Cyclin E2和Survivin在急性白血病的表达及其相关性
作者：王颖，徐世荣，林凤茹，郭晓楠，任金海
【摘要】 为了解细胞周期蛋白E2（cyclin E2）和存活蛋白（survivin）在急性白血病中基因表达及两者间的相关性，采用逆转录－聚合酶链反应的方法检测了84例成人急性白血病患者（16例复发患者，60例初治患者和8例持续缓解患者）和20例正常人的cyclin E2 和survivin mRNA的表达。结果表明： ①cyclin E2在急性白血病中表达的阳性率（70.24％）高于对照组（0％）；survivin在急性白血病中表达的阳性率（72.62％）高于对照组（30％）；②在急性白血病中cyclin E2的表达与survivin的表达呈正相关性，r＝0.65；③cyclin E2阳性组缓解率（55.81％）低于cyclin E2阴性组（88.24％），cyclin E2阳性组复发率（41.67％）高于阴性组（20％）；复发组cyclin E2表达率在三组中最高，持续缓解组cyclin E2表达率在三组中最低，④cyclin E2在急性髓系白血病患者表达率（59.32％）低于急性淋巴细胞白血病患者的表达率（96％）；与发病时白细胞计数未见有统计学意义的相关。结论：首次证实cyclin E2在急性白血病中有异常表达，且对临床预后有不良影响；cyclin E2在急性白血病中的表达与survivin有正相关，提示cyclin E2在急性白血病中有可能作为一种微小残留病变的检测指标。
【关键词】 存活蛋白
As a malignant hemophaty，acute leukemia (AL)，especially，acute lymphocytic leukemia （ALL） underwent a high relapse rate and a short disease-free survival (DFS)，in spite of the improvement in clinical outcome benefited from the development of hematopoietic stem cell transplantation (HSCT) and chemical therapy.Preventing relapse and prolonging DFS still is the focus of AL therapy.Minimal residual disease (MRD) became an effective indication for the foundation of post-remission therapy regulation since the routine morphological examination of the peripheral blood and bone marrow sample from the remission patient was normal.But the fusion gene such as AML1/MTG8 and PML/RARα as MRD marker，was only used in M2，M3，so it is valuable to search another new MRD marker for leukemia.On the other hand，RNA interference (RNAi) as a new and prospective gene therapy for cancer has become a new focus of the study of AL therapy［1］.Searching for an effective new target for a tumor-specific RNAi is worth developing for leukemia therapy.Recently，as a type of G1 cyclin，cyclin E2 was found out to be the potential marker of tumors since it presented a positive expression in the cell lines of solid tumors and a negative expression in proliferating normal cells［2］.However，there has been no report on its expression and role in AL.In order to find out the role of cyclin E2 in the progression and clinical outcome of Al patients，reverse transcription polymerase chain reaction (RT-PCR) is adopted to test the expression of cyclin E2 and survivin mRNA in 84 adult patients with AL，20 normal persons as controls and leukemia cell line K562.Subsequently，we investigated the relationship between the expression of cyclin E2 and the clinical progression of AL patients.The study of cyclin E2 can provide a preliminary theoretical basis for searching a new target of AL gene therapy and a new MRD marker.
Materials and Methods
Patients enrolled
84 adult inpatients with AL from April 2002 to March 2003 in the Second Affiliated Hospital of Hebei Medical University were enrolled in this study.The diagnoses of these patients [50 males and 34 females，14-62 years old (mean of 34.1 years)］were confirmed by morphological，cytochemical and immunologic marker analysis.According to the French-American-British (FAB) classification，59 patients were diagnosed as acute myelocytic leukemia (AML)，including 2 cases of M1，12 cases of M2，21 cases of M3，11 cases of M4，11 cases of M5，1 cases of M6，1 cases of M7.25 patients were diagnosed as acute lymphocytic leukemia (ALL) including 4 cases of L1，19 cases of L2，2 cases of L3; 60 patients with de novo AL，16 relapse cases，8 cases of continuously complete remission (CCR) ( time of disease-free survival≥3 years).20 normal persons aged 15-60 years (mean of 35 years old) containing 11 males and 9 females served as controls.Treatments: all these patients with AML，except M3，received standard ADE regimen (cytarabine arabinoside 150 mg/m2 for 7 days，daunorubicin 40 mg/m2 for 3 days and etoposide 75 mg/m2 for 3 days-7 days ).Patients with M3 received all-trans-retinoic acid (20-40 mg/day) and arsenic trioxide (10 mg/day) as remission induction therapy.Patients with ALL received VITP regimen (vincristine 1.4 mg/m2 for 1 day of each week，iphosphamide 1.2 g/m2 for 3 days of each week，pirarabicin 20 mg/m2 for 3 days of each 2 weeks，prednisone 1 mg/kg for 14 days and then gradually decrement until the end of regimen that lasts for about 28 days).
Cells and cell culture
Bone marrow samples from 84 patients with AL and 20 controls were used for mononuclear cells (MNC) extraction.4-5 ml of this fresh heparinized bone marrow samples were separated by Ficoll-Hypaque (Pharmacia Biotech，Uppsala，Sweden) and washed twice with phosphate-buffered saline (PBS)，followed by extraction of whole cell lysates，and the MNC concentration was 106 cells/ml.Leukemia cells amounted to over 80% in bone marrow samples from 84 AL patients assesed by Wright′ s staining，and then the MNCs were frozen after centrifugation and stored at-80℃ until further use.K562 cell line was obtained from the Institute of Hematology，Chinese Academy of Medical Sciences.Cells were maintained at 37℃ in a humidified atmosphere containing 5% CO2.Exponentially growing suspension cultures of leukemia cells were propagated by reseeding at 5×105 cells/ml every 3-5 days，in RPMI 1640 medium supplemented with 10% fetal bovine serum，50 U/ml penicillin G，and 50 μg/ml streptomycin sulfate.K562 cells were harvested at logarithmic growth phase，separated by Ficoll-Hypaque and washed twice with PBS，then frozen after centrifugation and stored at-80℃ until use.
RNA extraction
Total RNA was extracted from MNC and K562 cell by TRIZOL reagent (Gibco)，according to the recommendations of the manufacturer. The concentration and purity of total RNA was determined by UV spectrophotometry，The ratio of A260/A280 reached 1.8-2. The electrophoresis pattern on 0.1% agarose gel stained by ethidium bromide was used for determining the integrity of total RNA showing two bands of 18S and 28S in the gel.Total RNA was frozen at-80℃ until further use.
Semi-quantitative RT-PCR
Reverse transcription reaction 20 μl reverse transcription reaction system including total RNA 1 μg，M-MLV（Promega）200 U，dNTPs （Promega）2 mmol/L 4 μl，RNasin(Promega) 20 U，random primer （Promega）0.5 μg，5×RT buffer 4 μl（Tris-HCI 250 mmol/L，pH 8.3，KCl 375 mmol/L，MgCl2 25 mmol/L，DTT 50 mmol/L） was warmed for reaction at 37℃，60 min，then stop at 95℃ for 5 min.The products was frozen at -20℃ until further use.
Polymerase chain reaction 25 μl PCR system including reverse transcription products 2 μl，oligonucleotide primer 25 pmol，Taq DNA polymerase (Huamei）1 U，dNTPs （Promega）0.2 mmol/L，10×PCR buffer 2.5 μl (MgCl2 15 mmol/L，pH 8.4，Tris-HCl 100 mmol/L，KCl 500 mmol/L，BAS 20 μg/ml），reaction condition asfollows: 94℃ 40 sec，55℃ 40 sec，72℃ 60 sec，totally 29 cycles for cyclin E2 and β-actin; 94℃ 30 sec，55℃ 30 sec，72℃ 60 sec，totally 35 cycles，72℃ 10 min for survivin to stop the reaction.For each set of PCR reactions，parallel reaction using double-distilled water instead of the cDNA template solution as a negative control to assure the quality of the PCR.PCR primers were synthesized by Cybersyn.Survivin: sense primer: 5′-TTGGCAGGTGCCTGT TGAAT-3′，antisense primer: 5′-AGCCAGTCCCC CACAGCAT-3′.The am plified product should be 465 bp［3］. cyclin E2 primer was designed with oligo 6.2 according to gene gi3885975: sense primer 5′-TG GCTTTTAGAGGTATGTGAA-3′，antisense primer 5′-TAATGAATCAATGGCTAGAAT-3′ product 426 bp，β-actin sense primer 5′-TCATCACCATTGGCAAT GAG-3′，antisense primer 5-CACTGTGTTGGCGTA CAGGT-3′，product 155 bp。
PCR analysis of products
The electrophoresis of 10 μl PCR products was performed at 85 V for 90 min in 2% agarose gel contained 0.5 mg/L ethidium bromide.And the bands were viewed and photographed under UV illumination. The transcripts were estimated by the optical density ratio of cyclin E2 or survivin to β-actin.The ratio of positive standard was larger than 0.15.
Statistical analysis
The data was registered as mean±SD.Difference between different groups were evaluated by the chi-square test，correction for continuity chi-square test，and exact probabilities in 2×2 table.Correlation test: analysis of Pearson correlation，P values of less than 0.05 was considerd statistically significant.Statistical analysis was performed using SPSS 10.0 computer software.
Results
Expression of cyclin E2 and survivin mRNA in K562 cells
The expression of both cyclin E2 and survivin in the K562 cell line was positive，and could be used as a positive control，showed in Figure 1，2.
Expression of cyclin E2 in AL and the control
Expression of cyclin E2 mRNA was determined in 84 cases of AL（0.42±0.28)，out of which 59 cases were positive and 25 cases were negative，all 20 controls （0.10±0.02）were 20 negative (Figure 1).The rate of expression of cyclin E2 in AL was 70.24% and much higher than 0% in controls (χ2＝32.47，P<0.005）.
Expression of survivin in AL and the control
Expression of survivin mRNA was determined in 84 cases of AL（0.34±0.25），out of which 61 cases were positive and 23 cases were negative，that 6 in 20 controls （0.13±0.04）were positive and 14 were negative (Figure 2).The rate of expression of survivin in AL was 72.62% and much higher than that in control 30%，（χ2＝12.80，P<0.005）.
The correlation between expressions of cyclin E2 and survivin in AL
Expression of cyclin E2 was significantly positively correlated with expression of survivin in 84 cases of AL(Figure 3)( r =0.65，P<0.0001).
55.81% (24/43) of remission of 43 cyclin E2-positive patients with de novo AL were lower than 88.24% (15/17) of 17 cyclin E2-negative patients with de novo AL; the difference was significant (χ2＝5.63，P<0.05）.All of 14 patients with de novo M3 obtained compelte remission after receiving induction therapy.The rate of remission of 8 cyclin E2-positive patients with de novo M3 was 100% (8/8)，and that of 6 cyclin E2-negative patients with de novo M3 was also 100% (6/6)，and there were no difference between them. The remission rate of 35 cyclin E2-positive patients with de novo AL except M3 was 45.71% (16/35) lower than 81.82% (9/11) of 11 cyclin E2-negative patients with de novo AL except M3 ; and the difference was significant (χ2＝4.40，P<0.05）.39 of the 60 patients who obtained remission had been clinically observed for 24 months，and it proved that 24 remission patients with de novo AL in cyclin E2-positive group has a relapse rate of 41.67% (10/24)，which is higher than 20% (3/15) of the 15 remission patients with de novo AL in cyclin E2-negative group，but the difference was not statistically significant (exact probabilities in 2×2 table，P>0.05).16 remission patients with de novo AL except M3 in cyclin E2-positive group has a relapse rate of 62.5% (10/16)，which is higher than 33.33% (3/9) of the 9 remission patients with de novo AL except M3 in cyclin E2-negative group，P>0.05. Expression of cyclin E2 in relapse group，de novo AL group and CCR group was showed in table.The rate of cyclin E2 expression in relapse group was the highest in the three groups，(100% vs 71.67%，χ2＝4.32，P<0.05; 100% vs 0%，exact probabilities in 2×2 table，P<0.0001); the rate of cyclin E2 expression in CCR group was the lowest in the three groups，(0% vs 71.67%，χ2＝11.31，P<0.005).Table.Expression of cyclin E2 mRNA in relapse group，de novo AL group and CCR group（略）
Discussion
Cyclin E2，as a novel G1 cyclin，consists of 404 aminoacid residues，which shares 47% similarity with cyclin E1.Cyclin E2 shares 70％ identity in the domain of cyclin box with cyclin E1.Therefore，cyclin E2 plays a physiological role similar to cyclin E1，and cyclin E2 binds to Cdk2 to form the cyclin E2-Cdk2 kinase complex that promotes the transition of cell from G1 to S phase.The activity of cyclin E2-Cdk2 kinase complex is inhibited by p27Kip1 and p21Cip1.While the difference between cyclin E2 and cyclin E1 is the point that a low level of cyclin E1 mRNA，but no detectable cyclin E2 mRNA was present in normal proliferating cells; their mRNA variable levels were present in human cancer cells such as lung tumor cell lines，breast cancer cells，maybe cyclin E2 is a marker for cell transformation［2］.However，the expression of cyclin E2 in leukemia cells is unknown，and no report about it was published.In this study，the expression of cyclin E2 and survivin in leukemia cell line K562 was examined by RT-PCR.Both of their mRNA were present in K562 cells，and then we took K562 cells as the positive control.Subsequently，expression of cyclin E2 mRNA in bone marrow MNC from 84 adult patients with AL and 20 controls was examined by semi-quantitative RT-PCR.70.24% of cyclin E2-positive expression rate in AL was significantly higher than in controls 0%.The result indicated that the expression of cyclin E2 gene in Al was amplified，and the abnormal expression of cyclin E2 probably correlated with the malignant behavior of leukemia cells.The result was similar to that in lung，breast and uterus cancer reported by Gudas et al［2］.Survivin as a inhibitor of apoptosis plays a central role in cancer progression and resistance to apoptosis in diverse tumor types except for leukemia［3，4］.72.62% expression rate of survivin in AL was higher than 30% in controls.Our result was similar to that reported in literature［3］.
Subsequent studies showed that expression of cyclin E2 positively correlated with that of survivin in 84 Al (r=0.65)，and their synergetic expression was found in 68 cases out of 84 AL patients enrolled in the analysis.Study showed that retinoblastoma protein (pRb) can interact with the survivin promoter and repress survivin transcription，and E2F activators can bind to the survivin promoter and induce survivin transcription［4］. Cyclin E2 binds to Cdk2 to form cyclin E2-Cdk2 complex that phosphorylates pRb and inactivates pRb，then pRb releases E2F activator［5］.It is important that the activity of cyclin E2-Cdk2 complex positively correlates with the content of cyclin E2［2］.So，the positive correlation between expression of cyclin E2 and survivin suggests that the overexpression of cyclin E2 bound to Cdk2 forms more cyclin E2-Cdk2 complex which phosphorylated and inactivated pRb，and then pRb released more E2F activators; as a result，survivin transcription was increasingly induced.On the other hand，survivin also interacts with the cell cycle regulator Cdk4 leading to cyclin E2-Cdk2 activation，and plays the role of apoptosis inhibitors［6］.Overall，intensive studies of the interaction between cyclin E2 and survivin are valuable to illustrate the mechanism of cyclin E2 action.
Further clinical investigation showed that low remission rate and high relapse rate were presented in cyclin E2-positive AL patients with de novo AL; so did in patients with AL except M3.Overexpression of cyclin E2 was presented in relapse group，while no expression of cyclin E2 was presented in CCR group and controls.A number of studies have demonstrated a strong correlation between the elevated levels of the mRNA and protein of cyclin E1 and acute leukemia progression and mortality，while a detectable low level of cyclin E1 mRNA and protein was expressed in proliferating normal cells such as bronchial epithelial cells，bone marrow MNC［2，7］.Cyclin E2 mRNA was detectably expressed in diverse lung tumor cell lines and breast cancer cells.It was demonstrated that no detectable levels of cyclin E2 mRNA were in the proliferating normal cells like bronchial epithelial cells. Up to now，there have been no studies focusing on the expression of cyclin E2 and its influence on clinical progression of leukemia patients.The investigation result suggested that cyclin E2 was more often expressed in relapse and de novo AL，while no expression of cyclin E2 in CCR group and controls; the aberrant expression of cyclin E2 implicated that AL was in progression and mortality.So cyclin E2 might be a marker for MRD，and the correlation between cyclin E2 expression and progression of AL should be intensively carried out.No significant difference of relapse rate between cyclin E2-positive patients and cyclin E2-negative patients was found as the cases enrolled in analyses and the time of investigation was limited.Increasing the number of patients enrolled in analysis and prolonging the investigation time contribute to intensively evaluate the influence of cyclin E2 on relapse rate.
The study of cyclin E2 and clinical feature in AL patients showed that 59.32% of cyclin E2 expression rate in AML was lower than 96% of that in ALL.ALL was more apt to relapse，worse clinical outcome and less disease-free survival than AML; the aberrant overexpression cyclin E2 in ALL suggested that cyclin E2 correlated with the poor prognosis of ALL.Cyclin E2 as a positive regulator in cell cycle was aberrantly expressed in AL cells.Expression of cyclin E2 should have been correlated with the WBC counts of patients with AL，if the aberrant expression of cyclin E2 accelerated the proliferating of cells，and decreased the apoptosis of cells.But interestingly，the WBC counts of patients with AL at diagnosis were not correlated with the expression of cyclin E2，P>0.05.The interpretation on this result was that the fluctuation in the WBC counts was influenced by the occasion of diagnosis，and subsequently would influence the result accuracy; on the other hand，the result perhaps suggested a function of cyclin E2 outside of cell cycle control.Study reported by Zariwala［8］ has shown that cyclin E2 is expressed in brain，an organ contains predominantly post mitotic cells，while the cyclin E1 transcript is undetectable in the brain and mainly detected in tissues that contain actively dividing cells such as thymus，placenta and testis.So cyclin E2 perhaps plays a role outside of cell cycle control，which is not similar to cyclin E1.
Our study showed that cyclin E2 was aberrantly expressed in AL，and correlated with the poor prognosis of AL，and might become a tumor marker easily for MRD examination.The result of the positive correlation between cyclin E2 and survivin suggested that the role of cyclin E2 promoting the occurrence and development of AL would be correlated with the inhibition of apoptosis engaged by survivin.An in-depth elucidation of the value of cyclin E2 as a novel oncogene in leukemia should be given as soon as possibeo.
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1 Tuschl T，Borkhardt A.Small interfering RNAs: a revolutionary tool for the analysis of gene function and gene therapy.Mol Interv，2002; 2: 158-167
2 Gudas JM，Payton M，Thukral S，et al. Cyclin E2，a novel G1 cyclin that binds Cdk2 and is aberrantly expressed in human cancers.Mol Cell Biol，1999; 19: 612-622
3 Shinozawa I，Inokuchi K，Wakabayashi I，et al. Disturbed expression of the anti-apoptosis gene，survivin and Epr-1 in hematological malignancies.Leu Res，2000; 24: 965-970
4 Jiang Y，Saavedra HI，Holloway MP，et al.Aberrant regulation of survivin by the RB/E2F family of proteins.J Biol Chem，2004; 279: 40511-40520
5 Chiarugi V，Magnelli L，Cinelli M，et al. Apoptosis and the cell cycle.Cell Mol Biol Res，1994; 40: 603-612
6 Suzuki A，Ito T，Kawano H，et al. Survivin initiates procaspase 3/p21 complex formation as a result of interaction with Cdk4 to resist Fas-mediated cell death.Oncogene，2000; 19: 1346-1353
7 Iida H，Towatari M，Tanimoto M，et al. Overexpression of cyclinE in acute myelogenous leukemia.Blood，1997; 90: 3707-3713
8 Zariwala M，Liu J，Xiong Y.Cyclin E2，a novel human G1 cyclin and activating partner of CDK2 and CDK3，is induced by viral oncoprotiens.Oncogene，1998; 17: 2787-2798
