Silencing of Genes Required for Glycosylphosphatidylinositol Biosynthesis in a Burkitts’ Lymphoma Cell Line (Ramos) and Hematopoietic Stem Cells (HSC).

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-AP^lo/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-AP^lo/neg after staining with FLAER and that these GPI-AP^lo/neg cells do not harbor PIG-A mutations. After 2 days cultured in regular medium, the GPI-AP^lo/neg Ramos cells reverted to a mix of GPI-AP^lo/neg and GPI-AP^pos cells demonstrating that the GPI-AP^lo/neg cells appear to be precursors to the GPI-AP^pos cells. An in vitro assay for early steps in GPI anchor biosynthesis using UDP-[³H]GlcNAc found that the GPI-AP^lo/neg cells generated reduced amounts of the 1^st and 2^nd 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 FLAER^lo/neg Ramos cells. Furthermore, culturing the FLAER^lo/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-AP^lo/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-AP^lo/neg. Fr25lin⁻GPI-AP^lo/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⁻FLAER^lo/neg or Fr25Lin⁻FLAER^pos, respectively. 3/4 animals transplanted with Fr25lin⁻FLAER^lo/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-AP^lo/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.