The Impact Of RhIFN-γ On The Immune Escape In Leukemic K562 Cells

Chronic myeloid leukemia(CML) is a malignant hematopoietic stem cell disease characterized with the Philadelphia chromosome, and over-proliferation and apoptosis tolerance of malignant done lead to formation and development of CML. Patients with CML traditionally received chemotherapy, targeted therapy combined with hematopoietic stem cell transplantation (HCT). Although the clinical remission rate of was improved, the five-year survival rate is only about 15%～25%. Therefore, exploring new treatment strategy in the prevention and treatment of leukemia is a matter of priority and urgency. Cancer immunotherapy was named Science’s top ten breakthroughs of 2013, have thought for a promising approach to curing malignant tumor. Cytokine induced killer cells (cytokine-induced killer cell, CIK cells) are heterogeneous population. Just one of the mechanism in antitumoral action is the generation of cytokines (such as:interferon gamma, IL-2, TNF-alpha, GM-CSF) and other bioactive substances as well as regulation the function of the immune cell and immune system through the positive feedback loop. It was reported that IFN-γ plays a significant antitumor effect. While it has a negative role in immune regulation, promoting the development of tumor. However, the effect of rhIFN-γ on immune escape molecular labels of leukemia cell line K562 cells is remains unclear.K562 cell line comes from human CML(blastic phase) with the character of both acute leukemia cell and the specific essence of CML cell, which is a model for drug screen testing as well as the mechanism of leukemia in vitro. This project investigated the effects of recombinant human interfer gamma (rhIFN-γ) on the immune escape of K562 cells. We observed the time and dose inhibition efficacy of rhIFN-γ on K562 cells proliferation with MTT, trypan blue staining and colony formation assay; the phenotype of CD 123 was measured by flow cytometry; the expression of p53, SOCS1, Cyclin D1 and NSE mRNA was evaluated using RT-PCR assay; the expression of NSE protein was detected by Western blot (WB). Furthermore, we observed the effect of 200U/ml,104U/ml rhIFN-γ intervention on mentioned molecular Tags of K562 cells.β-Galactosidase Staining Kit can be using to estimate the senescence in K562 cells induced by rhIFN-y, and the synergistic effect of TNF alpha. Above all, the research explore the regulation effect of rhIFN-γ on K562 cells, in order to provide important information for elucidating the mechanism of CIK cells.Results:(1) The relative inhibition of K562 cells proliferation was hampered when the cells were treated with rhIFN-y for 24h,48h,72 h at the concentration from 103 to 105 U/ml rhIFN-γ demonstrated obvious dose-effect relationship. Whereas 50～800 U/ml rhIFN-γ at 48h～72 h, it got a significant raise, particularly with 200 U/ml rhIFN-γ. (2) After treatment with rhIFN-γ at 0,200 and 104 U/ml, the percentage of CD123 expression on K562 cells was (4.10±1.46)%、(7.2±2.50)% as well as(21.6±1.17)%, respectively. Compared with the blank control group,104U/ml rhIFN-γ increased a significant positive expression of CD123 on K562 cells (P<0.05). (3) RT-PCR analysis showed that the expression of SOCS1, CyclinD1 mRNA was up-regulated in K562 cells induced by rhIFN-γ. The expression of p53 mRNA in K562 cells was down-regualted by rhIFN-γ from 24-48h, while over expression induced by 104U/ml rhIFN-y at 7d. The NSE mRNA expression was up-regulated by 104U/ml rhIFN-y from 3-7d. While contrast to control group, there existed a significant difference (P<0.05). (4) 104U/ml rhIFN-γ at 2h significantly increased NSE protein level in K562 cells detected by Western blot (P<0.05). (5) Senescent cell stiffness increased in K562 cells induced by 2000U/ml rhIFN-γ assessed by staining for senescence-associated SA-β-Gal. At the same time, we obeserved the synergistic senescence effect of TNF-a. It has a significant difference in statistics (P<0.05).Conclusion:The double effect of rhIFN-y on the K562 cells proliferation appeared inhibition or promotion. The expression level of SOCS1, Cyclin D1, NSE mRNA was increased induced by rhIFN-y. The expression of p53 mRNA in K562 cells was down-regualted by rhIFN-y from 24-48h, while shown up-regulation at 7d. rhIFN-y elevated the protein expression level of CD 123 and NSE, as well as promotion K562 cells senescence. Above all, the two faces of rhIFN-γ suggest that the regulation of biological labels in K562 cells had variation. Exploring its optimal therapy time, dosage and duration will be done.