A single nucleotide polymorphism determines protein isoform production of the human c-FLIP protein

Blood ◽  
2009 ◽  
Vol 114 (3) ◽  
pp. 572-579 ◽  
Author(s):  
Nana Ueffing ◽  
Kusum K. Singh ◽  
Andrea Christians ◽  
Christoph Thorns ◽  
Alfred C. Feller ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
B Cell ◽  
Cell Lines ◽  
Splice Site ◽  
Death Receptor ◽  
Protein Translation ◽  
Nucleotide Polymorphism ◽  
Inhibitory Protein ◽  
Single Nucleotide ◽  
B Cell Homeostasis

Abstract The cellular FLICE-inhibitory protein (c-FLIP) is a modulator of death receptor-mediated apoptosis and plays a major role in T- and B-cell homeostasis. Three different isoforms have been described on the protein level, including the long form c-FLIPL as well as 2 short forms, c-FLIPS and the recently identified c-FLIPR. The mechanisms controlling c-FLIP isoform production are largely unknown. Here, we identified by sequence comparison in several mammals that c-FLIPR and not the widely studied c-FLIPS is the evolutionary ancestral short c-FLIP protein. Unexpectedly, the decision for production of either c-FLIPS or c-FLIPR in humans is defined by a single nucleotide polymorphism in a 3′ splice site of the c-FLIP gene (rs10190751A/G). Whereas an intact splice site directs production of c-FLIPS, the splice-dead variant causes production of c-FLIPR. Interestingly, due to differences in protein translation rates, higher amounts of c-FLIPS protein compared with c-FLIPR are produced. Investigation of diverse human cell lines points to an increased frequency of c-FLIPR in transformed B-cell lines. A comparison of 183 patients with follicular lymphoma and 233 population controls revealed an increased lymphoma risk associated with the rs10190751 A genotype causing c-FLIPR expression.

scholarly journals miR-146a promotes the initiation and progression of melanoma by activating Notch signaling

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Matteo Forloni ◽  
Shaillay Kumar Dogra ◽  
Yuying Dong ◽  
Darryl Conte ◽  
Jianhong Ou ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Notch Signaling ◽  
Cell Lines ◽  
Somatic Mutation ◽  
Melanoma Cell ◽  
Human Melanoma ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Oncogenic Mutations ◽  
Melanoma Cell Lines

Oncogenic mutations in BRAF and NRAS occur in 70% of melanomas. In this study, we identify a microRNA, miR-146a, that is highly upregulated by oncogenic BRAF and NRAS. Expression of miR-146a increases the ability of human melanoma cells to proliferate in culture and form tumors in mice, whereas knockdown of miR-146a has the opposite effects. We show these oncogenic activities are due to miR-146a targeting the NUMB mRNA, a repressor of Notch signaling. Previous studies have shown that pre-miR-146a contains a single nucleotide polymorphism (C>G rs2910164). We find that the ability of pre-miR-146a/G to activate Notch signaling and promote oncogenesis is substantially higher than that of pre-miR-146a/C. Analysis of melanoma cell lines and matched patient samples indicates that during melanoma progression pre-miR-146a/G is enriched relative to pre-miR-146a/C, resulting from a C-to-G somatic mutation in pre-miR-146a/C. Collectively, our results reveal a central role for miR-146a in the initiation and progression of melanoma.

scholarly journals Serotonin 5-HT2C Receptor Cys23Ser Single Nucleotide Polymorphism Associates with Receptor Function and Localization In Vitro

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Michelle A. Land ◽  
Holly L. Chapman ◽  
Brionna D. Davis-Reyes ◽  
Daniel E. Felsing ◽  
John A. Allen ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Subcellular Localization ◽  
Cell Lines ◽  
Intracellular Signaling ◽  
Calcium Release ◽  
Wild Type ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Recycling Pathway

Abstract A non-synonymous single nucleotide polymorphism of the human serotonin 5-HT2C receptor (5-HT2CR) gene that converts a cysteine to a serine at amino acid codon 23 (Cys23Ser) appears to impact 5-HT2CR pharmacology at a cellular and systems level. We hypothesized that the Cys23Ser alters 5-HT2CR intracellular signaling via changes in subcellular localization in vitro. Using cell lines stably expressing the wild-type Cys23 or the Ser23 variant, we show that 5-HT evokes intracellular calcium release with decreased potency and peak response in the Ser23 versus the Cys23 cell lines. Biochemical analyses demonstrated lower Ser23 5-HT2CR plasma membrane localization versus the Cys23 5-HT2CR. Subcellular localization studies demonstrated O-linked glycosylation of the Ser23 variant, but not the wild-type Cys23, may be a post-translational mechanism which alters its localization within the Golgi apparatus. Further, both the Cys23 and Ser23 5-HT2CR are present in the recycling pathway with the Ser23 variant having decreased colocalization with the early endosome versus the Cys23 allele. Agonism of the 5-HT2CR causes the Ser23 variant to exit the recycling pathway with no effect on the Cys23 allele. Taken together, the Ser23 variant exhibits a distinct pharmacological and subcellular localization profile versus the wild-type Cys23 allele, which could impact aspects of receptor pharmacology in individuals expressing the Cys23Ser SNP.

scholarly journals Prevalence of Early B-Cell Factor 1 Gene rs4704963 Single Nucleotide Polymorphism (T>C) in a Population of Type 2 Diabetic Patients with Obesity

2018 ◽  
Vol 86 (5) ◽  
pp. 310-316
Author(s):  
Mario B. Principato ◽  
Rocío Castilla ◽  
Alejandra Duarte ◽  
Julio C. Bragagnolo ◽  
Graciela Ortensi ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
B Cell ◽  
Diabetic Patients ◽  
Nucleotide Polymorphism ◽  
Type 2 Diabetic Patients ◽  
Single Nucleotide ◽  
Early B Cell Factor ◽  
Type 2 Diabetic

A Single Nucleotide Polymorphism in CD19 Defines a Novel Target for Immunotherapy of B Cell Malignancies with CD4+ Cytotoxic T Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1798-1798
Author(s):  
Robbert M. Spaapen ◽  
Henk M. Lokhorst ◽  
Kelly van den Oudenalder ◽  
Brith Otterud ◽  
Harry Dolstra ◽  
...  
Keyword(s):  
T Cells ◽  
Single Nucleotide Polymorphism ◽  
B Cell ◽  
Cd4 T Cells ◽  
Class Ii ◽  
Hla Class Ii ◽  
Malignant Cells ◽  
Nucleotide Polymorphism ◽  
B Cell Malignancies ◽  
Single Nucleotide

Abstract The Graft-versus-Tumor (GvT) effect of HLA-matched allogeneic stem cell transplantation (allo-SCT) is largely mediated by donor-derived alloreactive CD4+ and CD8+ T cells. Major targets of this curative effect are the minor Histocompatibility antigens (mHags) expressed on the malignant cells. Here we report the first mHag encoded by a hematopoietic gene and recognized by HLA class II (HLA-DQA1*05/B1*02)-restricted CD4+ T cells. This antigen is encoded by a single nucleotide polymorphism (SNP) in the B cell lineage-specific CD19 gene, a highly important target antigen for immunotherapy of almost all B cell malignancies. We identified this antigen using a novel and powerful genetic strategy, in which a phenotype-genotype correlation scanning was the key step for fine-mapping the genetic locus defined by pair-wise linkage analysis. In functional assays, CD4+ T cells specific for the CD19L-encoded mHag mediated effective peptide-dependent maturation of DCs and polarized them to produce significant levels of interleukin-12. In another assay, the CD19L-mHag-specific T cells facilitated the proliferation of a CD8+ mHag-specific T cell clone in an antigen-dependent manner. Even more important, they also lysed CD19L-positive malignant cells, illustrating the therapeutic advantages of targeting this CD19 L -derived, HLA class II-restricted mHag. The currently available immunotherapy strategies enable the exploitation of these therapeutic effects within and beyond allo-SCT settings.

A 3′ UTR Single Nucleotide Polymorphism 829C→T in Dihydrofolate Reductase Gene Results in an Increase in DHFR Protein Level and MTX Resistance.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2084-2084
Author(s):  
Prasunkumar J. Mishra ◽  
Giuseppe S.A. Longo ◽  
Lata G. Menon ◽  
Emine Abali ◽  
Debabrata Banerjee ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Cell Lines ◽  
Dihydrofolate Reductase ◽  
Protein Level ◽  
Gene Copy ◽  
Mrna Levels ◽  
Wild Type ◽  
Nucleotide Polymorphism ◽  
Dhfr Gene ◽  
Single Nucleotide

Abstract Dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate to tetrahydrofolate (THF) required for the synthesis of thymidylate and purines. Methotrexate (MTX) acts as a tight-binding inhibitor of DHFR and remains an important chemotherapeutic agent for treatment of leukemias and lymphomas. Increased DHFR confers resistance to antifolates in target cells. A previously reported single nucleotide polymorphism (SNP) 829C/C→829T/T (829C→T) found in the 3′- untranslated region of DHFR gene transcript (between the first and second polyadenylation site) was associated with higher expression of the DHFR transcript. The SNP was identified in 5.4% of the cases and 6.0% in the controls of Japanese patients with childhood leukemia/lymphomas (Goto et al. 2001, Clinical Cancer Research, Vol. 7, 1952-1956). The objective of the present study was to determine the role of the 3′ UTR SNP 829C→T in DHFR gene expression, DHFR protein level and resistance to MTX. The mutation 829C→T in the 3′ UTR of wild type DHFR was introduced by site directed mutagenesis and the mutant cDNA expressed in DHFR deficient CHO cells (DG-44), wild type DHFR and vector alone constructs were also transfected into DG44 as controls. After two weeks of selection in G418 containing media, several well-isolated surviving colonies were picked and expanded as cell lines in media containing G418. Real-time quantitative PCR was used to compare mRNA and genomic DNA level of the clones while Western blotting was used to compare the protein levels. MTX cytotoxicity assay was carried out in media lacking thymidine. Clones expressing the mutant 829C→T showed greater than two fold enhanced expression of DHFR transcripts as compared to wild type clones. Corresponding to the high mRNA levels, an increase in DHFR protein level was observed in the mutant clones without an increase in DHFR gene copy number. Cytotoxicity studies showed that cell lines with increased levels of DHFR were significantly more resistant to MTX than cells with wild type 3′ UTR. Of interest clonogenic efficiency of the mutants in medium lacking thymidine was greater than wild type and was directly proportional to the level of DHFR expressed in the clones. This study demonstrates that when SNP 829C→T is introduced in the 3′ UTR of wild type DHFR, the expression of the DHFR mRNA is enhanced with a corresponding increase in the protein level. The presence of a SNP 829C→T in patients with ALL may contribute to treatment failure, as MTX is a key drug in curative regimen for this disease. Future studies are directed toward determining the abundance of this SNP in other populations, and the correlation between this SNP and clinical methotrexate resistance and or decreased MTX toxicity.

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