Molecular Pathological Analysis Of The Protein Tyrosine Phosphatase: Shp1 Gene In The Pathogenesis Of Lymphoma And Leukemias

1622 The study of gene expression with cDNA microarrays make it easy to measure the transcripts for every gene at once. We analyzed various types of lymphomas/leukemias with the combination analysis of cDNA macroarray and tissue microarray to investigate the molecular basis of lymphomagenesis and leukemogenesis followed by RT-PCR and Western blotting. We detected significant changes in the gene expression of leukemia/lymphoma cells compared to that of normal PBMCs. Among these genes, we found a strong decrease in hematopoietic cell specific protein-tyrosine-phosphatase SHP1 mRNA by cDNA-macroarray and RT-PCR. With the further analysis with methylation specific PCR (MSP), bisulfite sequencing and restriction enzyme-mediated PCR analyses, aberrant methylation in the CpG island of the SHP1 gene was detected in many leukemia/lymphoma cell lines as well as in patient specimens, including diffuse large B-cell lymphoma (methylation frequency 93%), MALT lymphoma (82%), mantle cell lymphoma (75%), plasmacytoma (100%) and follicular lymphoma (96%). The methylation frequency was significantly higher in high-grade MALT lymphoma cases (100%) than in low-grade MALT lymphoma cases (70%), which correlate well with the frequency of no expression of SHP1 protein in high-grade (80%) and low-grade MALT lymphoma (54%). It suggests that the SHP1 gene silencing with aberrant CpG methylation relates to the lymphoma progression. Transfection of the wild type SHP1 gene to the hematopoitic cultured cells induced growth inhibition, indicating that gene silencing of the SHP1 gene by aberrant methylation is playing an important role to get the growth advantage of the malignant lymphomas/leukemias cells. The cDNA microarray experiments have shown the dramatical change of overall gene expression profile including Jak-Stat pathways with the transfection of wild type and several types of deletion mutants of the SHP1 gene. In the case of the transfection of the c-terminal truncated mutant construction, gene expression of some cluster of genes were up-regulated, on the other hand that of another cluster of genes were down-regulated compared to the expression profile of the wild type SHP1 transfectant, indicating that the c-terminal region of the SHP1 gene is playing critical roles in the regulation of downstream signal transductions. The extra-ordinal high frequency (75 to 100%) of CpG methylation of the SHP1 gene in lymphoma/leukemia patient specimen indicates that the SHP1 gene silencing is one of the critical events to the onset of malignant lymphomas/leukemias as well as important implications for the diagnostic or prognostic markers and the target of gene therapy. Our results suggest that reduced expression of the SHP1 gene associated with the promoter methylation is related to lymphoma/leukemia progression in addition to malignant transformation.