British Journal of Radiology 75 (2002),657-662 © 2002 The British Institute of Radiology
The combination of ionizing radiation and expression of a wild type p53 gene via recombinant adenovirus induced a prominent tumour suppressing effect in human oral squamous cell carcinoma
T Wakasa, DDS, DDSc1,
T Inoue, DDS, DDSc2,
N Kawai, DDS1,
J Murakami, DDS1,
K Kishi, DDS, MD1 and
K Fukui, DDS, MD2
Departments of 1 Oral and Maxillofacial Radiology and 2 Oral Microbiology, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama City, Okayama 700-8525, JAPAN
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Abstract
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Human oral squamous cell carcinoma (SCC) cell lines HSC4 and SAS were infected with wild type p53 (wt-p53)-encoding adenovirus (AxCAip53) and subsequently irradiated to investigate the effectiveness of p53 gene therapy in combination with radiation therapy for treating oral SCC. Western blot analysis using anti-p53 monoclonal antibody showed that a large amount of mutant p53 protein was accumulated in HSC4 cells, while no detectable p53 protein was observed in SAS cells. The induction of p53 expression by AxCAip53 infection was clearly observed in both HSC4 and SAS cells. A clonogenic cell survival assay demonstrated that AxCAip53 infection alone, or X-irradiation alone, significantly inhibited the growth of cancer cells, but that combined treatment was most effective, even in mutant p53-accumulated HSC4 cells. Flow cytometric analysis showed that the apoptotic pathway was induced in virus treated and radiation treated cells. Taken together, these findings suggest that the combination of p53 gene therapy and radiation therapy has a possibility to effectively treat oral SCC defective in p53 function.
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Introduction
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Oral cancers, which are mostly squamous cell carcinoma (SCC), have generally been treated by a combination of operation, radiation therapy and chemotherapy. However, surgical resection of tumours frequently causes profound defects in oral function, such as speech and swallowing, and in cosmetics. Radiation therapy has a great advantage in that patients' oral function and/or facial configuration can be well preserved. Therefore it is critical to reduce tumour size by radiation therapy to minimize damage to oral function.
Recently, gene therapy has received much attention. Non-replicating adenoviruses expressing the tumour suppressor gene p53 have been shown to suppress the growth of cancer cells in vitro and in vivo [1–3]. Furthermore, several reports have indicated that the combination of radiation therapy and adenovirus-mediated p53 expression has synergistic suppressive effects on various cancer cells, including colorectal [4], ovarian [5], nasopharyngeal [6] and head/neck [7] cancer cells.
In SCCs of the oral cavity, alternation of p53 production was reported to range 20–50% of cell lines and tumour tissues [8, 9]. Here, we investigated the effects of p53-expressing adenovirus transfection on radiation treatment in two human oral SCC cell lines defective in the p53 allele.
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Materials and methods
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Cells and culture conditions
Human oral SCC cell lines HSC4 and SAS, human embryonic fibroblast cell line TIG 3–20, and human embryonic kidney cell line 293 were obtained from the Cell Resource Center for Biomedical Research Institute of Development, Aging and Cancer, Tohoku University (Japan). Cells were maintained in Dulbecco's modified Eagle's medium, supplemented with 10% fetal bovine serum.
The experiments in this study were conducted when the cells were in an exponential phase of growth.
Recombinant adenovirus preparation and infection
The plasmid pProSp53 (registration form: YG-CO104) encoding full length wild-type p53 was obtained from the Japanese Collection of Research Bioresources gene bank (Japan). Full length p53 DNA was amplified by polymerase chain reaction with a pair of primers (5'-ATGGAGGAGCCGCAGTC-3', 5'-TCAGTCTGAGTCAGGCCCTTCT-3') and pProSp53 as a template.
Recombinant p53-expressing adenovirus was prepared using an adenovirus expression vector kit (Takara, Otsu, Japan), which was based on the DNA-terminal protein complex (DNA-TPC) method [10]. Briefly, the amplified p53 DNA was inserted into the Swa I site of the cosmid vector pAxCAwt. The resulting recombinant cosmid was packaged into phage lambda heads and transduced into E. coli VCS257 cells. Of the colonies grown on LB agar plates, the clones containing an adequate insert were selected. One of the isolated recombinant cosmids was designated pAxCAip53. The nucleotide sequence of p53 DNA in pAxCAip53 was then confirmed. To obtain a recombinant adenovirus encoding the p53 gene, the pAxCAip53 and DNA-TPC were cotransfected to 293 cells using the calcium phosphate method [11]. One of the recombinant adenovirus clones showing an appropriate restriction pattern was designated AxCAip53, and propagated in 293 cells to obtain a high titer virus solution. The control adenovirus AxCAiLacZ expressing ß-galactosidase was also prepared using pAxCAiLacZ and the method described above. Virus titers were estimated as 50% tissue culture infection dose (TCID50) [12], which corresponds to plaque forming units (PFUs) per millilitre (PFU ml-1).
Detection of ß-galactosidase expression
To evaluate the efficiency of gene expression from the infected adenovirus vector, ß-galactosidase activity was determined in AxCAiLacZ-infected cells using a quantitative galactosidase assay [13]. Following 24 h incubation, cells were treated with lysis buffer and Z-buffer, and reacted with ortho-nitrophenyl-ß-D-galactopyranoside solution. The OD415 values from the samples were then converted to ß-galactosidase activity using the standard curve and expressed as ß-galactosidase activity units per 105 cells.
Western blot analysis
24 h after AxCAip53 infection, cells were exposed to 2 Gy of X-irradiation in an X-ray generator (MBR-1520; Hitachi, Tokyo, Japan) (dose rate 2 Gy min-1). Cells were collected by centrifugation and treated with radioimmunoprecipitation buffer. The protein concentration was determined using the detergent compatible protein assay kit (Bio-Rad Laboratories, Hercules, CA). Samples containing 50 µg of protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and proteins were transferred to a nitrocellulose membrane using a semi-dry transfer kit (LKB, Sweden). Membranes were probed with a monoclonal anti-human p53 antibody (DO7; Novocastra, Newcastle-upon-Tyne, UK). Blots were incubated with a secondary antibody conjugated with horseradish peroxidase (Lsab kit; DAKO, Carpinteria, CA). Reactive bands were detected using Tris-HCl buffer containing 0.05% DAB.
Clonal survival assay after irradiation
24 h after infection, cells were irradiated with 0 Gy, 1 Gy, 2 Gy, 3 Gy, 4 Gy or 6 Gy using an X-ray generator. Immediately after irradiation, cells were re-plated in 60 mm petri dishes, in triplicate, and incubated at 37°C under 5% carbon dioxide for 14 days. Cultures were then washed, fixed and stained with Giemsa's solution, and the number of colonies counted.
Flow cytometry (apoptosis analysis)
24 h after infection, cells were exposed to X-irradiation with 2 Gy. At certain time points cells were treated with trypsin, pelleted and resuspended in binding buffer on ice. For fluorescence staining, cells were labelled with Annexin V-fluorescein isothiocianate (Annexin V-FITC kit; Bender MedSystems, Vienna, Austria), and propidium iodide (PI) on ice in the dark for 10 min, and analyzed by a flow cytometer (Epics XL; Beckman Coulter, Fullerton, CA). Flow cytometric analysis was repeated three times for each cell group. Data were obtained using System II software (Beckman Coulter, Fullerton, CA).
Statistical analysis
Analysis of variance was used for statistical analysis and confirmed using the F-test, with values of p<0.05 considered to indicate statistical significance.
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Results
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Quantitation of ß-galactosidase activity in AxCAiLacZ-infected cells
As indicated in Figure 1
, ß-galactosidase activity per unit cell increased proportionally with increasing multiplicity of infection (MOI) of AxCAiLacZ to HSC4 or SAS cells, in the range of 5–25 MOI. MOI is defined as the value of PFU per cell, when cells were infected with the virus. HSC4 cells required approximately two-fold higher MOI of AxCAiLacZ to achieve the same level of ß-galactosidase activity when compared with SAS cells. Therefore, the two cell lines were infected with iso-effective MOIs of either AxCAip53 or AxCAiLacZ (20 MOIs for HSC4 cells and 10 MOIs for SAS cells) in all the following experiments.
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Figure 1. ß-galactosidase (ß-gal) activity in AxCAiLacZ-infected HSC4 and SAS cells. Cells were infected with AxCAiLacZ at multiplicity of infection (MOI) ranging from 0–25 plaque forming units per cell (pfu/cell). After 24 h, cells were lyzed and ß-galactosidase activity was determined. One unit of ß-galactosidase is defined as the amount of enzyme that will hydrolyze 1 µmol of ortho-nitrophenyl-ß-D-galactopyranoside in 1 min at 37°C.
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Detection of p53 production by Western blotting
As shown in Figure 2, mock-infected normal fibroblast TIG 3-20 cells produced a small but fully detectable amount of p53 protein. In mock-infected SAS cells, no p53 band was detected. However, a band with smaller molecular size than p53 appeared in all samples from the SAS cell line, and was more clearly detected in AxCAip53-infected cells. In contrast, mock-infected HSC4 cells showed a prominent p53 band, probably containing mutated p53 proteins. There was no significant increase of p53 level at 24 h following exposure to X-irradiation in all cell lines. However, when infected with AxCAip53, an increase of p53 production was clearly observed in both HSC4 and SAS cells.
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Figure 2. Detection of p53 production in the HSC4 and SAS cells by Western blot analysis using p53-specific monoclonal antibody. Cells were mock- or AxCAip53-infected and subsequently irradiated with 2 Gy. 24 h after irradiation, cell lysates were prepared. 50 µg of protein was loaded in each lane.
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Effects of AxCAip53 infection on radiosensitivity of HSC4 and SAS cells
Radiosensitivity of HSC4 and SAS cells infected with AxCAip53, AxCAiLacZ or mock-infected, was assessed using the clonogenic assay (Figure 3). Although the survival fraction of AxCAiLacZ-infected cells was slightly lower than that of mock-infected cells, the difference was not significant in any dose (p<0.05). The survival fraction decreased to 8.2% in HSC4 cells and 15.4% in SAS cells, following 2 Gy of irradiation. The two cell lines mock-infected or infected with AxCAiLacZ showed overlapping clonogenic survival levels in response to irradiation; the D0 value was 1.84 for mock-infected HSC4 cells, 1.88 for mock-infected SAS cells, 1.82 for AxCAiLacZ-infected HSC4 cells and 1.86 for AxCALacZ-infected SAS cells. In contrast, the fraction of AxCAip53-infected cells that survived irradiation was significantly lower (p<0.05) than that of AxCAiLacZ-infected cells in both cell lines in any dose (Figure 3). Furthermore, the slope of the survival curve for AxCAip53-infected cells was significantly higher than that for AxCAiLacZ-infected cells; the Do value for AxCAip53-infected HSC4 cells was 1.02, and that for AxCAip53-infected SAS cells was 1.41.
Flow cytometric analysis
The results of flow cytometric analysis are shown in Figure 4. Annexin V-FITC protein connects to phosphatidyl serine (PS), which is released in the early stage of apoptosis [14]. PI is incorporated into necrotic cells but not into apoptotic cells. The combination of Annexin V-FITC and PI enables the differentiation between apoptosis and necrosis. In Figure 4, zone 2, zone 3 and zone 4 indicate necrotic cells, viable cells, and apoptotic cells, respectively.
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Figure 4. Flow cytometric analysis of HSC4 and SAS cells treated with AxCAip53 and irradiation. (a–d), HSC4 cells; (e–h), SAS cells. (a) and (e), mock-infected, non-irradiated cells; (b) and (f), mock-infected, irradiated cells; (c) and (g), AxCAip53-infected, non-irradiated cells; (d) and (h), AxCAip53-infected, irradiated cells.
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The percentages of mock-infected, non-irradiated cells that were apoptotic were 4.07±0.90 (mean±standard deviation)% in HSC4 cells and 4.60±1.22% in SAS cells. The percentages of mock-infected, irradiated cells that were apoptotic were 5.47±1.52% in HSC4 cells and 6.70±1.65% in SAS cells. Those of AxCAip53-infected, non-irradiated cells were 5.10±0.63% in HSC4 cells and 4.77±1.25% in SAS cells, and those of AxCAip53-infected, irradiated cells were 14.1±1.27% in HSC4 cells and 14.63±1.19% in SAS cells.
The combination of AxCAip53 infection and 2 Gy irradiation resulted in a significantly higher percentage of apoptotic cells compared with either AxCAip53 infection or irradiation alone (p<0.05).
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Discussion
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To date, the function of the p53 gene has not been fully understood. However, its product is known to be assembled into a tetramer and to function as a transcriptional factor that is involved in determining cell cycle arrest to repair DNA damage or cell death through the apoptosis pathway when cells are exposed to DNA-damaging agents [15]. Mutations or deletions have frequently been found in the p53 gene in a wide variety of human cancer cells, including oral cancers [16]. The reconstitution of wt-p53 function with p53-expressing adenovirus has been reported to have a significant tumour suppressing effect on various cancer cells [4–7], and it has been suggested that this improves the effects of radiation therapy through activation of the apoptotic pathway [17].
In this study, we examined in detail the effects of adenovirus-mediated p53-expression on X-irradiation of two oral cancer cell lines, HSC4 and SAS. HSC4 cells are known to have a mutation in one of the p53 alleles (a transition from guanine to adenosine at codon 248), resulting in production of a mutant p53 protein (amino acid change from arginine to glutamic acid) [18]. SAS cells also have a point mutation in one p53 allele (at codon 336 of exon 10), but this mutation causes no amino acid change [19]. In the Western blot shown in Figure 2
, HSC4 cells were found to produce a large amount of p53 protein, which may represent an accumulation of mutant p53 protein. In cancer cells, mutant p53 protein is known occasionally to accumulate owing to a decrease in p53 turnover [20]. In contrast, SAS cells had no detectable p53 protein, even under X-irradiation, which is a condition for induction of p53 expression. Instead, a band with smaller molecular size than p53 was observed in SAS cells. This band was more clearly detected in AxCAip53-infected cells. These findings indicate that degradation of p53 protein might be accelerated within SAS cells for unknown reasons.
Compared with mock-infected or AxCAiLacZ-infected cells, AxCAip53-infected HSC4 and SAS cells showed a significantly lower survival fraction, suggesting that the combination of adenovirus-mediated wt-p53 gene expression and X-irradiation could possibly treat oral SCCs effectively. In other reports [4–7] there was a reduction of at least 50% in the surviving fraction after 2 Gy (SF2) value when cancer cells were infected with wt-p53 expressing adenovirus. In the present study, we observed a similar reduction of SF2 values when cells were infected with AxCAip53 (Figure 3); a 58.6% reduction in the HSC4 cell line and a 51.8% reduction in the SAS cell line. Analysis of virus and irradiation treated cells by flow cytometry suggested that the apoptotic pathway was activated by these treatments.
In cancer cells, one p53 allele is frequently mutated, while another p53 allele is not. In this situation, the tetramer formed by mutant and normal p53 proteins fails to work correctly as a tumour suppressor factor, a phenomenon known as the dominant-negative effect [20]. Although in HSC4 cells a large amount of mutant p53 protein was accumulated, the radiation effect was improved by AxCAip53 infection, indicating that adenovirus-mediated wt-p53 gene expression can reduce tumour size even in oral SCC showing accumulation of the mutant p53 protein. In particular it should be noted that the slope of survival curve in AxCAip53-infected HSC4 cells was clearly higher than that in AxCAiLacZ-infected HSC4 cells. This may suggest that the radiation sensitivity of HSC4 cells was enhanced by p53 expression through activation of the apoptotic pathway, as reported in other cancer cells by Kastan et al [15].
In conclusion, our findings indicated that the combination of adenovirus-mediated wt-p53 expression and X-irradiation could be an effective treatment of oral human SCCs. However, experimental growth cycling cells were used in this study. In clinical cases, which contain high proportions of non-cycling cells, the strategy proposed in this study may not always be applicable. Further in vitro and in vivo studies will be needed to find an optimum therapeutic protocol for this combination therapy in order to treat oral cancers effectively and safely.
Received for publication November 12, 2001.
Revision received February 7, 2002.
Accepted for publication February 20, 2002.
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Abstract
Introduction
—, mock-infected cells; —
—, AxCAiLacZ-infected cells; —
—, AxCAip53-infected cells.