SHP-1 overexpression increases the radioresistance of NPC cells by enhancing DSB repair, increasing S phase arrest and decreasing cell apoptosis
- Authors:
- Published online on: April 28, 2015 https://doi.org/10.3892/or.2015.3939
- Pages: 2999-3005
Abstract
Introduction
Nasopharyngeal carcinoma (NPC) is one of the most common cancers in Southern China, Southeast Asia, the Arctic, and the Middle East/North Africa (1). Epstein-Barr virus infection, genetic factors, dietary and environmental factors are risk factors for the development of NPC (2). Due to the unique anatomical location of the nasopharynx where many important nerves and vessels are located, surgery is not the primary choice for NPC treatment. Since NPC cells are sensitive to ionizing radiation (IR), radiotherapy is now the primary therapy for NPC patients (3). However, some carcinoma cells are resistant to IR. These radioresistant cells remain the major cause of local recurrence and metastasis of NPC (4). Therefore, decreasing the radioresistance of NPC cells could help improve NPC patient prognosis.
SHP-1, also called PTPN6 (5), is an SH2 domain-containing protein tyrosine phosphatase (PTP). It consists of 17 exons and 16 introns and spans ~17 kb (6,7). SHP-1 is highly expressed in normal hematopoietic cells (8) and is weakly expressed in several hematological malignancies, including Burkitt’s (9), natural killer cell (10) and diffuse large cell lymphomas, Hodgkin’s disease (11) and chronic myeloid leukemia (12). However, some studies have found that SHP-1 is highly expressed in certain epithelial carcinoma cells, such as ovarian and breast cell lines (13). Although many studies have been conducted concerning SHP-1 in hematological tumors, the function of SHP-1 in solid tumors, particularly in NPC, is mostly unknown.
Our previous study found that SHP-1 is overexpressed in NPC tissues and is associated with local recurrence after radiotherapy (14). Knockdown of SHP-1 by siRNA in the NPC cell line CNE-2 and in the non-small cell lung cancer (NSCLC) cell line A549 resulted in increased radiosensitivity (15,16). These results suggest that overexpression of SHP-1 may be related to radioresistance.
In the present study, we aimed to further examine whether increased SHP-1 contributes to radioresistance of NPC CNE-2 cells. We also investigated how it is related to DNA double-strand break (DSB) repair, cell cycle arrest and cell apoptosis.
Materials and methods
Cell culture and irradiation procedure
The NPC cell line CNE-2 was obtained from the Cell Bank of the Sun Yat-Sen university (Guangzhou, China) and cultured in RPMI-1640 medium (HyClone, Logan, UT, USA) supplemented with 12% fetal bovine serum (Gibco, Grand Island, NY, USA) and 1% penicillin/streptomycin (HyClone). The cells were maintained at 37°C in a humidified incubator with 5% CO2 and 95% room air. Irradiation was performed at room temperature with single doses of X-rays using a linear accelerator (Primus K; Siemens, Munich, Bayern, Germany) with 6-Mv photons/100-cm focus-surface distance and a dose rate of 2.0 Gy/min.
SHP-1 is upregulated by lentiviral-mediated gene knock-in
Both the SHP-1 gene sequence and a negative oligo sequence were inserted into pEZ-Lv201-green fluorescent protein (GFP) lentiviral vectors (GeneCopoeia, Guangzhou, China). After confirmation of the constructed plasmids by DNA sequencing, the lentiviruses were then transfected into 293T cells. LP-H1802Lv201 is a lentivirus containing the SHP-1 gene and LP-NegLv201 is a negative control containing a negative oligo sequence. Supernatants containing the lentiviruses were harvested, purified and the titer of lentiviruses was determined. Both lentiviral stocks were transfected into the CNE-2 cells. Fifty microliters of lentivirus stock (LP-H1802Lv201 and LP-NegLv201) was added to the CNE-2 cells. Puromycin (Sigma-Aldrich, St. Louis, MO, USA) was used to screen cells transfected with the lentivirus at a concentration of 2 μg/ml. RNA was extracted and RT-qPCR was used to detect SHP-1 mRNA expression. Total protein was isolated and the expression of SHP-1 was detected by western blotting. The efficiency of infection was observed by fluorescence microscopy.
Colony formation assay
Cells were seeded into 6-well culture plates and irradiated the next day at distinct doses (0, 2, 4, 6 and 8 Gy). The plates were incubated for 14 days, fixed with methanol, and stained with Giemsa (both from Wuhan Google Biotechnology Ltd. Co., Wuhan, China), and colonies containing at least 50 cells were counted as a clone. A multi-target single-hit model was used to describe the survival fraction (SF). The equation SF = 1-(1 - e−D/D0)N (where D is the radiation dose; e is the bottom of the natural logarithm; D0 is the mean death dose; and N is the extrapolated number) was used to fit the cell survival curves.
Immunofluorescent assay (IFA)
Cells were irradiated with 2 Gy of X-rays, and incubated for specified times after IR. The cells were harvested and immunostained with anti-histone H2AX phosphorylation (γH2AX; Abcam, Cambridge, UK) or anti-RAD51 (Millipore, Billerica, MA, USA) antibodies. Then the cells were incubated with Dylight 549 goat-anti-rabbit IgG (Abbkine, Redlands, CA, USA). The nuclei were visualized by staining with Hoechst 33258 (Wuhan Google Biotechnology Ltd. Co.). Images were captured using an Olympus laser scanning confocal microscope (Olympus Optical Co., Tokyo, Honshu, Japan). For each treatment condition, fluorescently labeled γH2AX foci or RAD51 were assessed by fluorescence microscopy in at least 50 cells.
Cell cycle flow cytometry (FCM) analysis
Cells were irradiated with 6 Gy of X-rays and incubated for 24 h after IR. The cells were fixed overnight with 70% ethanol, and resuspended in PBS containing 1 mg/ml RNase and 50 μg/ml propidium iodide (both from Sigma-Aldrich). Cellular DNA content was determined using a FACScan flow cytometer (Becton-Dickinson, San Jose, CA, USA). Quantification of cells in the g1, S, and g2/M phases was performed using CellQuest software (BD Biosciences).
FCM analysis of apoptosis
Cells were exposed to 6 Gy of X-rays and incubated for 24 h after IR. The cells were then collected and resuspended in 200 μl binding buffer and stained with 2 μl Annexin V-APC and 4 μl of 7-AAD (all from Bestbio, Shanghai, China). Analyses were performed using a FACScan flow cytometer (Becton-Dickinson). Both APC-and 7-AAD-positive cells were considered apoptotic cells.
Western blotting
Cells were harvested and lysed after the different treatments. Protein lysate concentrations were determined using the BCA protein assay (Wuhan Google Biotechnology Ltd. Co.). Equal amounts of protein were separated by 8–15% SDS-PAGE (according to molecular weight; Wuhan Google Biotechnology Ltd. Co.) and transferred to PVDF membranes (Millipore). The membranes were blocked with 5% BSA (Wuhan Google Biotechnology Ltd. Co.) and then probed with anti-SHP-1 (Epitomics, Burlingame, CA, USA), anti-RAD51 (Millipore), anti-phospho-p53 (Cell Signaling Technology, Danvers, MA, USA), anti-γH2AX (Abcam), anti-phospho-ATM kinase and anti-phospho-ATR protein (both from Cell Signaling Technology), anti-phospho-CHK1 (Abcam), anti-phospho-CHK2 (Cell Signaling Technology) or anti-GAPDH (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) antibodies. After washing by TBST (Wuhan Google Biotechnology Ltd. Co.), the membranes were incubated with goat-anti-rabbit or goat-anti-mouse IgG (Invitrogen, Carlsbad, CA, USA) and visualized by a chemiluminescence detection system (UVP OptiCam 600; UVP Inc., Upland, CA, USA) using a chemiluminescence kit (Invitrogen). GAPDH protein levels were used as a control to verify equal protein loading. Image J 1.43b software (NIH, Bethesda, MD, USA) was used to scan the protein bands and to measure the optical density values.
RT-qPCR
Total RNA was extracted with TRIzol (Invitrogen), and reverse transcription was used to obtain cDNA, according to the manufacturer’s instructions for the Takara RT-PCR kit (Takara, Shiga, Japan). Then, qPCR was performed according to the manufacturer’s instructions using SYBR-Green in a PCR amplifier, ABI Prism 7000 (both from Applied Biosystems, Foster City, CA, USA). The StepOne™ software v2.1 was used to analyze the data. The primer sequences for SHP-1 were: forward, 5′-ACCATCATCCACCTCAAGT ACC-3′ and reverse, 5′-CTGAGCACAGAAAGCACGAA-3′. β-actin was used as an internal control, and the primer sequences were: forward, 5′-GATGAGATTGGCATGGC TTT-3′ and reverse, 5′-CACCTTCACCGTTCCAGTTT-3′.
Statistical analysis
Experimental data are expressed as the mean ± SD from at least three or more independent experiments. Differences in the measured variables of the experimental and control groups were assessed using a t-test (SPSS 21.0 software). The criterion for statistical significance was p<0.05.
Results
Overexpression of SHP-1 by lentiviral-mediated gene knock-in in CNE-2 cells
We transfected the NPC cell line CNE-2 with a lentivirus containing the SHP-1 gene or a nonsense sequence (referred to as LP-H1802Lv201 and LP-NegLv201 cells, respectively). Fluorescence microscopy was used to observe GFP intensity and transfection efficiency (Fig. 1A). RT-PCR and western blot analyses were used to detect SHP-1 expression levels at the mRNA and protein levels, respectively. RT-qPCR showed that in the LP-H1802Lv201 cells, SHP-1 mRNA expression levels were increased by 300-fold (Fig. 1B). Western blot analyses also indicated an increased expression of SHP-1 protein in the LP-H1802Lv201 cells (Fig. 1C).
Upregulation of SHP-1 results in enhanced radioresistance
To determine the relationship between SHP-1 and radio-resistance, colony formation assays were performed, and cell survival curves were used to analyze the results. The shoulder area under the survival curve was larger in the LP-H1802Lv201 cells (Fig. 2). In contrast to the control and LP-NegLv201 cells, LP-H1802Lv201 cells had increased D0, Dq, N and SF2 (Table I) values, which represented a higher radioresistance. The differences in these values were negligible between the control and LP-NegLv201 cells. Therefore, we concluded that upregulation of SHP-1 resulted in enhanced radioresistance.
DNA DSB repair is enhanced in the SHP-1-overexpressing cells
To determine DNA DSB repair, we used an anti-γH2AX to immunofluorescently stain γH2AX foci at different time-points after exposure to IR. As shown in Fig. 3A without exposure to IR, few γH2AX foci were observed in the three cell groups. After irradiation with 2 Gy IR, γH2AX foci rapidly increased. The numbers of γ-H2AX foci in the 3 groups were almost equal at 0.5 h after IR. However, foci in the LP-H1802Lv201 cells disappeared more quickly. In contrast to the control and LP-NegLv201 cells, γH2AX foci in the LP-H1802v201 cells were significantly decreased at 3, 6 and 24 h after IR. Cells having more than 10 γH2AX foci were scored as γH2AX-positive cells. We found that the percentage of γH2AX-positive cells was significantly decreased in the LP-H1802Lv201 cell group at 6 and 24 h after IR. In contrast, at 0.5 and 3 h after IR, the percentages of γH2AX-positive cells in the 3 groups did not differ significantly. We also assessed RAD51 foci at 6 h after IR or under the condition without IR. As shown in Fig. 3B, without IR, RAD51 foci did not show a significant difference in the 3 groups. At 6 h after IR, the number of RAD51 foci in the LP-H1802Lv201 cells was significantly less than that in the control and LP-NegLv201 cells. Cell having more than 10 RAD51 foci were scored as RAD51-positive cells. The percentage of RAD51-positive cells was also lower in the LP-H1802Lv201 cell group. Western blot analyses were also used to assess γH2AX and RAD51 expression at 24 h after IR or under the condition without IR (Fig. 3C). Without IR, expression levels of γH2AX and RAD51 in the 3 cell groups were almost equal. At 24 h after IR, expression levels of γH2AX and RAD51 were significantly increased in contrast to the condition without IR. However, expression levels of γH2AX and RAD51 were lower in the LP-H1802Lv201 cells. We concluded that IR caused DNA DSBs equally in the 3 cell groups. Yet, SHP-1-overexpressing cells showed an enhanced DSB repair capacity.
SHP-1-overexpressing cells undergo increased S phase arrest after IR
To evaluate how SHP-1 affects cell cycle distribution, we used FCM to estimate the cell cycle changes. Without IR, the cell fractions in the S phase did not show a significant difference in the 3 cell groups. At 24 h after IR, the percentage of S phase cells was significantly increased and the percentage of g2/M phase cell group was decreased in the LP-H1802Lv201 cells compared with the control and LP-NegLv201 cells (Fig. 4). The results suggest that overexpression of SHP-1 led to increased IR-induced S phase arrest and thus decreased g2/M phase cells.
Overexpression of SHP-1 causes an anti-apoptotic effect
Before IR, the apoptotic rate of the control, LP-NegLv201 and LP-H1802Lv201 cells had no significant differences. At 24 h after IR, the apoptotic rates of the 3 cell groups were increased. However, the apoptotic rate of the LP-H1802Lv201 cells was significantly lower than the rate in the control and LP-NegLv201 cells (Fig. 5). These data suggest that IR promoted apoptosis in the 3 cell groups. However, LP-H1802Lv201 cells were more resistant to IR-induced apoptosis. The results suggest that overexpression of SHP-1 had an anti-apoptotic effect.
SHP-1-overexpressing cells show increased activation of ATM and CHK2 and suppressed activation of p53 after IR
To explore how the ATM/CHK1 and ATR/CHK1 pathways were activated after IR, we determined the phosphorylation levels of ATM (p-ATM), CHK2 (p-CHK2), ATR (p-ATR), CHK1 (p-CHK1) and p53 (p-p53). Results of the western blot analyses showed that the phosphorylation levels of ATM, CHK2, ATR, CHK1 and p53 were extremely low when cells did not receive IR. After radiation, phosphorylation levels of these proteins were increased. Compared with the control and LP-Neglv201 cells, LP-H1802lv201 cells had relatively increased phosphor-ylation levels of ATM and CHK2, while the phosphorylation of p53 was decreased. Phosphorylation of ATR and CHK1 did not show a significantly difference (Fig. 6).
Discussion
In the past few decades, SHP-1 has been believed to be a tumor-suppressor in many malignancies (10,17,18). However, our previous research found that SHP-1 was overexpressed in NPC tissues and was associated with local recurrence and metastasis after radiotherapy (4). Suppression of SHP-1 expression resulted in a higher radiosensitivity (15,16). These results suggest that overexpression of SHP-1 may be related to radioresistance and that it may be a potential target to enhance NPC radiosensitivity.
In the present study, we investigated the effects of SHP-1 on the radioresistance of NPC cells. We showed that over-expression of SHP-1 enhanced DNA DSB repair, increased IR-induced S phase arrest and decreased cell apoptosis, thus resulting in radioresistance in CNE-2 cells. The result was consistent with previous observations (15,16).
IR-induced cell death is a result of irreparable DSBs (19). The repair response of DSBs is one of the factors that influences radiosensitivity. DSBs are mainly repaired by non-homologous end joining (NHEJ) and homologous recombination. Repair of DSBs appeared within 30–60 min after radiation. The majority of DSBs are repaired in 24 h. γH2AX is a hallmark of DSB recognition and repair. Fewer γH2AX foci represent a more rapid repair of DSBs and higher radio-resistance (19–24). RAD51 is an important protein involved in homologous recombination processes. Increased RAD51 expression is related to radioresistance of tumor cells (25). In the present study, we showed that overexpression of SHP-1 in NPC cells decreased the expression of γH2AX, which indicted enhanced repair of DSBs. Notably, RAD51 expression was decreased in the LP-H1802lv201 cells, which are relatively radioresistant cells. One reason for this result may be that we detected RAD51 expression only at one time-point after IR. According to γH2AX expression, LP-H1802Lv201 cells had an enhanced DSB repair capacity. Thus, the DSB repair peak of LP-H1802Lv201 cells should have appeared sooner than the control and LP-NegLv201 cells. When we detected the expression of RAD51, the DSB repair peak may have transpired in the LP-H1802lv201 cells while this peak was not yet achieved in the control and LP-Neglv201 cells.
Cells often respond to IR-induced DSBs by activating cell cycle checkpoints, which play an importance role in determining radiosensitivity. In general, S phase cells are the most radioresistant, while G2/M phase cells are the most sensitive to radiation (26). It has been reported that abrogation of the G2/M checkpoint promotes IR-induced cell death (27). The present data showed that overexpression of SHP-1 increased the fraction of S phase cells. At the same time, the cell fraction in the G2/M phase was decreased, which may have resulted from S phase arrest. Thus, we inferred that overexpression of SHP-1 contributed to the radioresistance of NPC cells by increasing S phase arrest.
ATM kinase plays vital roles in IR-induced DNA damage repair response (DDR). ATM is activated upon DNA damage, and downstream effector kinases of ATM, including CHK2 and p53, are also activated (28). Increased activation of ATM/CHK2 enhances DNA damage repair, thus leading to radioresistance. It has been reported that inhibition of ATM activation increases apoptosis and enhances radiosensitivity. The ATR/CHK1 pathway also has an influence on radio-resistance by regulating homologous recombination repair. Overactivation of the ATR/CHK1 pathway increased the radioresistance of tumor cells. When DNA damage is not repaired, cells will be eliminated though different mechanisms including p53-dependent apoptosis (28–32). In the present study, we found that SHP-1-overexpressing cells had an increased phosphorylation of the ATM/CHK2 pathway, while phosphorylation of the ATR/CHK1 pathway did not show a difference in the other two cell groups. These data suggest that overexpression of SHP-1 enhanced DNA damage repair by activating the ATM/CHK1 pathway, but not the ATR/CHK1 pathway, and enhanced DNA damage repair resulted in decreased p53-dependent apoptosis.
ATM also takes part in intra-S checkpoint response to IR-induced DSBs through two separate pathways (33,34). One pathway involves activation of CHK1, CHK2 and Cdc25A. CHK1 and CHK2 are phosphorylated and activated by ATM, which leads to phosphorylation and proteolysis of Cdc25A and then activates intra-S checkpoint response (35). The other pathway involves the cohesin subunits, Smc1 and Smc3. Smc1 and Smc3 are phosphorylated by ATM and bind to Scc1 and SA1 or SA2 to form cohesin, which plays important roles in homologous recombination repair of DSBs (33,34,36–38). In the present study, SHP-1-overexpressing cells showed an increased S phase arrest accompanied by increased ATM and CHK2 activation after IR. However, activation of CHK1 was not increased in the SHP-1-overexpressing cells. We inferred that IR-induced DSBs activated the ATM/CHK2 pathway. Then Cdc25A was phosphorylated and the intra-S checkpoint was activated, leading to S phase arrest. However, whether the ATR/CHK1 pathway and ATM/Smc1/Smc3 are involved in S phase arrest in SHP-1-overexpressing cells needs further study.
In the present study, we demonstrated that overexpression of SHP-1 was related to the acquired resistance to IR in the NPC cell line CNE-2. Enhanced DSB repair, increased S phase arrest and decreased apoptosis contributed to this acquired radioresistance. Examining the SHP-1 expression level in tumor tissues of NPC patients may help to predict prognosis.
Acknowledgments
The present study was supported by grants from the Natural Sciences Foundation of China (no. 81301976) and the Wu Jieping Medical Foundation.
Abbreviations:
|
NPC |
nasopharyngeal carcinoma |
|
IR |
ionizing radiation |
|
DSB |
double-strand break |
|
NSCLC |
non-small cell lung cancer |
|
GFP |
green fluorescent protein |
|
IFA |
immunofluorescent assay |
|
FCM |
flow cytometry |
|
DDR |
DNA damage repair response |
|
ATM |
ataxia telangiectasia mutated |
|
CHK2 |
checkpoint kinase 2 |
|
ATR |
ataxia telangiectasia and Rad3-related protein |
|
CHK1 |
checkpoint kinase 1 |
|
NHEJ |
non-homologous end joining |
|
TKI |
tyrosine kinase inhibitor |
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