Dysregulation of RUNX1 Plays a Critical Role in the Progression of Myelodysplastic Syndromes

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4108-4108
Author(s):  
Hiroko Sakurai ◽  
Yuka Harada ◽  
Hirotaka Matsui ◽  
Hideaki Nakajima ◽  
Toshio Kitamura ◽  
...  
Keyword(s):  
Splice Site ◽  
Myelodysplastic Syndromes ◽  
Critical Role ◽  
Gene Mutations ◽  
Splicing Factor ◽  
Expression Level ◽  
Dominant Negative Effect ◽  
Rapid Progression ◽  
Myeloid Neoplasms ◽  
Cd34 Cells

Abstract RUNX1/AML1 mutations have been frequently detected in patients with myeloid neoplasms, especially myelodysplastic syndromes (MDS) and chronic monocytic leukemia (CMML). Although the mutations have been analyzed thoroughly, its expression level has not been investigated. Therefore, we attempt to clarify the expression of RUNX1 in the pathogenesis of myeloid neoplasms. The study was approved by the institutional review board and patients gave written informed consent for the study, according to the Declaration of Helsinki. Several isoforms of RUNX1 mRNA are known and we analyzed RUNX1a (including exon 7a which has stop codon) and RUNX1b (skipping exon 7a and including exon 7b and 8). Expression levels of full length isoform (RUNX1b) and short isoform (RUNX1a which has a dominant negative effect on RUNX1b) in CD34+ cells from patients with myeloid neoplasms were examined. A part of patients with MDS or myelodysplastic syndrome / myeloproliferative neoplasms (MDS/MPN) including CMML showed RUNX1a overexpression. Average of relative RUNX1a expression level in MDS patients (n=34) and MDS/MPN patients (n=20) was 7.4-fold and 8.6-fold of the level in normal bone marrow (BM), respectively, whereas most of these patients showed almost same or slight increase of expression level of RUNX1b compared with normal BM. Interestingly, some patients showed high expression of RUNX1a and repression of RUNX1b. In both disease categories, patients with excess blasts displayed a significantly higher expression level of RUNX1a compared with normal BM and patients without excess blasts. During the disease progression in a single patient with MDS or MDS/MPN, the expression of RUNX1a became higher, while azacitidine treatment reduced RUNX1a expression. Genomic mutations of RUNX1 were also examined. RUNX1 mutations were detected in 16% of MDS and 35% of MDS/MPN. Surprisingly, a part of patients had both RUNX1 gene mutation and RUNX1a overexpression, and they showed rapid progression of disease. To evaluate the effects of RUNX1a overexpression, RUNX1a was transduced into CD34+ cells from MDS patients with low expression level of RUNX1a. RUNX1a-transduction resulted in cell proliferation on MS5 stromal cells. These results indicate that overexpression of RUNX1a may add growth advantage to CD34+ cells in patients with MDS or MDS/MPN. We next analyzed the mechanism of RUNX1a overexpression. Gene mutations affecting exon recognition were examined in the patients. Splicing factor mutations, SRSF2 and U2AF1, were detected frequently in MDS (15%) and MDS/MPN (50%). Patients with splicing factor mutations showed higher RUNX1a expression than patients without the mutations. To confirm that the splicing factor mutations affect the expression of RUNX1a, we performed enforced expression of SRSF2 p.P95H mutant using pMYs.IRES.EGFP retrovirus vector in a MDS-derived cell line, TF-1. After a single cell sorting, independent 13 expanding clones were analyzed. Most of the clones demonstrated higher expression of RUNX1a than mock cells, whereas RUNX1b expression was reduced in all clones. Increase of RUNX1a expression in SRSF2 mutant-transduced TF-1 cells was also confirmed by Western blot. Moreover, the clones with higher GFP intensity showed higher expression level of RUNX1a, suggesting that SRSF2 p.P95H expression level may affect the expression level of RUNX1a. Furthermore, SRSF2 mutant-transduced TF-1 cells showed phenotypic changes of higher CD11b and CD14 than mock TF-1 cells, suggesting that SRSF2 mutant may induce monocytic differentiation via RUNX1a overexpression. Gene mutations of RUNX1 in intron 6 and exon 7a were also analyzed. A 5' splice site change just after exon 6 was detected in a CMML patient with RUNX1a overexpression, which may be another mechanism of RUNX1a overexpression. Mutations of exon 7a or changes in 3' splice site just before exon 7a have not been detected yet. In conclusion, our data suggest that overexpression of RUNX1a may play a critical role in the progression of MDS and MDS/MPN, in addition to RUNX1 mutations. Splicing factor mutations are suspected to contribute to the mechanism of the dysregulation of RUNX1. Disclosures No relevant conflicts of interest to declare.

Lineage-Specific Aberrant mRNA Splicing By U2AF1 Mutation Alters Erythroid and Granulomonocytic Differentiation

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 142-142
Author(s):  
Bon Ham Yip ◽  
Swagata Roy ◽  
Hamid Dolatshad ◽  
Jacqueline Shaw ◽  
Seishi Ogawa ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Growth ◽  
Mouse Model ◽  
Progenitor Cells ◽  
Splice Site ◽  
Myeloid Cells ◽  
Mrna Splicing ◽  
Splicing Factor ◽  
Aberrant Splicing ◽  
Cd34 Cells

Abstract Splicing factor genes are the most common targets of somatic mutations in myelodysplastic syndromes (MDS). The splicing factor U2AF1 is an auxiliary factor that forms a heterodimer for the recognition of the 3′ splice site during pre-mRNA splicing. Heterozygous mutations of U2AF1 occur in ~10% of MDS patients and are predominantly located at S34 and Q157 within the zinc fingers domains. Recently an inducible transgenic mouse model expressing mutant U2AF1 S34F demonstrated altered hematopoiesis and aberrant pre-mRNA splicing in hematopoietic progenitor cells. MDS are clonal stem-cell disorders characterized by ineffective hematopoiesis in one or more myeloid lineages of the bone marrow. To investigate the effects of U2AF1 S34F mutation on hematopoiesis, U2AF1 S34F mutant (S34F) and U2AF1 wild type (WT) were overexpressed in human bone marrow CD34+ progenitor cells by retroviral transduction and the cells were differentiated along erythroid and granulomonocytic lineages. S34F erythroblasts exhibited impaired erythroid differentiation compared to WT and empty vector (EV) controls. A significant increase in CD71-CD235a- non-erythroid cells (p≤0.02, n=7) followed by a significant decrease in CD71+CD235a+ (p≤0.002, n=7) and CD71-CD235a+ (p=0.005, n=7) erythroid cells was observed in S34F erythroblasts from day 11 to 14 using flow cytometry, when compared to WT and EV controls. Moreover, S34F inhibited formation and hemoglobinization of BFU-E colonies from bone marrow CD34+ cells in colony forming cell (CFC) assays compared to WT (p=0.002, n=7) and EV (p=0.0006, n=7) controls. S34F erythroblasts also exhibited impaired cell growth and increased apoptosis (Annexin V+) compared to WT (p<0.05, n=6-8) and EV (p≤0.01, n=6-8) controls. Thus, the S34F mutation results in impaired erythropoiesis. S34F perturbed the granulomonocytic lineage by skewing differentiation of myeloid cells towards granulocytes. A reduction in the CD11b+ population was observed in S34F myeloid cells compared to WT (p≤0.001, n=9) and EV (p≤0.001, n=9) controls from day 11 to 14. An increase in granulocytes (CD15+, p≤0.001, n=5) followed by a concomitant decrease in monocytes (CD14+,p=0.026, n=5) was also observed in S34F myeloid cells on day 20 compared to WT and EV controls. Morphological analysis of myeloid cells confirmed a reduction in monocytes caused by an expansion of granulocyte eosinophils. Moreover, S34F bone marrow CD34+ cells produced a significantly higher number of CFU-G (p=0.035, n=5) with a decrease in the number of CFU-M (p≤0.03, n=5) in myeloid CFC assays compared to WT (p≤0.01, n=7) and EV (p≤0.01, n=7) controls. S34F myeloid cells exhibited impaired cell growth associated with G2/M cell cycle arrest compared to WT (p=0.0003, n=6) and EV (p=0.0002, n=6) controls. To investigate aberrant splicing events, we performed RNA sequencing on individual erythroid (BFU-E) and granulomonocytic (CFU-G and CFU-M) colonies formed by S34F, WT and EV transduced bone marrow CD34+ cells (n=3 each). By comparison with WT and EV colonies of the same lineage, we observed that S34F differentially alters the splicing pattern in different lineages. We have observed aberrant splicing of many genes, including BCOR and H2AFY, two genes previously shown to be aberrantly spliced in common myeloid progenitors from a U2AF1 S34F mouse model. The transcriptional co-repressor BCOR is commonly mutated in MDS/AML. Alternative 3' splice site usage in BCOR, resulting in reduced expression of its long isoform, was observed in S34F granulomonocytic colonies, but not in S34F erythroid colonies. In contrast, reduced expression of isoform 1.1 of H2AFY (a member of H2A histone family), due to mutually exclusive exons, was observed in both S34F erythroid and granulomonocytic colonies. Deregulation in isoform expression levels in BCOR and H2AFY was validated by isoform-specific qRT-PCR in S34F transduced cells compared to WT (p≤0.015, n=5) and EV (p≤0.045, n=5) controls. We are currently introducing these isoform imbalances into bone marrow CD34+ cells as they differentiate towards the erythroid and granulomonocytic lineages to elucidate the lineage-specific effect of S34F. Our results indicate that U2AF1 S34F mutant alters erythroid and granulomonocytic differentiation by inducing lineage-specific aberrant splicing patterns, providing new insights into the molecular pathogenesis of U2AF1 mutant MDS. Disclosures No relevant conflicts of interest to declare.

Elevated Basal Autophagy in SF3B1 Mutated Myelodysplastic Syndromes: Relationship with Survival Outcomes and Therapeutic Implications

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1647-1647 ◽  
Author(s):  
Valeria Visconte ◽  
Kevin R. Kelly ◽  
Steffan T. Nawrocki ◽  
Yingchun Han ◽  
Hetty E. Carraway ◽  
...  
Keyword(s):  
Iron Overload ◽  
Board Of Directors ◽  
Myelodysplastic Syndromes ◽  
Lower Risk ◽  
Critical Role ◽  
Cysteine Proteases ◽  
Splicing Factor ◽  
Refractory Anemia ◽  
Advisory Committees ◽  
Mitochondrial Iron

Abstract About 60-80% of patients with myelodysplastic syndromes (MDS) manifest with anemia. Red blood cell (RBC) transfusions are the most commonly used therapy to alleviate anemia in patients that are ineligible for other curative approaches. Transfusion-dependent patients frequently develop iron overload, which correlates with infections, mortality, leukemia, and organopathy. At the subcellular level, long-term iron exposure produces iron-catalyzed hydroxyl radicals that induce oxidative damage to mitochondria and disrupt bioenergetic homeostasis. The use of iron-chelating drugs to counter transfusion-related iron overload remains controversial due to the significant side effects that these agents cause. New therapies that effectively address iron overload in transfusion-dependent MDS patients are clearly needed. Mitochondrial dysfunction frequently occurs during MDS cell maturation and leads to abnormal iron distribution. However, the mechanistic basis of this biological phenomenon has not been rigorously studied. We previously linked the presence of Splicing factor 3b subunit 1 (SF3B1) mutations, which are frequent in patients with refractory anemia with ring sideroblasts (RARS), with abnormalities in mitochondrial iron. Transmission electron microscopy and flow cytometry showed that mitochondria of SF3B1 mutant RARS patients have higher iron content than those of wild type (WT) RARS patients and expressed an increased mRNA level of the iron transporter, Mitoferrin 1. Despite these prevalent mitochondrial abnormalities and transfusional dependence, SF3B1 mutant lower-risk MDS patients experienced significantly longer median survival compared to SF3B1 WT lower-risk MDS patients (34 mos vs. 13 mos; P = .002; N=16 vs. 101). Autophagy is an evolutionarily conserved lysosomal mechanism of protein degradation that plays a critical role in the elimination of damaged mitochondria and other organelles. We hypothesized that autophagic clearance of defective mitochondria may contribute to the superior survival of SF3B1 mutant patients suffering from transfusion-mediated iron overload. We conducted RNA sequencing analyses on a group of fresh bone marrow (BM) cells of SF3B1 mutant and WT RARS patients and healthy donors (n = 11) to investigate the basal autophagy status in this distinct patient population. In addition to confirming increased levels of mitochondrial transporters such as Mitoferrin 1 and 2 (FC = 2.0), we detected a striking increase in multiple genes involved in the proximal and distal regulation of autophagy in cells of SF3B1 mutant RARS compared to WT RARS patients. Genes controlling the early stages of autophagy including the protein kinases ULK1 (FC = 2.0) and ULK3 (FC = 3.9; P=.05), ATG complexes ATG2A/B (FC = 1.9), ATG9A (FC = 5.5; P=.05), ATG18 (FC = 4.8; P=.02) and the cysteine proteases ATG4A/C (FC = 2.0) were all elevated in SF3B1 mutant RARS vs. SF3B1 WT RARS patients. Key components of the late stages of the autophagic degradation cascade, including multiple members of the cathepsin family of lysosomal proteases [CTSL1: FC = 20.9; CTSD: FC = 5.8 (P=.06); CTSB: FC = 2.1; CTSE: FC = 5.9 (P=.01); CTSD: FC = 1.8], were also significantly increased. qRT-PCR confirmed higher expression levels in the BM cells of RARS patients carrying sole SF3B1 mutations compared to cells of SF3B1 WT RARS patients. The link between SF3B1, mitochondrial iron, and elevated autophagy was specific as evidenced by the unmutated status and lack of significant mRNA changes in any other splicing factor genes including PRPF8. Our data demonstrate that autophagy may play an important, previously unreported role in SF3B1 mutant RARS. Based on our findings, we hypothesize that SF3B1 mutant RARS cells stimulate autophagy to eliminate damaged mitochondria and alleviate iron overload and that further stimulation of autophagy will diminish the pathogenic effects of chronic transfusions. We are currently investigating the therapeutic benefit and pharmacodynamics of autophagy-modulating drugs (temsirolimus, metformin, arsenic trioxide) in in vitro (primary cells) and in vivo (SF3B1 haploinsufficient mice) models of MDS to facilitate the design of an investigator-initiated clinical trial that will test autophagy modulation as a new precision strategy for the treatment of transfusion-dependent patients with low-risk MDS carrying SF3B1 mutations. Disclosures Sekeres: TetraLogic: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Carew:Boehringer Ingelheim: Research Funding.

HSP70, the Key to Account for Erythroid Tropism of Diamond-Blackfan Anemia?

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 671-671
Author(s):  
Marc Gastou ◽  
Sarah Rio ◽  
Mickael Dussiot ◽  
Narjesse Karboul ◽  
Thierry Leblanc ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Critical Role ◽  
Erythroid Differentiation ◽  
Expression Level ◽  
Hsp70 Expression ◽  
Erythroid Cells ◽  
Diamond Blackfan Anemia ◽  
Cd34 Cells ◽  
Proliferation And Differentiation ◽  
P53 Activation

Abstract Diamond-Blackfan anemia (DBA) was the first ribosomopathy identified and is characterized by a moderate to severe, usually macrocytic aregenerative anemia associated with congenital malformations in 50% of the DBA cases. This congenital rare erythroblastopenia is due to a blockade in erythroid differentiation between the BFU-e and CFU-e stages. The link between a haploinsufficiency in a ribosomal protein (RP) gene that now encompass 15 different RP genes and the erythroid defect is still to be fully defined. Recently, mutations in TSR2 and GATA1 genes have been identified in a few DBA families. The GATA1 gene encodes for the major transcription factor critical for erythropoiesis and mutation in this gene that lead to loss of expression of the long form of the protein, necessary for the erythroid differentiation accounts for erythroblastopenia of DBA phenotype. Our group and others (Dutt et al., Blood 2011) have shown previously that p53 plays an important role in the DBA erythroblastopenia, inducing cell cycle arrest in G0/G1 and depending on the nature of RP gene mutation, a delayed erythroid differentiation and an increased apoptosis. Indeed, we identified two distinct DBA phenotypes (H. Moniz, M. Gastou, Cell Death Dis, 2012): a haploinsufficiency in RPL5 or RPL11 reduced dramatically the erythroid proliferation, delayed the erythroid differentiation, and markedly increased apoptosis, while RPS19 haploinsufficiency while reduced the extent of erythroid proliferation without inducing significant apoptosis. While p53 pathway has been found to be activated in RP haploinsufficient erythroid cells in DBA patients or shRNA-RPS19, -RPL5, or -RPL11 infected CD34+ erythroid cells, the intensity of the p53 activation pathway (p21, BAX, NOXA) is different depending on the mutated RP gene. Since the differences between the two phenotypes involved the degree of apoptosis we hypothesized that HSP70, a chaperone protein of GATA1 may play a key role in the erythroid defect of DBA. Indeed, HSP70 protects GATA1 from the cleavage by the caspase 3, a protease activated during erythroid differentiation and as such reduced levels of HSP70 related to a RP haploinsufficiency could account for increased apoptosis and delayed erythroid differentiation of erythroid cells in DBA. Indeed, a defect in RPL5 or RPL11 decreased dramatically the expression level of HSP70 and GATA1 in primary human erythroid cells from DBA patients and following in vitro knockdown of the proteins in CD34+ cells by RPL5 or RPL11 shRNA. Importantly, RPS19 haploinsufficiency did not exhibit this effect in conjunction with normal levels of HSP70 expression. Furthermore, we found that the decreased expression level of HSP70 was independent on the p53 activation. Strikingly, HSP70 was noted to be degraded by the proteasome since the bortezomib, the MG132, or the lactacystin were able to restore both the HSP70 expression level and intracellular localization in the cell. The lentiviral infection of haploinsufficient RPL5 or RPL11 cord blood CD34+ cells with a wild type HSP70 cDNA restored both the erythroid proliferation and differentiation confirming a critical role for HSP70 in the erythroid proliferation and differentiation defect in the RPL5 or RPL11 DBA phenotypes. The loss of HSP70 may explain the loss of GATA1 in DBA and also the erythroid tropism of the DBA disease. Restoration of the HSP70 expression level may be a viable and novel therapeutic option for management of this debilitating and difficult to manage erythroid disorder. Disclosures No relevant conflicts of interest to declare.

scholarly journals NPM1-Mutated Myeloid Neoplasms with <20% Blasts: A Really Distinct Clinico-Pathologic Entity?

2020 ◽  
Vol 21 (23) ◽  
pp. 8975
Author(s):  
Fabio Forghieri ◽  
Vincenzo Nasillo ◽  
Ambra Paolini ◽  
Francesca Bettelli ◽  
Valeria Pioli ◽  
...  
Keyword(s):  
Bone Marrow Cells ◽  
Myeloproliferative Neoplasm ◽  
Gene Mutations ◽  
Moderate Intensity ◽  
Rapid Progression ◽  
Myeloid Neoplasms ◽  
Blast Count ◽  
Aggressive Clinical Course ◽  
Myelomonocytic Leukemia ◽  
Acute Myeloid

Nucleophosmin (NPM1) gene mutations rarely occur in non-acute myeloid neoplasms (MNs) with <20% blasts. Among nearly 10,000 patients investigated so far, molecular analyses documented NPM1 mutations in around 2% of myelodysplastic syndrome (MDS) cases, mainly belonging to MDS with excess of blasts, and 3% of myelodysplastic/myeloproliferative neoplasm (MDS/MPN) cases, prevalently classified as chronic myelomonocytic leukemia. These uncommon malignancies are associated with an aggressive clinical course, relatively rapid progression to overt acute myeloid leukemia (AML) and poor survival outcomes, raising controversies on their classification as distinct clinico-pathologic entities. Furthermore, fit patients with NPM1-mutated MNs with <20% blasts could benefit most from upfront intensive chemotherapy for AML rather than from moderate intensity MDS-directed therapies, although no firm conclusion can currently be drawn on best therapeutic approaches, due to the limited available data, obtained from small and mainly retrospective series. Caution is also suggested in definitely diagnosing NPM1-mutated MNs with blast count <20%, since NPM1-mutated AML cases frequently present dysplastic features and multilineage bone marrow cells showing abnormal cytoplasmic NPM1 protein delocalization by immunohistochemical staining, therefore belonging to NPM1-mutated clone regardless of blast morphology. Further prospective studies are warranted to definitely assess whether NPM1 mutations may become sufficient to diagnose AML, irrespective of blast percentage.

scholarly journals Replication stress signaling is a therapeutic target in myelodysplastic syndromes with splicing factor mutations

Haematologica ◽  
2020 ◽  
pp. 0-0 ◽  
Author(s):  
Johanna Flach ◽  
Johann-Christoph Jann ◽  
Antje Knaflic ◽  
Vladimir Riabov ◽  
Alexander Streuer ◽  
...  
Keyword(s):  
Myelodysplastic Syndromes ◽  
Strong Association ◽  
Replication Stress ◽  
Splicing Factor ◽  
Attractive Potential ◽  
Splicing Factors ◽  
Pathway Activation ◽  
Cd34 Cells ◽  
Pladienolide B ◽  
Cord Blood Cd34

Somatic mutations in genes coding for splicing factors, e.g. SF3B1, U2AF1, SRSF2, and others are found in approximately 50% of patients with Myelodysplastic Syndromes (MDS). These mutations have been predicted to frequently occur early in the mutational hierarchy of the disease therefore making them particularly attractive potential therapeutic targets. Recent studies in cell lines engineered to carry splicing factor mutations have revealed a strong association with elevated levels of DNA:RNA intermediates (R-loops) and a dependency on proper ATR function. However, data confirming this hypothesis in a representative cohort of primary MDS patient samples have so far been missing. Using CD34+ cells isolated from MDS patients with and without splicing factor mutations as well as healthy controls we show that splicing factor mutation-associated R-loops lead to elevated levels of replication stress and ATR pathway activation. Moreover, splicing factor mutated CD34+ cells are more susceptible to pharmacological inhibition of ATR resulting in elevated levels of DNA damage, cell cycle blockade, and cell death. This can be enhanced by combination treatment with low-dose splicing modulatory compound Pladienolide B. We further confirm the direct association of R-loops and ATR sensitivity with the presence of a splicing factor mutation using lentiviral overexpression of wild-type and mutant SRSF2 P95H in cord blood CD34+ cells. Collectively, our results from n=53 MDS patients identify replication stress and associated ATR signaling to be critical pathophysiological mechanisms in primary MDS CD34+ cells carrying splicing factor mutations, and provide a preclinical rationale for targeting ATR signaling in these patients.

scholarly journals The anti-angiogenic isoforms of VEGF in health and disease

2009 ◽  
Vol 37 (6) ◽  
pp. 1207-1213 ◽  
Author(s):  
Yan Qiu ◽  
Coralie Hoareau-Aveilla ◽  
Sebastian Oltean ◽  
Steven J. Harper ◽  
David O. Bates
Keyword(s):  
Splice Site ◽  
Site Selection ◽  
Age Related Macular Degeneration ◽  
Splicing Factor ◽  
Splice Site Selection ◽  
Age Related ◽  
Normal Human ◽  
Radical Revision

Anti-angiogenic VEGF (vascular endothelial growth factor) isoforms, generated from differential splicing of exon 8, are widely expressed in normal human tissues but down-regulated in cancers and other pathologies associated with abnormal angiogenesis (cancer, diabetic retinopathy, retinal vein occlusion, the Denys–Drash syndrome and pre-eclampsia). Administration of recombinant VEGF165b inhibits ocular angiogenesis in mouse models of retinopathy and age-related macular degeneration, and colorectal carcinoma and metastatic melanoma. Splicing factors and their regulatory molecules alter splice site selection, such that cells can switch from the anti-angiogenic VEGFxxxb isoforms to the pro-angiogenic VEGFxxx isoforms, including SRp55 (serine/arginine protein 55), ASF/SF2 (alternative splicing factor/splicing factor 2) and SRPK (serine arginine domain protein kinase), and inhibitors of these molecules can inhibit angiogenesis in the eye, and splice site selection in cancer cells, opening up the possibility of using splicing factor inhibitors as novel anti-angiogenic therapeutics. Endogenous anti-angiogenic VEGFxxxb isoforms are cytoprotective for endothelial, epithelial and neuronal cells in vitro and in vivo, suggesting both an improved safety profile and an explanation for unpredicted anti-VEGF side effects. In summary, C-terminal distal splicing is a key component of VEGF biology, overlooked by the vast majority of publications in the field, and these findings require a radical revision of our understanding of VEGF biology in normal human physiology.

scholarly journals Contribution of Target Gene Mutations and Efflux to Decreased Susceptibility of Salmonella enterica Serovar Typhimurium to Fluoroquinolones and Other Antimicrobials

2006 ◽  
Vol 51 (2) ◽  
pp. 535-542 ◽  
Author(s):  
Sheng Chen ◽  
Shenghui Cui ◽  
Patrick F. McDermott ◽  
Shaohua Zhao ◽  
David G. White ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Efflux Pump ◽  
Critical Role ◽  
Gene Mutations ◽  
Efflux Pumps ◽  
Fluoroquinolone Resistance ◽  
Naturally Occurring ◽  
Serovar Typhimurium ◽  
Resistant Mutants ◽  
Increased Susceptibility

ABSTRACT The mechanisms involved in fluoroquinolone resistance in Salmonella enterica include target alterations and overexpression of efflux pumps. The present study evaluated the role of known and putative multidrug resistance efflux pumps and mutations in topoisomerase genes among laboratory-selected and naturally occurring fluoroquinolone-resistant Salmonella enterica serovar Typhimurium strains. Strains with ciprofloxacin MICs of 0.25, 4, 32, and 256 μg/ml were derived in vitro using serovar Typhimurium S21. These mutants also showed decreased susceptibility or resistance to many nonfluoroquinolone antimicrobials, including tetracycline, chloramphenicol, and several β-lactams. The expression of efflux pump genes acrA, acrB, acrE, acrF, emrB, emrD, and mdlB were substantially increased (≥2-fold) among the fluoroquinolone-resistant mutants. Increased expression was also observed, but to a lesser extent, with three other putative efflux pumps: mdtB (yegN), mdtC (yegO), and emrA among mutants with ciprofloxacin MICs of ≥32 μg/ml. Deletion of acrAB or tolC in S21 and its fluoroquinolone-resistant mutants resulted in increased susceptibility to fluoroquinolones and other tested antimicrobials. In naturally occurring fluoroquinolone-resistant serovar Typhimurium strains, deletion of acrAB or tolC increased fluoroquinolone susceptibility 4-fold, whereas replacement of gyrA double mutations (S83F D87N) with wild-type gyrA increased susceptibility >500-fold. These results indicate that a combination of topoisomerase gene mutations, as well as enhanced antimicrobial efflux, plays a critical role in the development of fluoroquinolone resistance in both laboratory-derived and naturally occurring quinolone-resistant serovar Typhimurium strains.

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