Silencing of genes required for glycosylphosphatidylinositol anchor biosynthesis in Burkitt lymphoma.
OBJECTIVE
To investigate the mechanism of glycosylphosphatidylinositol (GPI) anchor deficiency in Burkitt lymphoma cell lines.


METHODS
We identified a large GPI anchor protein deficient population in three different Burkitt lymphoma cell lines through proaerolysin treatment of the cells and flow cytometry analysis using a proaerolysin variant (FLAER). The mechanism of GPI anchor protein deficiency was studied by DNA gene sequencing, a cell-free assay to investigate the GPI anchor biosynthetic pathway, microarray analysis, and quantitative real-time polymerase chain reaction.


RESULTS
Burkitt lymphoma cell lines harbor large populations of FLAER(neg) cells, which are resistant to proaerolysin. In all three cell lines, silencing of a gene involved in an early step in GPI-anchor biosynthesis was responsible for the lack of GPI-anchored proteins on the cell surface. Quantitative polymerase chain reaction and microarray analysis demonstrate that the level of mRNA for PIGL and PIGY is lower in the FLAER(neg) Ramos cells and that mRNA levels of PIGY are reduced in the Akata and Daudi cells. Hypermethylation of these genes was associated with the low levels of mRNA and treatment of the cells with 5-aza-2' deoxycytidine restored cell surface GPI-anchored proteins to the FLAER(neg) cells.


CONCLUSIONS
GPI-anchored protein deficiency in Burkitt lymphoma cells is not due to a genetic mutation (e.g., PIGA); rather, the lack of GPI-anchored proteins results from transcriptional silencing of PIGL and PIGY.
Hu
Rong
R
Division of Hematology, Johns Hopkins University School of Medicine, Baltimore, MD 21205-2196, USA.
Mukhina
Galina L
GL
Lee
Soo Hee
SH
Jones
Richard J
RJ
Englund
Paul T
PT
Brown
Patrick
P
Sharkis
Saul J
SJ
Buckley
J Thomas
JT
Brodsky
Robert A
RA
D002051:Burkitt Lymphoma / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D005434:Flow Cytometry; D020868:Gene Silencing; D017261:Glycosylphosphatidylinositols / Q000096:biosynthesis* / Q000172:deficiency / Q000235:genetics; D006413:Hematopoietic System / Q000166:cytology; D006801:Humans; D007150:Immunohistochemistry; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017261:Glycosylphosphatidylinositols; D012333:RNA, Messenger