Silencing of Genes Required for Glycosylphosphatidylinositol Biosynthesis in a Burkitts’ Lymphoma Cell Line (Ramos) and Hematopoietic Stem Cells (HSC).
Bibliographic record
Abstract
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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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".