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121.
N El-Andaloussi S Bonifati JK Kaufmann L Mailly L Daeffler F Deryckère DM Nettelbeck J Rommelaere A Marchini 《Journal of virology》2012,86(19):10418-10431
In this study, our goal was to generate a chimeric adenovirus-parvovirus (Ad-PV) vector that combines the high-titer and efficient gene transfer of adenovirus with the anticancer potential of rodent parvovirus. To this end, the entire oncolytic PV genome was inserted into a replication-defective E1- and E3-deleted Ad5 vector genome. As we found that parvoviral NS expression inhibited Ad-PV chimera production, we engineered the parvoviral P4 early promoter, which governs NS expression, by inserting into its sequence tetracycline operator elements. As a result of these modifications, P4-driven expression was blocked in the packaging T-REx-293 cells, which constitutively express the tetracycline repressor, allowing high-yield chimera production. The chimera effectively delivered the PV genome into cancer cells, from which fully infectious replication-competent parvovirus particles were generated. Remarkably, the Ad-PV chimera exerted stronger cytotoxic activities against various cancer cell lines, compared with the PV and Ad parental viruses, while being still innocuous to a panel of tested healthy primary human cells. This Ad-PV chimera represents a novel versatile anticancer agent which can be subjected to further genetic manipulations in order to reinforce its enhanced oncolytic capacity through arming with transgenes or retargeting into tumor cells. 相似文献
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123.
Nik-Zainal S Alexandrov LB Wedge DC Van Loo P Greenman CD Raine K Jones D Hinton J Marshall J Stebbings LA Menzies A Martin S Leung K Chen L Leroy C Ramakrishna M Rance R Lau KW Mudie LJ Varela I McBride DJ Bignell GR Cooke SL Shlien A Gamble J Whitmore I Maddison M Tarpey PS Davies HR Papaemmanuil E Stephens PJ McLaren S Butler AP Teague JW Jönsson G Garber JE Silver D Miron P Fatima A Boyault S Langerød A Tutt A Martens JW Aparicio SA Borg Å Salomon AV Thomas G Børresen-Dale AL Richardson AL 《Cell》2012,149(5):979-993
124.
Q Wen B Goldenson SJ Silver M Schenone V Dancik Z Huang LZ Wang TA Lewis WF An X Li MA Bray C Thiollier L Diebold L Gilles MS Vokes CB Moore M Bliss-Moreau L Verplank NJ Tolliday R Mishra S Vemula J Shi L Wei R Kapur CK Lopez B Gerby P Ballerini F Pflumio DG Gilliland L Goldberg Y Birger S Izraeli AS Gamis FO Smith WG Woods J Taub CA Scherer JE Bradner BC Goh T Mercher AE Carpenter RJ Gould PA Clemons SA Carr DE Root SL Schreiber AM Stern JD Crispino 《Cell》2012,150(3):575-589
The mechanism by which cells decide to skip mitosis to become polyploid is largely undefined. Here we used a high-content image-based screen to identify small-molecule probes that induce polyploidization of megakaryocytic leukemia cells and serve as perturbagens to help understand this process. Our?study implicates five networks of kinases that?regulate the switch to polyploidy. Moreover, we find that dimethylfasudil (diMF, H-1152P) selectively increased polyploidization, mature cell-surface marker expression, and apoptosis of malignant megakaryocytes. An integrated target identification approach employing proteomic and shRNA screening revealed that a major target of diMF is Aurora kinase A (AURKA). We further find that MLN8237 (Alisertib), a selective inhibitor of AURKA, induced polyploidization and expression of mature megakaryocyte markers in acute megakaryocytic leukemia (AMKL) blasts and displayed potent anti-AMKL activity in?vivo. Our findings provide a rationale to support clinical trials of MLN8237 and other inducers of polyploidization and differentiation in AMKL. 相似文献
125.
Bonala S Lokireddy S Arigela H Teng S Wahli W Sharma M McFarlane C Kambadur R 《The Journal of biological chemistry》2012,287(16):12935-12951
Classically, peroxisome proliferator-activated receptor β/δ (PPARβ/δ) function was thought to be restricted to enhancing adipocyte differentiation and development of adipose-like cells from other lineages. However, recent studies have revealed a critical role for PPARβ/δ during skeletal muscle growth and regeneration. Although PPARβ/δ has been implicated in regulating myogenesis, little is presently known about the role and, for that matter, the mechanism(s) of action of PPARβ/δ in regulating postnatal myogenesis. Here we report for the first time, using a PPARβ/δ-specific ligand (L165041) and the PPARβ/δ-null mouse model, that PPARβ/δ enhances postnatal myogenesis through increasing both myoblast proliferation and differentiation. In addition, we have identified Gasp-1 (growth and differentiation factor-associated serum protein-1) as a novel downstream target of PPARβ/δ in skeletal muscle. In agreement, reduced Gasp-1 expression was detected in PPARβ/δ-null mice muscle tissue. We further report that a functional PPAR-responsive element within the 1.5-kb proximal Gasp-1 promoter region is critical for PPARβ/δ regulation of Gasp-1. Gasp-1 has been reported to bind to and inhibit the activity of myostatin; consistent with this, we found that enhanced secretion of Gasp-1, increased Gasp-1 myostatin interaction and significantly reduced myostatin activity upon L165041-mediated activation of PPARβ/δ. Moreover, we analyzed the ability of hGASP-1 to regulate myogenesis independently of PPARβ/δ activation. The results revealed that hGASP-1 protein treatment enhances myoblast proliferation and differentiation, whereas silencing of hGASP-1 results in defective myogenesis. Taken together these data revealed that PPARβ/δ is a positive regulator of skeletal muscle myogenesis, which functions through negatively modulating myostatin activity via a mechanism involving Gasp-1. 相似文献
126.
Cardinale A Racaniello M Saladini S De Chiara G Mollinari C de Stefano MC Pocchiari M Garaci E Merlo D 《The Journal of biological chemistry》2012,287(4):2618-2631
Accumulation of DNA damage and deficiency in DNA repair potentially contribute to the progressive neuronal loss in neurodegenerative disorders, including Alzheimer disease (AD). In multicellular eukaryotes, double strand breaks (DSBs), the most lethal form of DNA damage, are mainly repaired by the nonhomologous end joining pathway, which relies on DNA-PK complex activity. Both the presence of DSBs and a decreased end joining activity have been reported in AD brains, but the molecular player causing DNA repair dysfunction is still undetermined. β-Amyloid (Aβ), a potential proximate effector of neurotoxicity in AD, might exert cytotoxic effects by reactive oxygen species generation and oxidative stress induction, which may then cause DNA damage. Here, we show that in PC12 cells sublethal concentrations of aggregated Aβ(25-35) inhibit DNA-PK kinase activity, compromising DSB repair and sensitizing cells to nonlethal oxidative injury. The inhibition of DNA-PK activity is associated with down-regulation of the catalytic subunit DNA-PK (DNA-PKcs) protein levels, caused by oxidative stress and reversed by antioxidant treatment. Moreover, we show that sublethal doses of Aβ(1-42) oligomers enter the nucleus of PC12 cells, accumulate as insoluble oligomeric species, and reduce DNA-PK kinase activity, although in the absence of oxidative stress. Overall, these findings suggest that Aβ mediates inhibition of the DNA-PK-dependent nonhomologous end joining pathway contributing to the accumulation of DSBs that, if not efficiently repaired, may lead to the neuronal loss observed in AD. 相似文献
127.
Fu J Jin J Cichewicz RH Hageman SA Ellis TK Xiang L Peng Q Jiang M Arbez N Hotaling K Ross CA Duan W 《The Journal of biological chemistry》2012,287(29):24460-24472
Huntington disease (HD) is an inherited neurodegenerative disorder caused by an abnormal polyglutamine expansion in the protein Huntingtin (Htt). Currently, no cure is available for HD. The mechanisms by which mutant Htt causes neuronal dysfunction and degeneration remain to be fully elucidated. Nevertheless, mitochondrial dysfunction has been suggested as a key event mediating mutant Htt-induced neurotoxicity because neurons are energy-demanding and particularly susceptible to energy deficits and oxidative stress. SIRT3, a member of sirtuin family, is localized to mitochondria and has been implicated in energy metabolism. Notably, we found that cells expressing mutant Htt displayed reduced SIRT3 levels. trans-(-)-ε-Viniferin (viniferin), a natural product among our 22 collected naturally occurring and semisynthetic stilbenic compounds, significantly attenuated mutant Htt-induced depletion of SIRT3 and protected cells from mutant Htt. We demonstrate that viniferin decreases levels of reactive oxygen species and prevents loss of mitochondrial membrane potential in cells expressing mutant Htt. Expression of mutant Htt results in decreased deacetylase activity of SIRT3 and further leads to reduction in cellular NAD(+) levels and mitochondrial biogenesis in cells. Viniferin activates AMP-activated kinase and enhances mitochondrial biogenesis. Knockdown of SIRT3 significantly inhibited viniferin-mediated AMP-activated kinase activation and diminished the neuroprotective effects of viniferin, suggesting that SIRT3 mediates the neuroprotection of viniferin. In conclusion, we establish a novel role for mitochondrial SIRT3 in HD pathogenesis and discovered a natural product that has potent neuroprotection in HD models. Our results suggest that increasing mitochondrial SIRT3 might be considered as a new therapeutic approach to counteract HD, as well as other neurodegenerative diseases with similar mechanisms. 相似文献
128.
Advanced glycation endproducts (AGEs) are a group of modified molecular species formed by nonenzymatic reactions between the aldehydic group of reducing sugars with proteins, lipids, or nucleic acids. Formation and accumulation of AGEs are related to the aging process and are accelerated in diabetes. AGEs are generated in hyperglycemia, but their production also occurs in settings characterized by oxidative stress and inflammation. These species promote vascular damage and acceleration of atherosclerotic plaque progression mainly through two mechanisms: directly, altering the functional properties of vessel wall extracellular matrix molecules, or indirectly, through activation of cell receptor-dependent signaling. Interaction between AGEs and the key receptor for AGEs (RAGE), a transmembrane signaling receptor which is present in all cells relevant to atherosclerosis, alters cellular function, promotes gene expression, and enhances the release of proinflammatory molecules. The importance of the AGE-RAGE interaction and downstream pathways, leading to vessel wall injury and plaque development, has been amply established in animal studies. Moreover, the deleterious link of AGEs with diabetic vascular complications has been suggested in many human studies. Blocking the vicious cycle of AGE-RAGE axis signaling may be essential in controlling and preventing cardiovascular complications. In this article, we review the pathogenetic role of AGEs in the development, progression and instability of atherosclerosis, and the potential targets of this biological system for the prevention and treatment of cardiovascular disease. 相似文献
129.
130.
Nazim El-Andaloussi Barbara Leuchs Serena Bonifati Jean Rommelaere Antonio Marchini 《Journal of visualized experiments : JoVE》2012,(62)
Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 which regulate the expression of non-structural (NS1 and NS2) and capsid proteins (VP1 and VP2) respectively1. They attract high interest as anticancer agents for their oncolytic and oncosuppressive abilities while being non-pathogenic for humans2. NS1 is the major effector of viral cytotoxicity3. In order to further enhance their natural antineoplastic activities, derivatives from these vectors have been generated by replacing the gene encoding for the capsid proteins with a therapeutic transgene (e.g. a cytotoxic polypeptide, cytokine, chemokine, tumour suppressor gene etc.)4. The recombinant parvoviruses (recPVs) vector retains the NS1/2 coding sequences and the PV genome telomeres which are necessary for viral DNA amplification and packaging. Production of recPVs occurs only in the producer cells (generally HEK293T), by co-transfecting the cells with a second vector (pCMV-VP) expressing the gene encoding for the VP proteins (Fig. 1)4. The recPV vectors generated in this way are replication defective. Although recPVs proved to possess enhanced oncotoxic activities with respect to the parental viruses from which they have been generated, their production remains a major challenge and strongly hampers the use of these agents in anti-cancer clinical applications.We found that introduction of an Ad-5 derived vector containing the E2a, E4(orf6) and the VA RNA genes (e.g. pXX6 plasmid) into HEK293T improved the production of recPVs by more than 10 fold in comparison to other protocols in use. Based on this finding, we have constructed a novel Ad-VP-helper that contains the genomic adenoviral elements necessary to enhance recPVs production as well as the parvovirus VP gene unit5. The use of Ad-VP-helper, allows production of rec-PVs using a protocol that relies entirely on viral infection steps (as opposed to plasmid transfection), making possible the use of cell lines that are difficult to transfect (e.g. NB324K) (Fig. 2). We present a method that greatly improves the amount of recombinant virus produced, reducing both the production time and costs, without affecting the quality of the final product5. In addition, large scale production of recPV (in suspension cells and bioreactors) is now conceivable. 相似文献