The Expression Of Cyclin E2 In Acute Leukaemia Patients And Experimental Study Of Regulation Of Cyclin E2 Antisense Oligodeoxynucleotide For K562 Cell Proliferation

Objective: Almost all cyclins can act as proto-oncogene to be activitied and become oncogene in human tumors. Cyclin E2 is a new member of the G1 cyclin family, it encodes a 404-amino-acid protein that is most closely related to cyclin E1. The catalytic activity associated with cyclin E2 complexes is cell cycle regulated and peaks at the G1/S transition. Overexpression of cyclin E2 in mammalian cells accelerates G1, demonstrating that cyclin E2 may be rate limiting for G1 progression. Unlike cyclin E1, which is expressed in most proliferating normal and tumor cells, cyclin E2 levels were low to undetectable in nontransformed cells and increased significantly in tumor-derived cells. Many research observed that the cyclin E2 expression was positive correlated with human tumors such as lung cancer, and gastric,breast,cervix,ovary,brain tumors. One previous research have analysed the expression of cyclin E2 in patients with acute leukemia(AL), observed that the cyclin E2 mRNA expression was significantly elevated in AL patients relative to healthy controls and negtively correlated with prognosis, is the index of uncontrol magligant carcinoma cell growing and assiociated with prognosis. Therefore, we investigated the expression of cyclin E2 in patients with acute leukemia(AL), and relationship with CDK2(cyclin dependent kinase,CDK2)as well as its clinical value.Antisense nucleotides techonology is a method of inhibition the expression of the target gene by antisense nucleotide. This method have been used in the researching of tumor and genetic disorders. So we wish diagram a specially cyclin E2 antisense oligodeoxynucleotide (ASON) to inhibit cyclin E2 gene expression, and also study the effect of cyclin E2 on proliferation regulated and apoptosis of leukemia cell in vitro. We used lipid plastid transfection for improve transfection efficiency. We attempted to identify cyclin E2 effect on proliferating tumor cells on molculer level, and speculate to find a way for clinical therapy of leukemia.Method: The expression of Cyclin E2 and CDK2 mRNA were measured in 70 adult acute leukemia patients(including 41 de novo acute leukemia patients, 10 relapsed patients and 19 complete remission patients), and 14 samples of normal controls(NC) by semi-quantity reverse transcription polymers chain reaction(RT-PCR). cyclin E2 ASON were used in vitro culture K562 cell study. MTT assays were used to measure the growth inhibitory effect of transfection of ASON and lipofectamineTM2000. The mRNA expression levels of cyclin E2 were examined by semi-quantitative reverse transcription polymerase chain reaction RT-PCR. Apopsis were detected by flow cytometry and morphology method.Result:1. To detect expression of cyclin E2 and CDK2 mRNA by RT-PCR. Cyclin E2 mRNA and CDK2 mRNA has high expression in AL.The positive rate of cyclin E2 mRNA in newly diagnosed AL patients was(70.7%,M=51.00), cyclin E2 mRNA in healthy controls (HC) has no expression. The level and positive rate of cyclin E2 mRNA in newly diagnosed acute leukemia (AL) patients was higher than that of healthy controls (P<0.01,P<0.05). The positive rate and expression level of cyclin E2 mRNA was no significance difference between acute myelogenous leukemia(AML) (70%,M = 33.95) and acute lymphocytic of leukemia(ALL) (71%,M=32.63) (P>0.05). Two groups were all high than healthy controls .The cyclin E2 level in relapsed patients (70.0%,M=53.6) was higher than that of healthy controls, (P<0.05), with no significance difference compared to newly diagnosed AL and CR patients (P>0.05). The positive rate and expression level of patients in remission(47.4%, M = 34.89) was lower than AL group, (P<0.01,P<0.05). And the cyclin E2 level of patients in remission was higher than HC group's, with no statistical significance, (P>0.05).The positive rate of CDK2 mRNA in newly diagnosed AL patients was (78.1%, M=53.83), The level of CDK2 mRNA in newly diagnosed acute leukemia (AL) patients was higher than that of healthy controls(28.6%,M=20.79),(P<0.01,P<0.05). The positive rate of CDK2 level in relapsed patients (60.0%, M=39.95) was higher than that of healthy controls with no statistical significance, (P>0.05). The level of patients in remission(63.2%, M = 36.37) was lower than AL group, (P<0.05). And the CDK2 level of patients in remission was higher than HC group's with no statistical significance, (P>0.05).2. The therapeutic efficacy in 41 newly diagnosed acute leukemia patients was evaluated. 14 of 29 cyclin E2+ AL patients achieved complete hematological response(CR) after induction therapy with a CR rate of 48.3%. 11 of 12 cyclin E2- AL patients achieved CR with a CR rate of 91.7%. The CR rate in cyclin E2-AL was significantly higher as compared with cyclin E2+AL (χ2=5.016, P<0.05). In CR group cyclin E2+ acute leukemia patients had a higher relapse rate than cyclin E2- group with no statistical significance, (P>0.05).3. In all acute leukemia patients, the level of cyclin E2 mRNA was positively correlated with CDK2 (r=0.5097,P<0.01), 23 of 41 newly diagnosed AL had cyclin E2 and CDK2 mRNA positive coexpression, the positive rate is 56.1%, in which only 9 achieved CR with a CR rate of 38.1%. 8 newly diagnosed AL has cyclin E2 and CDK2 mRNA negative coexpression, in which 7 achieved CR with a CR rate of 87.5%.(P<0.05)。Cyclin E2 and CDK2 mRNA positive coexpression had a lower CR rate than cyclin E2 and CDK2 mRNA negative coexpression.4. In cyclin E2 ASON group the mRNA expression levels of cyclin E2 were significantly inhibited than those in SON,Random ASON and blank groups(p<0.01). The rates of K562 cell were significantly inhibited.The results showed that the antisense oligodeoxynucleotides were effective antisense oligodeoxynucleotides, which were able to inhibit K562 cell growth, to decrease the level of cyclin E2 mRNA, to induce apoptosis of K562 cells significantly.Conclusion:1. The level of cyclin E2 and CDK2 mRNA in newly diagnosed acute leukemia was significantly higher than that of healthy controls. Cyclin E2 and CDK2 in newly diagnosed AL was significantly higher than patients in remission. Relapsed patients was significantly higher than healthy controls. There was no significant difference between relapsed patients and patients in remission, no significant difference between relapsed patients and newly diagnosed acute leukemia either; Suggesting that overexpression of cyclin E2 result in cyclin disorder, which performs significant role in tumor development.2. The CR rate of cyclin E2~- group was significantly higher than that of cyclin E2~+, indicating that the high expression of cyclin E2 is an important factor for low CR rate in leukemia.3. The level of cyclin E2/CDK2 mRNA in AL patients was significantly overexpressioned. There was a positive correlation beween cyclin E2 and CDK2 mRNA. cyclin E2 and CDK2 mRNA positive coexpression AL patients had a lower CR rate than cyclin E2 and CDK2 mRNA negative group. Cyclin E2 is an important factor of poor prognosis in leukemia. Indicating that cyclin E2 and CDK2 cooperated in the development of leukemia. Cyclin E2 and CDK2 positive coexpression leukemia has high proliferation rate and resistence to chemistry therapy, and prone to relapse.4. cyclin E2 ASON can specifically inhibite K562 cell mRNA expression levels as well as the K562 cell proliferations. After transfected with cyclin E2 ASON, K562 cells developed apoptosis. Cyclin E2 gene is likely to be a new taget for antisense nucleotides techonology therapy of leukemia.