Silencing of Genes Required for Glycosylphosphatidylinositol Biosynthesis in a Burkitts’ Lymphoma Cell Line (Ramos) and Hematopoietic Stem Cells (HSC).
Notice bibliographique
Résumé
Abstract Glycosylphosphatidylinositol is an important means of anchoring many cell surface proteins. Glycosylphosphatidylinositol anchored proteins (GPI-AP) are distributed on all hematopoietic lineages, but are absent on hematopoietic cells from patients with paroxysmal nocturnal hemoglobinuria (PNH). The absence of GPI-AP in PNH is due to mutations in PIG-A, whose product is necessary for the 1st step in GPI biosynthesis. A small percentage of GPI-AP deficient cells can be found in cell lines and that these GPI-APlo/neg cells do not harbor PIG-A mutations. The significance and mechanism of the GPI-AP deficiency in these cells are unclear. We found that 25% of the Burkitts’ lymphoma cell line, Ramos, is GPI-APlo/neg after staining with FLAER and that these GPI-APlo/neg cells do not harbor PIG-A mutations. After 2 days cultured in regular medium, the GPI-APlo/neg Ramos cells reverted to a mix of GPI-APlo/neg and GPI-APpos cells demonstrating that the GPI-APlo/neg cells appear to be precursors to the GPI-APpos cells. An in vitro assay for early steps in GPI anchor biosynthesis using UDP-[3H]GlcNAc found that the GPI-APlo/neg cells generated reduced amounts of the 1st and 2nd GPI intermediates, GlcNAc-PI and GlcN-PI. RT-PCR of the 24 known genes involved in GPI anchor biosynthesis revealed silencing of PIGL and to a lesser extent, PIGY. Methylation specific PCR demonstrated that PIGL was hypermethylated in the FLAERlo/neg Ramos cells. Furthermore, culturing the FLAERlo/neg Ramos cells in 5-Aza-2′ deoxycytidine greatly increased the percentage of cells displaying surface GPI-AP, suggesting that demethylating PIGL and perhaps PIGY may restore surface expression of GPI-AP. We hypothesized that primitive HSC may also enriched for GPI-APlo/neg cells. Thus, we isolated small lineage depleted Fr25Lin− cells from C57Bl6/NCR (Ly 5.2) mice as described1 and found that 30% were GPI-APlo/neg. Fr25lin−GPI-APlo/neg cells were highly enriched for HSC/progenitor cells using hematopoietic colony forming assays. We next transplanted lethally irradiated female recipients with either 100 Fr25Lin−FLAERlo/neg or Fr25Lin−FLAERpos, respectively. 3/4 animals transplanted with Fr25lin−FLAERlo/neg cells survived 17 weeks and revealed 81%, 85% and 90% engraftment; 2/4 animals transplanted with 100 Fr25lin−FLAER pos cells survived 17 weeks with 17% and 48% engraftment. Similar to the Ramos cells, RT-PCR analysis of the Fr25lin−GPI-APlo/neg cells revealed silencing of pigl and to a lesser extent, pigy. In this study, we found that reduced surface expression of GPI-AP is a common feature of cancer cell lines and primitive hematopoietic progenitor cells. In contrast to the fixed GPI-AP deficiency in HSC from PNH patients, the absence of GPI-AP in certain cancer cell lines and normal HSC is not fixed; the progeny of these cells acquire GPI-AP upon cellular differentiation. Furthermore, our data suggest that silencing of the genes required for early steps in GPI anchor biosynthesis, most notably PIG-L and PIG-Y, are responsible for the paucity of GPI-AP on the HSC/progenitor cells.
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Comment cette classification a été obtenuedéplier
Prédiction machine sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Le volet Gemma est une étiquette directe du modèle pour chaque travail de la base, lue sur la notice réduite au titre. Le volet Codex est un classifieur appris des 10 348 étiquettes directes de Codex et calibré sur les taux pondérés de l'échantillon; les champs sans appui suffisant ne portent aucun appel Codex. Le mode candidate est l'union des deux volets; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont pas des étiquettes humaines.
Scores du classifieur distillé par catégorie (deux têtes)
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,000 | 0,000 |
| Méta-épidémiologie (sens strict) | 0,000 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,000 |
| Bibliométrie | 0,000 | 0,000 |
| Études des sciences et des technologies | 0,000 | 0,000 |
| Communication savante | 0,000 | 0,000 |
| Science ouverte | 0,000 | 0,000 |
| Intégrité de la recherche | 0,000 | 0,000 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,002 | 0,001 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule source (Gemma direct ou Codex distillé), pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».