| ObjectiveHuman acute myeloid leukaemia (AML) is the generic term for a group of myeloid leukaemias that are characterized by clonal expansion of immature myeloid progenitor stages in the bone marrow, blood or other tissues. All-trans retinoic acid (ATRA), the biologically active form of vitamin A, bypasses this arrest in differentiation, which is central to the successful clinical application of ATRA for the differentiation-based treatment of acute promyeloid leukemia (APL). Considerable attention has been paid to understand the exact mechanisms by which ATRA induces leukaemic cell differentiation. ATRA induces leukaemia cell differentiation and reduces proliferation, which requires cell cycle exit. Accordingly, various cell cycle regulators have been implicated in ATRA-mediated differentiation, including Cyclin-dependent kinase (CDK)-activating kinase (CAK), Cyclin D1, p21 WAF1/CiP-1, p27 Kip1, and Cyclin E. However, the role of CDKs, which reliably control the mammalian cell cycle, in ATRA-induced differentiation remains largely unclear.Cyclin-dependent kinase 2 (CDK2), a member of the serine/threonine kinase family of CDKs, is a key regulator of G1/S transition. CDK2 is activated during the G1 and S phases by binding to its partner, Cyclin E or Cyclin A, and the subsequent phosphorylation of threonine 160 (Thr-160) by CAK. Apart from its role in cell cycle, CDK2 has recently been found to participate in modulating the self-renewal capacity and differentiation. Down-regulation of CDK2 activity or expression is crucial for differentiation of mouse neuroblastoma cells, human and mouse embryonic stem cells (ESCs) and adult mouse central nervous system. These observations strongly support the significance of CDK2 reduction in the regulation of self-renewal capacity and cell differentiation. Therefore, a greater understanding about the functions of CDK2 and a re-evaluation of this kinase as a therapeutic target in some special types of cancer are needed.Cellular CDK2 levels are controlled in a dynamic state dictated by the balance between CDK2 synthesis and degradation. Although several studies have investigated the determinants of CDK2 synthesis, the factors regulating CDK2 degradation remain to be elucidated. The ubiquitin-proteasome system (UPS), a major system for controlled proteolysis in eukaryotes, is a potential candidate involved in selective CDK2 turnover. Our previous works found that the 26S proteasomal inhibitor attenuated ATRA-induced differentiation of AML cells, which was tightly coupled with the restoration of ATRA-mediated decreased CDK2 protein levels. These results strongly implied the involvement of the UPS-mediated destabilisation of CDK2 in the ATRA-mediated differentiation of AML. Consequently, a detailed assessment of the regulation and function of CDK2 reduction is warranted.The aim of this study was to investigate the involvement of ubiquitin-proteasomal degradation of CDK2 in ATRA-mediated differentiation of AML cells. The current study was divided into two sections:1) to prove that CDK2 is a substrate of UPS in heterologous expression systems and that ATRA promotes the ubiquitination and proteasomal degradation of CDK2 during differentiation of AML cells.2) to explore the role of CDK2 down-regulation in ATRA-triggered granulocytic differentiation.Methods1) Human acute myeloid leukemia cells NB4 and U937 were treated with ATRA. Cell surface marker CD11b and cell cycle profile were detected by flow cytometry. Morphologic changes in the cells were assessed by Wright-Giemsa staining. Cell lysates were subjected to western-blot analysis for the detection of CDK2 protein level. The transcriptional level of CDK2 was measured through Real-time PCR. Immunofluorescence was carried out to detect the colocalisation of CDK2 and Ubiquitin in cells. Immunoprecipitation was employed to detect polyubiquitinated CDK2. COS-7 cells were transfected to express CDK2 or Ubquitin protein. Ubiquitin-proteasomal degradation assays in vitro were performed in crude rabbit reticulocyte lysate (RRL), an established source of ubiquitinating enzymes and proteasome complexes in the presence of an ATP-regenerating system. Recombinant CDK2 protein was expressed in vitro using the TNT(?) T7 Coupled Reticulocyte Lysate System.2) Lentivirus-shRNA specific against CDK2 mRNA was transfected into the parental NB4 cells to silence the CDK2 expression. Cell growth curve was performed by counting cells each day. Giemsa staining was performed to evaluate morphologic changes. NBT-reducing activity as a potent differentiation marker of leukemia cells was used and the blue cells were counted by light microscopy for at least 200 cells. Cell-surface marker expression and cell cycle profile were detected by flow cytometry. Expressions of CDK2 Cyclin E, Cyclin A and myeloid differentiation-relevant transcription factors (PU.1, STAT1, p-STAT1 and C/EBPβ) were analysed by western-blot. Transcripts of CDK2, CSF3R, PU.1, STAT1 and C/EBPβwere determined by Real-time PCR.ResultsPart 1 ATRA promoted UPS-dependent degradation of CDK2 during myeloid differentiation1) CDK2 protein is down-regulated during ATRA-induced granulocytic differentiation and G0/G1 cell cycle arrestExponentially growing NB4 and U937 cells were treated with 0.1 and 1μM ATRA, separately, for 0,24,48 and 72 hrs. The number of NB4 and U937 cells expressing CD11b, a marker of myeloid differentiation, increased significantly in a time-dependent manner. Morphologic changes assessed by Wright-Giemsa staining revealed that the ATRA-treated NB4 cells acquired a granulocytic morphology and ATRA-treated U937 cells exhibited a monocytic morphology. Upon ATRA treatment, a time-dependent increase in the number of NB4 and U937 cells in G0/G1 phase was observed. In parallel, ATRA caused a substantial, time-dependent reduction in CDK2 protein levels in both NB4 and U937 cells, which correlated inversely with the levels of differentiation and G0/G1 arrest mediated by ATRA.Moreover, during TPA- and DMSO-induced differentiation of NB4 cells, intensive down-regulation of CDK2 was also observed. However, CDK2 level was relatively stable in NB4 cells with different cell cycle distribution, strongly implying an association between CDK2 reduction and differentiation of myeloid leukemia cells.2) Proteasome-dependent degradation of CDK2 during myeloid differentiationResults obtained from Real-time PCR demonstrated that CDK2 mRNA levels were rarely influenced by differentiation-inducing agents, including ATRA, TPA and DMSO, in both NB4 cells and U937 cells, implying the involvement of degradation in CDK2 reducation. Lysosomal inhibition resulted in little change in ATRA-triggered down-regulation of CDK.2, whereas proteasomal inhibitions led to a drastic abrogation of the decline in CDK2 levels triggered by ATRA treatment in both NB4 and U937 cells. Moreover, decreases in CDK2 level induced by TPA and DMSO were also abrogated by proteasomal inhibition. However, reciprocal change in CDK2 mRNA levels was not detected by Real-time PCR. These data indicated that CDK2 reduction during AML differentiation was dependent on proteasomal pathway.3) CDK2 was a target for ubiquitin-mediated degradation both in vivo and in vitro.Lysates of COS-7 cells co-transfected with pCMV-CDK2-HA and pEGFP-C1-ubiquitin (Ub) plasmids were subjected to immunoprecipitation and western-blot analysis. The data revealed that co-expression of GFP-ubiquitin decreased the levels of CDK2-HA, and the turnover of CDK2-HA was blocked by MG132. A smear of high-molecular-weight ubiquitinated products (polyubiquitinated CDK2-HA) was detected and the amount of polyubiquitinated CDK2-HA was accumulated upon treatment with MG132. Next, CDK2-HA was immunoprecipitated from transfected COS-7 cells and incubated with RRL. CDK2-HA-ubiquitin conjugates were detected as a ladder of bands, which was not observed after the reactions without RRL or CDK2-HA precipitates. Addtionaly, data acquired from in vitro RRL ubiquitination assay using in vitro-translated recombinant CDK2 (rCDK) indicated that the polyubiquitination of rCDK2 was enhanced by extending the incubation time, and the intensity of the rCDK2 bands correspondingly declined, clearly indicating the proteasomal degradation of rCDK2 via ubiquitination.Taken together, these observations provided evidence of the polyubiquitination and subsequent degradation of CDK2 by the 26S proteasome both in COS-7 cells and in vitro RRL system.4) ATRA promoted UPS-dependent degradation of CDK2 in differentiating NB4 and U937 cellsColocalisation of CDK2 and Ub in ATRA-treated NB4 cells was detected by immunofluorescence microscopic analysis. A time-dependent increase in ubiquitin staining and decrease in CDK2 staining were clearly observed. Remarkably, ubiquitin merged with CDK2 increased upon ATRA treatment, which was particularly obvious by 72 hrs. Immunoprecipitation analysis performed in both ATRA-treated NB4 and U937 cells revealed that the relative levels of ubiquitination (the ratio of ubiquitinated/total CDK2) increased in a time-dependent manner and was tightly coordinated with the time course of ATRA-induced granulocytic differentiation. Furthermore, the addition of MG132 inhibited CDK2 degradation and allowed for ubiquitinated CDK2 accumulation. Collectively, these data demonstrate that ATRA accelerated the ubiquitin-proteolysis of CDK2 in NB4 cells undergoing differentiation.5) Decrease in phosphorylation of CDK2 at Thr-160 accelerated UPS-dependent degradation of CDK2Western-blot analysis showed that the down-regulation of p-CDK2 (Thr-160) was faster and earlier than that of CDK2 in ATRA-treated NB4 and U937 cells. Hypophosphorylated form of CDK2 was contructed by applying a point mutation from threonine to alanine (ACC to GCC). Detection in COS-7 cells co-transfected by pCMV-CDK2-HA or pCMV-CDK2-T160-HA with pEGFP-Cl-Ub demonstrated that poly ubiquitinated CDK2-T160-HA was more than polyubiquitinated CDK2-HA. Moreover, immunoprecipitated CDK2-T160A-HA from transfected COS-7 cells was found easier to be ubiquitinated than CDK2-HA extracted the same way. Accordingly, western-blot analysis revealed that halflife of CDK2-T160A-HA was approximately 1.8 hrs, while that of CDK2-HA was about 3.4 hrs in COS-7 cells. Taken together, these data demonstrated that deline in phosphorylation of CDK2 at Thr-160 enhanced UPS-dependent degradation of CDK2, which might be involved in ATRA-triggered degradation of CDK2.Part 2 Reducion in CDK2 protein level enhanced ATRA-mediated AML differentiation1) Down-regulation of CDK2 contributed to differentiation induced by ATRA in NB4 cells.Lentivirus-pLKO.1 (Vector),-pLKO.1-CDK2 #1 shRNA,-pLKO.1-CDK2 #5 shRNA was transfected into parental NB4 cells, resulting in dramatic reduction in CDK2 at protein level, with about 100%,0% and 40% of CDK2 remained, relatively. NB4-Vector, NB4-CDK2 #1 shRNA and NB4-CDK2 #5 shRNA cells exposed to EtOH (vehicle) showed no difference in proliferation ability and cell viability. Nevertheless, significant increases in ATRA-induced proliferation inhibition were observed in both NB4-CDK2 #1 shRNA and NB4-CDK2 #5 shRNA cells compared with NB4-Vector cells (81.00%,44.88% and 63.05% of EtOH-treated NB4-Vector cells, relatively). After treatment with ATRA (5 nM) for 72 hrs, NB4-Vector, NB4-CDK2 #1 shRNA and NB4-CDK2 #5 shRNA cells showed 51.80%,76.28% and 66.44% increase in CD1 lb-positive cells compared with EtOH-treated NB4-Vector cells.Then NB4-CDK2#1 shRNA (NB4-CDK2 shRNA in the following paragragh) cells were employed in the following experiments as more intensive slience of CDK2 protein level. Data obtained from NBT reduction assay, Wright-Geimsa staining for morphologic change and Real-time PCR measurement of CSF3R mRNA revealed that NB4-CDK2 shRNA cells exhibited higher differentiation rate than NB4-Vector cells upon ATRA treatment.Taken together, these data demonstrated that CDK2 reduction enhanced ATRA-mediated granulocytic differentiation of NB4 cells.2) Alterations in CDK2 expression failed to impact ATRA-induced G0/G1 cell cycle arrestCell cycle profile analysis indicated that ATRA induced G0/G1 phase accumulation in both NB4-Vector and NB4-CDK2 shRNA cells and the cell cycle distribution in both of the cells was distinct whether with or without treatment of ATRA. Moreover, cell cycle synchronision assay revealed that the NB4-CDK2 shRNA cells did not exhibit changes in cell cycle progression, including the progressions from GO to S phase and G2 to S phase, compared with the NB4-vector cells. Taken together, these data suggested that the positive role of CDK2 reduction in ATRA-induced differentiation might be independent of its cell cycle function.3) Decline in CDK2 increased ATRA-induced expression of important myeloid transcription factorsReal-time PCR analysis showed that increase in mRNA levels of PU.1, C/EBPβ, STAT1 stimulated by ATRA treatment were stronger in NB4-CDK2 shRNA cells than in NB4-Vector cells. Measurements of PU.1, C/EBPβ, STAT1, the active form of STAT1 (p-STAT1, Tyr-701) protein levels through western-blot indicated that ATRA-caused up-regulation and nuclear translocation of PU.1, C/EBPβ, STAT1, and tyrosine-phosphorylated STATl were intensified in NB4-CDK2 shRNA cells in comparison with NB4-Vector cells. Collectively, these results illustrated that CDK2 reduction in NB4 cells enhanced ATRA-induced expression of myeloid transcription factors, which in turn facilitated ATRA-mediated granulocytic differentiation.ConclusionThe data presented herein demonstrated, for the first time that ATRA acts in a novel manner to decrease CDK2 expression by stimulating the ubiquitin-proteasomal degradation of CDK2. The reduction in CDK2 played an indispensable role in ATRA-mediated granulocytic differentiation. A better understanding of the post-translational modification and novel function of CDK2 might provide fresh insights into the design of new differentiation therapies against leukaemia. |
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