Sphingolipid metabolism and signaling pathway is differentially regulated in leukemic LGLs. The expression profile of LGL leukemia PBMCs (n = 30) was compared with that of activated normal PBMCs (n = 3) using Gene Set Enrichment Analysis (GSEA). Two of the 13 pathways enriched (FDR ≤ 15%) in leukemic LGLs are shown. The expression profile of the components of (A) EDG_Pathway (P = .006) and (B) ST_GA_12_pathway (P = .001) gene sets in leukemic LGLs compared with activated normal PBMCs. Each column represents individual sample from a LGL leukemia patient (gray) or healthy donor (yellow). Each row represents a gene. Red shows high expression, white denotes intermediate expression, and blue denotes low expression. (C) Microarray analysis of ASAH1 mRNA expression. The expression of ASAH1 mRNA in naive normal PBMCs (N PBMCs, n = 4), and activated normal PBMCs (AC PBMCs, n = 3) compared with LGL leukemia PBMCs (TLGLs, n = 30). Each bar represents mean relative fluorescence units, and error bars represent standard error of mean (SEM). (D) Differential expression of ASAH1 in LGL leukemia PBMCs. Expression of α-subunit of acid ceramidase in naive normal PBMCs (N PBMCs, n = 3) and activated normal PBMCs (AC PBMCs, n = 4) compared with LGL leukemia PBMCs (TLGLs, n = 6). Samples were subjected to SDS-PAGE followed by membrane transfer. The blot was probed with antibody to α-subunit of acid ceramidase or β-actin and developed using chemiluminescence. Western blot analysis suggests that acid ceramidase is expressed in naive PBMCs constitutively (N PBMCs, lanes 1-3). Following activation of lymphocytes, α-subunit of acid ceramidase is down-regulated to almost undetectable levels (AC PBMCs, lanes 4-7), whereas it is significantly up-regulated in all LGL leukemia PBMC samples (TLGLs, lanes 8-13). (E) Relative abundance of S1P receptors in naive enriched normal CD8⁺ cells (white dots, n = 4) and leukemic LGLs (black dots, n = 5) as analyzed by real-time PCR. The figure shows that S1P₅ is the predominant S1P receptor for leukemic LGLs. (F) Relative expression of S1P₁ (□), S1P₄ (), and S1P₅ (■) in leukemic LGLs (n = 5) compared with normal phenotypes (n = 3-5). A positive value indicates up-regulation, whereas a negative value indicates down-regulation of a gene in leukemic LGLs. Error bars represent standard deviation of expression. S1P₅ is up-regulated in LGL leukemia PBMCs compared with all normal phenotypes. S1P₁ is up-regulated in naive phenotypes compared with both activated phenotypes and leukemic LGL. (*P < .05; **P < .001.)