- Email: [email protected]
Ubiquitination-resistant p53 protein transduction therapy facilitates anti-cancer effect on the growth of human malignant glioma cells
FEBS 29728
FEBS Letters 579 (2005) 3965–3969
Ubiquitination-resistant p53 protein transduction therapy facilitates anti-cancer effect on the growth of human malignant glioma cells Hiroyuki Michiuea,b, Kazuhito Tomizawaa,*, Masayuki Matsushitaa, Takashi Tamiyac, Yun-Fei Lud, Tomotsugu Ichikawab, Isao Dateb, Hideki Matsuia,d a
Department of Physiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558, Japan b Department of Neurological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558, Japan c Department of Neurological Surgery, Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0793, Japan d Protein Therapy, New Techno-Venture Oriented R&D, Japan Science and Technology Agency, 2-5-1 Shikata-cho, Okayama 700-8558, Japan Received 11 May 2005; revised 14 June 2005; accepted 15 June 2005 Available online 27 June 2005 Edited by Varda Rotter
Abstract Protein transduction therapy using poly-arginine can deliver the bioactive p53 protein into cancer cells and inhibits the proliferation of the cells. However, one disadvantage of such therapy is the short intracellular half-life of the delivered protein. Here, we generated mutant proteins in which multiple lysine residues in the C-terminal were substituted by arginines. The mutant proteins were effectively delivered in glioma cells and were resistant to Mdm2-mediated ubiquitination. Moreover, the mutant proteins displayed higher transcription regulatory activity and powerful inhibition of the proliferation of glioma cells. These results suggest that ubiquitination-resistant p53 protein therapy may become a new effective cancer therapy. Ó 2005 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. Keywords: Poly-arginine; Gene therapy; Brain tumor; TAT; p53
24 h [8]. Therefore, repeated transduction of 11R-p53 is needed for the inhibition of cancer cell proliferation [8]. The development of protein therapy to transduce stable proteins or of methods to stabilize the delivered proteins is important for the initiation of clinical trials of protein therapy. Previous studies have shown that the COOH-terminal region of p53 modulates the susceptibility of p53 to Mdm2-mediated degradation [9–11]. p53 mutant in which lysine residues 370, 372, 373, 381, 382 and 386 in the COOH-terminal are replaced by arginine residues is resistant to ubiquitin-proteosomemediated degradation, and its transcriptional activity was higher than that of wild-type p53 [9]. In the present study, we developed a new protein transduction therapy with mutated p53 protein by substituting multiple lysine residues with arginine and investigated the effect of the substitution on the stability of the protein and the ability of the protein to inhibit the growth of malignant glioma cells.
2. Materials and methods 1. Introduction Protein transduction domains (PTDs) such as that in TAT protein from HIV-1 are capable of mediating the transfer of proteins across the plasma membrane into nearly all eukaryotic cells [1,2]. Poly-arginine (6–12 residues) also has the same transduction activity as the PTDs [3–5]. PTDs have become widely used as tools for the delivery of proteins into cells in culture, and even into the tissues of living animals [6]. Recent advances in molecular oncology have led to the identification of proteins that are quantitatively, qualitatively or genetically altered in cancer cells. P53 protein is one of the altered proteins in cancer cells, and is mutated or deleted in more than half of human tumors [7]. Eleven poly-arginine-fused wild-type p53 protein (11R-p53) effectively penetrates across the plasma membrane and inhibits the proliferation of human cancer cells [8]. However, the half-life of 11R-p53 is less than
* Corresponding author. Fax: +81 86 235 7111. E-mail address: [email protected] (K. Tomizawa).
Abbreviations: HIV-1, human immunodeficiency virus type-1
2.1. Cell lines and cell culture The human malignant glioma cell lines A172, U251-MG and T98G were provided by Health Science Research Resources Bank (Osaka, Japan). The malignant glioma cell line KR158 was a gift from Dr. T Jacks (MIT). All cell lines were maintained in DMEM (Invitrogen) supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 U/ml streptomycin. The cells were cultured in a 37 °C incubator with 5% CO2. Rat primary astrocytes were prepared from postnatal day 1 newborn Wistar rats (Japan SLC Inc.). 2.2. Site-directed mutagenesis of human p53 cDNA and the structure of 11R-p53 Human wild-type p53 cDNA was subcloned into p11R-HA vector (Supplementary Fig. 1A, Ref. [8]). The multiple lysine-to-arginine mutants of the p53 cDNA were generated by site-directed mutagenesis using a PCR strategy as described previously [12]. In the 3KR-p53 mutant, the lysine residues at 381, 382, and 386 were changed to arginine and in the 6KR-p53 mutant, the six lysine residues at 370, 372, 373, 381, 382, and 386 were changed to arginine (Supplementary Fig. 1B). 2.3. Purification of 11R-p53 fusion protein Purification of 11R-p53 proteins was performed as described previously [8]. There was no difference in the expression or the purity among the proteins (Supplementary Fig. 1C).
0014-5793/$30.00 Ó 2005 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. doi:10.1016/j.febslet.2005.06.021
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2.4. Recombinant adenovirus preparation The recombinant adenoviruses (pAdex-p53) were then purified and concentrated using the CsCl step-gradient method as described previously [8]. 2.5. Western blotting analysis Western blotting analysis for p53 was carried out at high stringency, essentially as described previously [13]. The blots were probed with primary antibodies against p53 (1:1000; Pab 1801; Santa Cruz Biotechnology, Inc.) and p21 (1:1000; H-164; Santa Cruz Biotechnology). 2.6. Detection of the ubiquitination of 11R-p53 proteins A172 cells and U251-MG cells were incubated with 100 nM WTp53, 3KR-p53, or 6KR-p53 for 12 h in the presence or absence of 25 lM MG132, a proteasome inhibitor. Whole-cell extracts (100 lg of protein) were analyzed by Western blotting analysis with anti-p53 antibody (Pab 1801; Santa Cruz Biotechnology). 2.7. Reporter assay for p53-driven transactivation The reporter assay was performed as described previously [8]. 2.8. Cell viability assay Cell viability was determined using a WST-1 (2-[2-methoxy-4-nitrophenyl]-3-[4-nitrophenyl]-5-[2, 4-disulfophenyl]-2H-tetrazolium, mono sodium salt) assay (Roche Applied Science) as described previously [8]. 2.9. TUNEL assay The TUNEL assay was performed following the protocol of the TUNEL kitÕs manufacturer (Roche Diagnostics). Before assessment, U251-MG cells were treated with each 11R-p53 at 0.1 lM for 24 h. Representative graphs are shown for experiments in which at least 6 randomly chosen fields with 50 cells were scored. 2.10. Statistical analysis Data are shown as the mean (±S.E.M.). Data were analyzed using either StudentÕs t test to compare two conditions or ANOVA followed by planned comparisons of multiple conditions, and P < 0.05 was considered to be significant.
3. Results 3.1. Long-term maintenance of 6KR-p53 and 3KR-p53 in malignant glioma cell lines Three malignant glioma cell lines, A172, U251-MG and T98G, were incubated with 100 nM poly-arginine-fused wildtype p53 protein (WT-p53) or two kinds of mutant p53 proteins (3KR-p53 and 6KR-p53) for 4 h and then washed with PBS three times. A high level of WT-p53 was detected 12 h after the protein transduction in all three cell lines, but the level was rapidly decreased by 24 h (Fig. 1A). WT-p53 was undetectable in U251-MG and T98 cells at 24 h and in A172 cells 48 h after the protein transduction (Fig. 1A and B). In contrast, both 3KR-p53 and 6KR-p53 were maintained at a high level 24 h after the protein transduction in all three cell lines (Fig. 1A). The residual levels of 3KR-p53 and 6KR-p53 48 h after the protein transduction were significantly higher than that of WT-p53 in all cell lines (Fig. 1B, n = 4 each, \ P < 0.01, \\P < 0.05 vs. WT-p53). These results suggest that both 3KR and 6KR are resistant to degradation in malignant glioma cells. 3.2. Resistance of 6KR-p53 to ubiquitination A172 cells were transduced with 100 nM WT-p53, 3KR-p53 or 6KR-p53 in the presence of a proteasome inhibitor,
Fig. 1. (A) Time-dependent degradation of WT-p53, 3KR-p53 and 6KR-p53 in malignant glioma cell lines, A172 (upper panel), U251 (middle panel) and T98G (lower panel). Cont., no transduction of any protein. As a control, A172 cells were infected with recombinant adenovirus of wild-type p53 (pAdex-p53) at MOI 100. (B) Comparison of the levels of WT-p53, 3KR-p53 and 6KR-p53 48 h after protein transduction in each malignant glioma cell line. Data are represented as % of the level 12 h after each protein transduction and are the means ± S.E.M. of four independent experiments. \, P < 0.01 and \\ , P < 0.05 vs. WT-p53.
MG132, and the cells were harvested 12 h after the transduction and subjected to Western blotting analysis using antip53 antibody. In the WT-p53-transduced A172 cells, forms of p53 whose migration was consistent with ubiquitination were observed (Fig. 2A). In the 6KR-p53-transduced cells, the slower migrating forms of p53 were virtually eliminated (Fig. 2A). In the WT-p53 transduced U251-MG cells, slower migrating forms of p53 were not observed in the absence of MG132, whereas significant amounts of the slower migrating forms were generated in the presence of MG132 (Fig. 2B). In the 6KR-p53-transduced cells, slower migrating forms of p53 were not detected in the presence or absence of MG132 (Fig. 2B). These results suggest that the 6KR-p53 protein with mutation of the COOH-terminal six lysine residues is resistant to poly-ubiquitination.
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Fig. 2. Ubiqutination of each type of 11R-p53 in A172 (A) and U251MG (B) cells. (A) A172 cells were incubated with 100 nM WT-p53, 3KR-p53 or 6KR-p53 for 12 h in the presence of 25 lM MG-132. The whole-cell extracts were resolved by SDS–PAGE and Western blotting analysis was performed with anti-p53 antibody. As a control, 10 ng of purified WT-p53 was electrophoresed on the same gel (purified WTp53). (B) U251-MG cells were incubated with 100 nM WT-p53 or 6KR-p53 for 12 h in the presence or absence of 25 lM MG-132. Intact, no protein tranduction.
3.3. Higher and longer-lasting transcription regulatory activity of 6KR-p53 in KR158 cells KR158 cells, which lack endogenous p53, were transfected with a p53-responsive reporter plasmid. After 24 h, the cells were transduced with 100 nM WT-p53, 3KR-p53 or 6KR-p53. As a positive control, the cells were infected with recombinant adenoviruses carrying the wild-type p53 gene (pAdex-p53) at an MOI of 100. Twenty-four hours after protein transduction, the p53 transcription regulatory activity showed no difference among WT-p53-, 3KR-p53- and 6KR-p53-transduced cells (Fig. 3A). The transcription regulatory activity of 3KR-p53 and 6KRp53 was significantly higher than that of WT-p53 36 h after protein transduction (Fig. 3A). Moreover, transduction of 6KR-p53 resulted in maintenance of higher p53 activity after 48 h compared with transduction of WT-p53 (Fig. 3A). We next examined whether transduction of each p53 protein induced endogenous p21 expression. The expression level of p21 was very low in U251-MG cells (Fig. 3B). WT-p53 protein transduction slightly induced the expression of endogenous p21 in the cells (Fig. 3B). 3KR-p53 was more effective for inducing p21 expression. In the 6KR-p53 transduced cells, endogenous p21 expression was most prominent (Fig. 3B). These results suggest that 6KR-p53 has higher and longerlasting p53 transcription regulatory activity compared with WT-p53 in malignant glioma cells.
Fig. 3. (A) Time-dependent changes of the transcription regulatory activity of WT-p53, 3KR-p53 and 6KR-p53 in KR158 cells. A luciferase reporter vector carrying the p21/WAF1 promoter was transfected into KR158 cells. After 24 h, the cells were either transduced with each type of 11R-p53 at 100 nM or were infected with pAdex-p53 at MOI 100. As a control (Cont.), the cells were only transfected with the luciferase reporter vector. n = 6 in each group. \ , P < 0.05 and \\, P < 0.01. (B) Effect of transduction of each 11R-p53 protein on endogenous p21 expression. U251-MG cells were transduced with 100 nM WT-p43, 3KR-p53 or 6KR-p53. The cells were harvested 48 h after protein transduction and Western blotting analysis using anti-p21antibody was performed. Cont., no protein transduction.
3.4. Inhibition of the growth of malignant glioma cell lines by administration of 6KR-p53 A previous study showed that a single administration of 100 nM WT-p53 failed to inhibit the growth of oral cancer cells (8). In the present study, the inhibitory effect of a single transduction of WT-p53, 3KR-p53 or 6KR-p53 on the proliferation of malignant glioma cell lines was investigated. T98G (Fig. 4A), U251-MG (Fig. 4B) and A172 (Fig. 4C) cells were incubated with various concentrations of WT-p53, 3KR-p53 or 6KR-p53 for 2 h. After the cells were washed with PBS, they were replaced in fresh medium in the absence of 11R-p53. The cell viability was assessed by the WST-1 assay 2 h (Day 0) and
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Fig. 5. Comparison of the effect of each protein transduction on the induction of apoptosis of U251-MG cells. U251-MG cells were transduced with 100 nM WT-p53, 3KR-p53 or 6KR-p53. After 24 h, the cells were stained with TUNEL and TUNEL-positive cells were counted. Representative graphs are shown for experiments in which at least 6 randomly chosen fields with 50 cells were scored. \, P < 0.001.
apoptosis was slight and the effect of 3KR-p53 protein transduction was not different from that of WT-p53 (Fig. 5). In contrast, 6KR-p53 significantly induced apoptosis of malignant glioma cells compared with WT-p53 (Fig. 5). The transduction of 3KR-p53 and 6KR-p53 did not inhibit the growth of primary astrocytes or induce the apoptosis of the cells, whereas recombinant adenovirus carrying the wild-type p53 gene at an MOI of 500 significantly induced apoptosis and inhibited the cell growth of primary astrocytes (Fig. 6).
4. Discussion Fig. 4. Comparison of the effect of a single transduction of each type of 11R-p53 on the growth of malignant glioma cell lines, T98G (A), U251-MG (B) and A172 (C) 96 h after protein transduction. The cells were transduced with various concentrations of WT-p53, 3KR-p53 or 6KR-p53 on Day 0 and were cultured for 96 h (Day 4). Cell viability was measured by the WST-1 assay on Day 0 and Day 4. Cont.; no protein transduction. Data are presented as the means; n = 8 in each group; bars, S.E.M. \, P < 0.01 and \\, P < 0.05 compared with the untreated cells of each cell line.
96 h (Day 4) after the protein transduction. Administration of WT-p53 (1–100 nM) did not inhibit the cell growth of any of the cell lines (Fig. 4A–C). In the T98G cells, 100 nM 3KRp53 significantly inhibited the proliferation (Fig. 4A). However, all tested concentrations of 3KR-p53 failed to inhibit the cell growth of U251-MG and A172 cell lines (Fig. 4B and C). In contrast, 100 nM 6KR-p53 significantly inhibited the growth of all cell lines 96 h after the protein transduction compared with the growth of control cells (Fig. 4A–C) but 1 and 10 nM 6KR-p53 did not affect the growth of any of the cell lines. We further examined whether 6KR-p53 protein transduction induced the apoptosis of malignant glioma cells. U251MG cells were treated with 100 nM WT-p53, 3KR-p53 or 6KR-p53 for 24 h and apoptotic cells were assessed by TUNEL staining. The effect of WT-p53 on the induction of
Advanced-stage carcinomatosis is often resistant to current chemotherapy treatment, and new strategies for treating the disease are clearly needed [14]. It has been thought that gene therapy with viral vectors and non-viral vectors may be an attractive tool to treat cancer [15]. However, a large number of studies have indicated that virus-mediated gene therapy has some safety problems, such as inflammatory response, toxicity of the virus vector itself and random integration of virus vector DNA into the host chromosomes [16,17]. Similarly, non-virus vectors also have a problem of the efficiency of gene transduction at present [16]. In the last few years, a number of protein transduction therapies have been tried for treating cancer in vitro and in an experimental murine model [5,8,18]. These studies have shown that protein transduction therapy is useful for the delivery of tumor suppressor proteins, peptides and drugs and effective for the inhibition of cancer growth. However, the transduced proteins and peptides show limited stability; for example, poly-arginine-fused wild-type p53 protein (11R-p53) is rapidly degraded in cancer cells [8]. In the present study, we generated 11R-p53 mutants (3KR-p53 and 6KR-p53), in which multiple lysine residues in the COOH-terminal region were substituted by arginines. The mutant proteins were resistant to ubiquitination and were maintained longer than 11R-p53 in glioma cell lines, and the anti-tumor effect of the transduction of the mutant proteins was maintained longer than that of 11R-p53. Moreover, the anti-tumor
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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.febslet.2005.06.021.
References
Fig. 6. Effect of WT-p53, 3KR-p53 and 6KR-p53 on the growth and apoptosis of normal glial cells. Rat primary astrocytes were transduced with 100 nM WT-p53, 3KR-p53 or 6KR-p53. (A) The WST assay was performed 4 days after protein transduction. Cont.; no protein transduction. As a control, the cells were infected with recombinant adenovirus carrying the wild-type p53 gene at an MOI of 500. n = 8 in each group. P < 0.001. (B) Quantitative analysis of the TUNELpositive cells. TUNEL staining was performed 24 h after protein transduction. n = 8 in each group. P < 0.001.
effect of 6KR-p53 was significantly stronger than that of 3KRp53. These results suggest that the six lysine residues in the COOH terminal of p53 are the main sites of ubiquitin ligation, which targets p53 for proteasome-mediated degradation, and indicate that protein transduction therapy of ubiquitinproteasome-mediated-degradation- resistant p53 proteins such as 6KR-p53 may avoid the disadvantages of p53 protein transduction therapy for cancer. Acknowledgements: We thank A. Kemori and T. Ogawa for technical assistance, T. Akiyama and K. Yoshikawa for luciferase reporter vector for p21/WAF1 promoter and H. Matsushita and T. Jacks for KR158 cells. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan.
[1] Schwarze, S.R., Hruska, K.A. and Dowdy, S.F. (2000) Protein transduction: unrestricted delivery into all cells?. Trends Cell Biol. 10, 290–295. [2] Wadia, J.S. and Dowdy, S.F. (2002) Protein transduction technology. Curr. Opin. Biotechnol. 13, 52–56. [3] Rothbard, J.B., Garlington, S., Lin, Q., Kirschberg, T., Kreider, E., McGrane, P.L., Wender, P.A. and Khavari, P.A. (2000) Conjugation of arginine oligomers to cyclosporin A facilitates topical delivery and inhibition of inflammation. Nat. Med. 6, 1253–1257. [4] Matsushita, M., Tomizawa, K., Moriwaki, A., Li, S.T., Terada, H. and Matsui, H. (2001) A high-efficiency protein transduction system demonstrating the role of PKA in long-lasting long-term potentiation. J. Neurosci. 21, 6000–6007. [5] Matsui, H., Tomizawa, K., Lu, Y.F. and Matsushita, M. (2003) Protein Therapy: in vivo protein transduction by polyarginine (11R) PTD and subcellular targeting delivery. Curr. Protein Pept. Sci. 4, 151–157. [6] Joliot, A. and Prochiantz, A. (2004) Transduction peptides: from technology to physiology. Nat. Cell Biol. 6, 189–196. [7] Greenblatt, M.S., Bennett, W.P., Hollstein, M. and Harris, C.C. (1994) Mutations in the p53 tumor suppressor gene: clues to cancer etiology and molecular pathogenesis. Cancer Res. 54, 4855–4878. [8] Takenobu, T., Tomizawa, K., Matsushita, M., Li, S.T., Moriwaki, A., Lu, Y.F. and Matsui, H. (2002) Development of p53 protein transduction therapy using membrane-permeable peptides and the application to oral cancer cells. Mol. Cancer Ther. 1, 1043–1049. [9] Rodriguez, M.S., Desterro, J.M., Lain, S., Lane, D.P. and Hay, R.T. (2000) Multiple C-terminal lysine residues target p53 for ubiquitin-proteasome-mediated degradation. Mol. Cell Biol. 20, 8458–8467. [10] Nakamura, S., Roth, J.A. and Mukhopadhyay, T. (2000) Multiple lysine mutations in the C-terminal domain of p53 interfere with MDM2-dependent protein degradation and ubiquitination. Mol. Cell Biol. 20, 9391–9398. [11] Nakamura, S., Roth, J.A. and Mukhopadhyay, T. (2002) Multiple lysine mutations in the C-terminus of p53 make it resistant to degradation mediated by MDM2 but not by human papillomavirus E6 and induce growth inhibition in MDM2overexpressing cells. Oncogene 21, 2605–2610. [12] Tomizawa, K., Sunada, S., Lu, Y.F., Oda, Y., Kinuta, M., Ohshima, T., Saito, T., Wei, F.Y., Matsushita, M., Li, S.T., Tsutsui, K., Hisanaga, S., Mikoshiba, K., Takei, K. and Matsui, H. (2003) Cophosphorylation of amphiphysin I and dynamin I by Cdk5 regulates clathrin-mediated endocytosis of synaptic vesicles. J. Cell Biol. 163, 813–824. [13] Tomizawa, K., Iga, N., Lu, Y.F., Moriwaki, A., Matsushita, M., Li, S.T., Miyamoto, O., Itano, T. and Matsui, H. (2003) Oxytocin improves long-lasting spatial memory during motherhood through MAP kinase cascade. Nat. Neurosci. 6, 384–390. [14] Parsons, S.L., Watson, S.A. and Steele, R.J.C. (1996) Malignant ascites. Br. J. Surg. 83, 6–14. [15] McCormick, F. () Cancer gene therapy: fringe or cutting edge? Nat. Rev. Cancer. 1, 130–141. [16] Verma, I.M. and Somia, N. (1997) Gene therapy – promises, problems and prospects. Nature 389, 239–242. [17] Check, E. (2002) Regulators split on gene therapy as patient shows signs of cancer. Nature 419, 545–546. [18] Snyder, E.L., Meade, B.R., Saenz, C.C. and Dowdy, S.F. (2004) Treatment of terminal peritoneal carcinomatosis by a transducible p53-activating peptide. PLoS. Biol. 2, 186–193.
FEBS Letters 579 (2005) 3965–3969
Ubiquitination-resistant p53 protein transduction therapy facilitates anti-cancer effect on the growth of human malignant glioma cells Hiroyuki Michiuea,b, Kazuhito Tomizawaa,*, Masayuki Matsushitaa, Takashi Tamiyac, Yun-Fei Lud, Tomotsugu Ichikawab, Isao Dateb, Hideki Matsuia,d a
Department of Physiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558, Japan b Department of Neurological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558, Japan c Department of Neurological Surgery, Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0793, Japan d Protein Therapy, New Techno-Venture Oriented R&D, Japan Science and Technology Agency, 2-5-1 Shikata-cho, Okayama 700-8558, Japan Received 11 May 2005; revised 14 June 2005; accepted 15 June 2005 Available online 27 June 2005 Edited by Varda Rotter
Abstract Protein transduction therapy using poly-arginine can deliver the bioactive p53 protein into cancer cells and inhibits the proliferation of the cells. However, one disadvantage of such therapy is the short intracellular half-life of the delivered protein. Here, we generated mutant proteins in which multiple lysine residues in the C-terminal were substituted by arginines. The mutant proteins were effectively delivered in glioma cells and were resistant to Mdm2-mediated ubiquitination. Moreover, the mutant proteins displayed higher transcription regulatory activity and powerful inhibition of the proliferation of glioma cells. These results suggest that ubiquitination-resistant p53 protein therapy may become a new effective cancer therapy. Ó 2005 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. Keywords: Poly-arginine; Gene therapy; Brain tumor; TAT; p53
24 h [8]. Therefore, repeated transduction of 11R-p53 is needed for the inhibition of cancer cell proliferation [8]. The development of protein therapy to transduce stable proteins or of methods to stabilize the delivered proteins is important for the initiation of clinical trials of protein therapy. Previous studies have shown that the COOH-terminal region of p53 modulates the susceptibility of p53 to Mdm2-mediated degradation [9–11]. p53 mutant in which lysine residues 370, 372, 373, 381, 382 and 386 in the COOH-terminal are replaced by arginine residues is resistant to ubiquitin-proteosomemediated degradation, and its transcriptional activity was higher than that of wild-type p53 [9]. In the present study, we developed a new protein transduction therapy with mutated p53 protein by substituting multiple lysine residues with arginine and investigated the effect of the substitution on the stability of the protein and the ability of the protein to inhibit the growth of malignant glioma cells.
2. Materials and methods 1. Introduction Protein transduction domains (PTDs) such as that in TAT protein from HIV-1 are capable of mediating the transfer of proteins across the plasma membrane into nearly all eukaryotic cells [1,2]. Poly-arginine (6–12 residues) also has the same transduction activity as the PTDs [3–5]. PTDs have become widely used as tools for the delivery of proteins into cells in culture, and even into the tissues of living animals [6]. Recent advances in molecular oncology have led to the identification of proteins that are quantitatively, qualitatively or genetically altered in cancer cells. P53 protein is one of the altered proteins in cancer cells, and is mutated or deleted in more than half of human tumors [7]. Eleven poly-arginine-fused wild-type p53 protein (11R-p53) effectively penetrates across the plasma membrane and inhibits the proliferation of human cancer cells [8]. However, the half-life of 11R-p53 is less than
* Corresponding author. Fax: +81 86 235 7111. E-mail address: [email protected] (K. Tomizawa).
Abbreviations: HIV-1, human immunodeficiency virus type-1
2.1. Cell lines and cell culture The human malignant glioma cell lines A172, U251-MG and T98G were provided by Health Science Research Resources Bank (Osaka, Japan). The malignant glioma cell line KR158 was a gift from Dr. T Jacks (MIT). All cell lines were maintained in DMEM (Invitrogen) supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 U/ml streptomycin. The cells were cultured in a 37 °C incubator with 5% CO2. Rat primary astrocytes were prepared from postnatal day 1 newborn Wistar rats (Japan SLC Inc.). 2.2. Site-directed mutagenesis of human p53 cDNA and the structure of 11R-p53 Human wild-type p53 cDNA was subcloned into p11R-HA vector (Supplementary Fig. 1A, Ref. [8]). The multiple lysine-to-arginine mutants of the p53 cDNA were generated by site-directed mutagenesis using a PCR strategy as described previously [12]. In the 3KR-p53 mutant, the lysine residues at 381, 382, and 386 were changed to arginine and in the 6KR-p53 mutant, the six lysine residues at 370, 372, 373, 381, 382, and 386 were changed to arginine (Supplementary Fig. 1B). 2.3. Purification of 11R-p53 fusion protein Purification of 11R-p53 proteins was performed as described previously [8]. There was no difference in the expression or the purity among the proteins (Supplementary Fig. 1C).
0014-5793/$30.00 Ó 2005 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. doi:10.1016/j.febslet.2005.06.021
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2.4. Recombinant adenovirus preparation The recombinant adenoviruses (pAdex-p53) were then purified and concentrated using the CsCl step-gradient method as described previously [8]. 2.5. Western blotting analysis Western blotting analysis for p53 was carried out at high stringency, essentially as described previously [13]. The blots were probed with primary antibodies against p53 (1:1000; Pab 1801; Santa Cruz Biotechnology, Inc.) and p21 (1:1000; H-164; Santa Cruz Biotechnology). 2.6. Detection of the ubiquitination of 11R-p53 proteins A172 cells and U251-MG cells were incubated with 100 nM WTp53, 3KR-p53, or 6KR-p53 for 12 h in the presence or absence of 25 lM MG132, a proteasome inhibitor. Whole-cell extracts (100 lg of protein) were analyzed by Western blotting analysis with anti-p53 antibody (Pab 1801; Santa Cruz Biotechnology). 2.7. Reporter assay for p53-driven transactivation The reporter assay was performed as described previously [8]. 2.8. Cell viability assay Cell viability was determined using a WST-1 (2-[2-methoxy-4-nitrophenyl]-3-[4-nitrophenyl]-5-[2, 4-disulfophenyl]-2H-tetrazolium, mono sodium salt) assay (Roche Applied Science) as described previously [8]. 2.9. TUNEL assay The TUNEL assay was performed following the protocol of the TUNEL kitÕs manufacturer (Roche Diagnostics). Before assessment, U251-MG cells were treated with each 11R-p53 at 0.1 lM for 24 h. Representative graphs are shown for experiments in which at least 6 randomly chosen fields with 50 cells were scored. 2.10. Statistical analysis Data are shown as the mean (±S.E.M.). Data were analyzed using either StudentÕs t test to compare two conditions or ANOVA followed by planned comparisons of multiple conditions, and P < 0.05 was considered to be significant.
3. Results 3.1. Long-term maintenance of 6KR-p53 and 3KR-p53 in malignant glioma cell lines Three malignant glioma cell lines, A172, U251-MG and T98G, were incubated with 100 nM poly-arginine-fused wildtype p53 protein (WT-p53) or two kinds of mutant p53 proteins (3KR-p53 and 6KR-p53) for 4 h and then washed with PBS three times. A high level of WT-p53 was detected 12 h after the protein transduction in all three cell lines, but the level was rapidly decreased by 24 h (Fig. 1A). WT-p53 was undetectable in U251-MG and T98 cells at 24 h and in A172 cells 48 h after the protein transduction (Fig. 1A and B). In contrast, both 3KR-p53 and 6KR-p53 were maintained at a high level 24 h after the protein transduction in all three cell lines (Fig. 1A). The residual levels of 3KR-p53 and 6KR-p53 48 h after the protein transduction were significantly higher than that of WT-p53 in all cell lines (Fig. 1B, n = 4 each, \ P < 0.01, \\P < 0.05 vs. WT-p53). These results suggest that both 3KR and 6KR are resistant to degradation in malignant glioma cells. 3.2. Resistance of 6KR-p53 to ubiquitination A172 cells were transduced with 100 nM WT-p53, 3KR-p53 or 6KR-p53 in the presence of a proteasome inhibitor,
Fig. 1. (A) Time-dependent degradation of WT-p53, 3KR-p53 and 6KR-p53 in malignant glioma cell lines, A172 (upper panel), U251 (middle panel) and T98G (lower panel). Cont., no transduction of any protein. As a control, A172 cells were infected with recombinant adenovirus of wild-type p53 (pAdex-p53) at MOI 100. (B) Comparison of the levels of WT-p53, 3KR-p53 and 6KR-p53 48 h after protein transduction in each malignant glioma cell line. Data are represented as % of the level 12 h after each protein transduction and are the means ± S.E.M. of four independent experiments. \, P < 0.01 and \\ , P < 0.05 vs. WT-p53.
MG132, and the cells were harvested 12 h after the transduction and subjected to Western blotting analysis using antip53 antibody. In the WT-p53-transduced A172 cells, forms of p53 whose migration was consistent with ubiquitination were observed (Fig. 2A). In the 6KR-p53-transduced cells, the slower migrating forms of p53 were virtually eliminated (Fig. 2A). In the WT-p53 transduced U251-MG cells, slower migrating forms of p53 were not observed in the absence of MG132, whereas significant amounts of the slower migrating forms were generated in the presence of MG132 (Fig. 2B). In the 6KR-p53-transduced cells, slower migrating forms of p53 were not detected in the presence or absence of MG132 (Fig. 2B). These results suggest that the 6KR-p53 protein with mutation of the COOH-terminal six lysine residues is resistant to poly-ubiquitination.
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Fig. 2. Ubiqutination of each type of 11R-p53 in A172 (A) and U251MG (B) cells. (A) A172 cells were incubated with 100 nM WT-p53, 3KR-p53 or 6KR-p53 for 12 h in the presence of 25 lM MG-132. The whole-cell extracts were resolved by SDS–PAGE and Western blotting analysis was performed with anti-p53 antibody. As a control, 10 ng of purified WT-p53 was electrophoresed on the same gel (purified WTp53). (B) U251-MG cells were incubated with 100 nM WT-p53 or 6KR-p53 for 12 h in the presence or absence of 25 lM MG-132. Intact, no protein tranduction.
3.3. Higher and longer-lasting transcription regulatory activity of 6KR-p53 in KR158 cells KR158 cells, which lack endogenous p53, were transfected with a p53-responsive reporter plasmid. After 24 h, the cells were transduced with 100 nM WT-p53, 3KR-p53 or 6KR-p53. As a positive control, the cells were infected with recombinant adenoviruses carrying the wild-type p53 gene (pAdex-p53) at an MOI of 100. Twenty-four hours after protein transduction, the p53 transcription regulatory activity showed no difference among WT-p53-, 3KR-p53- and 6KR-p53-transduced cells (Fig. 3A). The transcription regulatory activity of 3KR-p53 and 6KRp53 was significantly higher than that of WT-p53 36 h after protein transduction (Fig. 3A). Moreover, transduction of 6KR-p53 resulted in maintenance of higher p53 activity after 48 h compared with transduction of WT-p53 (Fig. 3A). We next examined whether transduction of each p53 protein induced endogenous p21 expression. The expression level of p21 was very low in U251-MG cells (Fig. 3B). WT-p53 protein transduction slightly induced the expression of endogenous p21 in the cells (Fig. 3B). 3KR-p53 was more effective for inducing p21 expression. In the 6KR-p53 transduced cells, endogenous p21 expression was most prominent (Fig. 3B). These results suggest that 6KR-p53 has higher and longerlasting p53 transcription regulatory activity compared with WT-p53 in malignant glioma cells.
Fig. 3. (A) Time-dependent changes of the transcription regulatory activity of WT-p53, 3KR-p53 and 6KR-p53 in KR158 cells. A luciferase reporter vector carrying the p21/WAF1 promoter was transfected into KR158 cells. After 24 h, the cells were either transduced with each type of 11R-p53 at 100 nM or were infected with pAdex-p53 at MOI 100. As a control (Cont.), the cells were only transfected with the luciferase reporter vector. n = 6 in each group. \ , P < 0.05 and \\, P < 0.01. (B) Effect of transduction of each 11R-p53 protein on endogenous p21 expression. U251-MG cells were transduced with 100 nM WT-p43, 3KR-p53 or 6KR-p53. The cells were harvested 48 h after protein transduction and Western blotting analysis using anti-p21antibody was performed. Cont., no protein transduction.
3.4. Inhibition of the growth of malignant glioma cell lines by administration of 6KR-p53 A previous study showed that a single administration of 100 nM WT-p53 failed to inhibit the growth of oral cancer cells (8). In the present study, the inhibitory effect of a single transduction of WT-p53, 3KR-p53 or 6KR-p53 on the proliferation of malignant glioma cell lines was investigated. T98G (Fig. 4A), U251-MG (Fig. 4B) and A172 (Fig. 4C) cells were incubated with various concentrations of WT-p53, 3KR-p53 or 6KR-p53 for 2 h. After the cells were washed with PBS, they were replaced in fresh medium in the absence of 11R-p53. The cell viability was assessed by the WST-1 assay 2 h (Day 0) and
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Fig. 5. Comparison of the effect of each protein transduction on the induction of apoptosis of U251-MG cells. U251-MG cells were transduced with 100 nM WT-p53, 3KR-p53 or 6KR-p53. After 24 h, the cells were stained with TUNEL and TUNEL-positive cells were counted. Representative graphs are shown for experiments in which at least 6 randomly chosen fields with 50 cells were scored. \, P < 0.001.
apoptosis was slight and the effect of 3KR-p53 protein transduction was not different from that of WT-p53 (Fig. 5). In contrast, 6KR-p53 significantly induced apoptosis of malignant glioma cells compared with WT-p53 (Fig. 5). The transduction of 3KR-p53 and 6KR-p53 did not inhibit the growth of primary astrocytes or induce the apoptosis of the cells, whereas recombinant adenovirus carrying the wild-type p53 gene at an MOI of 500 significantly induced apoptosis and inhibited the cell growth of primary astrocytes (Fig. 6).
4. Discussion Fig. 4. Comparison of the effect of a single transduction of each type of 11R-p53 on the growth of malignant glioma cell lines, T98G (A), U251-MG (B) and A172 (C) 96 h after protein transduction. The cells were transduced with various concentrations of WT-p53, 3KR-p53 or 6KR-p53 on Day 0 and were cultured for 96 h (Day 4). Cell viability was measured by the WST-1 assay on Day 0 and Day 4. Cont.; no protein transduction. Data are presented as the means; n = 8 in each group; bars, S.E.M. \, P < 0.01 and \\, P < 0.05 compared with the untreated cells of each cell line.
96 h (Day 4) after the protein transduction. Administration of WT-p53 (1–100 nM) did not inhibit the cell growth of any of the cell lines (Fig. 4A–C). In the T98G cells, 100 nM 3KRp53 significantly inhibited the proliferation (Fig. 4A). However, all tested concentrations of 3KR-p53 failed to inhibit the cell growth of U251-MG and A172 cell lines (Fig. 4B and C). In contrast, 100 nM 6KR-p53 significantly inhibited the growth of all cell lines 96 h after the protein transduction compared with the growth of control cells (Fig. 4A–C) but 1 and 10 nM 6KR-p53 did not affect the growth of any of the cell lines. We further examined whether 6KR-p53 protein transduction induced the apoptosis of malignant glioma cells. U251MG cells were treated with 100 nM WT-p53, 3KR-p53 or 6KR-p53 for 24 h and apoptotic cells were assessed by TUNEL staining. The effect of WT-p53 on the induction of
Advanced-stage carcinomatosis is often resistant to current chemotherapy treatment, and new strategies for treating the disease are clearly needed [14]. It has been thought that gene therapy with viral vectors and non-viral vectors may be an attractive tool to treat cancer [15]. However, a large number of studies have indicated that virus-mediated gene therapy has some safety problems, such as inflammatory response, toxicity of the virus vector itself and random integration of virus vector DNA into the host chromosomes [16,17]. Similarly, non-virus vectors also have a problem of the efficiency of gene transduction at present [16]. In the last few years, a number of protein transduction therapies have been tried for treating cancer in vitro and in an experimental murine model [5,8,18]. These studies have shown that protein transduction therapy is useful for the delivery of tumor suppressor proteins, peptides and drugs and effective for the inhibition of cancer growth. However, the transduced proteins and peptides show limited stability; for example, poly-arginine-fused wild-type p53 protein (11R-p53) is rapidly degraded in cancer cells [8]. In the present study, we generated 11R-p53 mutants (3KR-p53 and 6KR-p53), in which multiple lysine residues in the COOH-terminal region were substituted by arginines. The mutant proteins were resistant to ubiquitination and were maintained longer than 11R-p53 in glioma cell lines, and the anti-tumor effect of the transduction of the mutant proteins was maintained longer than that of 11R-p53. Moreover, the anti-tumor
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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.febslet.2005.06.021.
References
Fig. 6. Effect of WT-p53, 3KR-p53 and 6KR-p53 on the growth and apoptosis of normal glial cells. Rat primary astrocytes were transduced with 100 nM WT-p53, 3KR-p53 or 6KR-p53. (A) The WST assay was performed 4 days after protein transduction. Cont.; no protein transduction. As a control, the cells were infected with recombinant adenovirus carrying the wild-type p53 gene at an MOI of 500. n = 8 in each group. P < 0.001. (B) Quantitative analysis of the TUNELpositive cells. TUNEL staining was performed 24 h after protein transduction. n = 8 in each group. P < 0.001.
effect of 6KR-p53 was significantly stronger than that of 3KRp53. These results suggest that the six lysine residues in the COOH terminal of p53 are the main sites of ubiquitin ligation, which targets p53 for proteasome-mediated degradation, and indicate that protein transduction therapy of ubiquitinproteasome-mediated-degradation- resistant p53 proteins such as 6KR-p53 may avoid the disadvantages of p53 protein transduction therapy for cancer. Acknowledgements: We thank A. Kemori and T. Ogawa for technical assistance, T. Akiyama and K. Yoshikawa for luciferase reporter vector for p21/WAF1 promoter and H. Matsushita and T. Jacks for KR158 cells. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan.
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