Cancer Letters 356 (2015) 910–917
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Cancer Letters j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t
Original Articles
A small peptide derived from p53 linker region can resume the apoptotic activity of p53 by sequestering iASPP with p53 Shi Qiu a, Yun Cai a, Xing Gao a, Shou-Zhi Gu a,b, Ze-Jun Liu a,* a b
Southwest Cancer Center, Southwest Hospital, Third Military Medical University, Chongqing 400038, China School of Rehabilitation Sciences, Seirei Christopher University, Hamamatsu, 433-8558, Japan
A R T I C L E
I N F O
Article history: Received 14 May 2014 Received in revised form 27 October 2014 Accepted 31 October 2014 Keywords: iASPP p53 A34 Apoptosis Derepress
A B S T R A C T
One of the most important tumor suppression functions of p53 is its ability to induce apoptosis. iASPP is an inhibitory member of the ASPP protein family. It can specifically inhibit the normal function of p53 as a suppressor. The mechanism of iASPP suppressing the cell apoptotosis is through inhibiting the transactivation function of p53 on the promoters of proapoptotic genes by binding with p53. Therefore, relieving the combination of iASPP with p53 and leaving p53 free may be a useful strategy to activate p53 function. We therefore use A34, a small peptide derived from p53 linker region, to investigate the possibility of resuming the apoptosis activity of p53 by sequestering iASPP with p53 and derepressing p53. The results show that A34 can competitively combine with iASPP and therefore release p53 from iASPP; A34 can enhance the transcriptional activity of p53 on the promoters of Bax and PUMA; A34 can increase cell apoptosis and slow tumor growth in vitro and vivo. This study will open the way for using small molecule peptides that directly disturb the interaction of p53 with iASPP, thereby resume function of p53 as a suppressor. © 2014 Elsevier Ireland Ltd. All rights reserved.
Introduction The p53 protein is an important tumor suppressor [1–3]. One of the most important functions of p53 in suppressing tumor is apoptosis induction [4–7]. iASPP is an inhibitory member of the ASPP (apoptosis stimulating protein of p53) protein family [8–11]. It can specifically inhibit the normal function of p53 as a suppressor [9–11]. iASPP has been found overexpressed in many kinds of cancers such as leukemia [12,13], hepatocellular carcinoma [14], non-small cell lung cancer [15], ovarian cancer [16], pituitary tumorigenesis [17] etc. Therefore, the overexpression of iASPP may be the key factor in the loss of normal p53 tumor suppressor function, and p53 apoptosis activity can be resumed after RNA interference of iASPP in the cancer cells [14,18–21]. The mechanism of iASPP suppressing the cell apoptotosis is through inhibiting the transactivation function of p53 on the promoters of proapoptotic genes by binding with p53 [9,22]. Therefore, it may be a useful strategy to activate p53 function by relieving the combination of iASPP with p53 and leaving p53 free. Bell et al. described identification of a p53-derived peptide (37AA) that induces cell death by displacing iASPP from p73. Their
Abbreviations: ASPP, apoptosis-stimulating protein of p53; iASPP, inhibitory member of the ASPP family; ChIP, chromatin immunoprecipitation; FCM, flow cytometry; PUMA, p53 upregulated modulator of apoptosis. * Corresponding author. Tel.: +86 23 68754691; fax: +86 23 68754691. E-mail address: [email protected] (Z.-J. Liu). http://dx.doi.org/10.1016/j.canlet.2014.10.044 0304-3835/© 2014 Elsevier Ireland Ltd. All rights reserved.
analysis revealed that 37AA acts via sequestration of iASPP. Because iASPP functions as a negative regulator of whole p53 family members, including p53, p63 and p73, therefore, interference with iASPP binding may well induce cell death by p53, p63 and p73. 37AA might be especially useful in human cancers containing mutant p53 [23]. Ahn et al. reported that iASPP may interfere with the proapoptotic activity of p53 by engaging the linker region of p53 and thereby altering the precise positioning of the DNA core binding domains at the promoters of apoptotic genes [24]. They find that the p53 linker region is the highest affinity target for iASPP. Domains of p53 includes the transcription activation (TAD), the proline-rich (Pro), the DNA binding or core (CD), the linker (L), the oligomerization (OD), and the basic (BD) domains [24–26]. Up to now, all these domains, except the linker (L), have been identified to be involved in interactions with cellular factors which regulate the activity of p53 [1,24]. Therefore, designing small molecules or peptides that simulate the linker region will directly target the iASPP and should not affect other cellular factors. We therefore constructed A34 plasmid, which expresses a fragment that stimulates the linker (289–322) of p53 and investigated the ability of A34 to influence cell growth, proliferation and apoptosis, the interaction of iASPP with p53, both in vitro and in vitro, and the influence of A34 on the transcriptional activity of p53 on the promoter(s) of Bax and PUMA. The study will open the way for using small molecule peptides that directly target the iASPP-p53 interface, thereby reinforcing the function of p53 as a tumor suppressor.
S. Qiu et al./Cancer Letters 356 (2015) 910–917
Materials and methods
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ture for 1 h. The immunoreactive proteins were visualized by enhanced chemiluminescence.
Cell culture, plasmids and antibodies In vitro translation and in vitro immunoprecipitation The p53-wild cancer cell line used was U2OS. U2OS (p53−/−) cells were generated by CRISPR/Cas-mediated knockout of p53 in U2OS cells. The targeting nucleotide sequences were: SgRNA top: caccGGAGCGCACCATCTTCTTCA; SgRNA bottom: aaacTGAAGAAGATGGTGCGCTCC. Cells were cultured in DMEM supplemented with 10% fetal bovine serum at 37 °C in an atmosphere containing 5% CO2. TNT T7 quick coupled transcription/translation system was purchased from Promega Corporation (Madison, USA). The plasmids EX-p53-M01 and EX-A34-M01 were made by inserting p53, A34 (residues 289–322 from p53) fragments into pReceiver-M01 Expression Clone. The plasmids were constructed by FulenGen (Guangzhou, China) and tagged with His epitope. The reporter construct for Bax-Luc and PUMA-Luc was constructed by KangChen Bio-tech (Shanghai, China). The full-length coding sequence of iASPP was cloned in-frame into pcDNA3.1 (+) [27]. A peptide encoding iASPP amino acids 28–40 (KQMELDTAAAKVD) was used to generate a rabbit polyclonal antibody. Mouse monoclonal antibody DO-1, specific to p53, was purchased from Santa Cruz Biotechnology (CA, USA). His antibody was purchased from Beyotime Biotech, China. Western blot chemiluminescence reagent plus was purchased from PerkinElmer (Waltham, MA, USA). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo (Kumanoto, Japan). Chromatin immunoprecipitation (ChIP) assays A total of 1 × 107 cells were used for each ChIP experiment, and 270 μl of 37% formaldehyde was added directly to 1 ml of culture medium to a final concentration of 1%. The mixture was incubated at room temperature (RT) for 10 min. The crosslinking reaction was then stopped by adding 505 μl of 2.5 M glycine to a final concentration of 125 mM. Cells were washed with phosphate-buffered saline (PBS) and scraped into 1.5-ml microcentrifuge tubes. Cells were pelleted by centrifuging at 800 g for 5 min at 4 °C and the pellet was resuspended in 1 ml of lysis buffer. Cell suspensions were then sonicated using a Bioruptor (Diagenode) on “Mid” (M) setting. The tank was filled with cold water supplemented with 0.5 cm crushed ice and the cells were sonicated for 10 min, using 30 seconds ‘on’ and 30 seconds ‘off’, followed by spinning at 12,000 g for 10 min at 4 °C to pellet the debris. The supernatants were removed from each sample to new tubes for immunoprecipitation. Chromosomal DNA was precipitated using p53 antibody DO-1. The immunoprecipitated DNA was analyzed by polymerase chain reaction (PCR) using the following primers: Bax: forward, 5′-CCGGGAATTCCAGACTGCAGTGAGCC-3′; backward, 5′-AGCATGCT TCCAGGCAGGACGTTATAGATG-3′; PUMA: forward, 5′-CTCTGGGCTCTGCCTGCACG3′; backward, 5′-ATCGCCCAGACACCGGGACA-3′. PCR for the Bax promoter region was performed with 30 cycles of 30 s melting at 94 °C, 30 s annealing at 64 °C, and 30 s extension at 72 °C. PCR for the PUMA promoter region was performed under the same conditions as Bax, except annealing temperature 66 °C. The PCR products of Bax and PUMA were 317 bp and 134 bp, respectively. Transactivation assays U2OS cells were plated in 24-well plates 24 h prior to transfection. All transfection assays contained 1 μg of reporter plasmid, 0.05 μg pRL-SV40 vectors and 0.1 μg of expression plasmids for A34 or M01. Cells were lysed in passive lysis buffer 48 h after transfection and assayed using the Dual-luciferase Reporter Assay System (Promega). The fold activation of a particular reporter was determined by calculating the activation of the transfected plasmid compared to the activity of the control vector alone. Detection of apoptosis with flow cytometry (FCM) U2OS cells were plated in 6-well plates at 24–48 h prior to transfection. The cells were transfected with 4 μg of A34 plasmid or vector M01 alone. Cells were collected 48 h after transfection and washed twice with ice-cold PBS. The cell concentration was adjusted to 1 × 106/ml. One hundred microliters of cell suspension, 5 μl Annexin V/fluorescein isothiocyanate and 10 μl propidium iodide solution (20 ng/ml) were thoroughly mixed in an Eppendorf tube and incubated in the dark at RT for 15 min. PBS 400 μl was then added and the rate of apoptosis was measured by FCM within 1 hour. Western blot analysis For Western blotting, cells were collected and lysed in RIPA lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 1% NP-40, 0.1% sodium dodecyl sulfate (SDS), 5 mM benzamidine, 1 μM aprotinin A, 1 μM pepstatin, 1 μM leupeptin, 1 mM PMSF, pH 7.4). Supernatants were subjected to SDS–polyacrylamide gel electrophoresis (PAGE), and transferred to PVDF membranes. The membranes were blocked with PBS buffer containing 6% non-fat milk overnight, followed by incubation with rabbit anti-iASPP polyclonal antibody (working concentration, 1 μg/ml), mouse anti-p53 antibody DO-1 and His antibody respectively at RT for 1 h, and subsequently incubated with the secondary horseradish peroxidase (HRP)–IgG (1:2500) antibody at room tempera-
A34, p53 and iASPP were translated in vitro using the TNT T7 Quick Coupled Transcription/Translation System (Promega). One microgram of plasmid was added to 42 μl of mixing solution (including 40 μl TNT Quick Master Mix, 1 μl methionine and 1 μl enhancer). The above reaction mixture was made up to 50 μl with nuclease-free water and incubated at 30 °C for 90 min to produce the relevant protein. For immunoprecipitation, 10 μl of iASPP lysate, 10 μl of p53 lysate and A34 lysate (0 μl, 10 μl, and 35 μl) were incubated at 30 °C for 1 h. The iASPP antibody was added to the binding reaction mixtures and incubated with shaking at 4 °C overnight. The following day, 20 μl protein A + G agarose (Beyotime Biotech, Haimen, China) was added to the reaction mixtures and rotated at 4 °C for 3 h. The agarose was then washed with PBS. The bound proteins were released in SDS gel sample buffer and analyzed by 10% SDS-PAGE. iASPP, A34 and p53 were detected by Western blotting with anti-iASPP and anti-His antibodies. Cell proliferation assay After U2OS cells were transfected with A34 and M01 or injected with saline, cell proliferation was examined by CCK-8 assay. Cells were collected at days 1–4 and CCK-8 reagent was added to cells according to the manufacturer’s instructions. The absorbance was then read at 450 nm. Transplanted tumor studies Cultures of MKN-45 cells were injected into the flanks of nude mice to induce tumor formation. Tumors were palpable 6 days after subcutaneous injection. Once tumors were palpable (day 0), mice were treated by small tumor nodules injection of A34 or M01 expression plasmids on days 1, 3, 5, 7, 9 and 11. The experimental group were injected with the plasmid A34 (30 μg DNA) and control mice were injected with the plasmid M01 (30 μg DNA) or injected with saline. The tumor volume of nude mice (n = 6) was determined from caliper measurements and calculated as L × S2 × 0.5, where L represents longer diameter and S represents shorter diameter.
Results and discussion A34 can increase cell apoptosis of wild-type p53 In order to prove that A34 can increase cell apoptosis, we investigated the effects of A34 transfection in the p53-wild cell line, U2OS. Apoptosis was determined by FCM using the Annexin apoptosis detection kit. The number of apoptotic cells detected in cells transfected with A34 was (16.8 ± 2.36) %. The number of apoptotic cells detected in cells transfected with vector was (5.04 ± 2.48) %. Therefore, the number of apoptotic cells in cells transfected with A34 was higher than that in cells transfected with vector alone (p < 0.01, Fig. 1B). This result suggests that A34 can increase cell apoptosis of wild-type p53. In order to prove that these effects are dependent on p53, we also investigated the effects of A34 transfection in the U2OS (p53−/−) cells. The results showed that A34 did not induce cell death in U2OS (p53−/−) cells (Fig. 1C). Therefore, that A34 can increase cell apoptosis is dependent on p53. The influence of A34 to the interaction of iASPP with p53 in vitro and in vivo Reports have shown iASPP can interact with p53 and inhibit its apoptotic function [9,21]. To determine if A34 can influence the interaction of iASPP with p53 in vitro, we conducted in vitro translation and immunoprecipitation and western blot analysis (Fig. 2A). The results showed that A34 can interact with iASPP. When A34 was increased from 0 to 10 μl and then to 35 μl, p53 gradually decreased. This phenomenon indicates that A34 can competitively combine with iASPP and therefore release p53 from iASPP. To confirm that A34 can combine with iASPP in vivo, we further studied interaction between A34 and iASPP in the p53 wild cell, U2OS, which was transiently transfected with the A34 plasmids.
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Fig. 2. A34 interacts with iASPP in vitro (A) and in vivo (B). (A) A34, p53 and iASPP were translated in vitro using the TNT T7 Quick Coupled Transcription/Translation System. iASPP protein was immunoprecipitated with anti-iASPP antibody. Immunoprecipitates were fractionated on SDS–10% polyacrylamide gels. The presence of A34 and p53 complexed with iASPP was detected by Western blotting with His antibody. (B) U2OS cells were transiently transfected with the A34 plasmids. Sample of cells lysates was immunoprecipitated with iASPP antibody, p53 antibody and His antibody, respectively. The immunoprecipitates were separated on SDS–10% polyacylamide gels. The presence of p53 and A34 on the immunoblots was detected with mouse anti-p53 antibody DO-1 and His antibody, respectively. The presence of iASPP was detected with iASPP antibody. The control IgG antibody was used as unrelated antibody. IP: immunoprecipitation; Ab: antibody (the experiment was repeated twice).
Endogenous iASPP was immunoprecipitated by antibodies specific to iASPP (Fig. 2B). Consistent with our in vitro observation, the anti-iASPP antibody was able to coimmunoprecipitate A34 and endogenous p53. His antibody can coimmunoprecipitate A34 and endogenous iASPP. Under the same conditions, the control antibody failed to coimmunoprecipitate A34, p53 or iASPP. These results indicated that A34 can combine with iASPP, but not with p53. Overall, all these results suggested that A34 can interact with iASPP in vitro and in vivo. A34 can enhance the transcriptional activity of p53 on the promoters of Bax and PUMA iASPP is known to reduce the DNA-binding activities of p53 on the Bax and PUMA promoters [9]. We therefore studied if A34 can affect iASPP on the transactivation function of p53 in transient reporter assays. U2OS cells (p53-wild) and U2OS (p53−/−) cells were co-transfected with A34, Bax-Luc, PUMA-Luc and pRL-SV40 plasmids as indicated in Fig. 3. A34 expression enhanced the transactivation function of p53 on the Bax/PUMA promoters in U2OS cells (Fig. 3A) but not in U2OS (p53−/−) cells (Fig. 3B). Therefore, that A34 can affect the transactivation function is dependent on p53. The fold increase in Bax-luc activity detected in U2OS cells transfected with A34 was 3.86 times higher than that detected in cells transfected with vector (Fig. 3A). Fold increase in PUMA-luc activity detected in U2OS cells transfected with A34 was 4.57 times higher than that detected in cells transfected with vector (Fig. 3A). These results suggest that A34 can enhance the transactivation function
Fig. 3. A34 can enhance the transcriptional activity of p53 on the promoter of Bax and PUMA. U2OS cells (A) and U2OS cells (p53−/−) (B) were transfected with 0.1 μg A34 plasmid alongside 1 μg of Bax-luc (Puma-luc) reporter plasmid and 0.05 μg pRLSV40 plasmid. The graph shows the changes in relative transactivation activity. Mean values were derived from three independent experiments. (C) Schematic of the mode of action of A34. Introduction of A34 into p53-wild cells sequesters iASPP and thereby derepresses p53. Free p53 subsequently activates apoptotic target genes such as Bax and PUMA to bring about programmed cell death.
of p53 on the Bax/PUMA promoters. The reason may be that A34 has higher affinity with iASPP than the affinity of p53 with iASPP (Fig. 2A). Therefore, A34 can priorly combine with iASPP and let p53 free. Then free p53 members in the cell are increased and the p53 members combined with Bax and PUMA promoters are also
Fig. 1. A34 can increase cell apoptosis. (A) Domain organization of p53. The composition of the A34 peptide is from the linker domain. (B) A34 can increase cell apoptosis of U2OS. U2OS cells were transfected with A34 plasmid or control vector. The percentage of apoptotic cells was measured by cytofluorimetry using the Annexin Apoptosis Detection Kit. The bar graph shows the percentage of apoptotic cells 48 h after transfection. Mean values were derived from three independent experiments. (C) A34 cannot increase cell apoptosis of U2OS (p53−/−) cells, in which p53 was knocked out by CrispR/Cas9. (D) Western blot analysis of p53 protein expression in U2OS cells and U2OS (p53−/−) cells.
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Fig. 4. A34 can enhance the DNA binding function of p53 on the promoter of Bax and PUMA and increase the mRNA/protein levels of Bax and Puma. (A) ChIP of p53 at the Bax/Puma promoters. ChIP was performed using an anti-p53 antibody DO-1 or control antibody IgG, followed by PCR amplification with Bax primers or Puma primers. Lane No. 1, DNA derived from U2OS cells transfected with control vector; Lane No. 2, DNA derived from cells transfected with A34 plasmids. Bar graphs show the fold increase of PCR products detected. (B) The mRNA expression of Bax and Puma in U2OS after transfection with A34. Lane 1, U2OS cells; Lane 2, U2OS cells transfected with control vector; Lane 3, U2OS cells transfected with A34. The PCR products of Bax and PUMA were 424 bp and 520 bp, respectively. GAPDH was used as internal standard. DNA Marker, DL2000. (C) The protein levels of Bax and Puma in U2OS after transfection with A34. Lane 1, U2OS cells; Lane 2, U2OS cells transfected with control vector; Lane 3, U2OS cells transfected with A34. Rabbit antibodies bs-0127R and bs-1573R were used to immunoprecipitate Bax and Puma, respectively. GAPDH, sampling control.
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Fig. 5. A34 slows tumor growth in vitro and vivo. (A) CCK-8 assay for detecting the proliferation of U2OS cells after transfection with A34, M01 and saline, respectively. (B) Cultures of MKN-45 cells were injected into the flanks of BALB/c nude mice. Upon tumor formation (day 0), mice were treated by small tumor nodule injection with 13 μg of A34 and M01 or saline respectively on days 1, 3, 5, 7, 9 and 11. Tumor volume was measured, and tumor growth was plotted as relative to tumor size on day 0. (C) A34 can increase cell apoptosis of MKN-45. MKN-45 cells were transfected with A34 plasmid or control vector. The percentage of apoptotic cells was measured by cytofluorimetry using the Annexin Apoptosis Detection Kit. The bar graph shows the percentage of apoptotic cells 48 h after transfection. Mean values were derived from three independent experiments. (D) A34 can enhance the transcriptional activity of p53 on the promoter of Bax and PUMA. MKN-45 cells were transfected with 0.1 μg A34 plasmid alongside 1 μg of Bax-luc (Puma-luc) reporter plasmid and 0.05 μg pRL-SV40 plasmid. The graph shows the changes in relative transactivation activity. Mean values were derived from three independent experiments.
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increased, as a result, the transcriptional activity of p53 on the Bax and PUMA promoters is enhanced (Fig. 3C). A34 can enhance the DNA binding function of p53 on the promoter of Bax and PUMA and increase the mRNA/ protein levels of Bax and Puma To prove that if A34 can affect the DNA binding function of p53, we investigated the transactivation functions of p53 in U2OS cells transfected with control vector and cells transfected with A34 plasmids. Bax and PUMA promoters were used to measure the transactivation functions of p53. We studied the DNA-binding activity of p53 on the Bax and PUMA promoters in vivo using the chromatin immunoprecipitation technique. After various treatments, U2OS cells were exposed to formaldehyde to crosslink the DNA-binding proteins together with their target sequences in situ. p53 binds to DNA, and the promoter sequences of p53 target genes should therefore be detected by PCR in immunoprecipitates derived from anti-p53 antibodies (DO-1). Bax and PUMA promoter sequences were specifically detected in the immunoprecipitates derived from anti-p53 DO-1, but not from the control IgG antibody. The levels of Bax and PUMA promoter sequences detected in cells transfected with control vector was set as 1 and used to calculate the in vivo DNA-binding activity of p53. Transfection with A34 increased the DNA-binding activities of p53 on the Bax and PUMA promoters by 1.54- and 1.34-fold, respectively. Therefore, transfection with A34 clearly enhanced the DNA-binding abilities of p53 on the Bax and PUMA promoters (Fig. 4). To understand the changes of the expression of Bax and Puma, we investigated the mRNA/protein levels of Bax and Puma after adding A34. The results show that the mRNA/protein levels of Bax and Puma in U2OS are increased after transfection with A34 (Fig. 4B,C). All the experimental results in vitro suggest that A34 can enhance the transactivation function of p53 on the Bax/PUMA promoters, increase mRNA/protein expression levels of Bax and Puma and finally bring about programmed cell death. A34 can slow tumor growth in vitro and vivo Since A34 can increase cell apoptosis, we consider if A34 could slow cell proliferation. After U2OS cells were transfected with A34, M01 and saline, CCK-8 reagent was used to detect the cell proliferation. The result shows that the cell proliferation in the group transfected with A34 is slower than the group transfected with M01 or the group injected with saline (Fig. 5A). Next, we consider whether A34 would work as a therapeutic agent in vivo to cause cell death and tumor regression. As U2OS cells are difficult to form transplantation tumor in athymic mice [28], we decided to analyze the effects of A34 on tumors formed with MKN45 cells, which are easy to form transplantation tumors and are frequently used in xenograft studies. In addition, the similar experiment results of apoptosis assay and luciferase reporter assay in U2OS are also reproducible in MKN-45 (Fig. 5C,D), therefore, it is reasonable to use MKN-45 cells instead of U2OS cells in vivo tests. Cultures of MKN-45 cells were therefore injected into the flanks of mice to induce tumor formation. Once tumors were palpable, mice were treated by small tumor nodule injection of A34 or M01 expression plasmids on days 1, 3, 5, 7, 9 and 11. Analysis of tumor size during these treatments revealed that while tumors treated with M01 or saline continued to grow (Fig. 5B), tumor growth in mice treated with A34 was slowed, indicating that A34 can slow tumor growth in transplantation tumors (Fig. 5B). Since MKN-45 cells contain endogenous WT p53, and A34 can enhance the transcriptional activity of p53 on the promoter of Bax and PUMA (Fig. 3), therefore, the tumor growth in mice treated with
A34 slowed in this case is because more p53 was activated and p53 subsequently activates apoptotic target genes such as Bax and PUMA to bring about programmed cell death. Therefore, A34 might be used as a therapeutic agent in vivo to cause tumor cell death. In conclusion, the results show that A34, derived from the linker region of p53, can enhance DNA binding and transcriptional activity of p53 by binding to and sequestering iASPP away from p53. A34 is also shown to increase cell apoptosis and slow down tumor growth in vitro and vivo. This study will open the way for using small molecule peptides that directly disturb the interaction of p53 with iASPP, thereby resume the function of p53 as a suppressor. Acknowledgements We thank Takara Biothchnology (Dalian) CO., Ltd., GeneChem CO., Ltd. and KangChen Bio-tech Inc. for technical assistance. This work was supported by the National Natural Science Foundation of China (30971140). Conflict of interest The authors declare that they have no conflict of interest. References [1] K.H. Vousden, X. Lu, Live or let die: the cell’s response to p53, Nat. Rev. Cancer 2 (2002) 594–604. [2] C.J. Brown, S. Lain, C.S. Verma, A.R. Fersht, D.P. Lane, Awakening guardian angels: drugging the p53 pathway, Nat. Rev. Cancer 6 (2009) 862–873. [3] X. Lu, p53: a heavily dictated dictator of life and death, Curr. Opin. Genet. Dev. 15 (2005) 27–33. [4] E.A. Slee, D.J. O’Connor, X. Lu, To die or not to die: how does p53 decide?, Oncogene 23 (2004) 2809–2818. [5] A.R. Delbridge, L.J. Valente, A. Strasser, The role of the apoptotic machinery in tumor suppression, Cold Spring Harb. Perspect. Biol. 4 (2012) pii: a008789. [6] K.H. Vousden, Activation of the p53 tumor suppressor protein, Biochim. Biophys. Acta 1602 (2002) 47–59. [7] J.E. Chipuk, D.R. Green, Dissecting p53-dependent apoptosis, Cell Death Differ. 13 (2006) 994–1002. [8] Y. Samuels-Lev, D.J. O’Connor, D. Bergamaschi, G. Trigiante, J.K. Hsieh, S. Zhong, et al., ASPP proteins specifically stimulate the apoptotic function of p53, Mol. Cell 8 (2001) 781–794. [9] D. Bergamaschi, Y. Samuels, N.J.O. Neil, G. Trigiante, T. Crook, J.K. Hsieh, et al., iASPP oncoprotein is a key inhibitor of p53 conserved from worm to human, Nat. Genet. 33 (2003) 162–167. [10] Z.J. Liu, X. Lu, S. Zhong, ASPP – apoptotic specific regulator of p53, Biochim. Biophys. Acta 1756 (2005) 77–80. [11] A. Sullivan, X. Lu, ASPP: a new family of oncogenes and tumor suppressor genes, Br. J. Cancer 96 (2007) 196–200. [12] Z.J. Liu, Y. Zhang, X.B. Zhang, X. Yang, Abnormal mRNA expression of ASPP members in leukemia cell lines, Leukemia 18 (2004) 880. [13] X. Zhang, M. Wang, C. Zhou, S. Chen, J. Wang, The expression of iASPP in acute leukemias, Leuk. Res. 29 (2005) 179–183. [14] B.L. Lin, D.Y. Xie, S.B. Xie, J.Q. Xie, X.H. Zhang, Y.F. Zhang, et al., Down-regulation of iASPP in human hepatocellular carcinoma cells inhibits cell proliferation and tumor growth, Neoplasma 58 (2011) 205–210. [15] J. Chen, F. Xie, L. Zhang, W.G. Jiang, iASPP is over-expressed in human non-small cell lung cancer and regulates the proliferation of lung cancer cells through a p53 associated pathway, BMC Cancer 10 (2010) 694. [16] L. Jiang, M.K. Siu, O.G. Wong, K.F. Tam, X. Lu, E.W. Lam, et al., iASPP and chemoresistance in ovarian cancers: effects on paclitaxel-mediated mitotic catastrophe, Clin. Cancer Res. 17 (2011) 6924–6933. [17] E.M. Pinto, N.R. Musolino, V.A. Cescato, I.C. Soares, A. Wakamatsu, E. de Oliveira, et al., iASPP: a novel protein involved in pituitary tumorigenesis?, Front. Horm. Res. 38 (2010) 70–76. [18] G. Li, R. Wang, J. Gao, K. Deng, J. Wei, Y. Wei, RNA interference-mediated silencing of iASPP induces cell proliferation inhibition and G0/G1 cell cycle arrest in U251 human glioblastoma cells, Mol. Cell. Biochem. 350 (2011) 193– 200. [19] H. Liu, M. Wang, S. Diao, Q. Rao, X. Zhang, H. Xing, et al., siRNA-mediated down-regulation of iASPP promotes apoptosis induced by etoposide and daunorubicin in leukemia cells expressing wild-type p53, Leuk. Res. 33 (2009) 1243–1248. [20] Z.J. Liu, H.M. Xin, J. Chen, X. Lu, S. Zhong, S.Z. Gu, et al., A new strategy to resume the apoptosis activity of p53 in leukemia cell lines retaining wild-type p53, Leuk. Res. 31 (2007) 1156–1158.
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[21] Z.J. Liu, Y. Cai, L. Hou, X. Gao, H.M. Xin, X. Lu, et al., Effect of RNA interference of iASPP on the apoptosis in MCF-7 breast cancer cells, Cancer Invest. 26 (2008) 878–882. [22] M.J. Laska, U.B. Vogel, U.B. Jensen, B.A. Nexo, p53 and PPP1R13L(alias iASPP or RAI) form a feedback loop to regulate genotoxic stress responses, Biochim. Biophys. Acta 1800 (2010) 1231–1240. [23] H.S. Bell, C. Dufes, J.O. Prey, D. Crighton, D. Bergamaschi, X. Lu, et al., A p53-derived apoptotic peptide derepresses p73 to cause tumor regression in vivo, J. Clin. Invest. 117 (2007) 1008–1018. [24] J. Ahn, I.J. Byeon, C.H. Byeon, A.M. Gronenborn, Insight into the structural basis of pro- and antiapoptotic p53 modulation by ASPP proteins, J. Biol. Chem. 284 (2009) 13812–13822.
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[25] A.C. Joerger, A.R. Fersht, The tumor suppressor p53: from structures to drug discovery, Cold Spring Harb. Perspect. Biol. 2 (2010) a000919. [26] E.A. Slee, S. Gillotin, D. Bergamaschi, C. Royer, S. Llanos, S. Ali, et al., The N-terminus of a novel isoform of human iASPP is required for its cytoplasmic localization, Oncogene 23 (2004) 9006–9016. [27] Z.J. Liu, X. Gao, Y. Cai, X. Yang, X.L. Fu, J. Chen, et al., Construction of a full-length iASPP expression plasmid pcDNA3.1(+)/iASPP and its biological activity, Plasmid 62 (2009) 10–15. [28] P. Fei, W. Wang, S.H. Kim, S. Wang, T.F. Burns, J.K. Sax, et al., Bnip3L is induced by p53 under hypoxia, and its knockdown promotes tumor growth, Cancer Cell 6 (2004) 597–609.
Contents lists available at ScienceDirect
Cancer Letters j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t
Original Articles
A small peptide derived from p53 linker region can resume the apoptotic activity of p53 by sequestering iASPP with p53 Shi Qiu a, Yun Cai a, Xing Gao a, Shou-Zhi Gu a,b, Ze-Jun Liu a,* a b
Southwest Cancer Center, Southwest Hospital, Third Military Medical University, Chongqing 400038, China School of Rehabilitation Sciences, Seirei Christopher University, Hamamatsu, 433-8558, Japan
A R T I C L E
I N F O
Article history: Received 14 May 2014 Received in revised form 27 October 2014 Accepted 31 October 2014 Keywords: iASPP p53 A34 Apoptosis Derepress
A B S T R A C T
One of the most important tumor suppression functions of p53 is its ability to induce apoptosis. iASPP is an inhibitory member of the ASPP protein family. It can specifically inhibit the normal function of p53 as a suppressor. The mechanism of iASPP suppressing the cell apoptotosis is through inhibiting the transactivation function of p53 on the promoters of proapoptotic genes by binding with p53. Therefore, relieving the combination of iASPP with p53 and leaving p53 free may be a useful strategy to activate p53 function. We therefore use A34, a small peptide derived from p53 linker region, to investigate the possibility of resuming the apoptosis activity of p53 by sequestering iASPP with p53 and derepressing p53. The results show that A34 can competitively combine with iASPP and therefore release p53 from iASPP; A34 can enhance the transcriptional activity of p53 on the promoters of Bax and PUMA; A34 can increase cell apoptosis and slow tumor growth in vitro and vivo. This study will open the way for using small molecule peptides that directly disturb the interaction of p53 with iASPP, thereby resume function of p53 as a suppressor. © 2014 Elsevier Ireland Ltd. All rights reserved.
Introduction The p53 protein is an important tumor suppressor [1–3]. One of the most important functions of p53 in suppressing tumor is apoptosis induction [4–7]. iASPP is an inhibitory member of the ASPP (apoptosis stimulating protein of p53) protein family [8–11]. It can specifically inhibit the normal function of p53 as a suppressor [9–11]. iASPP has been found overexpressed in many kinds of cancers such as leukemia [12,13], hepatocellular carcinoma [14], non-small cell lung cancer [15], ovarian cancer [16], pituitary tumorigenesis [17] etc. Therefore, the overexpression of iASPP may be the key factor in the loss of normal p53 tumor suppressor function, and p53 apoptosis activity can be resumed after RNA interference of iASPP in the cancer cells [14,18–21]. The mechanism of iASPP suppressing the cell apoptotosis is through inhibiting the transactivation function of p53 on the promoters of proapoptotic genes by binding with p53 [9,22]. Therefore, it may be a useful strategy to activate p53 function by relieving the combination of iASPP with p53 and leaving p53 free. Bell et al. described identification of a p53-derived peptide (37AA) that induces cell death by displacing iASPP from p73. Their
Abbreviations: ASPP, apoptosis-stimulating protein of p53; iASPP, inhibitory member of the ASPP family; ChIP, chromatin immunoprecipitation; FCM, flow cytometry; PUMA, p53 upregulated modulator of apoptosis. * Corresponding author. Tel.: +86 23 68754691; fax: +86 23 68754691. E-mail address: [email protected] (Z.-J. Liu). http://dx.doi.org/10.1016/j.canlet.2014.10.044 0304-3835/© 2014 Elsevier Ireland Ltd. All rights reserved.
analysis revealed that 37AA acts via sequestration of iASPP. Because iASPP functions as a negative regulator of whole p53 family members, including p53, p63 and p73, therefore, interference with iASPP binding may well induce cell death by p53, p63 and p73. 37AA might be especially useful in human cancers containing mutant p53 [23]. Ahn et al. reported that iASPP may interfere with the proapoptotic activity of p53 by engaging the linker region of p53 and thereby altering the precise positioning of the DNA core binding domains at the promoters of apoptotic genes [24]. They find that the p53 linker region is the highest affinity target for iASPP. Domains of p53 includes the transcription activation (TAD), the proline-rich (Pro), the DNA binding or core (CD), the linker (L), the oligomerization (OD), and the basic (BD) domains [24–26]. Up to now, all these domains, except the linker (L), have been identified to be involved in interactions with cellular factors which regulate the activity of p53 [1,24]. Therefore, designing small molecules or peptides that simulate the linker region will directly target the iASPP and should not affect other cellular factors. We therefore constructed A34 plasmid, which expresses a fragment that stimulates the linker (289–322) of p53 and investigated the ability of A34 to influence cell growth, proliferation and apoptosis, the interaction of iASPP with p53, both in vitro and in vitro, and the influence of A34 on the transcriptional activity of p53 on the promoter(s) of Bax and PUMA. The study will open the way for using small molecule peptides that directly target the iASPP-p53 interface, thereby reinforcing the function of p53 as a tumor suppressor.
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Materials and methods
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ture for 1 h. The immunoreactive proteins were visualized by enhanced chemiluminescence.
Cell culture, plasmids and antibodies In vitro translation and in vitro immunoprecipitation The p53-wild cancer cell line used was U2OS. U2OS (p53−/−) cells were generated by CRISPR/Cas-mediated knockout of p53 in U2OS cells. The targeting nucleotide sequences were: SgRNA top: caccGGAGCGCACCATCTTCTTCA; SgRNA bottom: aaacTGAAGAAGATGGTGCGCTCC. Cells were cultured in DMEM supplemented with 10% fetal bovine serum at 37 °C in an atmosphere containing 5% CO2. TNT T7 quick coupled transcription/translation system was purchased from Promega Corporation (Madison, USA). The plasmids EX-p53-M01 and EX-A34-M01 were made by inserting p53, A34 (residues 289–322 from p53) fragments into pReceiver-M01 Expression Clone. The plasmids were constructed by FulenGen (Guangzhou, China) and tagged with His epitope. The reporter construct for Bax-Luc and PUMA-Luc was constructed by KangChen Bio-tech (Shanghai, China). The full-length coding sequence of iASPP was cloned in-frame into pcDNA3.1 (+) [27]. A peptide encoding iASPP amino acids 28–40 (KQMELDTAAAKVD) was used to generate a rabbit polyclonal antibody. Mouse monoclonal antibody DO-1, specific to p53, was purchased from Santa Cruz Biotechnology (CA, USA). His antibody was purchased from Beyotime Biotech, China. Western blot chemiluminescence reagent plus was purchased from PerkinElmer (Waltham, MA, USA). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo (Kumanoto, Japan). Chromatin immunoprecipitation (ChIP) assays A total of 1 × 107 cells were used for each ChIP experiment, and 270 μl of 37% formaldehyde was added directly to 1 ml of culture medium to a final concentration of 1%. The mixture was incubated at room temperature (RT) for 10 min. The crosslinking reaction was then stopped by adding 505 μl of 2.5 M glycine to a final concentration of 125 mM. Cells were washed with phosphate-buffered saline (PBS) and scraped into 1.5-ml microcentrifuge tubes. Cells were pelleted by centrifuging at 800 g for 5 min at 4 °C and the pellet was resuspended in 1 ml of lysis buffer. Cell suspensions were then sonicated using a Bioruptor (Diagenode) on “Mid” (M) setting. The tank was filled with cold water supplemented with 0.5 cm crushed ice and the cells were sonicated for 10 min, using 30 seconds ‘on’ and 30 seconds ‘off’, followed by spinning at 12,000 g for 10 min at 4 °C to pellet the debris. The supernatants were removed from each sample to new tubes for immunoprecipitation. Chromosomal DNA was precipitated using p53 antibody DO-1. The immunoprecipitated DNA was analyzed by polymerase chain reaction (PCR) using the following primers: Bax: forward, 5′-CCGGGAATTCCAGACTGCAGTGAGCC-3′; backward, 5′-AGCATGCT TCCAGGCAGGACGTTATAGATG-3′; PUMA: forward, 5′-CTCTGGGCTCTGCCTGCACG3′; backward, 5′-ATCGCCCAGACACCGGGACA-3′. PCR for the Bax promoter region was performed with 30 cycles of 30 s melting at 94 °C, 30 s annealing at 64 °C, and 30 s extension at 72 °C. PCR for the PUMA promoter region was performed under the same conditions as Bax, except annealing temperature 66 °C. The PCR products of Bax and PUMA were 317 bp and 134 bp, respectively. Transactivation assays U2OS cells were plated in 24-well plates 24 h prior to transfection. All transfection assays contained 1 μg of reporter plasmid, 0.05 μg pRL-SV40 vectors and 0.1 μg of expression plasmids for A34 or M01. Cells were lysed in passive lysis buffer 48 h after transfection and assayed using the Dual-luciferase Reporter Assay System (Promega). The fold activation of a particular reporter was determined by calculating the activation of the transfected plasmid compared to the activity of the control vector alone. Detection of apoptosis with flow cytometry (FCM) U2OS cells were plated in 6-well plates at 24–48 h prior to transfection. The cells were transfected with 4 μg of A34 plasmid or vector M01 alone. Cells were collected 48 h after transfection and washed twice with ice-cold PBS. The cell concentration was adjusted to 1 × 106/ml. One hundred microliters of cell suspension, 5 μl Annexin V/fluorescein isothiocyanate and 10 μl propidium iodide solution (20 ng/ml) were thoroughly mixed in an Eppendorf tube and incubated in the dark at RT for 15 min. PBS 400 μl was then added and the rate of apoptosis was measured by FCM within 1 hour. Western blot analysis For Western blotting, cells were collected and lysed in RIPA lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 1% NP-40, 0.1% sodium dodecyl sulfate (SDS), 5 mM benzamidine, 1 μM aprotinin A, 1 μM pepstatin, 1 μM leupeptin, 1 mM PMSF, pH 7.4). Supernatants were subjected to SDS–polyacrylamide gel electrophoresis (PAGE), and transferred to PVDF membranes. The membranes were blocked with PBS buffer containing 6% non-fat milk overnight, followed by incubation with rabbit anti-iASPP polyclonal antibody (working concentration, 1 μg/ml), mouse anti-p53 antibody DO-1 and His antibody respectively at RT for 1 h, and subsequently incubated with the secondary horseradish peroxidase (HRP)–IgG (1:2500) antibody at room tempera-
A34, p53 and iASPP were translated in vitro using the TNT T7 Quick Coupled Transcription/Translation System (Promega). One microgram of plasmid was added to 42 μl of mixing solution (including 40 μl TNT Quick Master Mix, 1 μl methionine and 1 μl enhancer). The above reaction mixture was made up to 50 μl with nuclease-free water and incubated at 30 °C for 90 min to produce the relevant protein. For immunoprecipitation, 10 μl of iASPP lysate, 10 μl of p53 lysate and A34 lysate (0 μl, 10 μl, and 35 μl) were incubated at 30 °C for 1 h. The iASPP antibody was added to the binding reaction mixtures and incubated with shaking at 4 °C overnight. The following day, 20 μl protein A + G agarose (Beyotime Biotech, Haimen, China) was added to the reaction mixtures and rotated at 4 °C for 3 h. The agarose was then washed with PBS. The bound proteins were released in SDS gel sample buffer and analyzed by 10% SDS-PAGE. iASPP, A34 and p53 were detected by Western blotting with anti-iASPP and anti-His antibodies. Cell proliferation assay After U2OS cells were transfected with A34 and M01 or injected with saline, cell proliferation was examined by CCK-8 assay. Cells were collected at days 1–4 and CCK-8 reagent was added to cells according to the manufacturer’s instructions. The absorbance was then read at 450 nm. Transplanted tumor studies Cultures of MKN-45 cells were injected into the flanks of nude mice to induce tumor formation. Tumors were palpable 6 days after subcutaneous injection. Once tumors were palpable (day 0), mice were treated by small tumor nodules injection of A34 or M01 expression plasmids on days 1, 3, 5, 7, 9 and 11. The experimental group were injected with the plasmid A34 (30 μg DNA) and control mice were injected with the plasmid M01 (30 μg DNA) or injected with saline. The tumor volume of nude mice (n = 6) was determined from caliper measurements and calculated as L × S2 × 0.5, where L represents longer diameter and S represents shorter diameter.
Results and discussion A34 can increase cell apoptosis of wild-type p53 In order to prove that A34 can increase cell apoptosis, we investigated the effects of A34 transfection in the p53-wild cell line, U2OS. Apoptosis was determined by FCM using the Annexin apoptosis detection kit. The number of apoptotic cells detected in cells transfected with A34 was (16.8 ± 2.36) %. The number of apoptotic cells detected in cells transfected with vector was (5.04 ± 2.48) %. Therefore, the number of apoptotic cells in cells transfected with A34 was higher than that in cells transfected with vector alone (p < 0.01, Fig. 1B). This result suggests that A34 can increase cell apoptosis of wild-type p53. In order to prove that these effects are dependent on p53, we also investigated the effects of A34 transfection in the U2OS (p53−/−) cells. The results showed that A34 did not induce cell death in U2OS (p53−/−) cells (Fig. 1C). Therefore, that A34 can increase cell apoptosis is dependent on p53. The influence of A34 to the interaction of iASPP with p53 in vitro and in vivo Reports have shown iASPP can interact with p53 and inhibit its apoptotic function [9,21]. To determine if A34 can influence the interaction of iASPP with p53 in vitro, we conducted in vitro translation and immunoprecipitation and western blot analysis (Fig. 2A). The results showed that A34 can interact with iASPP. When A34 was increased from 0 to 10 μl and then to 35 μl, p53 gradually decreased. This phenomenon indicates that A34 can competitively combine with iASPP and therefore release p53 from iASPP. To confirm that A34 can combine with iASPP in vivo, we further studied interaction between A34 and iASPP in the p53 wild cell, U2OS, which was transiently transfected with the A34 plasmids.
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Fig. 2. A34 interacts with iASPP in vitro (A) and in vivo (B). (A) A34, p53 and iASPP were translated in vitro using the TNT T7 Quick Coupled Transcription/Translation System. iASPP protein was immunoprecipitated with anti-iASPP antibody. Immunoprecipitates were fractionated on SDS–10% polyacrylamide gels. The presence of A34 and p53 complexed with iASPP was detected by Western blotting with His antibody. (B) U2OS cells were transiently transfected with the A34 plasmids. Sample of cells lysates was immunoprecipitated with iASPP antibody, p53 antibody and His antibody, respectively. The immunoprecipitates were separated on SDS–10% polyacylamide gels. The presence of p53 and A34 on the immunoblots was detected with mouse anti-p53 antibody DO-1 and His antibody, respectively. The presence of iASPP was detected with iASPP antibody. The control IgG antibody was used as unrelated antibody. IP: immunoprecipitation; Ab: antibody (the experiment was repeated twice).
Endogenous iASPP was immunoprecipitated by antibodies specific to iASPP (Fig. 2B). Consistent with our in vitro observation, the anti-iASPP antibody was able to coimmunoprecipitate A34 and endogenous p53. His antibody can coimmunoprecipitate A34 and endogenous iASPP. Under the same conditions, the control antibody failed to coimmunoprecipitate A34, p53 or iASPP. These results indicated that A34 can combine with iASPP, but not with p53. Overall, all these results suggested that A34 can interact with iASPP in vitro and in vivo. A34 can enhance the transcriptional activity of p53 on the promoters of Bax and PUMA iASPP is known to reduce the DNA-binding activities of p53 on the Bax and PUMA promoters [9]. We therefore studied if A34 can affect iASPP on the transactivation function of p53 in transient reporter assays. U2OS cells (p53-wild) and U2OS (p53−/−) cells were co-transfected with A34, Bax-Luc, PUMA-Luc and pRL-SV40 plasmids as indicated in Fig. 3. A34 expression enhanced the transactivation function of p53 on the Bax/PUMA promoters in U2OS cells (Fig. 3A) but not in U2OS (p53−/−) cells (Fig. 3B). Therefore, that A34 can affect the transactivation function is dependent on p53. The fold increase in Bax-luc activity detected in U2OS cells transfected with A34 was 3.86 times higher than that detected in cells transfected with vector (Fig. 3A). Fold increase in PUMA-luc activity detected in U2OS cells transfected with A34 was 4.57 times higher than that detected in cells transfected with vector (Fig. 3A). These results suggest that A34 can enhance the transactivation function
Fig. 3. A34 can enhance the transcriptional activity of p53 on the promoter of Bax and PUMA. U2OS cells (A) and U2OS cells (p53−/−) (B) were transfected with 0.1 μg A34 plasmid alongside 1 μg of Bax-luc (Puma-luc) reporter plasmid and 0.05 μg pRLSV40 plasmid. The graph shows the changes in relative transactivation activity. Mean values were derived from three independent experiments. (C) Schematic of the mode of action of A34. Introduction of A34 into p53-wild cells sequesters iASPP and thereby derepresses p53. Free p53 subsequently activates apoptotic target genes such as Bax and PUMA to bring about programmed cell death.
of p53 on the Bax/PUMA promoters. The reason may be that A34 has higher affinity with iASPP than the affinity of p53 with iASPP (Fig. 2A). Therefore, A34 can priorly combine with iASPP and let p53 free. Then free p53 members in the cell are increased and the p53 members combined with Bax and PUMA promoters are also
Fig. 1. A34 can increase cell apoptosis. (A) Domain organization of p53. The composition of the A34 peptide is from the linker domain. (B) A34 can increase cell apoptosis of U2OS. U2OS cells were transfected with A34 plasmid or control vector. The percentage of apoptotic cells was measured by cytofluorimetry using the Annexin Apoptosis Detection Kit. The bar graph shows the percentage of apoptotic cells 48 h after transfection. Mean values were derived from three independent experiments. (C) A34 cannot increase cell apoptosis of U2OS (p53−/−) cells, in which p53 was knocked out by CrispR/Cas9. (D) Western blot analysis of p53 protein expression in U2OS cells and U2OS (p53−/−) cells.
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Fig. 4. A34 can enhance the DNA binding function of p53 on the promoter of Bax and PUMA and increase the mRNA/protein levels of Bax and Puma. (A) ChIP of p53 at the Bax/Puma promoters. ChIP was performed using an anti-p53 antibody DO-1 or control antibody IgG, followed by PCR amplification with Bax primers or Puma primers. Lane No. 1, DNA derived from U2OS cells transfected with control vector; Lane No. 2, DNA derived from cells transfected with A34 plasmids. Bar graphs show the fold increase of PCR products detected. (B) The mRNA expression of Bax and Puma in U2OS after transfection with A34. Lane 1, U2OS cells; Lane 2, U2OS cells transfected with control vector; Lane 3, U2OS cells transfected with A34. The PCR products of Bax and PUMA were 424 bp and 520 bp, respectively. GAPDH was used as internal standard. DNA Marker, DL2000. (C) The protein levels of Bax and Puma in U2OS after transfection with A34. Lane 1, U2OS cells; Lane 2, U2OS cells transfected with control vector; Lane 3, U2OS cells transfected with A34. Rabbit antibodies bs-0127R and bs-1573R were used to immunoprecipitate Bax and Puma, respectively. GAPDH, sampling control.
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Fig. 5. A34 slows tumor growth in vitro and vivo. (A) CCK-8 assay for detecting the proliferation of U2OS cells after transfection with A34, M01 and saline, respectively. (B) Cultures of MKN-45 cells were injected into the flanks of BALB/c nude mice. Upon tumor formation (day 0), mice were treated by small tumor nodule injection with 13 μg of A34 and M01 or saline respectively on days 1, 3, 5, 7, 9 and 11. Tumor volume was measured, and tumor growth was plotted as relative to tumor size on day 0. (C) A34 can increase cell apoptosis of MKN-45. MKN-45 cells were transfected with A34 plasmid or control vector. The percentage of apoptotic cells was measured by cytofluorimetry using the Annexin Apoptosis Detection Kit. The bar graph shows the percentage of apoptotic cells 48 h after transfection. Mean values were derived from three independent experiments. (D) A34 can enhance the transcriptional activity of p53 on the promoter of Bax and PUMA. MKN-45 cells were transfected with 0.1 μg A34 plasmid alongside 1 μg of Bax-luc (Puma-luc) reporter plasmid and 0.05 μg pRL-SV40 plasmid. The graph shows the changes in relative transactivation activity. Mean values were derived from three independent experiments.
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increased, as a result, the transcriptional activity of p53 on the Bax and PUMA promoters is enhanced (Fig. 3C). A34 can enhance the DNA binding function of p53 on the promoter of Bax and PUMA and increase the mRNA/ protein levels of Bax and Puma To prove that if A34 can affect the DNA binding function of p53, we investigated the transactivation functions of p53 in U2OS cells transfected with control vector and cells transfected with A34 plasmids. Bax and PUMA promoters were used to measure the transactivation functions of p53. We studied the DNA-binding activity of p53 on the Bax and PUMA promoters in vivo using the chromatin immunoprecipitation technique. After various treatments, U2OS cells were exposed to formaldehyde to crosslink the DNA-binding proteins together with their target sequences in situ. p53 binds to DNA, and the promoter sequences of p53 target genes should therefore be detected by PCR in immunoprecipitates derived from anti-p53 antibodies (DO-1). Bax and PUMA promoter sequences were specifically detected in the immunoprecipitates derived from anti-p53 DO-1, but not from the control IgG antibody. The levels of Bax and PUMA promoter sequences detected in cells transfected with control vector was set as 1 and used to calculate the in vivo DNA-binding activity of p53. Transfection with A34 increased the DNA-binding activities of p53 on the Bax and PUMA promoters by 1.54- and 1.34-fold, respectively. Therefore, transfection with A34 clearly enhanced the DNA-binding abilities of p53 on the Bax and PUMA promoters (Fig. 4). To understand the changes of the expression of Bax and Puma, we investigated the mRNA/protein levels of Bax and Puma after adding A34. The results show that the mRNA/protein levels of Bax and Puma in U2OS are increased after transfection with A34 (Fig. 4B,C). All the experimental results in vitro suggest that A34 can enhance the transactivation function of p53 on the Bax/PUMA promoters, increase mRNA/protein expression levels of Bax and Puma and finally bring about programmed cell death. A34 can slow tumor growth in vitro and vivo Since A34 can increase cell apoptosis, we consider if A34 could slow cell proliferation. After U2OS cells were transfected with A34, M01 and saline, CCK-8 reagent was used to detect the cell proliferation. The result shows that the cell proliferation in the group transfected with A34 is slower than the group transfected with M01 or the group injected with saline (Fig. 5A). Next, we consider whether A34 would work as a therapeutic agent in vivo to cause cell death and tumor regression. As U2OS cells are difficult to form transplantation tumor in athymic mice [28], we decided to analyze the effects of A34 on tumors formed with MKN45 cells, which are easy to form transplantation tumors and are frequently used in xenograft studies. In addition, the similar experiment results of apoptosis assay and luciferase reporter assay in U2OS are also reproducible in MKN-45 (Fig. 5C,D), therefore, it is reasonable to use MKN-45 cells instead of U2OS cells in vivo tests. Cultures of MKN-45 cells were therefore injected into the flanks of mice to induce tumor formation. Once tumors were palpable, mice were treated by small tumor nodule injection of A34 or M01 expression plasmids on days 1, 3, 5, 7, 9 and 11. Analysis of tumor size during these treatments revealed that while tumors treated with M01 or saline continued to grow (Fig. 5B), tumor growth in mice treated with A34 was slowed, indicating that A34 can slow tumor growth in transplantation tumors (Fig. 5B). Since MKN-45 cells contain endogenous WT p53, and A34 can enhance the transcriptional activity of p53 on the promoter of Bax and PUMA (Fig. 3), therefore, the tumor growth in mice treated with
A34 slowed in this case is because more p53 was activated and p53 subsequently activates apoptotic target genes such as Bax and PUMA to bring about programmed cell death. Therefore, A34 might be used as a therapeutic agent in vivo to cause tumor cell death. In conclusion, the results show that A34, derived from the linker region of p53, can enhance DNA binding and transcriptional activity of p53 by binding to and sequestering iASPP away from p53. A34 is also shown to increase cell apoptosis and slow down tumor growth in vitro and vivo. This study will open the way for using small molecule peptides that directly disturb the interaction of p53 with iASPP, thereby resume the function of p53 as a suppressor. Acknowledgements We thank Takara Biothchnology (Dalian) CO., Ltd., GeneChem CO., Ltd. and KangChen Bio-tech Inc. for technical assistance. This work was supported by the National Natural Science Foundation of China (30971140). Conflict of interest The authors declare that they have no conflict of interest. References [1] K.H. Vousden, X. Lu, Live or let die: the cell’s response to p53, Nat. Rev. Cancer 2 (2002) 594–604. [2] C.J. Brown, S. Lain, C.S. Verma, A.R. Fersht, D.P. Lane, Awakening guardian angels: drugging the p53 pathway, Nat. Rev. Cancer 6 (2009) 862–873. [3] X. Lu, p53: a heavily dictated dictator of life and death, Curr. Opin. Genet. Dev. 15 (2005) 27–33. [4] E.A. Slee, D.J. O’Connor, X. Lu, To die or not to die: how does p53 decide?, Oncogene 23 (2004) 2809–2818. [5] A.R. Delbridge, L.J. Valente, A. Strasser, The role of the apoptotic machinery in tumor suppression, Cold Spring Harb. Perspect. Biol. 4 (2012) pii: a008789. [6] K.H. Vousden, Activation of the p53 tumor suppressor protein, Biochim. Biophys. Acta 1602 (2002) 47–59. [7] J.E. Chipuk, D.R. Green, Dissecting p53-dependent apoptosis, Cell Death Differ. 13 (2006) 994–1002. [8] Y. Samuels-Lev, D.J. O’Connor, D. Bergamaschi, G. Trigiante, J.K. Hsieh, S. Zhong, et al., ASPP proteins specifically stimulate the apoptotic function of p53, Mol. Cell 8 (2001) 781–794. [9] D. Bergamaschi, Y. Samuels, N.J.O. Neil, G. Trigiante, T. Crook, J.K. Hsieh, et al., iASPP oncoprotein is a key inhibitor of p53 conserved from worm to human, Nat. Genet. 33 (2003) 162–167. [10] Z.J. Liu, X. Lu, S. Zhong, ASPP – apoptotic specific regulator of p53, Biochim. Biophys. Acta 1756 (2005) 77–80. [11] A. Sullivan, X. Lu, ASPP: a new family of oncogenes and tumor suppressor genes, Br. J. Cancer 96 (2007) 196–200. [12] Z.J. Liu, Y. Zhang, X.B. Zhang, X. Yang, Abnormal mRNA expression of ASPP members in leukemia cell lines, Leukemia 18 (2004) 880. [13] X. Zhang, M. Wang, C. Zhou, S. Chen, J. Wang, The expression of iASPP in acute leukemias, Leuk. Res. 29 (2005) 179–183. [14] B.L. Lin, D.Y. Xie, S.B. Xie, J.Q. Xie, X.H. Zhang, Y.F. Zhang, et al., Down-regulation of iASPP in human hepatocellular carcinoma cells inhibits cell proliferation and tumor growth, Neoplasma 58 (2011) 205–210. [15] J. Chen, F. Xie, L. Zhang, W.G. Jiang, iASPP is over-expressed in human non-small cell lung cancer and regulates the proliferation of lung cancer cells through a p53 associated pathway, BMC Cancer 10 (2010) 694. [16] L. Jiang, M.K. Siu, O.G. Wong, K.F. Tam, X. Lu, E.W. Lam, et al., iASPP and chemoresistance in ovarian cancers: effects on paclitaxel-mediated mitotic catastrophe, Clin. Cancer Res. 17 (2011) 6924–6933. [17] E.M. Pinto, N.R. Musolino, V.A. Cescato, I.C. Soares, A. Wakamatsu, E. de Oliveira, et al., iASPP: a novel protein involved in pituitary tumorigenesis?, Front. Horm. Res. 38 (2010) 70–76. [18] G. Li, R. Wang, J. Gao, K. Deng, J. Wei, Y. Wei, RNA interference-mediated silencing of iASPP induces cell proliferation inhibition and G0/G1 cell cycle arrest in U251 human glioblastoma cells, Mol. Cell. Biochem. 350 (2011) 193– 200. [19] H. Liu, M. Wang, S. Diao, Q. Rao, X. Zhang, H. Xing, et al., siRNA-mediated down-regulation of iASPP promotes apoptosis induced by etoposide and daunorubicin in leukemia cells expressing wild-type p53, Leuk. Res. 33 (2009) 1243–1248. [20] Z.J. Liu, H.M. Xin, J. Chen, X. Lu, S. Zhong, S.Z. Gu, et al., A new strategy to resume the apoptosis activity of p53 in leukemia cell lines retaining wild-type p53, Leuk. Res. 31 (2007) 1156–1158.
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[21] Z.J. Liu, Y. Cai, L. Hou, X. Gao, H.M. Xin, X. Lu, et al., Effect of RNA interference of iASPP on the apoptosis in MCF-7 breast cancer cells, Cancer Invest. 26 (2008) 878–882. [22] M.J. Laska, U.B. Vogel, U.B. Jensen, B.A. Nexo, p53 and PPP1R13L(alias iASPP or RAI) form a feedback loop to regulate genotoxic stress responses, Biochim. Biophys. Acta 1800 (2010) 1231–1240. [23] H.S. Bell, C. Dufes, J.O. Prey, D. Crighton, D. Bergamaschi, X. Lu, et al., A p53-derived apoptotic peptide derepresses p73 to cause tumor regression in vivo, J. Clin. Invest. 117 (2007) 1008–1018. [24] J. Ahn, I.J. Byeon, C.H. Byeon, A.M. Gronenborn, Insight into the structural basis of pro- and antiapoptotic p53 modulation by ASPP proteins, J. Biol. Chem. 284 (2009) 13812–13822.
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[25] A.C. Joerger, A.R. Fersht, The tumor suppressor p53: from structures to drug discovery, Cold Spring Harb. Perspect. Biol. 2 (2010) a000919. [26] E.A. Slee, S. Gillotin, D. Bergamaschi, C. Royer, S. Llanos, S. Ali, et al., The N-terminus of a novel isoform of human iASPP is required for its cytoplasmic localization, Oncogene 23 (2004) 9006–9016. [27] Z.J. Liu, X. Gao, Y. Cai, X. Yang, X.L. Fu, J. Chen, et al., Construction of a full-length iASPP expression plasmid pcDNA3.1(+)/iASPP and its biological activity, Plasmid 62 (2009) 10–15. [28] P. Fei, W. Wang, S.H. Kim, S. Wang, T.F. Burns, J.K. Sax, et al., Bnip3L is induced by p53 under hypoxia, and its knockdown promotes tumor growth, Cancer Cell 6 (2004) 597–609.
