The Expression And Function Of HSP22 Gene In Hematological Malignances

Hsp22 gene was isolated by Craig and McCarthy from the Drosophila melanogaster genome in 1980. Then Smith et al. detected the HSP22 gene from the HeLa and melanoma cDNA libraries in 2000. Hsp22 (also known as HSPB8, H11, and E2IG1) was categorized as a member of the superfamily of sHSPs based on the presence of the conserved a-crystallin domain in this protein. It is composed of 196 amino acids with a calculated molecular mass of 21.6 kDa. Analysis of abundance of hHsp22 mRNA shows the highest expression of human Hsp22 was found in skeletal and smooth muscles, placenta, heart, and brain. Recent studies have demonstrated HSP22 displays chaperone activity and autokinase activity and was involved in regulation of cell proliferation, apoptosis. Abnormal expression of HSP22 correlates with development of cardiac hypertrophy, Alzheimer's disease and different neuromuscular diseases. Hsp22 is also involved in tumors and its expression is cell-type-specific and can be altered in human tumors. Recently, it was found that overload of HSP22 triggers melanoma cell apoptosis but contribute to breast carcinogenesis and progression. The mRNA and protein levels of Hsp22 were reduced in various tumor tissues and cell lines (i.e., prostate cancer, sarcoma) relative to their normal counterparts and the percentage of apoptotic cells increased with time after Hsp22 induction.In these cells, expression was restored by the demethylating agent 5-aza-2-deoxycytidine (decitabine, Aza-C, DacogenTM) suggesting that Hsp22 expression was inhibited by aberrant DNA methylation. To find its role in leukemia, we examined the expression of HSP22 by RT-PCR both in 13 human hematopoietic malignant cell lines and bone marrow samples of 20 patients and 10 healthy volunteers, however no expression was found. Then we tried cytarabine (Ara-c), Daunorubicin (DRN),17B-estradiol (E2) and decitabine to induce HSP22 expression, the results showed only 5-aza-2'-deoxycytidine worked and it was does-dependent. Then we tested the genomic DNA in 5 human hematopoietic malignant cell lines and bone marrow samples of 15 patients and 5 healthy volunteers and found DNA methylation in the promoter region of HSP22 gene, using the MSP method. Lentivector expression system-mediated delivery of HSP22 into K562 and Namalwa cells can inhibit proliferation of Namalwa in vitro and K562 in vivo. HSP22 transduction can also inhibit colony forming of K562 and Namalwa cells. However there was no significant difference in cell cycle and apoptosis.Aza-C is an effective antileukemic agent because of its reactivation of several types of tumor (growth) suppressor genes and tumor-associated antigen genes. The selective BCR-ABL tyrosine kinase inhibitor (TKI) imatinib mesylate is now frontline therapy for chronic myeloid leukemia (CML). Recently published data indicate that Decitabine and imatinib mesylate have a synergistic antitumor activity, and decitabine may be a potential candidate to overcome imatinib resistance in patients with CML. In this study, our results suggest decitabine treated leukemic cells may express HSP22. To detect the fuction of HSP22 in leukemia and to find if HSP22 is involved in the synergistic antitumor activity of decitabine and imatinib mesylate, we treated the transducted K562 (BCR/ABL+) cells with imatinib mesylate and found that HSP22 can enhance imatinib mesylate-induced apoptosis. To figure out the mechanism of enhanced apoptosis of K562 cells by HSP22, we detected the expression of mdrl and BCR/ABL gene by real-time PCR. Our results show that HSP22 can significantly reduce the expression level of mdrl and BCR/ABL gene, suggesting that HSP22 may enhance imatinib mesylate-induced apoptosis through down-regulation P-glycoprotein and Bcr/Abl levels.To investigate the involvement of HSP22 in the synergistic antitumor activity of decitabine and imatinib mesylate, wild type K562 cells were treated with decitabine to induce HSP22 expression, then cells were transfected with HSP22-specific siRNA and Non targeting siRNA, respectively. Compared to Non targeting siRNA, Anti-HSP22 siRNA treatment reduce imatinib induced apoptosis of K562 cells. These findings further support the hypothesis that HSP22 could be involved in mediating the synergistic antitumor activity of decitabine and imatinib mesylate.In conclusion, our work firstly demonstrated that the promoter methlation is the reason for HSP22 silence in hematopoietic system. The effective demethlating agent decitabine can induce HSP22 expression. Ectopic HSP22 expression inhibits the proliferation and colony forming of leukemic cells. HSP22 can enhance imatinib mesylate induced apoptosis through down-regulation P-glycoprotein and Bcr/Abl levels in K562 cells. HSP22 knockdown can reduce imatinib induced apoptosis of K562 cells, suggesting that HSP22 could be involved in mediating the synergistic antitumor activity of decitabine and imatinib mesylate.