OBJECTIVETo investigate the profile of promoter methylation and expression of SHP-1 gene in the progression of chronic myeloid leukemia (CML).

METHODSThe expression level of SHP-1 mRNA and protein in bone marrow or peripheral blood mononuclear cells from CML patients were detected by Western blot and SYBR Green-based qRT-PCR. The methylation status of SHP-1 were assessed by methylation-specific polymerase chain reaction (MSP) assay. K562 cells were infected with the lentiviral plasmids pEX-SHP-1-puro-Lv105 (K562-SHP-1) or pEX-EGFP-puro-Lv105 (K562-EGFP). The levels of proteins and phosphorylated proteins were detected by Western blot. qRT-PCR assay was used to test the level of BCR-ABL mRNA.

RESULTSThe relative levels of SHP-1 mRNA were sharply decreased in advanced stages CML compared to chronic phase (CP)-CML (0.79±0.37 vs 1.18±0.64, P= 0.009). The level of SHP-1 protein was lower in advanced stages CML compared to CP-CML (0.57±0.02 vs 1.02±0.04, P=0.039). The frequency of SHP-1 gene promoter methylation at selected loci in CP-CML was 23.8% (10/42), and the methylated regions were detected in all advanced CML samples (P<0.01). SHP-1 was stably transfected into K562 cells and selected with puromycin. Overexpression of SHP-1 inhibited the proliferation and induced the apoptosis of K562 cells, meanwhile leaded to G0/G1 phase arrest. After transfection, the level of BCR-ABL mRNA was not affected in K562-SHP-1 cells (1.32±0.34) compared to K562-EGFP cells (1.18±0.20, P=0.644), but overexpression of SHP-1 caused a slight decrease in BCR-ABL protein in K562-SHP-1 cells compared to K562 -EGFP cells (0.78±0.15 vs 1.27±0.24, P=0.040). Overexpression of SHP-1 resulted in a remarkable decrease in MYC protein, phosphorylated forms of JAK2, STAT5, Akt and MAPK. However, the un-phosphorylated forms of these molecules were not significantly affected.

CONCLUSIONDecreased expression of SHP-1 caused by aberrant promoter hypermethylation may play a key role in the progression of CML by dysregulation of BCR-ABL, Akt, MAPK, MYC, JAK2 and STAT5 signaling.

Apoptosis ; DNA Methylation ; Disease Progression ; Fusion Proteins, bcr-abl ; Humans ; Janus Kinase 2 ; K562 Cells ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; Leukocytes, Mononuclear ; Phosphorylation ; Protein Tyrosine Phosphatase, Non-Receptor Type 6 ; RNA, Messenger