Sertad1 antagonizes iASPP function by hindering its entrance into nuclei to interact with P53 in leukemic cells

In order to investigate the expression of iASPP and sertad1 in leukemic cells, several leukemic cell lines and bone marrow samples from AML patients were used to analyze the expressions of iASPP and sertad1 at transcriptional and translational levels by real-time RT-PCR and Western blot, and compared with that of 293 T cells. As shown in Fig. 1a, b and c, iASPP and sertad1 were both overexpressed in Raji, NB4, U937 and KG-1a cells, when compared with that in K562, Naml6 and HL60 cells at mRNA level. As the function of iASPP was closely related with P53, the expression of p53 in the above cell lines was also evaluated in Fig. 1d, the protein level of p53 was lower in K562, Nalm6 and HL60 compared with other cells. Integrated with mRNA level and protein level(Fig. 1d), the expression level of iASPP and sertad1 were obviously higher in U937 and KG1a, meanwhile lower in K562, that were further investigated in our study. When we performed the statistic analysis on the correlation of iASPP, Sertad1 and P53 in different cell lines. The data showed that only iASPP was correlated with P53, the Kendall’s tau_b was 0.643(p = 0.026) in mRNA level and 0.691(p = 0.018) in protein level, but the there was no correlation between Sertad1 and P53 or iASPP and Sertad1.The mRNA and protein level were also explored in primary AML cells, as the Fig. 1e and f implied, the expression level of sertad1 shown no obvious difference between normal donor and AML patients, but the expression level of p53 was lower in AML patients, particular in mRNA level.

In our previous experiments, Sertad1 was identified as one of the binding partners of iASPP by yeast two-hybrid screen in vitro. To further confirm the interaction between iASPP and Sertad1, Co-IP was performed in 293 T, K562, HL60 U937 and KG1a. Antibodies against iASPP and Sertad1 were used for IP reaction, and anti-iASPP antibody for WB. The result (Fig. 2a and b) clearly showed that the interaction between iASPP and Sertad1 did exist in 293 cell and leukemic cells. In addition, to better investigate the concrete sub-cellular colocalization of the binding proteins, the fluorescence confocal microscopic imaging of three suspension leukemic cell lines and adherent 293 cell were performed. The results (Fig. 2c) showed that both iASPP and Sertad1 scattered in the cytoplasm and nucleus, and their colocalizations were mainly in the cytoplasm, which encircled the nucleus. Besides the above endogenous colocalization results, the pcDNA3.1-iASPP and pcDNA3.1-Sertad1 plasmids were both transfected into 293 cells at the same time, colocalization of iASPP and Sertad1 was also in the cytoplasm (Data now shown).

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.

To explore the biological functions of iASPP and Sertad1 in the leukemic cell lines, pcDNA3.1-iASPP and pcDNA3.1-Sertad1 were transfected alone or co-transfected into K562 cells, the stable subclones that highly expressed iASPP, Sertad1 or both of them were then established by limiting dilution and named as K562-iASPP^hi, K562-Sertad1^hi, and K562-Dou^hi, respectively. Two subclones of each group were screened by protein for the following function studies, the protein level of each subclone were shown in Fig. 3a.

Firstly, the proliferation of three groups were explored, as shown in Fig. 3b, the proliferation rate of K562-iASPP^hi was 2-3 fold higher than that of the control cells, however, the K562-Sertad^hi proliferation rate was nearly the same with that of the control cells, interestingly, the proliferation rate of K562-Dou^hi cells was lowest, even lower than that of K562-Sertad1^hi 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-iASPP^hi cells had greater growth advantage than that of K562-Sertad1^hi cells and K562-Dou^hi 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-iASPP^hi 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-iASPP^hi 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-Dou^hi cells, but the cell proliferation rate of Dou^hi 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.

To explore the possible cause of difference between K562-Dou^hi cells and K562-iASPP^hi cells, the correlation between the subcellular relocalization of iASPP or Sertad1 proteins and this phenomenon was investigated. The subcellular distribution of iASPP and Sertad1 proteins in three transfected K562 cell lines was observed by fluorescence confocal microscopic imaging. As shown in Fig. 4a, in control (vector) and K562-Sertad1^hi cells, iASPP and Serad1 proteins scattered mainly in cytoplasm, partly in nuclei, the subcellular colocalization of the two proteins was also outside the nuclei, respectively. In K562-iASPP^hi cells, iASPP and Sertad1 scattered diffusely in the cytoplasm and nuclei, the subcellular colocalization of them was not limited to the cytoplasm, also observed in nuclei. But in K562-Dou^hi cells, both iASPP and Sertad1 were obviously located in the cytoplasm, which encircled the nuclei, the subcellular colocalization was nearly outside the nuclei. To further confirm the subcellular amount and distribution of iASPP and Sertad1 proteins, the nuclei and cytoplasm protein was extracted separately and analyzed by Western blot. As shown in Fig. 4b, the distribution of iASPP and Setad1 protein was in accord with the results of confocal images, iASPP was mainly located in cytoplasm in Dou^hi cells compared with vector cells. Because iASPP exerted its function mainly by P53 protein, the interaction between P53 and iASPP was investigated by immunoprecipitation in Sertad1^hi,Sertadl^low cells, as Fig. 4c shown, the interaction was the lower in Sertad1^hi cells compared with Sertadl^low cells, that supported the speculation that excess Sertad1 protein could tether iASPP protein in the cytoplasm, further reduced the binding between iASPP and P53 in the nucleus.

To better understand the mechanism of anti-apoptosis of K562-iASPP^hi, P53 and its target proteins including P21, Puma, Bcl-2, PARP, cleaved-PARP, caspase3 and cleaved-caspase3 were assayed to investigate whether they were involved in the apoptotic process. As shown in Fig. 5, K562-vector, K562-iASPP^hi and K562-Dou^hi cells were exposed to VP16 at different concentrations for 24 h (Fig. 5a-c) or exposed to VP16 at 5 μg/ml for different time (Fig. 5d-f), respectively. As p53 gene was mutated in K562 cells, two bands of P53 with different molecular weights were detected. It was observed that P53 protein decreased gradually as VP16 concentration increased from 0 to 20 μg/ml after 24 h exposure in all the three cell lines (Fig. 5a-c), while P53 protein increased gradually as the exposure time increased from 0 h to 24 h under 5 μg/ml of VP16 treatment (Fig. 5d-f). When looking over the expression trend of p53 target proteins, an interesting phenomenon was observed, namely the Puma protein increased in a time- and dose-dependent manner in VP16 treated K562-iASPP^hi cells accompanied by the Bcl-2, cleaved-PARP increment, irrespective of the expression level of P53. But this phenomenon was not observed in K562-vector and K562-Dou^hi cells when subjected to the same situation. Because the K562-iASPP^hi cells could resist to apoptotic stimuli effectively in a time- and dose-dependent manner (Fig. 3e-f), it suggested that Puma expression might participate in the anti-apoptotic process when considering apoptosis cells decreased obviously in the same situation. As we discussed above, though iASPP exerted its anti-apoptosis ability in some extent, but Puma expression could be activated by the stimuli irrespectively of apoptosis cells number.

As the above results showed that overexprssion of iASPP and Sertad1 could play a role in the biological function of leukemic cells, therefore, silence of the two proteins was performed to further confirm their functions. The expression of iASPP and Sertad1 proteins was knocked down in K562,U937 and KG1a respectively by relevant shRNA(Fig. 6a and b). After that, the cell proliferation, cell cycle and anti-apoptosis ability of above cells were investigated. The results showed that the cell proliferation,cell cycle and cell apoptosis did not change after iASPP was knocked down in all the three leukemic cells (Fig. 6c,e and g).Thus, it is speculated that iASPP is dispensable for maintenance of anti-apoptotic function, cell cycle and cell proliferation. But when Sertad1 was knocked down, to our surprised, the cell proliferation was inhibited(Fig. 6d), the cell cycle were prone to more blocked in G0/G1 stage(Fig. 6f) and leukemic cells became more susceptible to chemotherapy(Fig. 6h). Therefore, the above results suggested the absence of iASPP had no impact on cell biology, but the absence of Sertad1 could change the cell function of leukemic cells, thas was in accord with the oncoprotein function of Seratad1 reported in other solid tumor.

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.

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.

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.

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.

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.

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′.