The Expression Of CyclinE, CDK2 And Ki-67 And Their Clinical Significance In Leukemia

IntroductionTumorigenesis and its development are closely related to the molecular mechanism of cell cycle regulation. Through the investigation of the regulation of cell cycle, people realized that activation of cyclin dependent kinases (CDKs) is pivotal part of this process finishing the cell cycle by phosphorylation of the substrate. And the activation of these protein kinases depend upon a group of protein that specifically expressed, accumulated and discomposed in the cell cycle. This group of protein called cyclins. CyclinE is one of which expressed in the G1 phase. The expression of cyclinE is important in the cell proliferation. Cyclin dependent kinase 2 (CDK2) is another important one. They both are important regulator in the G, and S phase of cell cycle. Their abnormal expression is closely related to the tumor development and progression. Ki-67 in a non ?historic protein exists in the malignant and normal proliferation cells which contains two polypeptide chain of 395 KD and 345 KD respectively. It is the cell proliferation related antigen, expressed in the proliferation stage, but not in the quiescent stage. It can be used for measuring cell proliferation on tumor. We used immunohistochemistry method examined the expression of cyclinE, CDK2 and Ki-67 in 50 leukemia patients bone marrow (acute 40, chronic 10) and combined the clinical data analysis to investigate the abnormal expression of cyclinE , CDK2 and Ki-67 in the development and progression in leukemia.MaterialA total of 50 leukemia cases from 1st Teaching Hospital in China Medical University were studied, as obtained from Jan 1998 to Jan 2003, 31 males, and 19 females, ranging in age from 15 to 70, mean age 40.3. Acute non - lympho-cyte leukemia (ANLL) 22 cases, (M1, one, M2 four, M3 four, M4 two, M5 six, M6 three and M7 two cases) , acute lymphocyte leukemia (ALL) 18 cases ( Lj one, L2 fifteen and L3 two) and 10 cases of chronic myeloid leukemia (CML). All cases were consistent with hematology diagnosis and treatment standard. All 50 patients had collected bone marrow before chemotherapy using 0.2 ~0.3 ml heparin, Ficoll ( density 1. 077 ) isolate ( 2000 rpm/min 20 min) monocyte. Centrifuge making slides, store at -20. Control group is bone morrows from 10 normal people.Reagents; Immunohistochemistry, using S-P method, first antibody is mouse anti human CDK2 monoclonal antibody ( working concentration is 1: 200 ). Mouse anti human cyclinE monoclonal antibody, mouse anti human Ki-67 monoclonal antibody (instant version), S-P kit.MethodS-P immunohistochemistry: Bone marrow slides fixed with cold acetone plus 0.5% NP-40, placed in room temperature, added first antibody recognize cy-clinE, CDK2 (working concentration 1:200) , Ki-67 mouse to human monoclonal antibody respectively, 4 degree over night, then used biotin conjugated second antibody ( reagent C). PBS washed in each step. Fresh made DAB solution added to visualize. Finally washed with tap water. Hematoxylin countered stain, dried the slides and mounted with cover slips. Use PBS as negative controls as primary antibody, ovarian tumor cell line OVCAR3 as positive control for cyclinE, breast cancer cell line MCF-7 as positive control for CDK2 and Ki-67 , respectively. Result observation: the positive for cyclinE, CDK2 and Ki-67 is showing yellow -brown granules in the leukemia cells. Count 400 cells under light - microscope. Count percentage of positive cells. If positive equal or above 15% , we consider it is positive expression. The method of statistic for this research used is SPSS10.0 statistic software.ResultsI The expression of cyclinE, CDK2 and Ki-67 in normal and leukemia patientsIn 50 leukemia patients, cyclinE, CDK2 and Ki-67 positive rates were 38.0% (19/50), 64.0% (32/50), and 16.0% (8/50), respectively. In normal control group we didnt detect cyclin E and Ki-67 expression. CDK2 positive rate in normal control was 20. 0% (2/10). Both cyclinE and CDK2 expression in patients was higher than normal control (P <0.05). Ki-67 expression in...