Correlativity study between expression of DNA double-strand break repair protein and radiosensitivity of tumor cells

Abstract

DNA double-strand break (DSB) is generally regarded as the most lethal of all DNA lesions after radiation. Ku80, DNA-PK catalytic subunit (DNA-PKcs) and ataxia telangiectasia mutated (ATM) proteins are major DSB repair proteins. In this study, survival fraction at 2Gy (SF2) values of eight human tumor cell lines (including four human cervical carcinoma cell lines HeLa, SiHa, C33A, Caski, three human breast carcinoma cell lines MCF-7, MDA-MB-231, MDA-MB-453, and one human lung carcinoma cell line A549) were acquired by clone formation assay, and western blot was applied to detect the expressions of Ku80, DNA-PKcs and ATM protein. The correlativity of protein expression with SF2 value was analyzed by Pearson linear correlation analysis. We found that the expression of same protein in different cell lines and the expression of three proteins in the same cell line had a significant difference. The SF2 values were also different in eight tumor cell lines and there was a positive correlativity between the expression of DNA-PKcs and SF2 (r ＝0.723, P = 0.043), but Ku80 and ATM expression had no correlation with SF2 (P>0.05). These findings suggest that the expression level of DNA-PKcs protein can be an indicator for predicting the radiosensitivity of tumor cells.

Keywords

Ku80 / DNA-PK(cs)-binding protein, human / ataxia telangiectasia mutated protein / tumor cell lines / radiosensitivity

Introduction

Radiotherapy is the major treatment for tumor patients, and about 70% of patients would accept irradiation during the whole therapy. But different patients show different curative effects. The most probable reason is about the diverse DNA lesion repair abilities of tumor cells after irradiation. DNA Double-strand break (DSB) is generally regarded as the most lethal of all DNA lesions after radiation and is repaired by two major repair pathways: homologous recombination (HR) and non-homologous end-joining (NHEJ) [1,2]. Ataxia telangiectasia mutated (ATM) protein plays the main role in HR [3]. Correspondingly, DNA-dependent protein kinase (DNA-PK), a known major NHEJ protein, is a serine threonine kinase containing Ku heterodimer (Ku70 and Ku80) and DNA-PK catalytic subunit (DNA-PKcs) [4]. In this study, in order to explore which protein could prognosticate the radiosensitivity of tumor cells, the expressions of Ku80, DNA-PKcs and ATM protein and survival fraction at 2Gy (SF2) values in eight tumor cell lines were acquired.

Materials and methods

Cell lines and cell culture

Four human cervical carcinoma cell lines HeLa, SiHa, C33A, Caski, three human breast carcinoma cell lines MCF-7, MDA-MB-231, MDA-MB-453, and one human lung carcinoma cell line A549 were obtained from the China Center for Type Culture Collection (CCTCC, Wuhan, China). The cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, USA) supplemented with 10% fetal bovine serum (Invitrogen, USA) or in RPMI 1640 (Invitrogen, USA) supplemented with 10% fetal bovine serum, and were maintained in a humidified 37°C incubator with 5% CO₂.

Western blot analysis

Cells in the exponential phase of growth were harvested and whole cell proteins were isolated by using M-PER^TM mammalian protein extraction reagent (Pierce, USA). Protein concentration was determined by using a BCA protein assay kit (Pierce, USA) according to the manufacturer’s instructions. An equal amount of total protein (50 µg) from each lysate was loaded onto 6%–10% SDS-polyacrylamide gel. After electrophoresis, separated proteins were transferred to nitrocellulose membranes, which then were blocked with TBS-T (0.1 mol/L Tris-HCl, pH 7.5; 0.9% sodium chloride; 0.05% Tween 20) containing 5% powdered non-fat milk for 1 h at 37°C and then incubated with mouse anti-human monoclonal antibodies against Ku80 (NeoMarkers, USA, 1∶2 000 dilution), ATM (NeoMarkers, USA, 1∶200 dilution), DNA-PKcs (NeoMarkers, USA, 1∶2000 dilution) or actin (Santa Cruz, USA, 1∶1,000 dilution) at 4°C overnight. After washing with TBS-T for 4 × 10 min, nitrocellulose membranes were incubated with 1∶500 dilution horseradish peroxidase (HRP)-labeled goat anti-mouse second antibody (Pierce, USA) for 1 h at room temperature and washed with TBS-T for 4 × 10 min. Immunodetection was performed using SuperSignal West Femto Maximum Sensitivity Substrate (Pierce, USA) and the density of bands in the resulting film was quantified using NIH image analysis.

Clonogenic survival assay

Cells at the exponential phase of growth were plated in triplicate onto 60-mm dishes at the required concentration to get 50-100 colonies per dish and allowed to attach for 4–5 h. The cells were exposed to 0, 2, 3, 4, 6 and 8 Gy X-ray (6 MV, 300 cGy/min), then were cultured for 10 to 14 days in 5% CO₂ to get viable colonies. Colonies were stained with 0.5 mL of 0.1% crystal violet (Sigma-Aldridge, USA) solution for 20 min and counted by using microscope (× 40 magnification). A viable colony was defined as having at least 50 cells after 10 days of growth. Colonies were counted from each triplicate samples and presented as \overline{x}\pm s. The surviving fraction of treated cells was normalized to the plating efficiency of control (un-irradiation) cells. Cell survival was plotted as a function of dose and fitted using the linear quadratic model SF= exp(-αD–βD²), where SF is the cell survival fraction, D is the dose of radiation, and a and b are constants. SF2 was calculated from the actual data.

Statistical analysis

SPSS v11.5 software was used for all statistical procedures. Data was presented as \overline{x}\pm s of at least triplicate experiments. The correlativity was analyzed by Pearson linear correlation analysis. A P value less than 0.05 was considered significant.

Results

The expressions of Ku80, DNA-PKcs and ATM protein in eight cell lines

As shown in Figure 1 and Table 1, no significant differences of same protein expression were found in three different kinds of tumors. So, the expressions in eight cell lines were put together to be analyzed. The relative expressions of Ku80, DNA-PKcs and ATM protein in eight cell lines were diverse in the same cell line, and the same protein expressed differently in different cell lines. The average expressions of Ku80 and DNA-PKcs were both higher than that of ATM. There were no relativities among three proteins in eight cell lines by using Pearson linear correlation analysis (P>0.05).

Radiosensitivity analysis of eight cell lines after 6 MV x ray radiation

After irradiation, the radiosensitivity diversified in eight cell lines and the SF2 values varied range from 0.316±0.019 to 0.692±0.053. Among these cell lines, C33A was most sensitive to x ray, while A549 was the most resistant (Table 1).

The relativity between DSB repair protein expression and SF2 value

Analyzed by Pearson linear correlation analysis, there was a positive relationship between the expression of DNA-PKcs and SF2 (r = 0.723, P = 0.043), but no relationships were found between Ku80 and SF2 (r = 0.386, P = 0.345) or ATM and SF2 (r = 0.061, P = 0.887).

Discussion

DNA lesion repair ability is the most important influence on radiosensitivity, while DNA double-strand break is the most lethal of all DNA lesions after radiation. It is generally believed that NHEJ plays the most important role in mitotically replicating cells, especially in human cells [5]. When DSB is produced by irradiation, Ku heterodimer which has high affinity to DNA can bind preferentially to the free DNA ends [6,7]. Then, the conformation of Ku changes, allows it to interact with DNA-PKcs [8,9]. After autophosphorylation, DNA-PKcs recruits XRCC4/ligase IV complex to process the DNA ends and initiate re-ligation to form a single DNA molecule [10,11]. Our data shows that expressions of NHEJ protein Ku80 and DNA-PKcs were higher than that of HR protein ATM in all eight cell lines and confirmed the above conclusion that NHEJ played the major role in DSB repair. But in all cell lines studied, Ku80 and DNA-PKcs, two components of DNA-PK, had no correlation in eight cell lines. It is not in agreement with our hypothesis, indicating that Ku80 and DNA-PKcs not only play cooperative roles in DSB repair, but also have different contribution in other functions, just as Ku80 also has effect on cell proliferation [12,13], chromosomal stabilization [14,15] and so on [3].

SF2 value is one of the best parameter which can represent the radiosensitivity. In this study, SF2 values were detected by Clonogenic survival assay and varied in eight human tumor cell lines, which confirmed the fact that different cells have different radiosensitivities. At the same time, we also found that the DSB repair proteins expressed differently in these cell lines, the relativities between protein expression and SF2 value were analyzed by Pearson linear correlation analysis, and a positive correlation between the expression of DNA-PKcs and SF2 was found. It indicated that the high expression of DNA-PKcs might be able to prognosticate the resistance to the radiation and vice versa, which was similar to the result [16–18] reported before. However, no relativity was found between Ku80 expression and SF2 or ATM expression and SF2. The possible reasons for these results are shown below: First, the NHEJ repair pathway is more important than HR repair pathway, so HR protein ATM only plays few roles in DSB repair and could not prognosticate the radiosensitivity [19]; Second, it is generally believed [20] that Ku protein, including Ku80, contacts with the broken end of DNA at first. But some investigators had discovered that DNA-PKcs can also bind to, and is activated by free DNA ends in the absence of Ku [21,22]. This is answer to the question that DNA-PKcs, the catalytic subunit, is more important than Ku80, the structure subunit, in DSB repair.

As described above, DNA-PKcs may be able to prognosticate the radiosensitivity of tumor cells. However, this conclusion should be validated in vivo. At least, DNA-PKcs may be an ideal target to enhance radiosensitivity. We could apply small interfering RNA (siRNA), antisense oligonucleotide or selective inhibitors to inhibit DNA-PKcs expression or activity of radioresistant tumor cell, and this is currently an important ongoing investigation [23,24].

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