An archaeal histone-like protein mediates efficient p53 gene transfer and facilitates its anti-cancer effect in vitro and in vivo

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Abstract

The improvement of the transfection efficiency of the non-viral-based gene delivery systems is a key issue for the application in gene therapy. We have previously described an archaeal histone-like protein-based (HPhA) gene delivery system and showed that HPhA formed stable non-covalent complexes with nucleic acids and improved their delivery by using β-galactosidase as a reporter gene. In this study, the wild-type p53 gene was transfected into the cancer cells using the HPhA as a vector, and the expression level and the activity of p53 gene were evaluated both in vitro and in vivo. Gene expression was determined by real-time reverse transcriptase-PCR and western blotting analysis. The cellular growth inhibition and apoptosis of HPhA-mediated p53 transfection were assessed by XTT (sodium 3′-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate) assay and annexin V–FITC (fluorescein isothiocyanate) staining, respectively. Further more, transfection of HPhA/p53 into CNE (nasopharyngeal carcinoma cell line)-xenografted nude mice was performed and tumor growth was measured. The present study demonstrates that HPhA enhances the efficiency of p53 gene transfer and antitumor activity compared with the widely used Lipofectamine. These results demonstrate that HPhA enhances the in vitro and in vivo efficiency of p53 gene transfer and suggest that it may be served as a promising tool for gene delivery and gene therapy.

Introduction

Gene therapy has become an increasingly important strategy for treating a variety of human acquired and inherited diseases.1 The development of widely applicable DNA transfection systems is a primary focus of current molecular biological research. Generally, there are two main types of gene delivery vectors: viral and non-viral. Viruses are the most effective DNA delivery vehicles for gene therapy, but they suffer from a number of undesirable properties for therapeutic applications, such as uncertainties about safety, immunogenicity, limited packaging capacity for genetic material and manufacturing difficulties. As an alternative approach, the non-viral gene delivery system is a non-infectious and non-immunogenic, with low in vivo toxicity, carrier that may also provide a safe, inexpensive and easily handled technique for gene therapy.2

Among non-viral gene delivery systems, natural DNA-binding proteins, histones, as a vehicle for gene transfer have been reported by different authors.3, 4, 5, 6, 7, 8 Various preparations of histones are polycationic molecules forming stable ionic complexes with plasmid DNA via electrostatic interactions between the negatively charged phosphate DNA backbone and positively charged residues of lysine and arginine.9 Histones have nuclear localization signals that facilitate their nuclear import.4 The presence of both nuclear localization signals and the positively charged residues of lysine and arginine makes them good candidates for efficient gene delivery. The penetration of histone into tissue-cultured cells occurring by direct translocation through the cell plasma membrane, and not by a typical endocytosis, may facilitate the escape of DNA from the endosome.10, 11 Therefore, it is thought to be one of the most attractive alternative vectors.

In our previous studies, we used HPhA, an archaeal histone-like protein from hyperthermophilic archaeal bacteria Pyrococcus horikoshii OT3 strain.12, 13 An optimal protocol for HPhA-mediated transfection in vitro was established to improve transfection efficiency.14 The mass ratio of HPhA to DNA, the incubation time for the DNA–HPhA complex with the cells and the concentration of CaCl2 in the cell culture medium were all optimized for efficient transfection. Serum has no effect for the efficiency of HPhA-mediated transfection. Most importantly, the cytotoxicity of HPhA is lower than that of commonly used cationic liposome-based gene delivery systems.14 These features make HPhA potential good carriers of gene deliveries.

The p53 gene has been extensively studied and is known to play a critical role in cell regulation and control of apoptosis. Gene therapy using the p53 gene has been proposed for and performed with inactivation of p53 function. However, efficient and safe gene delivery remains a key issue for p53-based gene therapy.

In the present study, we attempted to use HPhA with naked plasmid DNA, which contains a wt-p53 tumor suppressor gene, and we evaluated the transfection efficiency and antitumor activity in human p53-deleted nasopharyngeal carcinoma (CNE) cells in vitro and in vivo. We observed that p53 delivery resulted in a more differential growth inhibition pattern in cancer cells in vitro and in vivo. Our data suggest that HPhA enhances the in vitro and in vivo efficiency of p53 gene transfer and is a promising new strategy for p53 gene therapy.

Materials and methods

Microbiological culture media were purchased from Oxoid, Hampshire, UK. Lipofectamine 2000 (1 mg ml−1) was obtained from Invitrogen, NY and used according to the manufacturer's recommendations. Cell culture media, RPMI 1640, heat-inactivated fetal calf serum and trypsin–EDTA were supplied by Gibco BRL, NY. Other chemicals were purchased from Sigma, Sydney, Australia.

Cells and culture conditions

The nasopharyngeal carcinoma cell line, CNE, was kindly provided by Dr Ping Zhu (Academy of Military Medical Science, Changchun, China) and has been characterized previously.15 The cells were maintained in RPMI 1640 supplemented with 4 mM L-glutamine supplemented with 10% heat-inactivated fetal calf serum, and 100 mg ml−1 of penicillin and streptomycin under 5% CO2 in a humidified atmosphere.

Animals and tumor model

Four- to five-week-old, 18–20 g female BALB/c nude mice were used and housed at the Institute of Laboratory Animals, the China–Japan Union Hospital. CNE cells (1 × 107) were inoculated subcutaneously on the right flank of nude mice. After the tumor size reached between 5 and 6 mm2 in size, treatment of the animals was initiated (day 0).

Plasmids

The plasmid pCMV/p53 containing wild-type human p53 cDNA under control of a cytomegalovirus promoter was a kindly gift from Dr Ming-Hua Zhu (Second Military Medical University, Shanghai, China). The plasmids were expanded in DH-5α Escherichia coli (Invitrogen) and isolated using V-gene endotoxin-free plasmid kit (V-gene, Hangzhou, China), according to the supplier's protocol. E. coli strain BL21-Codon Plus (DE3)-RIL, bearing the HPhA gene, was used to produce the recombinant HPhA.

Purification of recombinant HPhA protein

The recombinant HPhA protein was expressed in E. coli BL21-Codon Plus, as reported previously.12 Briefly, after growth and cellular disruption, most proteins from the E. coli cell lysate were precipitated by heating at 85°C. The recombinant HPhA remained in the supernatant and was then applied to a HiTrap heparin-sepharose column and eluted with a linear gradient of 0.1–1 M NaCl. The purified HPhA was dialyzed overnight at 4 °C in a 0.1 M Tris–HCl buffer (pH 8.0) and stored at −20 °C until used.

Protein analysis

The concentration of the expressed protein was determined by quantitative amino-acid analysis. Approximate protein concentrations were established by Bradford assays. Bovine serum albumin fraction V (Sigma) was used as the standard protein. The levels of cellular protein in transfection experiments were determined using the Bio-Rad Protein Assay kit (Bio-Rad, Richmond, CA).

Transfection procedure

The CNE cells were plated at a density of 1 × 105 per well on 24-well plates 24 h before transfection. The culture medium was removed before transfection and the cells were washed twice with RPMI 1640 medium. The HPhA–DNA complexes were prepared at a mass ratio of 6:1 and added to 1.4 ml of culture medium containing 10% fetal calf serum and 2 mM Ca2+. The transfection mixture was then added to the cells. After 4 h incubation under 5% CO2 at 37 °C, the transfection mixture was removed and replaced by 2 ml of RPMI 1640 medium containing 10% fetal bovine serum. In vitro transfection of Lipofectamine–DNA complex was according to the manufacturer's recommendations.

Immunohistochemistry

Transfected cell monolayers on round cover glass were fixed with 3.8% formaldehyde in phosphate-buffered saline (PBS) for 10 min, washed with PBS for 5 min per wash and incubated with mouse monoclonal anti-human p53 antibody DO-1 (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at 37 °C. Cells were rinsed twice in PBS for 5 min per wash and then incubated with anti-mouse IgG horseradish peroxidase-conjugated secondary antibody (Santa Cruz) for 1 h at 37 °C. After washing twice in distilled water, events were visualized using diaminobenzipin (DAB) as a chromophore.

Real-time reverse transcriptase-PCR and classical reverse transcriptase-PCR

The p53 mRNA expression was analyzed using real-time quantitative PCR with β-actin as a reference housekeeping gene. The cells were seeded in six-well plates at a confluence of 2 × 105 cells per well. After transfection, the medium was removed and the six-well plate was washed with PBS. Untreated cells were used as control. Total RNA was extracted and purified from the cells using EZ-10 spin column mRNA isolation kit (TakaRa, Dalian, China) as recommended by the manufacturer. Purified RNA was dissolved in 30 μl RNAse-free water.

First-strand cDNA was synthesized using 1 μl purified RNA, 0.5 μl AMV reverse transcriptase (RT) (TaKaRa), 1 μl 10 × RT buffer (TaKaRa), 2 μl MgCl2, 1 μl dNTP mixture (TaKaRa), 0.5 μl Oligo-dT-Adoptor-Primer (TaKaRa) and 4 μl RNAse-free water. After incubation for 30 min at 42 °C, the RT was inactivated at 99 °C for 5 min.

Classical PCR was performed to test the specificity of the amplification. The primers for PCR were p53 (5′-IndexTermTTTGCGTGTGGAGTATTTGG-3′ and 5′-IndexTermGTTTTTTATGGCGGGAGG-3′) and β-actin (5′-IndexTermGGATCAGCAAGCAGGAGTATG-3′ and 5′-IndexTermCACCTTCACCGTTCCAGTTT-3′), the amplified products of 549 and 225 bp, respectively. In brief, 10 μl of cDNA template was mixed with 1 μl dNTP, 10 μl 5 × PCR buffer (TaKaRa), 0.25 μl ExTaq HS polymerase (TaKaRa), 0.5 μl 20 μM each primer and RNAse-free water for volume of 50 μl. Amplification was performed as follows: 1 cycle at 94 °C for 2 min and followed 30 cycles at 94 °C for 30 s, 55 °C for 30 s and 72 °C for 1 min. The PCR products were detected by electrophoresis through a 2% agarose gel stained with ethidium bromide.

Real-time PCR were performed using the TaKaRa SYBR PCR kit and ABI Prism 7000 sequence detection system according to the manufacturer's specifications. The lightCycler system was used to monitor the SYBR Green signal at the end of each extension period for 40 cycles. The primers for amplification were p53 (5′-IndexTermCCCTCCTCAGCATCTTATCCG-3′ and 5′-IndexTermGGCACAAACACGCACCTCA-3′) and β-actin (the same primer with classical PCR), the amplified products of 262 and 225 bp, respectively. Total reaction volume was 50 μl including 25 μl SYBR Premix Ex Taq with SYBR Green I, 300 nM forward and reverse primers and 2 μl cDNA. The thermal cycler program was 1 cycle at 95 °C for 10 s, followed by 40 cycles at 95 °C for 5 s and 60 °C for 30 s. The threshold cycle (CT) for p53 gene and for β-actin housekeeping gene, and the difference between their CT values (ΔCT) were determined. For p53 gene of each group, normalization was performed against the pCMV/p53 sample as the reference with its ΔCT value subtracted from the ΔCT value of HPhA/p53 and Lipofectamine/p53 samples to obtain the ΔΔCT value. Finally, the 2 - Δ Δ C T value was calculated to reflect the relative expression of each transfection method. Each experiment was repeated either two or three times.

Western blotting analysis

The expression level of the p53 protein in the transfected cells was determined by western blotting analysis with β-actin as a reference housekeeping protein. The transfected cells were washed twice with cold PBS and lysed at 4 °C for 30 min in lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1% nonidet-P40, 50 mM NaF, 1 mM Na3VO4, 10 μg ml−1 leupeptin, 1 mM phenylmethanesulfonyl fluoride (PMSF) in ice for 20 min. Insoluble materials were removed by centrifugation at 4 °C for 30 min at 8000 r.p.m., and 40 μg protein was subjected to western blotting analysis in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis under denaturing and transferred to polyvinylidene difluoride membranes (Immobilon-pSQ; Millipore, Billerica, MA) using the phast-system (Bio-Rad). Blotted membranes were blocked with 10% dried milk in PBS plus 0.1% Tween 20 for 1 h at room temperatures and reacted with antibodies specific for p53, β-actin (mouse monoclonal anti-human antibodies; Santa Cruz) diluted 1:500 in PBS plus 0.1% Tween 20. After incubation, the membranes were extensively washed in PBS plus 0.1% Tween 20 and incubated with anti-mouse IgG horseradish peroxidase-conjugated secondary antibody (Santa Cruz) diluted 1:5000. The membrane was then processed and developed as the manufacturer's suggestions.

Cell proliferation assay

Cells were seeded on 96-well plates at a density of 5 × 103 cells per well. After culturing for 24 h, the cells were treated with four different agents (100 μl) as follows: (a) RPMI 1640 only (as the control); (b) pCMV/p53; (c) HPhA/p53; (d) Lipofectamine/p53. After culturing for 5 h, the cells were washed with PBS and the medium was replaced by RPMI 1640 supplemented with 10% fetal bovine serum. Cell proliferation was assessed at each time point by measuring the conversion of the tetrazolium salt XTT to formazan, according to the manufacturer's instruction (Roche, Mannheim, Germany).

Induction of apoptosis after p53 gene transfer

Analysis of apoptosis induction was performed by flow cytometry (FACScalibur, Becton Dickinson, Mountain View, CA) using annexin V–propidium iodide (PI) double labeling assay kit (KeyGene, Nanjing, China). Briefly, the cells were cultured on 24-well dishes and allowed to grow for 24 h. Three groups of CNE cells were transfected with pCMV/p53 alone, HPhA/p53 complexes or Lipofectamine/p53 complexes, separately. Forty-eight hours after transfection, both attached and floating cells were collected and washed twice with PBS. The cells were then stained with the annexin V–fluorescein isothiocyanate (FITC) and PI. The samples were analyzed using flow cytometer. Viable cells were not labeled while apoptosis and necrotic cells were labeled with annexin V–FITC and PI, respectively.

Cell cycle analysis after p53 gene transfer

The transfected cells were washed with PBS and resuspended in 0.1 ml of medium. For fixing, 3 ml of cold 70% ethanol was added to each sample. Samples were stored overnight at 4 °C. Cells were analyzed to determine cell cycle arrest. After overnight fixation, samples were centrifuged and ethanol was removed. Cells were washed with PBS and then resuspended in PI staining buffer and incubated for 30 min at 37 °C. The cells were analyzed by flow cytometry (FACScalibur, Becton Dickinson).

Treatment of tumor xenografts in nude mice

The CNE cells were harvested with trypsin–EDTA and centrifuged. Freshly harvested cells were rinsed and resuspended in PBS at 1 × 107 cells ml−1. Female BALB/c nude mice were injected with CNE tumor cells (106 cells/100 μl) subcutaneously on the right flank. When the tumors reach 4–5 mm2 in size, the animals were randomly divided into four groups with six mice per group, and each group was treated with one of the following: (1) untreated, (2) pCMV/p53 alone, (3) Lipofectamine/p53 complex, (4) HPhA/p53 complex. Each agent (100 μl) was injected along the tumor margin using a 27-gauge needle. The initial day of administration was defined as day 0. Administration was then repeated six times at 5-day interval such that day 30 was the final day of administration. The tumor volume was measured during the administration period and for 25 further days after day 30. And tumor volumes were calculated by using the formula V=a × b2/2, where a is the largest diameter and b is the perpendicular diameter and V is given in cubic millimeters.

Statistical analysis

Unless indicated, all experiments were performed in triplicate and the data were analyzed using Student's t-test. P<0.05 was considered statistically significant.

Results

HPhA-mediated efficient p53 gene transfer in CNE cells

The ability of HPhA gene transfer into cells was evaluated as percentage of p53-staining positive cells 24 h by standard immunohistochemical techniques. Immunohistochemistry showed prominent staining of human p53 protein using mouse monoclonal anti-human p53 antibodies DO-1. No p53 protein staining was observed in the untreated cells. However, when cells were transfected with the HPhA/p53, a strong staining was noted (Figure 1). Those transfected cells showed even intensity of staining. More than 60% cells transfected with HPhA/p53 expressed the human p53 protein.

Figure 1
figure1

Immunohistochemical staining for p53 protein 48 h after transfection. The presence of human p53 protein (stained by mouse monoclonal anti-human p53 antibodies DO-1) using immunohistochemical stain. (a) HPhA/p53 transfected cells, (b) untreated cells. Bar, 20 μm. HPhA, an archaeal histone-like protein.

To compare the transfection efficiency, p53 gene transfer was performed separately in CNE cells, using HPhA and other vectors. The expression of p53 mRNA was measured using RT-PCR, and p53 protein expression was assayed using western blotting 48 h after transfection.

The p53 mRNA relative expression value was calculated using real-time PCR (Table 1). In untreated cells, no expression of p53 mRNA was detected (Figure 2), and data were consistent with its p53 gene status. p53 mRNA expression was restored in transfected cells using pCMV/p53 alone, HPhA/p53 or Lipofectamine/p53. By real-time PCR, it showed that p53 mRNA expression in CNE cells transfected with HPhA/p53 was 1.35 times higher (P<0.05) than the cells transfected with pCMV/p53 alone. Although the expression of p53 following the Lipofectamine/p53 treatment was slightly higher than that following the p53 plasmid DNA alone, it was not statistically significant.

Table 1 p53 mRNA expression in CNE cells by real-time RT-PCR analysis
Figure 2
figure2

Classical RT-PCR analysis of p53 in the CNE cells. The pCMV/p53 plasmid (2 μg) was transfected with HPhA (12 μg) or Lipofectamine 2000 to the CNE cells. Forty-eight hours later, DNA was extracted from the cells and analyzed using RT-PCR. Lane 1, DL2000 maker; lane 2, control; lane 3, transfected with pCMV/p53 plasmid alone; lane 4, Lipofectamine-mediated pCMV/p53 transfection; lane 5, HPhA-mediated pCMV/p53 transfection. β-actin was used as an internal control. RT, reverse transcriptase; HPhA, an archaeal histone-like protein.

Western blotting analysis was performed to evaluate the amount of p53 protein produced after transfection. The p53 band, recognized by the mouse monoclonal anti-human p53 antibodies DO-1, was observed in cellular extracts from transfected cells. The band intensities of HPhA/DNA-transfected samples and that of Lipofectamine/p53-transfected samples were compared, and the result indicated that the former cells expressed significantly higher amount of p53 proteins (Figure 3). The protein level of p53 was slightly detected in cells transfected with the plasmid alone. Samples isolated from untreated cancer cell exhibited no p53 protein. These results indicate that the exogenous p53 gene introduced by HPhA is efficiently expressed in the cells.

Figure 3
figure3

Western blotting of the exogenous p53 protein expressions in the CNE cells. Lane 1, control; lane 2, transfected with pCMV/p53 plasmid alone; lane 3, HPhA-mediated pCMV/p53 transfection; lane 4, Lipofectamine-mediated pCMV/p53 transfection. β-actin was used as a loading control. HPhA, an archaeal histone-like protein.

Inhibition of cell growth in vitro

We studied whether HPhA/p53 complexes inhibited the proliferation of cancer cells. Western blotting analysis revealed that the HPhA/p53-transfected cells expressed the p53 proteins. We compared the effect of the p53 proteins to inhibit the cell proliferation in four groups (untreated, pCMV/p53 treated, HPhA/p53 treated, Lipofectamine/p53 treated, respectively) in different point. The degree of cell growth was measured via XTT (sodium, 3′-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate) assay. The growth rate of HPhA/p53-transfected cells was significantly inhibited compared with control or Lipofectamine/p53-transfected cells (Figure 4). The HPhA/p53 group showed a statistically significant decrease in cell growth as compared with Lipofectamine/p53 (P<0.05). Transfection of CNE cells with pCMV/p53 alone had little effect on cell growth compared to the untreated cell.

Figure 4
figure4

Effect of different transfection complex on the inhibition of the proliferation of CNE cells. Control (); transfected with pCMV/p53 plasmid alone (); HPhA-mediated pCMV/p53 transfection (•); Lipofectamine-mediated pCMV/p53 transfection (▪). Results are mean value of eight independent determinations. Means±s.d. are shown. HPhA, an archaeal histone-like protein.

Induction of apoptosis and cell cycle arrest following p53 gene transfer

Apoptosis induction was studied by flow cytometry using annexin V–FITC labeling for the detection of phosphatidylserine externalization occurring as an early step during apoptosis. The fractions of apoptotic, that is, annexin V–FITC-positive and PI-negative, were determined in CNE cell line (Figure 5). In untreated CNE cells, little apoptosis was found in less than 2% of the total cell population. The Lipofectamine/p53 group had significantly more apoptosis than the pCMV/p53 group (P<0.05), but less than the HPhA/p53 group (P<0.05). When p53 is transfected with HPhA, apoptosis and necrosis were increased due to the increase of both PI-positive and annexin-positive cells. The annexin-positive cells were significantly increased for the HPhA/p53-transfected cells compared with the untreated cells. Figure 6 showed a graphical summary of proportion of CNE cells in the G1 phase of cell cycle under different treatment conditions. Further analysis revealed that a higher percentage of cells in G1 in the HPhA/p53-treated group than the untreated, pCMV/p53 alone or Lipofectamine/p53-treated group with statistical significance (P<0.05). Thus, HPhA-mediated functional wild-type p53 protein expression effectively inhibited the cellular growth by p53-mediated apoptotic pathway.

Figure 5
figure5

(a) Flow cytometric analysis of induction of apoptosis of CNE cells treated with different carriers mediated the transfection of wt-p53 gene after 48 h. (b) The apoptotic and necrotic cell populations were determined using detection of annexin V–FITC- and PI-labeled cells, respectively. Group 1, control; group 2, pCMV/p53 plasmid alone; group 3, HPhA-mediated pCMV/p53 transfection; group 4, Lipofectamine-mediated pCMV/p53 transfection; annexin V–FITC-labeled cells (); PI-labeled cells (). FITC, fluorescein isothiocyanate; PI, propidium iodide; HPhA, an archaeal histone-like protein.

Figure 6
figure6

Percentage of G1 phases of CNE cells treated with different transfection complex after 48 h. Group 1, control; group 2, transfected with pCMV/p53 plasmid alone; group 3, HPhA-mediated pCMV/p53 transfection; group 4, Lipofectamine-mediated pCMV/p53 transfection. Results are expressed as mean values of three independent experiments (s.d.). *P<0.05. HPhA, an archaeal histone-like protein.

Inhibition of tumor growth in vivo

To evaluate whether the in vitro observations described above can translate into similar in vivo response, CNE tumor cells were xenografted subcutaneously in female BALB/c nude mice. After 7 days, the mice were randomly divided into four groups (untreated, pCMV/p53 alone, lipofectamine/p53 or HPhA/p53) of six animals. Tumor volumes were recorded every 5 days. No significant inhibition of tumor growth was observed in untreated mice. In contrast, the inhibition rates of tumor growth were 3.23% (pCMV/p53), 13.26% (Lipofectamine/p53) and 41.23% (HPhA/p53), as shown in Figure 7. The HPhA/p53 group exhibited a statistically significant decrease in tumor growth as compared with Lipofectamine/p53 and plasmid alone (P<0.01). Data from these animal experiments showed that HPhA-mediated p53 gene transfer was capable of suppressing the growth of established tumors derived from human p53-deleted cancer cells.

Figure 7
figure7

Tumor volume analysis of tumor-bearing mice after intratumoral injection of various agents. Each group included six mice. Administration was started on day 0 and repeated on days 5, 10, 15, 20 and 25, for a total of six times. Tumor growth was followed over 30 additional days.

Discussion

In the present study, we evaluated the effect of HPhA carrier system-mediated p53 gene therapy in a p53-deleted nasopharyngeal carcinoma cell line in vitro and in vivo. The results showed a significant efficiency of gene transfer and gene expression. Higher transfection of HPhA/p53 complexes in CNE cells seemed to be responsible for higher expression of p53 mRNA as shown in Figure 2. Serum did not exert any inhibition of the transfection mediated by HPhA. The HPhA/p53 complexes could improve the stability of plasmid DNA in the presence of serum.5, 14 In contrast, many cationic lipid-based transfection systems were inactivated by medium containing as low as 5–10% serum.16, 17 Thus, the higher transfection efficiency of plasmid DNA may be attributed to the enhanced stability of plasmid HPhA/DNA complexes. Moreover, the enhanced mRNA expression of p53 may increase the protein levels of p53 in the cells transfected with HPhA/p53 complexes compared to the cells treated with Lipofectamine complexes or the plasmid alone (Figure 3).

Efficient cell growth inhibition by HPhA/p53 complexes could be due to the increase of the expression of p53 proteins as well as due to the effect of histones on the proliferation of CNE cells. When using histones as DNA vectors, excess of the added histones could potentially interfere with cellular histones and play regulatory function in transcription and slowed the cell cycle progression.18, 19, 20, 21 Flow cytometric analysis suggested that the cytotoxic effect of HPhA/p53 was the result of apoptosis and cell cycle arrest. Encouraging results were also obtained in studies using tumor xenografted nude mice. Local HPhA/p53 treatment by intratumoral injection led to a significant inhibition of tumor growth consistent with the in vitro effect of HPhA/p53 on human cancer cells. Thus, it was supposed that functional exogenous p53 could be efficiently delivered in cancer cell lines using HPhA as a non-viral vector.

Because the functional loss of p53 gene was now well recognized as the most common event in carcinogenesis, it was not surprising that wild-type p53 gene introduction had recently been used as a cancer gene therapy. Many investigations and clinical trials using transfer of viral genes encoding wt-p53 had been performed.22, 23, 24 Viral vectors, such as genetically engineered adenovirus, had been shown to be capable of gene delivery both in vitro and in vivo. However, they still suffered from some problems in clinical use. On the other hand, non-viral gene transfer systems such as lipoplexes, polyplexes, calcium phosphate and electroporation have been developed.25, 26 The potential advantages of protein and peptide gene transfer were easy to produce and use, high purity and homogeneity, ability to target nucleic acids to specific cell types, attachment of targeting ligands and lack of limitation to the size or type of nucleic acid that could be delivered.9

Efficient gene delivery is one of the most important tasks in current non-viral gene therapy research. An ideal vector may find its application for in vivo and in vitro transfection of cells or grafts for transplantation and for various protocols of a localized gene therapy. Nuclear proteins, such as histone-mediated gene transfer system might be an ideal system for gene transfer. Histones, which were natural DNA binding and condensing proteins, offered good advantages to serve as a safe and efficient gene delivery tool. Histones had been demonstrated to have higher efficiency of gene transfer compared with other non-viral transfer systems.8, 12 Therefore, it was thought to be one of the most attractive alternative vectors.

HPhA, an archaeal histone-like protein, shares the properties of eukaryal histones: small, basic, toroidally wrap DNA, protect DNA from nuclease digestion and/or form nucleosome-like structures. These features make it potential carrier of gene deliveries. Another important feature that supports interest in HPhA for gene delivery is its low cytotoxicity. One of the major obstacles to effective non-viral gene delivery is vector cytotoxicity. Microscopic examination of cells treated with Lipofectamine revealed gross morphological alteration, cell disruption and reduction in viability, while the HPhA was less toxic and better tolerated than the Lipofectamine-based transfection reagents used.14 Combining with the advantages of higher transfection efficiency and lower toxicity, HPhA may serve as a good alternative tool for gene delivery.

In summary, this study was designed to evaluate the efficiency and potential applicability of HPhA carrier system-mediated p53 gene therapy. The results showed that the HPhA-mediated transfection of tumor suppressor gene p53 was effective in inducing apoptosis and inhibiting tumor growth. Our results suggested that the HPhA-mediated p53 delivery might have the potential for clinical application of non-viral vector-mediated cancer therapy.

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Acknowledgements

We are grateful to Li-Xue Shi for her helpful contributions. This study was supported by grants from National Natural Science Foundation of China (30571642) and the 973 programs (2004CB719606).

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Correspondence to X X Fang or Y Feng.

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Li, Y., Wang, R., Zhang, G. et al. An archaeal histone-like protein mediates efficient p53 gene transfer and facilitates its anti-cancer effect in vitro and in vivo. Cancer Gene Ther 14, 968–975 (2007) doi:10.1038/sj.cgt.7701086

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Keywords

  • p53
  • transfection
  • histone-like protein
  • non-viral gene transfer

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