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Effect of Exogenous Wild-Type P53 on Melanoma Cell Death Pathways Induced By Irradiation at Different Linear Energy Transfer

Posted on: Friday, 20 January 2006, 06:00 CST

By Min, Feng-Ling; Zhang, Hong; Li, Wen-Jian; Gao, Qing-Xiang; Zhou, Guang-Ming

SUMMARY

We investigated the effect of exogenous wild-type p53 on the radiation-induced cells apoptosis and necrosis at different levels of linear energy transfer (LET) to evaluate its mechanisms. The human melanoma cell line A375, which bears wild-type p53 gene status, was used, as well as the transfectant A375 cells (A375/p53) with adenoviral vector containing the wild-type p53 gene. We exposed these cells to X-rays and to accelerated carbon-ion (C-) beams. Cellular sensitivities were determined by using clonogenic assay. Apoptotic and necrotic cell deaths were determined morphologically by dual staining (acridine orange and ethidium bromide) using fluorescence microscopy. We discovered that (1) there was no significant difference in survival fraction between A375 cells and A375/p53 cells irradiated by C-beams with greater than 32 KeV/,m LET, (2) although apoptosis in the two kinds of cells increased in an LET-dependent manner, exogenous wild-type P53 induced cell apoptosis efficiently in A375/p53 relative to A375 cells with X- rays or high-LET irradiation, and (3) by high-LET irradiation, the number of necrosis in A375 cells increased significantly (P < 0.05) in comparison with A375/p53 cells. These results indicate that in high-LET irradiation apoptosis induction is p53 dependent partly and exogenous wild-type P53 plays an important role in modulating cell death type, although there was no significant difference in cellular radiosensitivities. Our observation in the study offers the potential application of high-LET radiation combined with p53 in the management of human patients with melanoma.

Key words: p53 gene; carbon-ion beams; LET; cell death; melanoma.

INTRODUCTION

Although traditional X- and γ-rays treatment is still the main therapy method for a wide variety oi malignant human cancer at present, the effect of different cancer cells to radiotherapy is greatly different. High linear energy transfer (LET) charged particle radiation offers several potential merits over traditional X- and γ-rays. These merits are as follows: (1) a reduction in the oxygen enhancement ratio with proportionately greater killing of hypoxic cells, (2) less variation in cell cycle-related radiosensitivity, and (3) less capability for radiation-induced deoxyribonucleic acid (DNA) damage repair. High-LET radiation induces more cancer cells apoptosis resulting from much more severe damage to DNA molecules compared with that by low-LET radiation (Asakawa et al., 2002). Some cancer cells with radioresistence to X- rays turned very sensitive to high-LET radiation-induced cell killing (Iwadate et al., 2001). In Japan and Germany, more than 1000 patients with chordomas, low-grade chondrosarcomas of the skull base, unfavorable adenoid cystic carcinomas, and other cancers were treated with carbon ion radiotherapy. The results are promising and carbon ion therapy is safe with respect to toxicity and offers high, local control rates (Nakano et al., 1999; Kamada et al., 2002; Schulz-Ertner et al., 2003, 2004). The melanoma, coming from the melanocyte in the skin, has high metastatic potential. Although melanoma cells are usually detected as wild-type p53 gene status, they have a wide range of resistance to radiation and are characteristically unresponsive to conventional chemotherapy (Xu et al., 2002). The reasons why melanoma is radioresistant and reiractory to chemotherapeutic agents may he a reduced ability to undergo apoptosis after treatment (Soengas et al., 2001). Cancer suppression gene p53 is well known that which involved in the cells apoptosis via DNA damage induced by X- and γ-rays (Tsuboi et al., 1998). However, the document about LET dependence of p53 activity is sparse, and the information on the relationship of the p53 status of cancer cells and their effect on cell death after exposure to high-LET radiation is not unanimous as yet (Asakawa et al., 2002; Oohira et al., 2004). In this study, we examined how the exogenous wt p53 gene transferred into human melanoma cell line A375 (wild-type p53) affects the cell death type such as apoptosis and necrosis after treatment with different LET radiation because such information will be a valuable input to the understanding of the mechanisms underlying an apparent association between DNA damage induced by different radiation modalities and subsequent cell kill. In this study, an accelerated carbon-ion beam (C-beam) generated by the heavy-ion accelerator at the Institute of Modern Physics, Chinese Academy of Science, was used for our experiment.

MATERIALS AND METHODS

Cells and gene, transduction. Human melanoma cell line A375 obtained from Chinese Medical University (confirmed to he wild-type p53) was grown up in Roswell Park Memorial Institute 1640 medium supplemented with 10% fetal bovine serum (FBS, GIBCO-BRL, Carlsbad, CA). Both p53 recombinant adenoviral vector (Ad5CMV-p5-3) and its control recombinant adenoviral vector (AdSCMV-GFP) are replication- deficient adenovirus vectors, which contain recombinant human wt p53 and green fluorescent protein (GFP) gene, respectively. The A375 cells were exposed to the graded doses of AdCMV-GFP infection (0- 200 multiplicity of infection). Gene transfer efficiency was measured according to the percentage of GFP-positive cells using a fluorescence microscope. Appropriate viral vector dosages were chosen according to the gene transduction efficiency of AdSCMV-GFf The p53 gene was transduced into A37S cells by AdCMV-p53 vector.

The p53 messenger ribonucleic acid and P53 expression analysis. The reverse transcriptase-polymerase chain reaction (RT-PCR) was used to examine the effects of Ad5CMV-p5.3 infection on the expression of the messenger ribonucleic acid (mRNA) of p53. TRIzol (life Technology) was used to extract the total RNA from the cells. The complementary DNA was composed with OligdT primer. All the required reagents were obtained from Promega. The polymerase chain reaction (PCR) primers were obtained from Sangon Shanghai Bio Co. (Shanghai, China) p53 (expanded products 310 bp): sense (CMV3): 5'- GGT GCA TTG GAA CGC GGA TT-3; antisense (p53 exon 8): 5'-CAA ATC ATC CAT TGC TTG GGA-3'. The PCR reaction conditions were as follows: 94 C, 2 min; 94 C, 1 min; 60 C, l min; and 72 C, 5 min; 36 cycles. The PCR products were separated by electrophoresis with 1% agar gel and then scanned by the fluorescent imaging system. The level of P53 expressions on AdSCM V-p.5.3-A375 cells was assessed by flow cytometry. Basically, A375 and A375//)53 were washed twice with cold phosphate-buffered saline (PBS), then fixed with 4% formaldehyde. They were hatched in PBS containing 0.1% (w/v) Tween 20 and 3% (w/ v) bovine serum albumin for 10 mm, then washed twice with cold PBS. Subsequently,l:100 diluted P53 Ab-I (mouse monoclonal antibody, DO- I) was added and left overnight before the cells were washed twice again. Then, 1:100 immunoglobulin G (SouthernBiotech; Southern Biotechnology Associates Inc., Birmingham, AL) conjugated fluoresoein isothiocyanate was added and exposure to sunlight avoided. The measurements of cells were made with flow cytometry (FAC-Scan), 30 min after the washing.

Sample radiation. The cells were exposed to X-rays or carbon-ion (C-) beams in 24 h after Ad5CMV-/)53 vector infection. For X-ray radiation, cells were exposed to an X-ray machine, 200 kVp, 19.0 mA, with 0.5-mm Cu and 0.5-mm Al shielding. The exposure rate was approximately 400 cGy/min. Carbon ions were accelerated by a heavy- ion accelerator at Institute of Modern Physics, Chinese Academy of Science, up to 80.55 MeV/u. Dosimetry of the beam at the biological experiment port was carried out by two independent methods: one using an ionization chamber and the other using a plastic scintillator for fluence measurement. The LET values were obtained by fitting the Bragg curve obtained by measurement to an already established theoretical reference. Plastic film absorbers were used to reduce the beam energy to obtain optimum LET beams. Initial energy of 80.55 MeV/u C-beams with different LET values selected for the experiments were 32, 70, 100 KeV/m. Cells attached in 10-cm^sup 2^ dishes were situated in a specially designed rack to correctly position them in the uniform exposure field of the beam. Irradiation was conducted using horizontal C-beams with a dose rate of approximately 2 Gy/min.

Clonogenie survival assay. Immediately after exposure to various doses of either carbon beams or X-rays, cells were trypsinized and counted with an electronic particle counter. Cell suspensions were plated in 60-mm diameter dish with various types of density in accordance with cytotoxicity of this therapy. Fourteen days later, plates were fixed and stained by the addition of 0.01% crystal violet in 1.5% acetic acid. Colonies containing more than 50 cells were scored. The surviving fraction values were fitted by a single- hit multitarget model: S/S^sub 0^ = 1 - (1 - e^sup -D/DO^)n, where S/ S^sub 0^ is a surviving fraction and D is the dose (Gy). The parameter n and DO were calculated by Kaleida Graph software.

Determination of apoptotic, necrotic, and live cells. The relative percentages of apoptotic, necrotic, and live cells were determined using a well-established assay.For Hoechst staining of cell nuclei, attached cells were harvested by trypsinization, combined with the floating cell population, fixed with 1% glutaraldehyde in PBS at 4 C, washed with PBS, and resuspended in 20 l PBS containing 8 g/ml Hoechst 33258 (Sigma Chemical Co., St. Louis, MO). After 15 min at room temperature in the dark, the cells were mixed with an equal volume of mowiol, mounted on glass slides, and investigated by fluorescence microscope. The detection and quantification of apoptosis and necrosis were also evaluated and compared by acridine orange-ethidium bromide (AO-EB) double staining. In brief, 1 l of dye mixture (100 μg/ml AO and 100 g/ ml EB) diluted in PBS was mixed gently with 24 l of cell suspension (1 10^sup 6^ cells/ml) and put onto a clean microscope slide. The morphologies of the cells were viewed under fluorescent microscope. A viable cell possesses a uniform bright green nucleus and orange cytoplasm. An early apoptotic cell still has a green nucleus, but its chromatin becomes condensed and manifests bright green patches. A late apoptotic cell shows bright orange areas of condensed chromatin in the nucleus, and a necrotic cell manifests uniform bright orange nucleus. At least 500 cells were counted in each slide.

FIG. 1. (a) The RT-PCR analysis mRNA expression of p53 gene in A375 cells transfected by AdCMV-p55 viral vector. M, marker; Lane 7, A375/p53 cells; lane 2, uninfected A375 cells, (b] Assay of P53 expression in A375 cells 24 h after AdCMV-p53 vector infection by flow cytometry (FACS).

RESULTS

Analysis of transfectants. We analyzed p53 gene mRNA by RT-PCR method to determine whether the Ad5CMV-p53 vector was expressed in A375 cells. As shown in Fig. Ia, after A375 cells were infected by Ad5CMV-/;53, p53 mRNA expressed instead of in uninfected A375 cells. Figure 16 illustrates the results of activation of P53 protein by flow cytometry analysis. The P53 expressed highly at 76.76% on d 1 after Ad5CMV-p53 infection, whereas uninfected A375 cells had no P53 expression.

Surviving fraction after X-rays or C-beam irradiation. Survival curves for X-rays or C-beams are shown in Fig. 2. The radiosensitivity of A375/p53 cells against X-rays (SF2) was about 1.4-fold (P < 0.05) higher than that of A375 cells. The D30 (the dose required to reduce survival to 30%) in A375 and A375/p53 was 7.6 and 4.2 Gy for X-rays, respectively. Although pronouncedly shoulders were observed on the curves with X-rays and 32 KeV/m C- beams, the shoulder was reduced and the curve became steeper as the LET value increased. The C-beams with 70 and 100 KeV/m LET values showed exponential dose responses. In A375 and A375/ p53 cells, there was a modest difference in survival curves for Cbeams with 32 KeV/m LET value. However, there was no significant difference in survival between A375 cells and A375/p53 cells irradiated by C- beams with 70 and 100 KeV/m LET values. The relative biological effectiveness (RBE) of C-beams against X-rays was calculated for the surviving fraction of clonogenic cells after 2 Gy irradiation (SF2), ranging from 1.29 (32 KeV/m) to 2.37 (100 KeV/m) in A375 cells, whereas A375/p,53 cells showed low RBE, from 1.1 (32 KeV/m) to 1.68 (100 KeV/m).

FIG. 2. Colony-forming ability of A375 and A375/p53 cells after exposure to graded dose of X-rays and carbon beams radiation at different linear energy transfer. Left panel: A375 cells. Right panel: A.375/p53 cells. X-ray: ([black square]), 32 KeV/m; ([black circle]), 70 KeV/m; ([black triangle up]), 100 KeV/m ([black triangle down]).

FIG. 3. Effect of treatment with X-rays or 70 KeV/m carbon beams at isosurvival dose (D30) irradiation for various times up to 96 h on the apoptosis of A375 and A375/p53 cells. The A375 irradiated with X-ray ([black square]) A375 irradiated with 70 KeV/m carbon beams ([white square]); A375/p53 irradiated with X-ray ([black triangle up]); A375/p5.3 irradiated with 70 KeV/m carbon beams ([white triangle up]).

FIG. 4. The relative number of apoptotic and necrotic cells 48 h after exposure to graded dose of different linear energy transfer (LET) irradiation. The LET of X-rays was taken to be 1.5 KeV/m. The pair bars in every LET irradiation represent the percentage of apoptosis and necrosis in A375 cells (left) and that in A375/p5.3 cells (right), respectively. Each date was the mean of three different experiments.

Time-dependent apoptosis after low- or high-LET irradiation. We have made time-course studies of apoptosis induction at various time points after exposure to different LET radiation at isosurvival dose (D30) by Hoechst 33258 staining (Fig. 3). The peak of apoptosis fraction was at 48-72 h after irradiation with low LET or high LET. Although a difference was observed in the percentage of apoptosis induction in the two kinds of cells, the kinetic patterns of apoptosis after treatment with low-LET and high-LET radiation were alike. The 70 KeV/m C-beams irradiation induced the higher proportion of apoptosis in A375/p5,3 than that in A375 cells. In contrast, low-LET radiation-induced apoptosis was observed more frequently in A375/J0.53 cells than in A375 cells. The apoptosis in A375//)53 cells was about 3.3- and 1.3-fold higher than that of A375 cells 72 h after isosurvival dose (D30) X-rays or 70 KeV/m C-beams exposure.

Apoptotic versus necrotic cell death. Both A375 cells and A375/ p53 cells exhibited increasing number of apoptotic and necrotic cells in a dose-dependent and LET-dependent manner. Figure 4 showed the percentage of apoptotic and of necrotic in the two kinds of cells 48 h after different irradiation treatment. In A375//>53 cells, the apoptotic cells increased distinctly relative to A375 cells after irradiation with X-rays, although there was no significant difference in necrotic cells. In contrast, with similar total dead cells fraction, the proportion of apoptosis and necrosis composing in A375 cells was different from that in A375/p53 cells after C-beams with a greater than 32 KeV/m LET radiation. The A375 cells showed similar proportion in the two parts of apoptosis and necrosis, whereas the apoptotic cells predominated over necrotic cells in the A375/ p53 cells. The percentages of apoptosis and necrosis after irradiation with C-beams of 70 KeV/m at 4 Gy in A375 cells were 10.8 versus 10.2% and in A375/p53 cells, 15.8 versus 5.6%.

DISCUSSION

Melanoma, with the characteristic of high malignance, often occurring in skin, and conventional radioresistance, is therefore quite a suitable candidate for receiving high-LET radiotherapy. The fact that melanoma cells with wild-type p53 gene status insensitive to radiation might relate to the defects in some phosphokinase and phosphorylase that result in lack of protein activation from the endogenesis wild-type P53 (Satyamoorthy et al., 2000). Therefore, we selected a radioresistant human melanoma cell line A375 (wt p53) with transfection of exogenous wild p53 gene by adenoviral-mediated gene transfer for experiment. The A375 cell line can be infected efficiently by AdCMV-p.5.3 vector, constitutively expressing large amounts of P53. Although there was no significant apoptosis induced by AdCMV-p,5,3 infection treatment alone (data not shown), more A375/ ju53 cells were arrested in Gl phase (data not shown) and had lager amount of apoptotic cells than A375 cells when irradiated by X- rays. The results indicated that cell apoptosis may depend on the exogenous function of P53. In other words, DNA damage can sensitize the cells to p,53-mediated apoptosis (Sah et al., 2003). These results were different from those of Satyamoorthy et al. (2000) in that both endogenous and ectopically expressed wild-type p53 are functionally defective in melanoma cells.

High-LET radiation is more effective than low-LET X- or γ- rays radiation in inducing biological damage. The clonogenic survival curves showed no obvious difference between A375 and A375/ wt p53 cells with a greater than 32 KeV/m C-beams radiation. The RBE of C-beams was larger in A375 cells than that in A375/wt p53 cells because the latter are sensitive to X-rays treatment. These results supported previous reports that the odds in cellular radiosensitivities will be diminished as LET increases (Aoki et al., 2000; Iwadate et al., 2001; Takahashi et al., 2001; Coelho et al., 2002).

Because of the possibility of apoptotic cells undergoing a subsequent secondary necrosis in the absence of phagocytosis occurring in vitro, it is generally advisable in cell death research to perform time-kinetics experiments of the cell death parameters (Vanden et al., 2004). In this study, the time-kinetic experiments were performed for evaluating the apoptotic and necrotic cells after irradiation at different LET. Apoptosis as a mechanism of radiation- induced cell death, whether in time occurrence or frequency, has been found to vary greatly according to cell types (Stapper et al., 1995; Filippovich et al., 1997; Sasaki et al., 2001). In A375 and A375/ wt p53 cells, apoptosis was induced in an LET-dependent manner. Both the cells showed a similar apoptotic wave peaking 48- 72 h after irradiation. Moreover, in A375/wt p53 cells, after exposure to high-LET radiation, apoptosis was induced more efficiently than that in A375 cells, although there was no significant apoptosis induced by AdCMV-p5,3 infection treatment alone. In addition, 12 h after irradiation with C-beams of greater than 32 KeV/m, there were slight differences in the number of apoptotic and necrotic cells between A375 and A375/wt p53 cells. However, the difference was significant (P < 0.05) at 48 h after irradiation. Because of secondary necrosis and cell loss resulting from cells collapsing 96 h after irradiation, the proportions of apoptotic and necrotic cells were not suitable for analysis. These results indicated that on the one hand, unrepairable DNA damage induced by high-LET radiation (range of 32-100 K\eV/m) probably results in partly /)53-dependent apoptosis because apoptosis in A375 cells induced by C-beams was significantly increased compared with X- rays. On the other hand, the functional P53 may have some relation to the mechanism of cell death at high-LET radiation. Regarding cancer cells necrosis induced by irradiation, both apoptotic and necrotic cell death are likely to contribute to target cell killing after irradiation. Quto and Ng (2002) reported that there were more necrotic cells than apoptosis in a human melanoma cell line (Sk-Mel- 3) after treatment with X-ray. Takahashi et al. (2004) concluded that high-LET radiation enhanced apoptosis, not necrosis regardless of p53 status. In this study, the proportion of apoptosis and necrosis in A375 cells was different from that in A375/p53 cells, although both cells had same surviving fractions after C-beams with greater than 32 KeV/ m LET radiation. The A375 cells showed similar proportion in the two parts of apoptosis and necrosis, whereas the apoptotic cells predominated over necrotic cells in the A375/p5,3 cells. The discrepancy of the relative contribution of apoptosis and necrosis might be dependent on tumor type and stage. In recent years, the understanding of cell death, and particularly the distinctions between apoptotic and necrotic pathways, has considerably evolved. Although previously, it was believed that cell death proceeds through one of these two major, mutually exclusive pathways, which can be distinguished by morphological, biochemical, and genetic criteria, more recent findings suggest that some protooncogenes (bcl-2/bcl-xl) and interleukin-1&946;) converting enzyme (ICE)-like proteases may modulate both apoptotic and necrotic cell death, indicating that apoptosis and necrosis may share some common pathways or mediators, or both (Shimizu et al., 1996; Eguchi et al., 1997; Tsujimoto et al., 1997). The unrepairable DNA damage induced by high-LET radiation will trigger the mechanism of cell death, so we presume that the cancer cells are inclined to undergo apoptosis with the exogenous wt P53.

Extensive phase I/II clinical trials have already been conducted with Ad5CMV-p53 (INGN 201). Although the results of preclinical and clinical studies have demonstrated that adenovirus-mediated gene therapy is a safe and efficient method for the introduction of the wild-type p53 in a variety of human cancers, the effect of melanoma with Ad5CMV-p53 gene therapy alone is not satisfactory. In this study, we reported the findings of an in vitro study that high-LET charged particle radiation combined with Ad5CMV-p53 treatment induced more apoptosis in A375 cells than that with irradiation alone. Because apoptosis is the most desirable way of eliminating cancer cells with a minimum of normal tissue side effects, our observation in the study offered the potential application of high- LET radiation combined with p53 in the management of human patients with melanoma.

ACKNOWLEDGMENTS

We thank Dr. Wu Zuze for providing the p53 recombinant adenoviral vector (Ad5CMV-/;5,3) and its control recombinant adenoviral vector (Ad5CMV-GFP]. The study was supported by the One Hundred Person Project Foundation of Chinese Academy of Sciences and the Dedicated Project of Science and Technology Ministry of China (2003CCB00200).

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FENG-LING MIN, HONG ZHANG, WEN-JIAN LI, QING-XlANG GAO,1 AND GUANG-MING ZHOU

Institute of Modern Physics, Chinese Academy of Science, Lanzhou, Gansu province 730000, People's Republic of China (M. F.-L., Z. H., L. W.-J., Z. G.-M.), Graduate School, Chinese Academy of Sciences, Beijing 10039, People's Republic of China (M. F.-L), and School of Life Science, Lanzhou University, Lanzhou, Gansu province 730000, People's Republic of China (G. Q.-X.)

(Received 9 May 2005; accepted 20 June 2005)

1 To whom correspondence should he addressed at E-mail: gaoqx@lzu. edu.cn

Copyright Society for In Vitro Biology Sep/Oct 2005

Source: In Vitro Cellular & Developmental Biology; Animal

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