Inhibition of p53 and/or AKT as a new therapeutic approach specifically targeting ALT cancers
While the majority of all human cancers counteract telomere shortening by expressing telomerase, ~15% of all cancers maintain telomere length by a telomerase-independent mechanism known as alternative lengthening of telomeres (ALT). Here, we show that high load of intrinsic DNA damage is present in ALT cancer cells, leading to apoptosis stress by activating p53-independent, but JNK/c-Myc-dependent apoptotic pathway. Notably, ALT cells expressing wild-type p53 show much lower apoptosis than p53-deficient ALT cells. Mechanistically, we find that intrinsic DNA damage in ALT cells induces low level of p53 that is insufficient to initiate the transcription of apoptosis-related genes, but is sufficient to stimulate the expression of key components of mTORC2 (mTOR and Rictor), which in turn leads to phosphorylation of AKT. Activated AKT (p-AKT) thereby stimulates downstream anti-apoptotic events. Therefore, p53 and AKT are the key factors that suppress spontaneous apoptosis in ALT cells. Indeed, inhibition of p53 or AKT selectively induces rapid death of ALT cells in vitro, and p53 inhibitor severely suppresses the growth of ALT-cell xenograft tumors in mice. These findings reveal a previously unrecognized function of p53 in anti-apoptosis and identify that the inhibition of p53 or AKT has a potential as therapeutics for specifically targeting ALT cancers.
KeywordsALT p53 AKT DNA damage apoptosis telomeres
The majority of human cancers maintain telomere length by telomerase, ~15% of all cancers, including some of the more aggressive cancers, elongate telomeres by alternative lengthening of telomeres (ALT) (Kim et al., 1994; Bryan et al., 1995). It has been reported that anti-cancer therapeutics targeting telomerase may activate ALT, inducing drug resistance (Hu et al., 2012). Therefore, cancer treatment specifically targeting ALT, although not available so far, would be very valuable.
Accumulating evidence supports that ALT cancer cells bear large amounts of intrinsic DNA damage that may cause chromatin instability (Lovejoy et al., 2012). For example, ALT cells are characterized by high frequency of telomeric homologous recombination (HR) (Dunham et al., 2000) and frequently observed DNA damage foci (53BP1 or γ-H2AX) at telomeres termed telomere dysfunction-induced foci (TIFs) (Pickett and Reddel, 2015). In addition, ALT-associated promyelocytic leukaemia (PML) bodies, which are dynamic sensor of DNA damage and cellular stress, are widely observed in ALT cancers, but not in non-ALT cells (Dunham et al., 2000; Dellaire and Bazett-Jones, 2004). With such DNA damage stress, it has been interpreted that ALT cells are competent to mount a DNA damage response (DDR).
Wild-type (wt) p53 is a tumor suppressor that, when phosphorylated and activated by ATM or ATR in cells with DNA damage, triggers downstream events leading to cell cycle arrest, cell senescence, or apoptosis (Bieging et al., 2014). Consistent with this, mutations/alterations in p53 are present in 5 to 80 percent of human cancer cells, although the strength of the association between p53 status and the cancer phenotype varies with cancer type, stage, and etiology (Petitjean et al., 2007; Bouaoun et al., 2016). P53 acts as a transcription factor through binding to DNA targets, leading to the expression of many genes that participate in a variety of biological processes including cell-cycle arrest, DNA damage repair, apoptosis, reactive oxygen species (ROS) accumulation, and so on (Schwartzenberg-Bar-Yoseph et al., 2004; Bensaad et al., 2006; Matoba et al., 2006; Lane and Levine, 2010; Hager and Gu, 2014; Ubil et al., 2014). Transactivation of genes by p53 is sensitive to both DNA sequence and p53 amount, with some sequences being responsive to much lower p53 levels than others (Inga et al., 2002; Jordan et al., 2012). For example, DNA recognition elements (REs) of cell-cycle arrest genes appear to bind to p53 with much higher affinities than most REs related to apoptosis (Weinberg et al., 2005). Therefore, it has been reported that the cellular level of p53 dictates the cell response such that lower levels of p53 result in cell cycle arrest whereas higher levels lead to apoptosis (Chen et al., 1996).
In contrast to the conventional function of p53 in pro-apoptosis, here we report that wt-p53 plays an anti-apoptotic pro-proliferative role in ALT cancer cells. ALT cells bear persistent DNA damage that induces a low-concentration of wt-p53. P53 stimulates the transcription of genes encoding mTOR and Rictor, which are key components of mTORC2 responsible for phosphorylating AKT. Activated AKT thereby suppresses the apoptosis of ALT cells. However, when a high concentration of p53 is accumulated, p53 has opposite effect by directly stimulating the transcription of apoptosis initiating genes. Identified DNA damage-p53-AKT pathway may be a new target for therapeutics of ALT cancers.
Intrinsic DNA damage, apoptotic stress and expression of p53 in ALT cells
With a high load of persistent DDR in ALT cells, we speculated that these cells may bear higher apoptotic stress than non-ALT cells. We observed that human normal cells (MRC5 and BJ fibroblast) display the lowest apoptotic stress, as expected. Telomerase positive cancers (MCF7 and A549) have a similar apoptosis level as ALT cells that express wt-p53 (U2OS and VA13) (Fig. 1C and 1D). However, ALT cells with deficient p53 (SAOS2 and SKLU-1) contain the highest fraction of apoptotic cells (Fig. 1C and 1D). These results led to the hypothesis that p53 may play a role in suppressing apoptosis of ALT cells.
Although p53 is reported to be dysfunctional in many ALT cells (Sood et al., 2002; Mangerel et al., 2014), in checking all available ALT cancer cell lines, ~50% of ALT cancers were found to express wt-p53 (Fig. S1D). Interestingly, p53 positive VA13 and U2OS cells also proliferated faster than p53-deficient SAOS2 and SKLU-1 cells (Fig. 1E). The positive correlation between faster proliferation and expression of wt-p53 was also observed in another ALT cancer cell lines (Fig. S1E). These results suggested that wt-p53 may have an anti-apoptotic pro-proliferative function in ALT cells, which is conflict with the dogma that wt-p53 is generally a pro-apoptotic, anti-proliferative factor (Wu and Deng, 2002); thus, it is an unusual observation that we explore further below.
We then explored the mechanism by which apoptosis is induced in ALT cells. It is recognized that cancer cells can undergo both p53-dependent and p53-independent apoptosis upon severe cell stress, including DNA damage (Roos et al., 2016). JNK and c-Myc could induce apoptosis independently of p53 (Askew et al., 1991; Verheij et al., 1996). We found that JNK is activated/phosphorylated and c-Myc is accumulated not only in U2OS and VA13 cells that bear wt-p53, but also in p53-deficient SAOS2 and SKLU-1 cells, demonstrating the activation of apoptosis in ALT cells regardless of the status of p53 (Fig. 1F). In contrast, phosphorylated JNK and c-Myc accumulation were not observed in non-ALT MRC5, MCF7 and A549 cells (Fig. S1F).
Suppression of apoptosis by p53 is specific for ALT cells
We then examined non-ALT cells. For human normal BJ and MRC5 fibroblast cells, knockdown of p53 have a limited/no effect on cell apoptosis and proliferation (Figs. 2I–L and S2E–H). Similar results were obtained for telomerase positive cancer cells MCR7 and A549 (Figs. 2M–P and S2I–L). We thus concluded that suppression of apoptosis by p53 is specifically present in ALT cells.
Low level of p53 in ALT cells is insufficient to induce apoptosis
p53-dependent activation of AKT
Wt-p53 is a transcription factor. However, mutant p53-S269E is a dominant-negative p53 variant that lacks the transcriptional activity (Wu et al., 2011). Here, we observed that while expression of wt-p53 slightly increased the level of p-AKT, expression of p53-S269E decreased p-AKT level, suggesting that transcription activity of p53 is required for phosphorylation of AKT (Fig. 4F). Consistently, the exposure of cells to PFTα, an inhibitor of the transcription activation activity of p53 (Komarov et al., 1999), decreased the abundance of p-AKT in U2OS and VA13 cells (Figs. 4G and S3B).
p53 stimulates the expression of mTORC2 that is responsible for phosphorylation of AKT
P53 serving as a transcription factor of mTOR and Rictor was further examined by a standard ChIP assay, which demonstrated a physical interaction between p53 and targeted genes. Three pairs of primers were designed to detect the presence of p53 on DNA over 1 kb region surrounding the transcription start site (TSS) of mTOR and Rictor gene (Fig. 5F). ChIP assay showed that p53 primarily associates with a 0.5 kb upstream promoter region near the transcription start site of mTOR and Rictor gene (Fig. 5G and 5H).
Inhibition of p53 and/or AKT induces rapid death of ALT cancer cells
Similar to the effect induced by PFTα, the allosteric AKT inhibitors MK-2206 increased apoptosis significantly in VA13 and U2OS cells, but not in BJ cells (Figs. 6E, 6F and S5A). Other AKT inhibitor KRX-0401 slightly increased apoptosis of BJ cells, but dramatically increased apoptosis of VA13 and U2OS cells (Figs. 6E, 6F and S5A). Moreover, cancer cells treated with a low dose of PFTα (20 μmol/L) in combination with AKT inhibitor (MK-2206 or KRX-0401) rapidly lost viability (Fig. 6G).
Interestingly, p53-negative ALT cancers are also hypersensitive to and rapidly undergo apoptosis in the presence of MK-2206 or KRX-0401 (Fig. S5B–D). KRX-0401 and MK-2206 induced mass death of SAOS2 and SKLU-1, respectively (Fig. S5E). We reasoned that p-AKT, which is very limited in number in p53-deficient ALT cells, is absolutely required to suppress apoptosis induced by intrinsic DNA damage (Fig. 4A). Therefore, agents targeting the AKT pathway have a potential for therapeutics of all ALT cancers, independent of their p53 status.
The growth of ALT positive xenograft tumors in mice treated with p53 inhibitor
The pro-apoptotic function of p53 is well recognized. However, it has been recently recognized that wt-p53 suppresses metabolic stress-induced ferroptosis (non-apoptotic iron-dependent cell death), suggesting that wt-p53, under specific circumstances, may function to help cancers to cope with cellular stress (Tarangelo et al., 2018). Recently, Jinchul Kim et al. reported that wt-p53 plays an oncogenic role by suppressing oxidative phosphorylation in hepatocarcinoma (HCC) (Kim et al., 2019). It is certainly possible that wt-p53 may also promote the metabolic switch from oxidative phosphorylation to glycolysis in ALT cancer cells as it does in HCCs. This, however, does not explain how ALT cancers overcome high stress of apoptosis induced by intrinsic telomeric DNA damage. Here, our study documents a previously unrecognized p-AKT-mediated anti-apoptotic function of wt-p53 in ALT cancer cells. We suggest that in these cells, on account of high load of DNA damage, ALT cells suffer from high apoptotic stress through activating p53 independent pathways such as JNK or c-Myc; meanwhile, intrinsic DNA damage leads to low level of p53 that suppresses apoptosis through activation/phosphorylation of AKT; however, high level of p53 induced by either exogenously imposed DNA damage or exotic expression of p53 would result in apoptosis of ALT cells (Fig. 7H).
Anti-apoptosis activity of p53 specifically exists in ALT cells
It has been previously reported that p53-deficient mouse fibroblasts are more sensitive to genotoxins and DSB-triggered apoptosis than p53 wild-type cells (Lackinger et al., 2001; Lips and Kaina, 2001), raising the possibility that in specific cell systems and/or under given circumstances, p53 may function to protect against DSB induced apoptosis. In accord with this, our results showed that although low level of p53 in human ALT cells is insufficient to activate the expression of apoptosis-initiating genes such as BAX, NOXA and PUMA (Fig. 3H and 3I), it is adequate for stimulating the transcription of mTOR and Rictor (Fig. 5B and 5C), thus resulting in activation/phosphorylation of AKT that acts to suppress DNA damage-induced apoptosis. We thus proposed that the anti-apoptotic function of p53 represents a new mechanism for ALT cells to counteract intrinsic apoptotic stress. This mechanism may also exist in mice because mice cells are ALT positive (Neumann et al., 2013). However, in telomerase positive cancer and human normal cells, p53 does not display anti-apoptosis activity (Figs. 2I–P and S2E–L).
P53 is a short-lived protein that is maintained at low, often undetectable, levels in normal cells. In response to an activating signal, such as DNA damage, p53 protein is stabilized by ATM/ATR mediated phosphorylation and its level increases. The function of p53 is dependent on both amount and type of DNA damage (Lassus et al., 1996; Roos and Kaina, 2006). There are two unique features of ALT cells that may dictate anti-apoptosis function of p53. First, ALT cells bear persistent apoptotic stress in p53-indepednent manner. We observed that apoptosis exists not only in p53-positive U2OS and VA13 cells, but also in p53-deficient SAOS2 and SKLU-1 cells (Fig. 1C and 1D), and that both JNK and C-myc pathways are activated in these cells that could lead to cell apoptosis independently of p53 (Fig. 1F). Second, DNA damage in ALT cells stimulates consistent but relative low level of p53 that is not enough to initiate p53-mediated apoptosis (Fig. 3A–D). The nature of DNA damages in ALT cells is still not fully understood. Increasing evidence suggests that the defect in DNA replication machinery in ALT cells may cause incompletely replicated DNA that induces persistent DDR (Clynes et al., 2015; Dilley et al., 2016; Roumelioti et al., 2016). Whether similar DDR is present in other cell type and is capable to stimulate a pro-apoptotic function of p53 remains for further investigation.
P53 activates AKT that prevent cells from undergoing apoptosis
Our data show that p53 activates AKT by increasing the expression of mTORC2 component mTOR and Rictor (Fig. 5B and 5C). Activated AKT could prevent DNA damage-induced apoptosis in two ways. On the one hand, p-AKT could directly phosphorylate apoptosis execution factors (BAD and caspase-9) and suppress their function (del Peso et al., 1997; Cardone et al., 1998). Alternatively, p-AKT could activate NF-κB, thereby stimulating the transcription of anti-apoptotic BCL-2 family proteins BFL1/A1, BCL-XL and neuroligin 3 (NRL3) (Pahl, 1999). On the other hand, p-AKT could upregulate the expression of MRE11 through activating GSK3β/β-catenin pathway (Deng et al., 2011). Therefore, increased capacity for DNA damage repair relieves apoptotic stress.
It has been previously reported that p-AKT binds to, and phosphorylates MDM2, thereby increasing ubiquitin ligase activity of MDM2 and promoting its import into nucleus (Mayo and Donner, 2001). Because MDM2 plays an essential role in inactivation/degradation of p53, increased amount of MDM2 in nucleus would result in decrease of p53 (Zhou et al., 2001). As such, in ALT cells AKT and p53 may form a negative regulation mechanism that keeps p53 at a relatively low level: intrinsic DNA damage induces p53 → p53 activates AKT → p-AKT stimulates MDM2 → MDM2 reduces the amount of p53.
P53 and AKT as a target for ALT cancer therapy
Because of its near universal alteration in cancer, p53 is an attractive target for the development of new cancer therapeutics. Current strategy focuses on increasing the level of wt-p53 in cancer cells by either restoring wild-type properties to mutant p53 or preventing the binding of MDM2/MDM4 to wt-p53, thereby blocking its degradation (Duffy et al., 2014). In the context of ALT cancers, potential side effects from the use of these treatments should be taken into consideration. Under certain conditions, p53 may help cancer cells to escape the apoptosis induced by chemotherapy or radiotherapy.
Because ALT cancers are known to be aggressive and difficult to treat and conventional anti-cancer therapeutics are known to convert some telomerase-positive cells to ALT cells (Hu et al., 2012), effective therapeutics for ALT cancers, although not yet available, are urgently needed and would be extremely valuable. The discovery of DNA damage-p53-AKT pathway provides strong rationale for treating ALT cancers that express wt-p53 with agents that inhibit p53 and/or AKT without concomitant exposure to DNA damaging agents (Fig. 6). In addition, because of high apoptosis stress induced by persistent DNA damage, p53 deficient ALT cancers are also hypersensitive to suppression of AKT, suggesting that inhibitors of AKT may be especially beneficial in treatment of these ALT cancers (Fig. S5C–E). Further studies are needed to explore the clinical potential of these treatments.
MATERIALS AND METHODS
Cell culture, plasmids and compounds
U2OS, VA13, SAOS2, SKLU-1, A549, MCF-7, MRC5, BJ cells were obtained from the Cell Resource Center of Peking Union Medical College and were grown in DMEM (Gibco) with 10% FBS (Gibco) and cultured at 37 °C under 5% CO2. All cell lines were negative for mycoplasma contamination and they were authenticated using STR (Short Tandem Repeat) profiling method. Wild-type p53 gene was amplified from HEK293 mRNA and cloned into Phage-tet-SFB (moderate expression) and pLenti-HA/Flag (full expression). The mutant (S269E) of p53 was constructed. KU60019, VE-821, Pifithrin-α (PFTα), MK-2206, KRX-0401, rapamycin, KU0063794 were purchased from Selleck. Zeocin was purchased from ThermoFisher.
Gene silencing and knockout
SiRNA was transfected into target cells in a 6-well plate using Lipofectamine® RNAiMAX Transfection Reagent (Invitrogen), according to the manufacturer’s instructions. siRNA against p53 (si-1: 5′-GCAUGAACCGGAGGCCCAUdTdT-3′; si-2: 5′-GACUCCAGUGGUAAUCUACdTdT-3′), mTOR (5′-GTAAATGCTTCCACTAAACdTdT-3′), Rictor (5′-GCAGCCUUGAACUGUUUAAdTdT-3′), TBK1 (5′-UGACAGAGAUUUACUAUCAdTdT-3′), IKBKE (5′-GCUGAACCACCAGAACAUUdTdT-3′) were provided by Suzhou GenePharma Co., Ltd. The scrambled sequence was used as a control. For p53 gene knockout, the sgRNA sequence (5′-CGCTATCTGAGCAGCGCTCA-3′ and 5′-CCATTGTTCAATATCGTCCG-3′) were cloned into lenti-CRISPRv2 consisting of Flag-Cas9. Lentivirus was packaged into 293T cells using calcium phosphate transfection. Viral supernatants were collected to infect target cells. Lenti-CRISPRv2 with scrambled sequence was used as a control.
Cells were directly lysed in 2× SDS loading buffer and boiled for 15 min. Proteins were separated by SDS–PAGE, transferred to PVDF membrane, and probed with antibodies specific for, p-AKT (1:2000 dilution, s473, 4060, Cell Signaling Technology), AKT (1:2000 dilution, ab32505, Abcam), p-p53 (1:2000 dilution, s15, 9284, Cell Signaling Technology), p-JNK (1:2000 dilution, 9251, Cell Signaling Technology), JNK (1:2000 dilution, 9252, Cell Signaling Technology), c-Myc (1:2000 dilution, ab32072, Abcam), p53 (1:1000 dilution, sc-126, Santa Cruz), and Flag (1:5000 dilution, F1804, Sigma), mTOR (1:2000 dilution, 2983, Cell Signaling Technology), Rictor (1:2000 dilution, 2114, Cell Signaling Technology), TBK1 (1:2000 dilution, 3504, Cell Signaling Technology), IKBKE (1:2000 dilution, 3416, Cell Signaling Technology). β-Actin (1:5000 dilution, 66009-1-Ig, Proteintech) antibody was used as a loading control. Samples derived from the same experiment and gels/blots were processed in parallel.
Cell cycle synchronization
U2OS cells were synchronized with thymidine (2 mmol/L) for 19 h, washed with pre-warmed PBS (3 times), then released into fresh medium for 9 h; thymine (2 mmol/L) was then added for 17 h followed by washing with pre-warmed PBS (3 times) before release into fresh medium for 0, 3, 6 or 9 h.
Immunofluorescence and fluorescence in situ hybridization (IF-FISH)
Briefly, cells on the coverslip were fixed with 4% paraformaldehyde, and then permeabilized with 0.5% Triton X-100 (in 1× PBS). Cells were incubated overnight at 4 °C with primary antibodies against 53BP1 (1:2000 dilution, NB100-304, Novus), washed three times with 1× PBST, and incubated with secondary antibodies (1:2000 dilution, DyLight 549-conjugated anti-mouse, KPL). Cells were washed three times with 1× PBST, re-fixed with 4% paraformaldehyde for 30 min, dehydrated by ethanol series solution, denatured at 85 °C for 5 min, and then hybridized with FITC-labeled (CCCTAA)3 PNA probe (Panagene) at 37 °C for 4 h. The cells were washed and mounted with DAPI. Fluorescence was detected and imaged using Zeiss Axion Imager Z1 microscope.
Quantitative real-time PCR
Total RNA was extracted from cells using RNAiso Plus Reagent (Takara) according to manufacturer’s instructions. 1.0 μg of total RNA was reverse-transcribed to cDNA using PrimeScript RT reagent Kit (Takara). cDNA was used for real-time PCR using 2× RealStar Green Fast Mixture (GenStar). β-Actin was used as internal control for all experiments. The following primers were used for amplification: β-actin-forward: 5′-CATGTACGTTGCTATCCAGGC-3′; β-actin-reverse: 5′-CTCCTTAATGTCACGCACGAT-3′; BAX-forward: 5′-CCCGAGAGGTCTTTTTCCGAG-3′; BAX-reverse: 5′-CCAGCCCATGATGGTTCTGAT-3′; NOXA-forward: 5′-ATGCTGCGTTTCACCAGGG-3′; NOXA-reverse: 5′-TCCATGCTACTTGCACTTGTTCCT-3′; PUMA-forward: 5′-GACCTCAACGCACAGTACGAG-3′; PUMA-reverse: 5′-AGGAGTCCCATGATGAGATTGT-3′; mTOR-forward: 5′-GCAGATTTGCCAACTATCTTCGG-3′; mTOR-reverse: 5′-CAGCGGTAAAAGTGTCCCCTG-3′; Rictor-forward: 5′-AGAAGCACGATTTCTAGCCAGT-3′; Rictor-reverse: 5′-AGTAGACCTCGCCTTATTTCCA-3′.
U2OS stably expressing p53 were cross-linked with 1% formaldehyde for 10 min at room temperature. Reaction was terminated by 1.25 mmol/L Glycine, and cells were washed twice with cold PBS, re-suspended in SDS lysis buffer (50 mmol/L Tris-HCl, pH = 8.1, 10 mmol/L EDTA, 1% SDS) and sonicated until DNA fragmentation reached sizes of 200 bp to 1 kb. Chromatin immunoprecipitation (ChIP) was carried out overnight at 4 °C with flag-beads (M8823, sigma). Beads were washed three times, and eluted with 0.1 mol/L NaHCO3 & 1% SDS, followed by reverse cross-linking and phenol-chloroform extraction. DNA fragments were precipitated by ethanol. The following primers were used to identify fragments of mTOR and Rictor promoters: M-0.5-forward: 5′-CTGCATCAAAAGACGTTTCGGGCT-3′; M-0.5-reverse: 5′-ACTTGGCTATGGTTTCTCTGGAGTG-3′; M-TS-forward: 5′-TTCGGCCAAGGCCTCAGCTG-3′; M-TS-reverse: 5′-GCTTCTCTTCTTCATTACTATTGGCC-3′; M+1-forward: 5′-GACAGAGCGAAACTCCGTCTCA-3′; M+1-reverse: 5′-CCAGGTTTTTCCTCTGTGCCTTAC-3′; R−1-forward: 5′-TCCAGGGCACTTACTCATCCAACA-3′; R−1-reverse: 5′-ACTGACCCAGCAGCTTTCTCTTGA-3′; R−0.5-forward: 5′-TCTATGGCAGGGCTTCAGAGCAA-3′; R−0.5-reverse: 5′-AGTTCCCACGAGGAAAGTCCCATT-3′; R+0.6-forward: 5′-TGCAGGAGGATGTTTGAGGGAAGA-3′; R+0.6-reverse: 5′-AAAGGGAAGCAGAAGGGAAACAGC-3′.
Cell apoptosis assay
To detect apoptotic cells, cells treated with siRNA or indicated concentrations of compounds were co-stained with Annexin-V (FTIC) and PI (Annexin-V/PI apoptosis detection kit, KGA105, KeyGen BioTech) according to manufacturer’s instructions. Stained cells were analyzed by FACS Calibur (BD Bioscience) and data were analyzed with FlowJo software.
Constant-field gel electrophoresis (CFGE)
CFGE assay was performed as described previously (Mao et al., 2016). Cells were imbedded in 0.7% agarose, lysed with 0.5% SDS in Tris-HCl and digested with RNase A (100 μg/mL) and proteinase K (250 μg/mL) at 37 °C overnight. Gel electrophoresis was performed using 0.7% agarose in TAE buffer. Genomic DNA was stained by gel-red, and telomeric DNA was detected by hybridization with C-rich probe.
Establishment of xenograft models using human U2OS cells
A total of 3.0 × 106 U2OS cells were inoculated subcutaneously into the dorsal flank of the four-week-old female BALB/c nude mice in 100 μL PBS. Mice were randomly divided into control, and PFTα treatment groups. 2.2 mg/kg of PFTα dissolved in phosphate-buffered saline (PBS) was injected (Intraperitoneal injection) two times a week for 4 weeks. No animal died during the experimental period. The tumor size was estimated from the measurements of the longest diameter across the tumor and the corresponding perpendicular diameter.
Histology staining of p-AKT and apoptotic cells in xenograft tumor
Tumors were fixed overnight in 4% paraformaldehyde at room temperature, transferred to 70% ethanol and embedded in paraffin. 4 μm sections on slides were dewaxed in xylene, and then sequentially rehydrated in 100%, 95%, 70% ethanol and PBS buffer. For immunohistochemistry, sections were blocked with 5% goat serum in PBS buffer, then incubated overnight with primary antibodies against p-AKT (1:1000 dilution, s473, 4060, Cell Signaling Technology) at 4 °C, washed three times with 1× PBST, and incubated with secondary antibodies (HRP-conjugated anti-rabbit, KPL). Sections were washed three times with 1× PBST and stained with DAB and hematoxylin (for DNA staining) according to standard protocols. To assess the frequency of apoptosis, tissue was analyzed using a TdT-mediated dUTP-digoxigenin nick end labeling (TUNEL) method. Staining was performed according to manufacturer’s instructions (TUNEL assay Kit, KGA7061, KeyGEN BioTECH).
Statistical analysis was performed using GraphPad Prism version 5 (GraphPad Software). Data are expressed as the mean ± SEM. Comparisons between groups were made using a 2-tailed unpaired Student’s t test. A P value of less than 0.05 was considered statistically significant.
All animal experiments were approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University (Guangzhou, China).
This work was supported by the National Natural Science Foundation of China (Grants Nos. 31571410, 31701196, 81702756, 31570827 and 81771506), the National Key R&D Program of China (2018YFA0107000), and Guangzhou Municipal People’s Livelihood Science and Technology Plan (201803010108 and 201604016111), the China Postdoctoral Science Foundation (2016M600696), and the Fundamental Research Funds for the Central Universities (171gpy97 and 18lgpy52).
ALT, alternative lengthening of telomeres; DDR, DNA damage response; HR, homologous recombination; PFTα, pifithrin-α; PML, ALT-associated promyelocytic leukaemia; REs, DNA recognition elements; ROS, reactive oxygen species; TIFs, telomere dysfunction-induced foci
COMPLIANCE WITH ETHICS GUIDELINES
Yuanlong Ge, Shu Wu, Zepeng Zhang, Xiaocui Li, Feng Li, Siyu Yan, Haiying Liu, Junjiu Huang and Yong Zhao declare that they have no potential conflicts of interest. This article does not contain any studies with human subjects performed by any of the authors. All institutional and national guidelines for the care and use of laboratory animals were followed.
- Mangerel J, Price A, Castelo-Branco P, Brzezinski J, Buczkowicz P, Rakopoulos P, Merino D, Baskin B, Wasserman J, Mistry M et al (2014) Alternative lengthening of telomeres is enriched in, and impacts survival of TP53 mutant pediatric malignant brain tumors. Acta Neuropathol 128:853–862CrossRefGoogle Scholar
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