| iASPP is a key inhibitor of tumor suppressor p53 and is proved to be an oncoprotein.Our previous study showed that iASPP expression level was significantly higher in acute leukemia(AL) and leukemia cell lines compared with normal control,it implied that iASPP might play an important role in the leukemogenesis and/or disease progression of AL.To explore the effects of iASPP on leukemia cells and determine if the p53 pathway plays a critical role in this process, we blocked the expression of iASPP by RNA interference(RNAi) in two iASPP high expressing leukemic cell lines,p53 wild-type cell line Nalm6 and p53-null cell line K562.1.Reduction of iASPP mRNA and protein expression by siRNATo determine the activity of RNAi in Nalm6 and K562 cells,the mRNA and protein levels of two iASPP isoforms were analyzed.One of the isoforms was iASPP(828) encoding a 828-amino-acid(aa) protein,the other was iASPP-SV encoding a 407aa protein.After the cells were treated with siRNA for 24h,the mRNA levels of both isoforms in Nalm6 and K562 cell lines were down-regulated(P＜0.05). In Nalm6 cells,iASPP(828) and iASPP-SV mRNA levels of the iASPP siRNA treated group were reduced to(41.2±7.7)%and(23.6±6.9)%of those of the control group, respectively.In K562 cells,iASPP(828) and iASPP-SV mRNA levels of the iASPP siRNA treated group were reduced to(59.8±8.1)%and(36.3±7.9)%of those of the control group,respectively.To ensure that iASPP protein was silenced in leukemia cells following the reduction of mRNA,72h after transfection iASPP protein levels were examined by Western blot using the iASPP specific monoclonal antibody prepared in our laboratory. Our result showed that both isoforms were downregulated to some extent after iASPP siRNA treatment(P＜0.05).In Nalm6 cells,iASPP(828) and iASPP-SV protein levels of the iASPP siRNA treated group were reduced to(59.7±6.5)%and(29.5±9.2)%of those of the control group,respectively.Similarly,in K562 cells,iASPP(828) and iASPP-SV protein levels of the iASPP siRNA treated group were reduced to (73.4±6.9)%and(53.4±8.8)%of those of the control group,respectively.2.Effect of siRNA on the growth and proliferative potency of Nalm6 cells and K562 cellsTo investigate whether reduction of iASPP had some effect on cell growth and proliferation,the cell cycle distribution assay and methylcellulose colony-formation assay were performed.48h after transfection,cell cycle of siRNA treated Nalm6 and K562 cells were analyzed by FACScalibur flow cytometer.The result showed that the proportion of Nalm6 cells in G0/G1 increased slightly,from(34.4±0.6)%for the control siRNA treated group to(37.9±0.4)%for the iASSP siRNA treated group,but the proportion of K562 cells in G0/G1 hardly changed,from(42.4±2.9)%to (42.9±0.4)%.The proliferative potency of the two cell lines was then analyzed by cell colony formation assay.The colony number of siRNA treated Nalm6 cells decreased from 484.7±14.6 colonies/well for the control group to 158±7.0 colonies/well for the iASSP siRNA group(P＜0.05).For K562 cells,the number of colonies decreased from 908±21.0 colonies/well to 663.3±10.6 colonies/well(P＜0.05),the degree of reduction was much lower than that of Nalm6 cells.Following the down-regulation of iASPP, cell growth and proliferation were inhibited in leukemia cells,especially in the cells expressing wild-type p53.3.Induction of apoptosis by siRNA and etoposide or daunorubicinAs the tumor suppressor function of p53 is tightly linked to its ability to induce apoptosis,,effect of iASPP inhibition on apoptosis was examined in Nalm6 and K562 cells.The result revealed that the rate of apoptosis in Nalm6 cells treated with iASPP siRNA alone increased slightly compared with that in the control,,but significantly increased apoptotic cells were observed in groups treated with both the iASPP siRNA and etoposide or daunorubicin compared with that of groups treated with iASPP siRNA,etoposide or daunorubicin alone.The rate of apoptosis increased from(9.4±3.9)%and(34.1±9.9)%in iASPP siRNA and etoposide treated alone groups to(52.3±6.8)%in iASPP siRNA plus etoposide treated group(P＜0.01, P＜0.05,respectively),and from(8.4±3.0)%and(23.7±2.7)%in iASPP siRNA and daunorubicin treated alone groups to(45.1±4.5)%in iASPP siRNA plus daunorubicin treated group(P＜0.01,P＜0.01,respectively).Down-regulation of iASPP rendered Nalm6 cells susceptible to induction of apoptosis by etoposide and daunorubicin.For K562 cells,the rate of apoptosis changed from(7.7±2.0)%and(15.6±2.2)%in iASPP siRNA and etoposide treated alone groups to(20.0±3.7)%in iASPP siRNA plus etoposide treated group(P＞0.05,P＞0.05,respectively),and from(6.6±1.7)%and (18.9±4.1)%in iASPP siRNA and daunorubicin treated alone groups to(21.3±2.9)% in iASPP siRNA plus daunorubicin treated group(P＞0.05,P＞0.05,respectively). However,unlike in Nalm6 cells,down-regulation of iASPP in K562 cells had no significant effect on the enhancement of etoposide and daunorubicin chemosensitivity.4.Etoposide and daunorubicin have no significant effect on iASPP mRNA expression.To investigate whether etoposide and daunorubicin treatment could regulate the iASPP expression,Nalm6 and K562 cells treated with different combination of siRNA and chemotherapeutic drugs were harvested and subjected to iASPP mRNA assay by RT-PCR.For Nalm6 cells,when the value of iASPP mRNAs expression level in control siRNA treated group was set to 100%,the relative expression levels of iASPP(828) in iASPP siRNA treated group,control siRNA plus etoposide treated group,iASPP siRNA plus etoposide treated group,control siRNA plus daunorubicin treated group and iASPP siRNA plus daunorubicin treated group were(26.9±7.9)%, (98.7±7.3)%,(28.3±5.9)%,(95.4±8.1)%and(30.7±6.2)%,respectively.The relative expression levels of iASPP-SV in the above groups were(44.7±7.4)%,(101.6±7.9)%, (47.2±6.5)%,(96.4±9.1)%,(51.5±7.7)%,respectively(Fig.4C).For K562 cells,when the value of iASPP mRNAs expression level in control siRNA treated group was set to 100%,the relative expression levels of iASPP(828) in iASPP siRNA treated group, control siRNA plus etoposide treated group,iASPP siRNA plus etoposide treated group,control siRNA plus daunorubicin treated group and iASPP siRNA plus daunorubicin treated group were(39.6±8.8)%,(110.4±7.1)%,(32.7±6.9)%, (106.8±8.8)%and(35.3±7.3)%,respectively.The relative expression levels of iASPP-SV in the above groups were(65.9±7.4)%,(96.6±7.8)%,(60.9±6.3)%, (103.1±8.0)%and(64.2±7.1)%,respectively.No significant effect of etoposide or daunorubicin treatment on iASPP mRNA expression was detected(P＞0.05).5.Change in mRNA expression level of two apoptosis-related gene c-Myc,bcl-2To explore other mechanisms involved in iASPP-related apoptosis,mRNA expression levels of two apoptosis-related gene c-Myc and bcl-2 were analyzed 48h after transfection.Following the reduction of iASPP mRNA,mRNA levels of c-Myc and bcl-2 were also downregulated,the inhibition ratios of c-Myc and bcl-2 were (76.8±6.8)%and(34.1±5.4)%for Nalm6 cells(P＜0.05),(61.8±7.3)%and(10.3±2.1) %for K562 cells(P＜0.05).In conclusion,our study suggested that iASPP regulated the biologic function of leukemia cells especially in the aspect of apoptosis and cell proliferation.Since unbalance of these two pathways is a major cause of cancer,iASPP might play an important role in leukemogenesis,iASPP involved in many cell pathways,among them the p53 related pathway was particularly important.The result also indicated that inhibition of iASPP might have an important function in cell apoptosis activation, proliferation down-regulation and cellular response to cancer therapy,it might provide a new route to restore or modulate the tumor suppression function of p53 and therefore enable us to develop new therapeutic drugs and strategies to kill leukemia cells effectively especially cells with wild-type p53.iASPP might be a molecular target in AL therapy. DJ-1 is a multifunctional protein that has been implicated in pathogenesis of some solid tumors.In this study,we found that DJ-1 was overexpressed in acute leukemia (AL) patient samples and leukemia cell lines,which gave the first clue that DJ-1 overexpression might be involved in leukemogenesis and/or disease progression of AL.Inactivation of DJ-1 by RNA-mediated interference(RNAi) in leukemia cell lines K562 and HL60 resulted in inhibition of the proliferation potential and enhancement of the sensitivity of leukemia cells to chemotherapeutic drug etoposide.Further investigation of DJ-1 activity revealed that phosphatase and tensin homologue (PTEN),as well as some proliferation and apoptosis related genes,was regulated by DJ-1.Thus,DJ-1 might be involved in leukemogesis through regulating cell growth, proliferation and apoptosis.It could be a potential therapeutic target for leukemia.Results:1.Overexpression of DJ-1 mRNA in AL patients and leukemia cell linesRelative quantification of the transcript level for DJ-1 gene using semi-quantitative RT-PCR revealed a striking increase in AL patients and leukemia cell lines,in comparison with healthy donors(P＜0.01).Additionally,the median level of DJ-1 in 10 MDS patients also increased,but there was no significant difference when compared with normal control group(P＞0.05).With regard to the distribution profile of DJ-1 expression among different subtypes of AL,no difference was observed between AML and ALL(P＞0.05).DJ-1 gene expression in AL patients was not associated with gender,age,initial white blood cell count,CD34 expression and chromosomal karyotype.2.Reduction of DJ-1 mRNA expression by DJ-1 siRNATo determine the efficiency of RNAi in K562 and HL60 cells,levels of DJ-1 mRNA were analyzed by semi-quantitative RT-PCR after the cells were treated with siRNA for 48h.In K562 and HL60 cells,DJ-1 mRNA levels of the DJ-1 siRNA treated groups were reduced to(47.7±5.5)%and(64.7±6.7)%of those of the control siRNA treated groups,respectively(P＜0.01).3.Decrease of proliferative potency and serum starvation tolerance of K562 and HL60 cells by DJ-1 siRNA treatmentThe doubling times of K562 and HL60 cells were prolonged by DJ-1 siRNA treatment, from(33.4±0.5)h and(24.0±0.4)h in control siRNA treated groups to(44.4±1.8)h and (29.1±0.9)h in DJ-1 siRNA treated groups,respectively(P＜0.05).When cells were maintained in serum-free medium,DJ-1 siRNA treatment led to lower numbers of viable cells,suggesting that these cells became less tolerant to serum starvation.Cell cycle analysis showed that DJ-1 expression inhibition caused a slight up-regulation of cells in G0-G1 phase.Additionally,the colony numbers of siRNA treated K562 and HL60 cells decreased from 416.3±19.6 and 928±36.7 colonies/well for the control siRNA treated groups to 229.3±20.8 and 560±27.7 colonies/well for the DJ-1 siRNA treated groups,respectively(P＜0.01).Following the down-regulation of DJ-1,cell proliferation and cell tolerance to serum starvation were inhibited.4.No significant effect of DJ-1 down-regulation on differentiation of K562 or HL60 cellsTo examine the effect of DJ-1 inhibition on cell differentiation,the expression levels of three myeloid lineage markers CD11b,CD14 and CD15 on K562 and HL60 cells were analyzed 72h after siRNA transfection.For K562 cells,CD11b,CD14 and CD15 changed from(2.2±0.2)%,(1.6±0.6)%and(48.5±3.1)%in control siRNA treated group to(2.4±0.9)%,(1.9±0.9)%and(47.3±2.7)%in DJ-1 siRNA treated group, respectively.For HL60 cells,they were changed from(2.4±0.6)%,(0.15±0.05)%and (15.5±0.5)%to(2.3±0.3)%,(0.19±0.04)%and(17.9±3.3)%,respectively.No significant change after RNAi was revealed from the result(P＞0.05).Additionally no apparent morphologic change of differentiation was observed for both cell lines.5.Induction of apoptosis by DJ-1 siRNA and etoposideTo investigate whether inhibition of DJ-1 expression would affect the apoptosis induction of leukemia cells,K562 and HL60 cells were treated with siRNA and analyzed by Annexin V-FITC staining.The results revealed no difference between the DJ-1 siRNA treated group and the control siRNA treated group.However,if the siRNA treated cells further exposed to etoposide,significantly increased apoptotic cell population could be observed in comparison with that treated with siRNA or etoposide alone(P＜0.05).The apoptosis rate increased from(3.7±0.5)%and (17.3±0.6)%in DJ-1 siRNA and etoposide treated alone groups to(35.71±1.7)%in DJ-1 siRNA plus etoposide treated group(P＜0.01 and P＜0.05,respectively) for K562 cells,from(1.3±0.3)%and(24.6±0.9)%to(33.8±1.6)%(P＜0.01 and P＜0.05, respectively) for HL60 cells.Down-regulation of DJ-1 rendered K562 and HL60 cells susceptible to induction of apoptosis by etoposide.6.Effect of DJ-1 down-regulation on apoptosis-related and cell cycle-related genes expressionTo explore the mechanisms involved in DJ-1-related apoptosis and cell growth,the effect of DJ-1 knockdown on the expression of cell cycle and apoptosis related genes, including p21,Cdk2,Cyclin D1,Cyclin D2,c-Myc,NFκB and Bcl-2,was analyzed. Following the inhibition of DJ-1,mRNA levels of Cdk2,Cyclin D1,c-Myc,NF-kB and Bcl-2 were reduced to(80.2±4.5)%,(48.2±4.2)%,(72.1±5.6)%,(61.1±4.7)%and (38.9±5.7)%of their controls,respectively(P＜0.05).No significant changes of p21 and Cyclin D2 were observed(P＞0.05).7.Up-regulation of PTEN mRNA expression by inhibition of DJ-1DJ-1 is a negative regulator of PTEN,and our previous study showed that the expression of PTEN in AL patients was lower than that of normal control.In this study,we observed that DJ-1 expression was significantly higher than that of healthy controls.Therefore,we analyzed the expression PTEN gene after DJ-1 expression was blocked by siRNA.It revealed that down-regulation of DJ-1 led to increases of PTEN mRNA levels by(78.3±6.3)%in K562 cells and by(27.3±8.7)%in HL60 cells (P＜0.01 and P＜0.05,respectively). 8.Effect of DJ-1 overexpression on the growth and proliferative potency of K562 cellsFour independent G418-resistant clones were isolated and expanded,the one transfected with empty pIRES-EGFP plasmid was used as a control,the other three transfected with pIRES-EGFP-DJ-1 plasmid(K562 DJ-1 clto3) were DJ-1 high expressing.To investigate whether DJ-1 overexpression had effect on cell growth and proliferation of leukemia cells,viable cell count by typan blue dye exclusion method, the cell cycle distribution assay and methylcellulose colony-formation assay were performed in K562 control and K562 DJ-1 clto3.Compared with the K562 control clone,the DJ-1 high expressing clones K562 DJ-1 clto3 grew fast,and the population doubling time was shortened(p＜0.05),from 48.16±2.97 h to 36.21±2.4,32.76±3.56 and 38.27±3.01h,respectively.Similar result was also observed when cells were maintained in medium without FCS,K562 DJ-1 clto3 clones revealed an enhanced tolerance to serum starvation,the numbers of viable cells were higher.Analysis of cell cycle distribution showed that DJ-1 caused a slight up-regulation of cells in S phase, from(44.61±6.6)%for the control K562 cells to(48.25±7.98)%,(51.53±4.69)%and (49.74±0.21)%for the K562 DJ-1 clto3 cells.Cells in G0-G1 phase decreased from (50.94±0.3)%for the control K562 cells to(48.62±3.86)%,(42.11±1.51)%and (43.6±5.78)%for the K562 DJ-1 clto3 cells.Additionally,cells from K562 DJ-1 clto3 clones formed more colonies than cells from the control clone(p＜0.05),the colony numbers were 2.19±0.46,2.55±0.54 and 2.52±0.35 times as many as that of the control clone,respectively.All the above results indicated that the growth potency of K562 cells was enhanced by DJ-1 overexpression.9.High expression of DJ-1 caused a down-regulation of PTEN mRNA level,but couldn't change the methylation status of PTEN promoter. In our study high expression of DJ-1 leaded to a down-regulation of PTEN mRNA level and the reduction ratios for K562 DJ-1 clto3 clones were(26.13±4.45)%, (36.81±4.6)%and(36.94±5.61)%,respectively.Methylation is thought to be one possible way through which DJ-1 regulates the function of PTEN,to identify it,the methylation status of PTEN was examined by MSP.The results revealed that the promotors of PTEN in K562 cells were tmmethylated.Our result showed that high expression of DJ-1 fail to cause methylation of PTEN promoter in K562 cells.In conclusion,our results suggested that the aberrant up-regulation of DJ-1 expression might be an important step in the pathogenesis of human AL,where growth advantage acquisition occurred.The down-regulation of DJ-1 rendered leukemia cells vulnerable to apoptosis,so DJ-1 could serve as a potential molecular target for leukemia treatment.
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