In one form of -thalassemia, a genetic blood disorder, a mutation in intron 2 of the -globin gene (IVS2-654) causes aberrant splicing of -globin pre-mRNA and, consequently, -globin deficiency. Treatment of mammalian cells stably expressing the IVS2-654 human -globin gene with antisense oligonucleotides targeted at the aberrant splice sites restored correct splicing in a dose-dependent fashion, generating correct human -globin mRNA and polypeptide. Both products persisted for up to 72 hr posttreatment. The oligonucleotides modified splicing by a true antisense mechanism without overt unspecific effects on cell growth and splicing of other pre-mRNAs. This novel approach in which antisense oligonucleotides are used to restore rather than to down-regulate the activity of the target gene is applicable to other splicing mutants and is of potential clinical interest.-Thalassemia, a genetic blood disorder, affects a large number of people in the Mediterranean basin, Middle East, South East Asia, and Africa. Close to 100 thalassemic mutations causing defective -globin gene expression and -globin deficiency have been identified, but no more than 10 mutations are responsible for Ϸ90% of cases worldwide (1). Of the frequently occurring mutations, the ones that cause aberrant splicing of intron 1 of the human -globin gene are predominant in South Eastern Europe, Cyprus, Lebanon (mutation IVS1-110), India, Malaysia, and Indonesia (IVS1-5). Additional splicing mutations in intron 1 (IVS1-6) as well as in intron 2 of the -globin gene (IVS2-745) are also common in the above countries, while IVS2-654 is frequent among -thalassemia patients in China and Thailand (1-8). All of these mutations activate aberrant splice sites and change the splicing pathway even though the correct splice sites remain potentially functional. We hypothesized that blocking of the aberrant splice sites or other sequence elements involved in splicing with antisense oligonucleotides may force the splicing machinery to reselect the correct splice sites and induce the formation of -globin mRNA and polypeptide, hence restoring the gene function.Although we have previously effected correction of splicing by antisense oligonucleotides in cell-free extracts from HeLa cells (9), it was not at all clear whether the oligonucleotides delivered into the cell could enter the nucleus, hybridize to the aberrant splice sites in competition with the splicing factors, and promote the formation of the spliceosome and subsequent splicing at the correct splice site. Here we report that correct splicing was efficiently restored when phosphorothioate 2Ј-Omethyl-oligoribonucleotides were targeted to the aberrant splice sites of IVS2-654 pre-mRNA expressed in mammalian cells stably transformed with this mutated human -globin gene. This is a novel approach since antisense oligonucleotides have been used mostly as sequence specific down-regulators of gene expression (10). MATERIALS AND METHODSCells. Human -globin gene carrying a thalassemic mutation IVS2-654 w...
Metastatic melanoma is one of the most aggressive forms of cutaneous cancers. Although recent therapeutic advances have prolonged patient survival, the prognosis remains dismal. C-MER proto-oncogene tyrosine kinase (MERTK) is a receptor tyrosine kinase with oncogenic properties that is often overexpressed or activated in various malignancies. Using both protein immunohistochemistry and microarray analyses, we demonstrate that MERTK expression correlates with disease progression. MERTK expression was highest in metastatic melanomas, followed by primary melanomas, while the lowest expression was observed in nevi. IntroductionAlthough early cutaneous melanoma is usually curable with surgery, distant metastatic melanoma is an aggressive cancer with a median overall survival time of less than 1 year. In 2012, over 75,000 new melanoma diagnoses were expected and over 9,000 deaths were projected (1). Advances in the understanding of distinct melanoma subtypes as well as melanoma immunobiology have resulted in 2 FDA-approved therapies for metastatic melanoma in 2011: vemurafenib, an inhibitor of mutant BRAF -an oncogene present in approximately 50% of melanomas -and ipilimumab, a monoclonal antibody that targets CTLA-4 (2-4). Despite these rather impressive developments, the overall clinical benefit is limited to either small subgroups of patients who
Purpose-To assess the relative radiosensitivities of a large collection of melanoma cell lines and to determine whether pharmacologic inhibition of mutant B-RAF with PLX-4032 can radiosensitize B-Raf+ melanoma cells.Methods and Materials-A large collection of melanoma cell lines (n=37) were treated with 0 -8 Gy IR and clonogenic survival assays used to generate survival curves to rank relative radiosensitivities among the cell lines. The ability of a B-RAF inhibitor, PLX-4032, to radiosensitize highly radioresistant, B-Raf+ cells was also assessed by clonogenic cell survival and spheroid invasion assays and the effects of treatment on the cell cycle assessed by FACS. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Results-MelanomaConflict of Interest Statement: The authors declare no conflicts of interest exist. NIH Public Access
V-ATPases acidify multiple organelles, and yeast mutants lacking V-ATPase activity exhibit a distinctive set of growth defects. To better understand the requirements for organelle acidification and the basis of these growth phenotypes, 0074ف yeast deletion mutants were screened for growth defects at pH 7.5 in 60 mm CaCl 2 . In addition to 13 of 16 mutants lacking known V-ATPase subunits or assembly factors, 50 additional mutants were identified. Sixteen of these also grew poorly in nonfermentable carbon sources, like the known V-ATPase mutants, and were analyzed further. The cwh36⌬ mutant exhibited the strongest phenotype; this mutation proved to disrupt a previously uncharacterized V-ATPase subunit. A small subset of the mutations implicated in vacuolar protein sorting, vps34⌬, vps15⌬, vps45⌬, and vps16⌬, caused both VmaϪ growth phenotypes and lower V-ATPase activity in isolated vacuoles, as did the shp1⌬ mutation, implicated in both protein sorting and regulation of the Glc7p protein phosphatase. These proteins may regulate V-ATPase targeting and/or activity. Eight mutants showed a VmaϪ growth phenotype but no apparent defect in vacuolar acidification. Like V-ATPase-deficient mutants, most of these mutants rely on calcineurin for growth, particularly at high pH. A requirement for constitutive calcineurin activation may be the predominant physiological basis of the VmaϪ growth phenotype.
The yeast cwh36⌬ mutant was identified in a screen for yeast mutants exhibiting a Vma ؊ phenotype suggestive of loss of vacuolar proton-translocating ATPase (VATPase) activity. The mutation disrupts two genes, CWH36 and a recently identified open reading frame on the opposite strand, YCL005W-A. We demonstrate that disruption of YCL005W-A is entirely responsible for the Vma ؊ growth phenotype of the cwh36⌬ mutant. YCL005W-A encodes a homolog of proteins associated with the Manduca sexta and bovine chromaffin granule V-ATPase. The functional significance of these proteins for V-ATPase activity had not been tested, but we show that the protein encoded by YCL005W-A, which we call Vma9p, is essential for V-ATPase activity in yeast. V-ATPases 1 are highly conserved proton pumps responsible for acidification of multiple organelles, including the Golgi apparatus, endosomes, and lysosomes, in all eukaryotic cells (1, 2). All V-ATPases are multisubunit complexes comprised of a peripheral sector, V 1 , attached to a membrane sector, V 0 . In yeast cells, the V 1 sector contains eight different subunits, and the V 0 sector contains five subunits; all of these subunits have homologs in other eukaryotic cells (1, 2). Genetic deletion of any V-ATPase subunit results in a well defined set of growth defects in yeast, including sensitivity to elevated pH and calcium concentrations, inability to grow on non-fermentable carbon sources, and sensitivity to a variety of heavy metals (3, 4). This Vma Ϫ growth phenotype has been invaluable in determining the subunit composition of the yeast V-ATPase and in assessing the level of V-ATPase function in various mutants, and it has largely driven the emergence of yeast as the model system of choice in many studies of eukaryotic V-ATPases (5-7).Although V-ATPase subunit composition is very similar among eukaryotes, highly purified preparations of the Manduca sexta and bovine chromaffin granule V-ATPases both contain membrane proteins of 9 -10 kDa that had not been observed in other systems, including yeast (8, 9). This protein associated tightly with the V 0 sector in both preparations and appeared to be present at a comparable stoichiometry with the other V-ATPase subunits, prompting the investigators to name it the "e" subunit. However, it was impossible to determine whether these proteins played a critical role in V-ATPase function or were "accessory" proteins involved in assembly or regulation in an organelle-and/or species-specific manner. In fact, the bovine protein (8) showed limited homology to the yeast Vma21 protein, which is an endoplasmic reticulum protein essential for V-ATPase assembly (10). This suggested that higher eukaryotes had evolved a version of Vma21p that might play a similar role in assembly but remained attached to the assembled enzyme.We screened a library of yeast deletion strains for mutants exhibiting the Vma Ϫ phenotype characteristic of loss of VATPase activity. 2 One mutant, cwh36⌬, exhibited a very strong Vma Ϫ phenotype and no vacuolar acidification. Similar...
Patients with breast cancer brain metastases have extremely limited survival and no approved systemic therapeutics. Triple negative breast cancer (TNBC) commonly metastasizes to the brain and predicts poor prognosis. TNBC frequently harbors BRCA mutations translating to platinum sensitivity potentially augmented by additional suppression of DNA repair mechanisms through poly(ADP-ribose)polymerase (PARP) inhibition. We evaluated brain penetrance and efficacy of Carboplatin +/− the PARP inhibitor ABT888, and investigated gene expression changes in murine intracranial (IC) TNBC models stratified by BRCA and molecular subtype status. Athymic mice were inoculated intra-cerebrally with BRCA-mutant: SUM149 (basal), MDA-MB-436 (claudin-low), or BRCA-wild-type: MDA-MB-468 (basal), MDA-MB-231BR (claudin-low) TNBC cells and treated with PBS control (IP, weekly), Carboplatin (50mg/kg/week, IP), ABT888 (25mg/kg/day, OG), or their combination. DNA-damage (γ-H2AX), apoptosis (cleaved-Caspase-3(cC3)) and gene expression were measured in IC tumors. Carboplatin+/−ABT888 significantly improved survival in BRCA-mutant IC models compared to control, but did not improve survival in BRCA-wild-type IC models. Carboplatin+ABT888 revealed a modest survival advantage versus Carboplatin in BRCA-mutant models. ABT888 yielded a marginal survival benefit in the MDA-MB-436, but not in the SUM149 model. BRCA-mutant SUM149 expression of γ-H2AX and cC3 proteins was elevated in all treatment groups compared to Control, while BRCA-wild-type MDA-MB-468 cC3 expression did not increase with treatment. Carboplatin treatment induced common gene expression changes in BRCA-mutant models. Carboplatin+/−ABT888 penetrates the brain and improves survival in BRCA-mutant IC TNBC models with corresponding DNA damage and gene expression changes. Combination therapy represents a potential promising treatment strategy for patients with TNBC brain metastases warranting further clinical investigation.
Summary Melanoma cell lines and normal human melanocytes were assayed for p53-dependent G1 checkpoint response to ionizing radiation-induced DNA damage. Sixty six percent of melanoma cell lines displayed a defective G1 checkpoint. Checkpoint function was correlated with sensitivity to ionizing radiation with checkpoint-defective lines being radio-resistant. Microarray analysis identified 316 probes whose expression was correlated with G1 checkpoint function in melanoma lines (P≤0.007) including p53 transactivation targets CDKN1A, DDB2 and RRM2B. The 316 probe list predicted G1 checkpoint function of the melanoma lines with 86% accuracy using a binary analysis and 91% accuracy using a continuous analysis. When applied to microarray data from primary melanomas, the 316 probe list was prognostic of four year distant metastases-free survival. Thus, p53 function, radio-sensitivity and metastatic spread may be estimated in melanomas from a signature of gene expression.
A series of HeLa cell lines which stably express b-globin pre-mRNAs carrying point mutations at nt 654, 705, or 745 of intron 2 has been developed. The mutations generate aberrant 59 splice sites and activate a common 39 cryptic splice site upstream leading to aberrantly spliced b-globin mRNA. Antisense oligonucleotides, which in vivo blocked aberrant splice sites and restored correct splicing of the pre-mRNA, revealed major differences in the sensitivity of these sites to antisense probes. Although the targeted pre-mRNAs differed only by single point mutations, the effective concentrations of the oligonucleotides required for correction of splicing varied up to 750-fold. The differences among the aberrant 59 splice sites affected sensitivity of both the 59 and 39 splice sites; in particular, sensitivity of both splice sites was severely reduced by modification of the aberrant 59 splice sites to the consensus sequence. These results suggest large differences in splicing of very similar pre-mRNAs in vivo. They also indicate that antisense oligonucleotides may provide useful tools for studying the interactions of splicing machinery with pre-mRNA.
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