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

• Published in: Blood 2006-11, Vol.108 (11), p.1191-1191
• Main Authors: Hu, Rong, Mukhina, Galina L., Collector, Michael I., Lee, Soo Hee, Jones, Richard J., Yuan, Xuan, Englund, Paul T., Buckley, Thomas, Sharkis, Saul J., Brodsky, Robert A.
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V108.11.1191.1191

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,