a-practical-novel-method-to-extract-genomic-dna-from-blood-collection

A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation

Laboratory tests can be done on the cellular or  fluid portions of the blood. The use of different blood collection tubes  determines the portion of the ...

Emory University School of Medicine and Children's Health Care of Atlanta

The fusion tyrosine kinase NPM-ALK is central to the pathogenesis of ALK-positive anaplastic large cell lymphoma (ALK(+)ALCL). We recently identified that MSH2, a key DNA mismatch repair (MMR) protein integral to the suppression of tumorigenesis, is an NPM-ALK-interacting protein. In this study, we found in vitro evidence that enforced expression of NPM-ALK in HEK293 cells suppressed MMR function. Correlating with these findings, six of nine ALK(+)ALCL tumors displayed evidence of microsatellite instability, as opposed to none of the eight normal DNA control samples (P = 0.007, Student's t-test). Using co-immunoprecipitation, we found that increasing levels of NPM-ALK expression in HEK293 cells resulted in decreased levels of MSH6 bound to MSH2, whereas MSH2Â·NPM-ALK binding was increased. The NPM-ALKÂ·MSH2 interaction was dependent on the activation/autophosphorylation of NPM-ALK, and the Y191 residue of NPM-ALK was a crucial site for this interaction and NPM-ALK-mediated MMR suppression. MSH2 was found to be tyrosine phosphorylated in the presence of NPM-ALK. Finally, NPM-ALK impeded the expected DNA damage-induced translocation of MSH2 out of the cytoplasm. To conclude, our data support a model in which the suppression of MMR by NPM-ALK is attributed to its ability to interfere with normal MSH2 biochemistry and function.

Fusion tyrosine kinase NPM-ALK Deregulates MSH2 and suppresses DNA mismatch repair function novel insights into a potent oncoprotein.

La kinase de tyrosine de fusion NPM-ALK est au c&oelig;ur de la pathogen&egrave;se du lymphome anaplasique &agrave; grandes cellules de ALK-positive (ALK(+)ALCL). R&eacute;cemment, nous avons identifi&eacute; que MSH2, une DNA mismatch repair (MMR) prot&eacute;ine cl&eacute; faisant partie int&eacute;grante de la r&eacute;pression de la tumorigen&egrave;se, est un NPM-ALK-interacting protein. Dans cette &eacute;tude, nous avons trouv&eacute; la preuve in vitro qui appliquaient les expression de NPM-ALK en cellules HEK293 supprim&eacute;e fonction RRO. Mise en corr&eacute;lation avec ces r&eacute;sultats, six des neuf tumeurs ALCL ALK (+) affich&eacute; preuve d&#39;instabilit&eacute; des microsatellites, par opposition &agrave; aucun de ces huit &eacute;chantillons de contr&ocirc;le ADN normales (P = 0, 007, test t de Student). &Agrave; l&#39;aide de co-immunopr&eacute;cipitation, nous avons constat&eacute; qu&#39;augmenter les niveaux d&#39;expression de NPM-ALK dans les cellules HEK293 conduit &agrave; une baisse des taux de MSH6 li&eacute; &agrave; MSH2, alors que MSH2&middot;La fixation de NPM-ALK a &eacute;t&eacute; accrue. La NGP-ALK&middot;MSH2 interaction d&eacute;pend de l&#39;activation/autophosphorylation de NPM-ALK, et le r&eacute;sidu de Y191 de NPM-ALK est un site crucial pour cette interaction et r&eacute;pression m&eacute;di&eacute;e par NPM-ALK RRO. MSH2 s&#39;est av&eacute;r&eacute; tyrosine phosphoryl&eacute;e en pr&eacute;sence de NPM-ALK. Enfin, NPM-ALK entrav&eacute; l&#39;attendus ADN induite par le dommage translocation de MSH2 hors du cytoplasme. Pour conclure, les donn&eacute;es supportent un mod&egrave;le dans lequel la suppression de ROR par NPM-ALK est attribu&eacute;e &agrave; sa capacit&eacute; d&#39;interf&eacute;rer avec la fonction et de biochimie de MSH2 normal.

Kinase de tyrosine de fusion NPM-ALK d&eacute;r&eacute;glemente MSH2 et supprime l&#39;ADN incompatibilit&eacute; r&eacute;paration fonction nouveaux aper&ccedil;us une oncoprot&eacute;ine puissant.

La chinasi della tirosina di fusione NPM-ALK &egrave; centrale nella patogenesi del linfoma a grandi cellule anaplastico di ALK-positivo (ALK(+)ALCL). Abbiamo recentemente identificato che MSH2, una chiave del DNA mismatch repair (MMR) proteina integrale alla soppressione della tumorigenesi, &egrave; una proteina interagente NPM-ALK. In questo studio, abbiamo trovato prove in vitro che applicate espressione di NPM-ALK in cellule HEK293 soppressa funzione MMR. Correlazione con questi risultati, sei dei nove tumori ALCL ALK (+) visualizzato prova di instabilit&agrave; dei microsatelliti, al contrario di nessuno degli otto campioni di controllo del DNA normali (P = 0.007, test t di Student). Utilizzo di co-immunoprecipitazione, abbiamo trovato che aumentando i livelli di espressione di NPM-ALK in cellule HEK293 portato a diminuzione dei livelli di associati a MSH2, MSH6, considerando che MSH2&middot;Associazione NPM-ALK &egrave; stato aumentato. Il NPM-ALK&middot;MSH2 interazione dipendeva l&#39;attivazione/autophosphorylation del NPM-ALK, e il residuo di Y191 di NPM-ALK &egrave; stato un sito fondamentale per questa interazione e soppressione MMR NPM-ALK-mediata. MSH2 &egrave; stato trovato per essere la tirosina fosforilata in presenza di NPM-ALK. Infine, NPM-ALK ostacolato l&#39;attesa DNA indotta da danni traslocazione di MSH2 fuori dal citoplasma. Per concludere, i nostri dati supportano un modello in cui la soppressione del MMR di NPM-ALK &egrave; attribuita alla sua capacit&agrave; di interferire con il normale MSH2 biochimica e funzione.

Adam Benjamin

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