Sertad1 antagonizes iASPP function by hindering its entrance into nuclei to interact with P53 in leukemic cells
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As the important suppressor of P53, iASPP is found to be overexpressed in leukemia, and functions as oncogene that inhibited apoptosis of leukemia cells. Sertad1 is identified as one of the proteins that can bind with iASPP in our previous study by two-hybrid screen.
Co-immunoprecipitation and immunofluorescence were perfomed to identified the interaction between iASPP and Sertad1 protein. Westernblot and Real-time quantitative PCR were used to determine the expression and activation of proteins. Cell proliferation assays, cell cycle and cell apoptosis were examined by flow cytometric analysis.
iASPP combined with Sertad1 in leukemic cell lines and the interaction occurred in the cytoplasm near nuclear membrane. iASPP could interact with Sertad1 through its Cyclin-A, PHD-bromo, C terminal domain, except for S domain. Overexpression of iASPP in leukemic cells resulted in the increased cell proliferation and resistance to apoptosis induced by chemotherapy drugs. While overexpression of iASPP and Sertad1 at the same time could slow down the cell proliferation, lead the cells more vulnerable to the chemotherapy drugs, the resistance to chemotherapeutic drug in iASPPhi leukemic cells was accompanied by Puma protein expression. Excess Sertad1 protein could tether iASPP protein in the cytoplasm, further reduced the binding between iASPP and P53 in the nucleus.
Sertad1 could antagonize iASPP function by hindering its entrance into nuclei to interact with P53 in leukemic cells when iASPP was in the stage of overproduction.
KeywordsiASPP Sertad1 P53 Apoptosis Leukemic cell
Real-time quantitative polymerase chain reaction
Reverse transcription-polymerase chain reaction
At present, the incidence of various tumor increased gradually year by year, that had largely threatened the health of human, therefore, lots of researches involved of the pathogenesis and therapy of tumors were performed all over the world. P53, an important tumor suppressor, played an indispensable role in regulation of cell proliferation through induction of growth arrest or apoptosis . Alteration of p53 was frequent in a variety of solid tumors, such as lung, brain. But interestingly, the frequency of that was very low in acute myeloid leukemia (AML), only about 3-8% . But once p53 was mutated or absence in hematological maliganancies, the outcome would be dismal [3, 4].Therefore, it was conceivable that overexpression of oncogenes may be one way to bypass the requirement for p53 mutation in leukemogenesis.
iASPP belonged to the ASPP family consisting of three members, ASPP1, ASPP2 and iASPP. iASPP was described as a shorter protein and identified as a p65 rel A binding protein. iASPP could bind with p53, and prevented it from inducing apoptosis [5, 6, 7]. To date, iASPP has been found to be overexpressed in human breast carcinomas, ovarian cancers and so on, it has been confirmed to be related with poor prognosis [8, 9].We had previously detected the expression of iASPP in acute leukemia, and found that the expression of iASPP was significantly higher in patients compared with healthy donors or patients in complete remission . Further we identified a novel isoform of iASPP, named iASPP-SV, and demonstrated that iASPP-SV could inhibit the transactivation of p53 on transcription of its target genes Bax and P21 . By establishing iASPP transgenic mouse model, we found that iASPP could increase the number and reconstitution capacity of hematopoietic stem cells (HSCs), facilitated their resistance to chemotherapy and irradiation . All our previous results suggested that iASPP could play a distinguished role in the pathogenesis of acute leukemia.
To better understand iASPP function and search additional binding partners, the amino terminus of iASPP was used as bait in yeast two-hybrid screen of a cDNA library from human HeLa Matchmaker cDNA library (Clontech). Sertad1 was identified as one of the iASPP binding partners. Sertad1 was known as TRIP-Br1, p34SEI-1, positively regulated cell division by binding to cyclin-dependent kinase CDK4. It was also involved in gene transcription, could act as a transcriptional regulator that interacted with the PHD-bromodomain of corepressors and coactivators/adaptor p300/CBP. It possessed transcriptional domains and was differentially overexpressed during the G1 and S phases of the cell cycle [13, 14, 15]. Previous studies had shown that Sertad1 was highly expressed in carcinomas from pancreas , that considered Sertad1 as an oncoprotein. Hong SW et al. found that Sertad1 could also prevent the ubiquitination and degradation of X-lined inhibitor of apoptosis protein through a direct association, thus, it was suggested that Sertad1 could be a promising target for new antitumor therapy .
From the above information, we speculated that the interaction between iASPP and Sertad1 may play a role in the pathogenesis of acute leukemia. In this study, we explored the cell biology of leukemic cell lines when iASPP or Sertad1 was unregulated or downregulated, also binding position and relevant molecular pathways were investigated.
Results and discussion
Sertad1 expression level varied in leukemic cell lines and AML patients
iASPP binds directly to Sertad1 mainly through PHD-bromo domain
The structure of Sertad1 includes four domains, namely Cyclin-A, Serta, PHD-Bromo and C terminal. To understand which domain of Sertad1 can binds directly to iASPP, myc-tagged plasmids which contained the full-length Sertad1, as well as four separated domains of Sertad1 (CylcinA, Serta, PHD-Bromo, C terminal) were constructed, respectively. After co-transfecting 293 cells with each Sertad1 constructs and iASPP, antibodies against myc-tag and iASPP were used for IP and WB, respectively. As shown in Fig. 2d, iASPP binds directly to Sertad1 through its PHD-bromo domain, C-terminal domain and Cyclin-A domain in a reduced order, and Serta domain failed to bind to iASPP.
Sertad1 antagonizes the function of iASPP
Firstly, the proliferation of three groups were explored, as shown in Fig. 3b, the proliferation rate of K562-iASPPhi was 2-3 fold higher than that of the control cells, however, the K562-Sertadhi proliferation rate was nearly the same with that of the control cells, interestingly, the proliferation rate of K562-Douhi cells was lowest, even lower than that of K562-Sertad1hi cells. This finding implied that iASPP overexpression could promote the proliferation of leukemic cell, while Sertad1 overexpression had no impact on the proliferation, but when both iASPP and Sertad1 were up-regulated, the proliferation was slowed down.
Secondly, the anti-apoptosis ability of three groups was further explored. The proliferation of living cells and percentage of apoptosis cells after treatment with chemotherapeutic drugs were detected. Fig. 3c and d showed that K562-iASPPhi cells had greater growth advantage than that of K562-Sertad1hi cells and K562-Douhi cells when they were treated with 0.5 μg/ml and 4 μg/ml of etoposide for 48 h and 72 h, which implied that iASPP exerted its anti-apoptosis function only in an appropriate stimuli range. Further, we explored the percentage of apoptotic cells in the three groups when treated with etoposide at different concentrations for 48 h or 72 h. From the results of Fig. 3e and f, it was clearly found that the percentage of early apoptosis in K562-iASPPhi cells was lower than other groups in appropriate concentrations (1 μg/ml-3 μg/ml) of VP16, but when the concentration increased to 5 μg/ml, the percentage of early apoptosis in K562-iASPPhi cells was almost the same as other groups. So it is conceivable that iASPP overexpression could exert its anti-apoptotic ability in appropriate ranges. Besides, both the overexpression of iASPP and sertad1 can promote the leukemic cell blocked more in G2/M stage as the Additional file 1: Figure S1 shown. All above results strongly suggested that iASPP could promote the proliferation, inhibit the apoptosis induced by chemotherapy. Sertad1 only affect the cell cycle in leukemic cells, had no effect on proliferation and apoptosis. To our surprise, iASPP was overexpressed in K562-Douhi cells, but the cell proliferation rate of Douhi cells was lower than that of control group and the percentage of apoptotic cells was the same with other groups. Thus, it is speculated that the function of iASPP may be antagonized by Sertad1.
Sertad1 tetherd iASPP protein in the cytoplasm
The resistance of iASPP to chemotherapeutic drug was accompanied by puma protein expression in a p53-independent manner
The absence of iASPP had no impact on cell biology
P53 was one of the most important tumor suppressor genes, it lay at the center of a number of regulatory pathways. iASPP, as the important inhibitor of p53, was found to facilitate cancer progression in more cancer recently [18, 19]. iASPP was considered as an oncogene that not only inhibited the transactivation function of p53 on the promoters by binding with p53, but also promoted carcinogenesis through p53-independent mechanisms [20, 21, 22].The overexpression of iASPP in primary mouse embryonic fibroblasts promoted p53 degradation and stimulated cell migration and metastasis . In our study, we found a important partner of iASPP, namely Sertad1. iASPP can bind with Sertad1 protein in multiple leukemic cell lines. When iASPP expression was downregulated by shRNA technology, the biological function of the leukemic cells did not change. When iASPP and Sertad1 were both overexpressed in the leukemic cells, Sertad1 could tether iASPP outside the nucleus, resulting the leukemic cells vulnerable to chemotherapy. It implied that Sertad1 could block iASPP entrance into nucleus when iASPP was in the stage of overproduction.
Because iASPP inhibited the transactivation function of p53 on the promoters by binding with p53, it was conceivable to restore p53 function by relieving the interaction between p53 and iASPP. iASPP silencing by siRNA or shRNA had reduced the proliferation of cancer cells in vitro [24, 25, 26, 27]. Some small peptide has been developed to inhibit the apoptotic activity of p53 successfully,such as A34, JNJ-7706621 [18, 28, 29]. Therefore, increasing inhibitors were explored to release p53 from iASPP as the treatment of human tumors through activation of p53 . Our data provided a new insight to inhibt iASPP protein, namely through its binding partner Sertad1, when the similar proteins or drugs that can change the location of iASPP were transfected into the leukemic cells, it may restore p53 function to eliminate the leukemic cells.
Integrating our results, we proposed that in normal situation, the protein iASPP and Sertad1 scattered in both the nucleus and cytoplasm, mainly in the cytoplasm. iASPP could function as oncogene through its binding with P53 protein in the nucleus. When iASPP and Sertad1 were both overexpressed in the leukemic cells, Sertad1 could tether iASPP outside the nucleus mainly through its PHD-bromo domain, reduced the binding between iASPP and P53, eventually prevented iASPP from inhibiting P53 function.
Currently, intensive efforts have been made to restore wildtype p53 activity as an anticancer therapeutic pathway .iASPP, as the inhibitor of p53, have arouse enough attentions in the therapeutic target of acute leukemia. Though our study provided valuable evidence about the interaction between iASPP, sertad1 and p53, there were many obstacles awaiting for us to overcome, such as the following fate of iASPP when it was blocked by Sertad1, and whether the impact of Sertad1 on the biology of iASPP could be further confirmed in mouse model. These problems will be the aim of our future study. Above all, the interaction between iASPP and Sertad1 gave us more insights about the regulation of iASPP, including the impact on p53, these results were beneficial to understanding of pathogenesis of acute leukemia and targeted treatment for patients.
Bone marrow samples from primary acute myeloid leukemia (AML) patients were obtained from Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College. All patients provided written informed consent for analyses and the investigation was approved by the ethical advisory board of Institute of Hematology and Blood Diseases Hospital. The age range of patients in this study was from 25 to 67. Diagnoses of AML were established according to the criteria of the French-American-British (FAB) co-operative study group.
Cell culture and plasmid construction
Human leukemic cell lines K562, HL60, U937, KG1a were routinely maintained in RPMI1640 medium supplemented with 10% fetal calf serum (FCS), penicillin (100 U/ml) and streptomycin (100 μg/ml) in a humidified atmosphere of 5% CO2 at 37°C. 293 T cells and 293 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FCS without antibiotics. The pcDNA3.1-iASPP-V5 plasmid was kindly provided by Prof. Xin Lu from University of Oxford. The pcDNA3.1-p53-Flag plasmid was purchased from Addgene company. Plasmids containing different Sertad1 domains were constructed by PCR and cloned into pcDNA3.1-myc plasmid. iASPP and Sertad1 shRNA fragments were synthesized (Invitrogen) and cloned into PLKO.1 plasmid.
Cell proliferation assays
The cells were seeded at 10,000 cells per well in a 96-well plates in normal growth medium. 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) reagent was added to each well and incubated for an additional 4 h at 37°C with 5% CO2. Media was aspirated and the precipitate was solubilized in 100 μl 10%SDS. The absorbance of each well was measured at 547 nm by Synergy Hybrid Reader (BioTek Instruments, Inc) according to the manufacturer’s instructions. The percentage of viable cells was calculated relative to control wells.
Cell cycle analysis
The transfected cells were washed twice with PBS and fixed with 70% ethanol for at least 24 h, the cells were then treated with 0.5 mg/ml RNase in 0.1% sodium azide, and incubated at room temperature for 10-15 min followed by staining with 50 μg/ml PI for 10 min. The cells were analyzed for their DNA content using a FACS LSRII flow cytometer (BD Biosciences). Histograms were analyzed for cell-cycle compartments using ModFit version2.0. A minimum of 20,000 cells were collected to maximize statistical validity of the compartmental analysis.
The cells were incubated with etoposide (VP16) or adriamycin (ADM) for 48 h and 72 h, washed twice with PBS, and stained with propidium iodide (PI) and annexin V-647 in 100 μl of staining solution at room temperature for 10-15 min in the dark. Samples were then diluted with the binding buffer and analyzed by FACS LSRIIflow cytometer (BD Biosciences) within 0.5 h.
Co-immunoprecipitation and immunoblotting analysis
To analyze the interaction between iASPP and Sertad1, co-immunoprecipitation (Co-IP) was performed. Briefly, cell lysates were pre-cleared with protein A agarose (Santa Cruz). The supernatants were immunopreciptated with anti-iASPP antibody, followed by incubation with protein A-agarose. Protein A-agarose-antigen-antibody complexes were pelleted by centrifugation. Bound proteins were resolved by SDS-PAGE, followed by Western blot (WB) with antibodies against Sertad1 or myc-tag. The immunoreactive proteins were visualized using SuperSignal chemiluminescent detection system (Pierece).
Leukemic cell lines were transfected with gene specific shRNA or scrambled shRNA. iASPP shRNA sequence: 5′-CCAACTACTCTATCGTGGATT-3′; iASPP scrambled sequence: 5′-GCACTTAACTCGTAGTCCTAT-3′; Sertad1 shRNA sequence: 5′- AACGGGTCTGAAGGGAAACGG-3′; Sertad1 scrambled sequence: 5′- GAGCGA- GTGCAACGAGAGAGT-3′. All above PLKO.1-shRNA plasmids were constructed in our laboratory. The cell lines expressing shRNAs were maintained in 0.8 μg/ml puromycin.
For co-localization of iASPP and Sertad1, immunofluorescence staining was performed. The cells were subjected to two washes in PBS, and then were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.25% Triton X-100 in PBS for 10 min and blocked with 2% horse serum for 30 min at room temperature. Cells were then incubated with primary antibody diluted at 1:100 at 4°C overnight. After three washes in PBS, cells were incubated with DyLight™ 448 donkey anti-rabbit IgG or DyLight™ 649 goat anti-mouse IgG diluted at 1:100 for 1 h at room temperature, DAPI was used for nuclear staining. Observations were made using Leica TCS P2 microscope.
RNA isolation, RT-PCR and RQ-PCR
iASPP and Sertad1 mRNA were assessed by RT-PCR or RQ-PCR. Total RNA from de novo AML samples and leukemic cell lines were isolated, cDNA was synthesized using a reverse transcription kit (Invitrogen). Primers used for PCR were listed as follows:(1) iASPP 5′-CGCGGGACTTTCTGGACATG-3′ and 5′- TGCCGAAGGGC TCAGGAATC-3′ (2)Sertad1 5′-CTCATGGATGTGCTGGTGG-3′ and 5′-AGGAC GGATGTGAAGTTGC-3′.
Statistical difference between experimental groups was calculated and analyzed using Student’s t test. All experiments were performed in triplicate and averaged from >3 independent experiments. All tests were two-tailed and considered significant at a p < 0.05. All calculations were performed with SPSS version13.0.
The authors thank members of Shared Core Facility of State Key Laboratory of Experimental Hematology, especially Mei Zhang and Xuelian Chen for Immunofluorescence, Haoyue Liang for flowcytometry analysis.
This work was supported by grants from the National Natural Science Foundation of China (81,270,635,81,430,004), and Tianjin Major Science and Technology Project (12ZCDZSY17500).
Availability of data and materials
The datasets supporting the conclusions of this article are included within the article.
Performed all experimental validation, computational and statistical data analysis:SQ. Performed cell biological function: SL, TY and JY. Analyzed the data:HX and KT. Provided clinical samples and related data: Y.M. Wrote the manuscript:SQ. Conceived and designed this project, interpreted data, revised and proved manuscript: QR, MW and JW. All authors read and approve the final manuscript.
Ethics approval and consent to participate
All patients gave informed consent, and the investigation was approved by the ethical advisory board of Institute of Hematology and Blood Diseases Hospital.
Consent for publication
Jianxiang Wang acts as consultant of Novartis and Bristol Myers Squibb.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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