive regulatory element for BRCA1 expression. BRCA1 showed dispersed nuclear localization in non-cardiomyocytes while predominantly cytoplasmic localization in H9C2 cells. Meanwhile, NPR3 knockdown significantly increased TNF-α gene expression. These data show that NPR3 knockdown in H9C2 cells triggered both extrinsic and intrinsic apoptotic pathways. NPR3 protects cardiomyocytes from apoptosis through inhibition of cytosolic BRCA1 and TNF-α, which are regulators of apoptosis. Our studies demonstrate anti-apoptosis role of NPR3 in protecting cardiomyocytes and establish the first molecular link between NP system and programmed cell death.