Affection Of Ara-c In Expression Of Cflip_l Mrna In Primary Acute Leukemic Cells

Backgroud and ObjectiveApoptosis is a programmed cell death controlled by genes,which is important in maintenance of the development and homeostasis of tissues. Inhibition of apotosis leads the cell survival prolonged and the accumulation of mutated cells, which end in the development of cancers. Cellular apoptosis is regulated by genes, enzymes and signal pathways. The genes modulating apoptotsis can be divided into two class: Proapoptosis such as Reaper,Smac/DIABLO,Bax and HtrA2,and antiapoptosis such as Bcl-2,CrmA,P53,XIAP and cFLIP (Fas-associated death domain-like interleulin-1-converting enzyme (FLICE)-like inhibitory protein,cFLIP).cFLIP is a noval anti-apoptosis protein. It has two subtypes: long form cFLIP and short form cFLIP defined as cFLIP_L and cFLIP_S, respectively. Although its structure is homologous to caspace-8, the cFLIP protien does not have the proteinase activity of caspase-8. After it combines with the FADD, the cFLIP protein can competed with caspase-8 and block the binding of caspase-8 to FADD. Therefore, the activation of caspase-8 and the following cascade reaction are inhibited. As a result,tumor cells fail apoptosis. Acute leukemia is a malignant disease of hematopoietic system, accounting 5% of the overall cancer morbidity. Recent years,morbidity and mortality of acute leukemia has been increasing, but its mechanism has not been clear. So, the aetiology and pathological mechanisms of leukemia are the research focuses. In order to evaluate the role of cFLIP in primary acute leukemia, the expression of cFLIP_L mRNA was detected by RT-PCR and the effect of cytosine arabinoside (Ara-C) on expression of cFLIP_L mRNA in acute leukemia cells was explored. The findings,may be helpful in understanding the pathogenesis of acute leukemia.Material and methods1 . Patients: Total 26 patients with acute leukemia were enrolled for the research, who hospitalized in the Department of Hematology of the First Affliated Hopsital of ZhengZhou Univesrity from April,2008 to December,2008.These patients have a mean age of 40 years old(ranged from 16 to 63 years old), with 16 males and 10 females. The diagnosis of acute leukemia is made based on complete blood count, bone marrow aspiration, cytochemistry, cytogenetic and immunophenotype, according to the criteria for diagnosis and curative effects of hematological disease (3^rd edition) by Zhang Zhinan. Fifteen patients with non-malignant hematological diseases with relative normal bone marrow were set up as control. These patients have a mean age of 38 years old(ranged from 21 to 57 years old), with 4 males and 6 females.2. Total 5ml heparinized bone marrow were aspirated from 26 cases with acute leukemia. Bone marrow mononuclear cells (BMMNC) were separated by density centrifugation and washed twice. The BMMNCs were aliquoted into 2 flasks marked as untreated and Ara-C treated (cells treated with Ara-C at concentration is 120μg/ml). Meanwhile, BMMNC from 10 cases of benign hematological diseases were set up as control. BMMNC were separated by density centrifugation and washed twice. Adjust cell concentration at 1×10⁷/ml for culture. After cells wers cultured at 37℃,5%CO₂ and saturated moisture incubator for 24h ,they were harvested for RT-PCR assay.3.RT-PCRassay The cultured cells were harvested and washed for total RNA extract by TRIZOL. The cDNA was synthesized by a retrotranscript kit. Chemical synthesized primers targeted cFLIP_L sequence were used to amplify cFLIP_L mRNA, andβ-actin was set up as internal control. Total 25 cycles of PCR were run in a PCR amplifier. The products of PCR were separated by electrophoresis on 2% agarose gel. The bands were visualized by UV light and the optic density of each band was measured. The optic density of cFLIP_L mRNA andβ-actin mRNAwas calculated,the ratio of cFLIP mRNA/β-action mRNA was defined as the express level of cFLIP_L mRNA.4.Sotfware SPSS13.0 was applied to statisticly analyze the data. The measurement data was analyzed by x²-test. The the data were expressed asmean±standard deviative ((x|-)±s). Paired means were analyzed by student t test.Multiple means were analyzed by ANOVA. The standard of singificant level wasα=0.05.Result1. Twenty out of 26 cases (76.9%)of de novo acute leukemia expressed cFLIP_L mRNA in BMMNC, One out of ten cases (10%) of non-malignant hematological diseases expressed cFLIP_L mRNA in BMMNC. The expression rate of cFLIP_L mRNA in de novo acute 1 eukemia patients was significantly higher than that of controls (P<0.05). The mean expression level is 0.47±0.140.1 in 20 cases of de novo acute leukemia with cFLIP_L mRNA expression. The only case of control expressed low level of cFLIP_L mRNA (0.19).2. Expression of cFLIP_L mRNA in different acute leukemia: Fifteen out of 19 cases (81.3%) with AML expressed cFLIP mRNA in BMMNC. While five out of seven cases (71.4%) with ALL expressed cFLIP mRNA in BMMNC. There was no statistical significance between two groups (P>0.05). The mean expression level of cFLIP_L mRNA in 15 cases of AML was 0.49±0.152 and in 5 ALL patients was 0.45±0.127. There was no statistical significance between two groups (x²=0.022, p=0.883). However, The expression levels of cFLIP_L mRNA in both AML and ALL were significantly higher than that in controls (x²=19.81, P=0.00) .3.Affection of Ara-C on the expression of cFLIP_L mRNA in acute leukemia: After BMMNCs from acute leukemia were treated with Ara-c (120μg/ml)for 24h, the expression of cFLIP_L mRNA was significant downregulated as assayed by RT-PCR. Compared to untreated, the expression level of cFLIP_L mRNA was significantly decreased(P<0.01). However, the expression level in Ara-C treated BMNC was still higher than that in controls with no statistical significance(P>0.05).4. The relationship between the expression of cFLIP_L mRNA in BMMNC and clinical response: Total 26 patients with acute leukemia were clinically evaluable after they received remission induction therapy for one cycle. The complete response rate was 60.00% in 20 cases with cFLIP_L mRNA expression. On the other hand, the The complete response rate was 83.33% in 6 cases without cFLIP_L mRNA expression. There was significant difference between two groups(x²=7.231, P=0.026).Conclusions1 .Most de novo acute leukemia express high level cFLIP_L mRNA, while the non-malignant hematological diseases with relative normal bone marrow express no or marginal level of cFLIP_L mRNA. The expression level of cFLIP_L in acute leukemia was significantly higher than that in non-malignant hematological diseases.2. There are no difference in the expression rate of cFLIP_L mRNA between AML and ALL (P>0.05).3.The expression of cFLIP_L mRNA in acute leukemia is down-regulated by Ara-C.4.The expression of cFLIP_L mRNA in acute leukemia is negatively correlated with clinical response. The higher expression of cFLIP_L mRNA is found in BMMNC, the lower clinical response to chemotherapy is observed. These findings suggest that cFLIP_L mRNA level may act as a predicator for the clinical response of acute leukemia.