The NH2 Terminus of Influenza Virus Hemagglutinin-2 Subunit Peptides Enhances the Antitumor Potency of Polyarginine-mediated p53 Protein Transduction

Protein transduction therapy is a newly developing method that allows proteins, peptides, and biologically active compounds to penetrate across the plasma membrane by being fused with cell-penetrating peptides such as polyarginine. Polyarginine-fused p53 protein penetrates across the plasma membrane of cancer cells and inhibits the growth of the cells. However, the protein is often entrapped inside macropinosomes in the cytoplasm. Therefore, high dose concentrations of the protein are needed for it to function effectively. To overcome this problem, in the present study, polyarginine-fused p53 was linked with the NH2-terminal domain of influenza virus hemagglutinin-2 subunit (HA2), which is a pH-dependent fusogenic peptide that induces the lysis of membranes at low pH levels. The protein was capable of efficiently translocating into the nucleus of glioma cells and induced p21WAF1 transcriptional activity more effectively than did polyarginine-fused p53 protein. Moreover, low concentrations of the protein significantly inhibited the growth of cancer cells. These results suggest that protein transduction therapy using polyarginine and HA2 may be useful as a method for cancer therapy. Protein transduction therapy is a newly developing method that allows proteins, peptides, and biologically active compounds to penetrate across the plasma membrane by being fused with cell-penetrating peptides such as polyarginine. Polyarginine-fused p53 protein penetrates across the plasma membrane of cancer cells and inhibits the growth of the cells. However, the protein is often entrapped inside macropinosomes in the cytoplasm. Therefore, high dose concentrations of the protein are needed for it to function effectively. To overcome this problem, in the present study, polyarginine-fused p53 was linked with the NH2-terminal domain of influenza virus hemagglutinin-2 subunit (HA2), which is a pH-dependent fusogenic peptide that induces the lysis of membranes at low pH levels. The protein was capable of efficiently translocating into the nucleus of glioma cells and induced p21WAF1 transcriptional activity more effectively than did polyarginine-fused p53 protein. Moreover, low concentrations of the protein significantly inhibited the growth of cancer cells. These results suggest that protein transduction therapy using polyarginine and HA2 may be useful as a method for cancer therapy. The cellular delivery of various biological compounds such as bioactive protein has been improved recently by conjugating the compounds to short peptides known as cell penetrating peptides (CPPs) 1The abbreviations used are: CPP, cell penetrating peptide; PTD, protein transduction domain; HA, hemagglutinin; PBS, phosphate-buffered saline; WST-1, 2-2-methoxy-4-nitrophenyl-3-4-nitrophenyl-5-2,4-disulfophenyl-2H-tetrazolium, monosodium salt; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling; mtHA, mutant HA; m.o.i., multiplicity of infection; Rb, retinoblastoma.1The abbreviations used are: CPP, cell penetrating peptide; PTD, protein transduction domain; HA, hemagglutinin; PBS, phosphate-buffered saline; WST-1, 2-2-methoxy-4-nitrophenyl-3-4-nitrophenyl-5-2,4-disulfophenyl-2H-tetrazolium, monosodium salt; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling; mtHA, mutant HA; m.o.i., multiplicity of infection; Rb, retinoblastoma. or protein transduction domains (PTDs) (1Prochiantz A. Curr. Opin. Cell Biol. 2000; 12: 400-406Crossref PubMed Scopus (268) Google Scholar, 2Vives E. J. Mol. Recognit. 2003; 16: 265-271Crossref PubMed Scopus (145) Google Scholar). The PTD of human immunodeficiency virus type-1 TAT protein, which consists of an 11-amino acid polypeptide, is one of the most well known CPPs (3Schwarze S.R. Hruska K.A. Dowdy S.F. Trends Cell Biol. 2000; 10: 290-295Abstract Full Text Full Text PDF PubMed Scopus (515) Google Scholar, 4Wadia J.S. Dowdy S.F. Curr. Opin. Biotechnol. 2002; 13: 52-56Crossref PubMed Scopus (446) Google Scholar). Despite their broad acceptance as molecular carriers, the mechanism of internalization of CPPs and their cargo are still being discussed. Previous studies (5Vives E. Brodin P. Lebleu B. J. Biol. Chem. 1997; 272: 16010-16017Abstract Full Text Full Text PDF PubMed Scopus (2005) Google Scholar, 6Futaki S. Suzuki T. Ohashi W. Yagami T. Tanaka S. Ueda K. Sugiura Y. J. Biol. Chem. 2001; 276: 5836-5840Abstract Full Text Full Text PDF PubMed Scopus (1406) Google Scholar, 7Suzuki T. Futaki S. Niwa M. Tanaka S. Ueda K. Sugiura Y. J. Biol. Chem. 2002; 277: 2437-2443Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar) have demonstrated that the internalization of CPPs and PTD do not involve endocytosis or specific protein transporters. However, recent studies (2Vives E. J. Mol. Recognit. 2003; 16: 265-271Crossref PubMed Scopus (145) Google Scholar, 8Fittipaldi A. Ferrari A. Zoppe M. Arcangeli C. Pellegrini V. Beltram F. Giacca M. J. Biol. Chem. 2003; 278: 34141-34149Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar, 9Richard J.P. Melikov K. Vives E. Ramos C. Verbeure B. Gait M.J. Chernomordik L.V. Lebleu B. J. Biol. Chem. 2003; 278: 585-590Abstract Full Text Full Text PDF PubMed Scopus (1449) Google Scholar) have shown that the cellular internalization occurs through a temperature-dependent endocytic pathway. A very recent study (10Wadia J.S. Stan R.V. Dowdy S.F. Nat. Med. 2004; 10: 310-315Crossref PubMed Scopus (1375) Google Scholar) has shown that TAT-PTD fusion proteins are internalized rapidly by lipid raft-dependent macropinocytosis. After internalization via the macropinocytotic pathway, the proteins are carried to macropinosomes, where most of them are then degraded (10Wadia J.S. Stan R.V. Dowdy S.F. Nat. Med. 2004; 10: 310-315Crossref PubMed Scopus (1375) Google Scholar). In order for the molecules delivered by CPPs to function in the cell, they generally must reach the cytosol. Therefore, protein delivery into the cytosol of target cells via macropinosomal escape is an important route of delivery. The CPP, consisting of an 11-mer polyarginine (11R), efficiently delivers peptides and proteins into cells (11Matsushita M. Tomizawa K. Moriwaki M. Li S.-H. Terada H. Ohmoto T. Matsui H. J. Neurosci. 2001; 21: 6000-6007Crossref PubMed Google Scholar, 12Matsui H. Tomizawa K. Lu Y.-F. Matsushita M. Curr. Protein Pept. Sci. 2003; 4: 151-157Crossref PubMed Scopus (65) Google Scholar). The 11R-fused p53 protein (p53-11R) is delivered effectively into cancer cells and has transcriptional regulatory activity there (13Takenobu T. Tomizawa K. Matsushita M. Li S.T. Moriwaki A. Lu Y.F. Matsui H. Mol. Cancer Ther. 2002; 1: 1043-1049PubMed Google Scholar). Moreover, p53-11R inhibits the proliferation of the cancer cells (13Takenobu T. Tomizawa K. Matsushita M. Li S.T. Moriwaki A. Lu Y.F. Matsui H. Mol. Cancer Ther. 2002; 1: 1043-1049PubMed Google Scholar). However, a high concentration (>1 μm) and repeated administration of p53-11R are needed for transcriptional activation and the growth inhibition of cancer cells (13Takenobu T. Tomizawa K. Matsushita M. Li S.T. Moriwaki A. Lu Y.F. Matsui H. Mol. Cancer Ther. 2002; 1: 1043-1049PubMed Google Scholar). Entrapment of the transduced protein in macropinosomes may weaken the effect of p53-11R. For protein delivery to become a promising method for clinical cancer therapy, this problem must be overcome. Several viruses have acquired endosomal escape mechanisms that take advantage of the low pH in mature endosomes of mammalian cells (14Skehel J.J. Cross K. Steinhauer D. Wiley D.C. Biochem. Soc. Trans. 2001; 29: 623-626Crossref PubMed Scopus (61) Google Scholar). The NH2-terminal 20-amino acid peptide of the influenza virus hemagglutinin-2 protein (HA2) is well characterized as a pH-sensitive fusogenic peptide that destabilizes lipid membranes at low pH levels (14Skehel J.J. Cross K. Steinhauer D. Wiley D.C. Biochem. Soc. Trans. 2001; 29: 623-626Crossref PubMed Scopus (61) Google Scholar, 15Han X. Bushweller J.H. Cafiso D.S. Tamm L.K. Nat. Struct. Biol. 2001; 8: 715-720Crossref PubMed Scopus (397) Google Scholar). A recent study (10Wadia J.S. Stan R.V. Dowdy S.F. Nat. Med. 2004; 10: 310-315Crossref PubMed Scopus (1375) Google Scholar) has shown that TAT PTD fused with HA2 markedly enhances the release of fusion proteins from macropinosomes and that this approach has the beneficial aspect of disrupting only macropinosomes but no other types of vesicles. In the present study, we investigated whether the linking of HA2 with polyarginine-fused p53 protein induced delivery into the nucleus of glioma cells and enhanced the anticancer effect of p53-11R. Cell Lines and Cell Culture—A human malignant glioma cell line, U251-MG, was provided from Health Science Research Resources Bank (Osaka, Japan), and a malignant glioma cell line, KR158, was a gift from Dr. T. Jacks (Massachusetts Institute of Technology, Cambridge, MA). U251-MG cells contain a homozygous missense mutation corresponding to a His/Arg transition in codon 273 in p53. KR158 is a p53 gene-deficient cell line. Both cell lines were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 units/ml streptomycin. The cells were cultured in a 37 °C incubator with 5% CO2. Rat primary astrocytes were prepared from a newborn Wistar rat (Japan SLC, Inc.). The cortex of the rat on postnatal day 1 was dissected, and the meninges were removed. The cortical tissues were then treated with 0.25% trypsin (Invitrogen) for 15 min at 37 °C and further treated with 0.004% DNase-I (Sigma) for 10 min. The pieces of the cortex were dissociated mechanically. The dissociated cells were plated onto collagen-coated glass slides and 96-well collagen-coated dishes. The plated cell density was approximately 10,000 cells/ml. The cells were maintained in Dulbecco's modified Eagle's medium with 10% fetal calf serum (Invitrogen) and 5% horse serum. Cultures were maintained at 37 °C in a 95% air, 5% CO2 humidified incubator. Construction of p53-11R and HA2-p53-9R—Human wild-type p53 cDNA was subcloned into p11R-HA and p9R-HA vectors to produce genetic in-frame 11 (11R) and 9 polyarginine (9R) fusion proteins in the COOH-terminal, as described previously (13Takenobu T. Tomizawa K. Matsushita M. Li S.T. Moriwaki A. Lu Y.F. Matsui H. Mol. Cancer Ther. 2002; 1: 1043-1049PubMed Google Scholar). Influenza virus HA2 cDNA encoding 23 amino acids (GLFEAIEGFIENGWEGMIDGWYG) was tagged on the NH2 terminus of p53 cDNA in p53-9R plasmids (HA2-p53-9R). Site-directed Mutagenesis of Human p53 and HA2 cDNAs—The mutations of p53 and HA2 cDNAs were generated by site-directed mutagenesis using a PCR strategy as described previously (16Tomizawa 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. Matsui H. J. Cell Biol. 2003; 163: 813-824Crossref PubMed Scopus (160) Google Scholar). For dominant-negative p53, Arg at the site of 273 was changed to His as described previously (17Marutani M. Tonoki H. Tada M. Takahashi M. Kashiwazaki H. Hida Y. Hamada J. Asaka M. Moriuchi T. Cancer Res. 1999; 59: 4765-4769PubMed Google Scholar). An HA2 mutant that has no destabilization activity of the lipid membrane at low pH was produced by replacing Gly at site 13 and Met at site 17 with Pro and Leu, respectively, as described previously (18Hsu C.H. Wu S.H. Chang D.K. Chen C. J. Biol. Chem. 2002; 277: 22725-22733Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). The sequences of the mutants were confirmed with an ABI 3100 sequencer. Expression and Purification of 11R and 9R Fusion Proteins—Expression and purification of 11R and 9R fusion proteins were performed as described previously (13Takenobu T. Tomizawa K. Matsushita M. Li S.T. Moriwaki A. Lu Y.F. Matsui H. Mol. Cancer Ther. 2002; 1: 1043-1049PubMed Google Scholar). Briefly, the constructed plasmids were transformed into BL21-DE3 Escherichia coli cells. The proteins were expressed in these cells after induction with 0.1 mm isopropyl 1-thio-β-d-galactopyranoside. The expressed proteins were purified using a column of nickel-nitrilotriacetic acid-agarose (Invitrogen). After dialysis against PBS, the proteins were stored at -80 °C until they were used. Preparation of Fluorescein Isothiocyanate-conjugated p53-11R and HA2-p53-9R and the Localization of the Proteins in Living Malignant Glioma Cells—The p53-11R and HA2-p53-9R proteins were conjugated with a green fluorescent tag using a fluorescein-EX protein labeling kit (catalog no. F-10240, Molecular Probes). After purification of the proteins, U251-MG cells were incubated with each protein (0.1 μm), and the localization of the transduced proteins was observed with a confocal laser microscope (FluoView, Olympus, Japan). Western Blotting Analysis—U251-MG cells were incubated with 0.1 μm p53-11R and HA2-p53-9R. After 2 h, the cells were washed with PBS twice and placed in fresh medium in the absence of the proteins. The cells were harvested at each of the time points indicated in the text, and Western blot analysis was performed using anti-p53 monoclonal antibody. Western blot analysis for p53 was carried out at high stringency, essentially as described previously (13Takenobu T. Tomizawa K. Matsushita M. Li S.T. Moriwaki A. Lu Y.F. Matsui H. Mol. Cancer Ther. 2002; 1: 1043-1049PubMed Google Scholar). Briefly, the harvested cells were homogenized by sonication in a boiled buffer containing 1% SDS. Samples containing 100 μg of total protein were electrophoresed by SDS-PAGE and then transferred to nitrocellulose membranes (Hybond ECL, Amersham Biosciences). The blots were probed with primary antibody against p53 (1:1000) (catalog no. Pab 1801, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and developed with an enhanced chemiluminescence detection system (Amersham Biosciences). Reporter Assay for p53-driven Transactivation—The reporter assay was performed as described previously (13Takenobu T. Tomizawa K. Matsushita M. Li S.T. Moriwaki A. Lu Y.F. Matsui H. Mol. Cancer Ther. 2002; 1: 1043-1049PubMed Google Scholar). Briefly, the luciferase reporter vector pGL2-basic (Promega) containing a 2.4-kbp fragment of human p21WAF1 promoter was a gift from Drs. T. Akiyama (Tokyo University) and K. Yoshikawa (Osaka University). The luciferase reporter vector was transfected into 70% confluent KR158 cells in 35-mm dishes by the calcium phosphate method. After 24 h, the cells were incubated with 100 nm p53-11R or HA2-p53-9R. The cells were then harvested 6, 12, 18, 24, and 30 h after the protein and the luciferase were with a using a kit (Tokyo Japan). The luciferase activity was in were performed for each Cell was using a assay as described previously (13Takenobu T. Tomizawa K. Matsushita M. Li S.T. Moriwaki A. Lu Y.F. Matsui H. Mol. Cancer Ther. 2002; 1: 1043-1049PubMed Google Scholar). After glioma cells were on 96-well they were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum for 24 The cells were then supplemented with various concentrations of p53-11R or HA2-p53-9R and further incubated for 2 h After with PBS, the cells were placed in fresh medium in the absence of and HA2-p53-9R and further incubated for h The cell was using the assay on day and day to the of cells were confirmed by and as described previously Tomizawa K. Oda Y. Wei F.-Y. Lu Y.-F. Matsushita M. Li S.-T. Moriwaki A. Matsui H. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). Briefly, after with KR158 cells were incubated with 0.1 of (Sigma) for 1 min. The of the nucleus was observed with a cells were by the of or For KR158 cells in 35-mm dishes were incubated with p53-11R or HA2-p53-9R for 2 After twice with PBS, the cells were further incubated in fresh medium for 24 The cells were then with and was performed with an in cell detection kit After the cells were incubated with (1:1000) for 1 h to The was observed using a confocal laser microscope no. are shown for in which at containing 100 cells each were are shown as the were using to or analysis of by of and was of HA2-p53-9R and p53-11R in Glioma produced p53-11R protein. However, the protein was after dialysis against PBS not Therefore, p53 protein was fused with HA2 and which has protein transduction activity (11Matsushita M. Tomizawa K. Moriwaki M. Li S.-H. Terada H. Ohmoto T. Matsui H. J. Neurosci. 2001; 21: 6000-6007Crossref PubMed Google Scholar). Both HA2-p53-9R and p53-11R were delivered effectively into U251-MG cells h After h, the of proteins in a HA2-p53-9R more rapidly than p53-11R in the cells. p53 of HA2-p53-9R and the transcriptional activity HA2-p53-9R and p53-11R in glioma each protein (0.1 μm) was transduced into KR158 which p53, and the p53 transcriptional activity was The transcriptional activity in cells was significantly than that in cells at of the The transcriptional activity of HA2-p53-9R was h after the protein and it then in a To whether the of the transcriptional activity of HA2-p53-9R was the of the function of an HA2 mutant that has no activity was fused with and the transcriptional activity of the fusion protein was The transcriptional activity of was significantly than that of HA2-p53-9R and was the as that of p53-11R. Moreover, the transcriptional activity of dominant-negative p53 protein fused with HA2 and 9R was than that of HA2-p53-9R. These results that the with HA2 results in transcriptional activity of p53 protein using polyarginine of Localization of p53-11R and whether HA2-p53-9R effectively into the p53-11R and HA2-p53-9R were conjugated with a green fluorescent and U251-MG cells were transduced into each protein. The localization of each transduced protein was 30 min and 2 h after protein transduction in cells. Moreover, to the that the proteins were to the of the cell of the of the cells were then with a confocal Both p53-11R and HA2-p53-9R were observed in the cell membrane and after 30 and there was no of the protein observed in the nucleus not After 2 h, HA2-p53-9R was observed in the nucleus and cell p53-11R in the cell membrane and These results that HA2-p53-9R more effectively into the nucleus than p53-11R. of on the of Glioma study (13Takenobu T. Tomizawa K. Matsushita M. Li S.T. Moriwaki A. Lu Y.F. Matsui H. Mol. Cancer Ther. 2002; 1: 1043-1049PubMed Google Scholar) has shown that a administration of 1 μm p53-11R to the growth of cancer cells and that repeated administration 24 h is needed for the inhibition of their The present study that a of 1 μm p53-11R did not the growth of U251-MG cells or after the protein transduction In HA2-p53-9R inhibited the growth of the cells in a concentrations and 0.1 μm) of HA2-p53-9R no effect on the growth of glioma cells 1 or 2 after the protein However, HA2-p53-9R at the concentrations inhibited the cell growth after protein transduction The effect on the growth of cells was with 1 μm HA2-p53-9R To that the effect of 1 μm HA2-p53-9R was not by a effect of HA2 peptides fused with 11R were transduced into the cells. μm) did not the growth of glioma cells Moreover, protein transduction of 1 μm or 1 μm no effect on the cell growth of the of Glioma by The effect of on the induction of the of KR158 cells was investigated with 1 μm p53-11R did not of the glioma 1 μm HA2-p53-9R markedly induced of the cancer cells In the transduction of HA2-p53-9R did not the of primary astrocytes A and or the growth of the cells Moreover, p53-11R did not the growth or the of primary at an of significantly induced and inhibited the cell growth of primary astrocytes Protein transduction therapy of p53 is useful for the inhibition of the proliferation of cancer cells (13Takenobu T. Tomizawa K. Matsushita M. Li S.T. Moriwaki A. Lu Y.F. Matsui H. Mol. Cancer Ther. 2002; 1: 1043-1049PubMed Google Scholar, F. M. S. Cancer Ther. 2002; PubMed Scopus Google Scholar). However, the is of the of high for the inhibition and only occurs in the cells H. Tomizawa K. Lu Y.-F. Matsushita M. Curr. Protein Pept. Sci. 2003; 4: 151-157Crossref PubMed Scopus (65) Google Scholar, T. Tomizawa K. Matsushita M. Li S.T. Moriwaki A. Lu Y.F. Matsui H. Mol. Cancer Ther. 2002; 1: 1043-1049PubMed Google Scholar). A study (13Takenobu T. Tomizawa K. Matsushita M. Li S.T. Moriwaki A. Lu Y.F. Matsui H. Mol. Cancer Ther. 2002; 1: 1043-1049PubMed Google Scholar) that repeated protein transduction of p53-11R was needed for the inhibition of cancer cell the of transduced proteins has not been in repeated administration of protein therapy may an clinical of protein transduction therapy, the of protein therapy must be overcome. In the present study, we that the of HA2 peptides with p53-11R significantly enhanced the transcriptional activity and the effect of p53-11R. A of μm HA2-p53-9R inhibited the growth of glioma a of 1 μm p53-11R to cell Moreover, did not the cell growth of cells. These results suggest that transduction therapy using p53 protein fused with HA2 may overcome the of p53 protein therapy for it to become a promising of cancer therapy. The mechanism of protein transduction of p53-11R into cells A recent study (10Wadia J.S. Stan R.V. Dowdy S.F. Nat. Med. 2004; 10: 310-315Crossref PubMed Scopus (1375) Google Scholar) that TAT fusion proteins were internalized rapidly by lipid raft-dependent and most of the internalized proteins were entrapped in The present results that the transduction of p53-11R in transcriptional activity of p53-11R h, the transduction of the fusion with an mutant with no to macropinosomes no effect on the transcriptional Moreover, HA2-p53-9R more effectively into the nucleus with p53-11R. These results suggest that the of polyarginine fusion proteins such as p53-11R may be entrapped in macropinosomes and that the fusion of HA2 may effectively release these proteins from macropinosomes in cancer there was no of performed in the present The present results that astrocytes low to p53 protein and inhibition of cell In p53 therapy at an of induced and inhibited the cell growth of the cells. we did not the molecular mechanism for of glioma cells and astrocytes in the present study, studies J. Ther. PubMed Scopus Google Scholar, M. S. A. Ther. 1997; 4: PubMed Scopus Google Scholar, A. M. S. A. S. J. Med. 2000; PubMed Google Scholar) have to a the Previous studies have shown that the of wild-type p53 protein has activity in it not in cells. which is a a with p53, and this the function of p53 D. V. F. S. Lu X. Mol. Biol. 2002; PubMed Scopus Google Scholar). In the activity of is by the inhibits the activity of p53 through with D. V. F. S. Lu X. Mol. Biol. 2002; PubMed Scopus Google Scholar). In in the is often in the of activity Full Text PDF PubMed Scopus Google Scholar). The then with and p53 to may the of glioma cells to p53 protein transduction therapy. The present results may a for the of p53 protein therapy to the to target which is one of the most in protein therapy with The that HA2-p53-9R was more rapidly degraded in glioma cells with p53-11R is well known that wild-type p53 protein is rapidly degraded by the in the nucleus D. M. Cancer Biol. 2003; 13: PubMed Scopus Google Scholar). These results suggest that from macropinosomes may into the where it may be rapidly by the In p53-11R may be in macropinosomes than protein and may not by the Moreover, these results suggest that p53 protein transduction therapy to p53 protein be further is the transduction of p53 protein that is to mutation of and of the p53 protein to a p53 with transcriptional activity that is to S. Mol. Biol. 2000; PubMed Scopus Google Scholar). to a transduction method for proteins fused with HA2 are in in A. and T. for T. Akiyama and K. Yoshikawa for luciferase reporter vector and p21WAF1 and H. Matsushita and T. Jacks for KR158 cells.