L- and D-lactate enhance DNA repair and modulate the resistance of cervical carcinoma cells to anticancer drugs via histone deacetylase inhibition and hydroxycarboxylic acid receptor 1 activation
© Wagner et al. 2015
Received: 15 May 2015
Accepted: 16 July 2015
Published: 25 July 2015
The consideration of lactate as an active metabolite is a newly emerging and attractive concept. Recently, lactate has been reported to regulate gene transcription via the inhibition of histone deacetylases (HDACs) and survival of cancer cells via hydroxycarboxylic acid receptor 1 (HCAR1). This study examined the role of L- and D-lactate in the DNA damage response in cervical cancer cells.
Three cervical cancer cell lines were examined: HeLa, Ca Ski and C33A. The inhibitory activity of lactate on HDACs was analysed using Western blot and biochemical methods. The lactate-mediated stimulation of DNA repair and cellular resistance to neocarzinostatin, doxorubicin and cisplatin were studied using γ-H2AX, comet and clonogenic assays. HCAR1 and DNA repair gene expression was quantified by real-time PCR. DNA-PKcs activity and HCAR1 protein expression were evaluated via immunocytochemistry and Western blot, respectively. HCAR1 activation was investigated by measuring intracellular cAMP accumulation and Erk phosphorylation. HCAR1 expression was silenced using shRNA.
L- and D-lactate inhibited HDACs, induced histone H3 and H4 hyperacetylation, and decreased chromatin compactness in HeLa cells. Treating cells with lactate increased LIG4, NBS1, and APTX expression by nearly 2-fold and enhanced DNA-PKcs activity. Based on γ-H2AX and comet assays, incubation of cells in lactate-containing medium increased the DNA repair rate. Furthermore, clonogenic assays demonstrated that lactate mediates cellular resistance to clinically used chemotherapeutics. Western blot and immunocytochemistry showed that all studied cell lines express HCAR1 on the cellular surface. Inhibiting HCAR1 function via pertussis toxin pretreatment partially abolished the effects of lactate on DNA repair. Down-regulating HCAR1 decreased the efficiency of DNA repair, abolished the cellular response to L-lactate and decreased the effect of D-lactate. Moreover, HCAR1 shRNA-expressing cells produced significantly lower mRNA levels of monocarboxylate transporter 4. Finally, the enhancement of DNA repair and cell survival by lactate was suppressed by pharmacologically inhibiting monocarboxylate transporters using the inhibitor α-cyano-4-hydroxycinnamic acid (α-CHCA).
Our data indicate that L- and D-lactate present in the uterine cervix may participate in the modulation of cellular DNA damage repair processes and in the resistance of cervical carcinoma cells to anticancer therapy.
KeywordsLactate DNA repair HDACs HCAR1 Cervical cancer
The model of lactate as an active metabolite has emerged as an attractive concept. The role of lactate as a signalling factor is supported by observations that lactate mimics hypoxic conditions, stimulates connective tissue synthesis and enhances endothelial cell mobility and tumour angiogenesis [1–3]. Locally produced L-lactate may play a role in hormone function in an autocrine and paracrine fashion via hydroxycarboxylic acid receptor 1 (HCAR1, also referred to as GPR81/FKSG80) to exert antilipolytic effects on adipose tissue  or to modulate the activity of primary cortical neurons . Recently, HCAR1 has been implicated in the regulation of lactate transport mechanisms. HCAR1 presence enhances pancreatic cancer cell growth and metastasis , and is necessary for survival of the HER2-positive and the triple-negative breast cancer cells . The intracellular biological activity of lactate depends on its cellular uptake, which is facilitated by monocarboxylate transporters (MCTs) . Functional studies have identified MCT1-4 in the plasma membrane of various cell types, including uterine cervical cells, and have demonstrated that the bidirectional transport of monocarboxylates (e.g., lactic acid, pyruvic acid and acetic acid) across the plasma membrane is directed by substrate and proton concentration gradients . Cells with high glucose metabolism (e.g., most cancer cells) export lactic acid to maintain intracellular homeostasis, whereas other cells, such as astrocytes and heart and skeletal muscle cells, import lactic acid for mitochondrial respiration or as a substrate for gluconeogenesis (hepatocytes). The weak inhibitory activity of both L- and D-lactate on histone deacetylases (HDACs) was also recently reported . Thus, lactate, a natural fermentation product (e.g., butyrate, an established potent HDAC inhibitor), is an important effector of the epigenetic regulation of chromatin function. HDACs are involved in acetylation, an important posttranslational protein modification, and their activity opposes that of histone acetyltransferases (HATs). In general, an increasing level of histone acetylation (hyperacetylation) results in a more relaxed, transcriptionally permissive chromatin conformation, whereas the reverse action (hypoacetylation) results in a more condensed, transcriptionally repressive chromatin state. One of the primary implications of chromatin-HAT/HDAC remodelling complex interactions is its important but poorly characterised role in regulating the DNA damage response (DDR) [10, 11]. HATs and HDACs are recruited to DNA double-strand break (DSB) sites to create a repair-proficient chromatin state that orchestrates the activity of repair and signalling proteins, thereby promoting DNA repair processes [12–14]. Furthermore, recent evidence suggests that the coordinated action of HATs/HDACs may directly affect the DDR by modulating the activity of key proteins involved in DNA damage detection and repair, such as DNA-PK  and ATM .
The lower female genital tract is an internal structure of the body in which an extremely high concentration of lactate is maintained by symbiotic lactic acid bacteria under physiological conditions . Vaginal secretions may contain 10–50 mM lactate; approximately 55 % of vaginally secreted lactate is the D isoform . Therefore, the modulation of mucosal epithelial cell activity in the female reproductive tract via the inhibition of HDACs by lactate represents an appealing topic of investigation because these cells are constantly exposed to L-/D-lactate of bacterial origin. This potential modulation is particularly important in the context of patients with cervical cancer, in which lactate may modulate the activity of the cervical cancer cells in a manner that alters the effectiveness of chemotherapeutic agents. The present study reports a novel biological activity of lactate, specifically the modulation of cellular DDR processes, in cervical cancer cells. We demonstrated that concentrations of L- and D-lactate consistent with those observed in the uterine cervix inhibit class I and II HDACs, induce the hyperacetylation of H3 and H4 histones, increase chromatin accessibility and significantly enhance the DNA repair rate in cervical cancer cells, as evaluated by γ-H2AX and comet assays. The observed increase in the activity of the DNA repair machinery was accompanied by a significant enhancement of the survival of three different cervical cancer cell lines after chemotherapeutic treatment. In addition, we showed that all three examined cervical cancer cell lines display surface expression of HCAR1, which is known to be involved in cell survival. Furthermore, we demonstrated the essential role of HCAR1 and MCTs in the lactate-mediated enhancement of cellular DNA repair capacity and in the resistance of the examined cervical cancer cell lines to anticancer chemotherapeutics. Importantly, the present study provides new insight into the role of microorganism-mammalian cell interactions in the female genital tract and demonstrates a novel mechanism underlying the regulation of cellular resistance to genotoxins/chemotherapeutics.
L- and D-lactate stimulate the acetylation of histones via the inhibition of histone deacetylases
Lactate- and butyrate-induced histone hyperacetylation corresponds to decreased chromatin compactness
Increased histone acetylation at lysine residues due to the dominance of HAT activity over HDAC activity reduces the positive charge of histones and disrupts electrostatic interactions between DNA and histones. Extensive acetylations occur on numerous histone tail lysines, including H3K9, H3K14, H3K18, H4K5, H4K8 and H4K12 . This process leads to chromatin relaxation. Because both lactate and butyrate are HDAC inhibitors that contribute to H3 and H4 pan-acetylation, we examined the effect of lactate on chromatin compactness. We studied lactate and butyrate at concentration ranges of 10–20 mM and 0.25–2.5 mM, respectively. After 24 h of treatment, we observed a decrease in chromatin compactness for both compounds tested, which was observed as a reduction in the number of distinct spaces within the nucleus (Fig. 1c) after image transformation using the Sobel edge detection algorithm. Raw images of Hoechst-stained cells were used to calculate the coefficient of variation (CV) for Hoechst nuclear fluorescence and to quantify chromatin compactness (Fig. 1d). Incubating cells with 2.5 mM butyrate significantly decreased the Hoechst CV by 8 %, whereas cells treated with 20 mM L-lactate or D-lactate showed a less pronounced decrease in the Hoechst CV, as their respective Hoechst CVs were 3.2- and 1.7-fold lower than that of 2.5 mM butyrate.
L- and D-lactate enhance DNA double-strand break repair following neocarzinostatin, doxorubicin and cisplatin treatment
Incubating HeLa cells in L- or D-lactate for 24 h induces the expression of genes involved in DNA repair
Effect of L-lactate and D-lactate on DNA repair gene expressiona
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0.83 ± 0.15
1.76 ± 0.28*
1.54 ± 0.26
1.01 ± 0.18
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1. 54 ± 0.82
Lactate initiates the nuclear activation of DNA-PKcs
L- and D-lactate enhance cervical cancer cell survival after chemotherapeutic treatment
Modulation of the chemoresistance of cervical cancer cells by lactate
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HCAR1 abundance and functionality in cervical cancer cell lines
Pertussis toxin compromises L-lactate-, D-lactate- and 3,5-dihydroxybenzoic acid- stimulated γ-H2AX foci resolution in cells treated with chemotherapeutics
HCAR1 and MCT activity is required for the L- and D-lactate-mediated enhancement of DNA repair and of HeLa cell survival
Recent evidence has identified lactate as an active metabolite and a pseudo-hormone that coordinates metabolic processes at the systemic and cellular levels. L-lactate produced by malignant tumours may be a crucial component enabling significant cancer cell growth and resistance [6, 7, 24]. Latham and colleagues have demonstrated that both L- and D-lactate inhibit HDACs and promote changes in gene expression in a manner similar to the established HDAC inhibitors butyrate and trichostatin A . This evidence strongly suggests that lactate is involved in the posttranslational modifications of histone and non-histone proteins and that lactate participates in the modulation of protein signalling and activity. Our study is the first to demonstrate that pretreating cervical cancer cells with lactate improves their DNA repair capacity and enhances cell survival following chemotherapeutic treatment. In the present study, we incubated cervical cancer cells with 10–20 mM lactate to mimic physiological lactate concentrations in the lower female genital tract environment. Lower female genital tract epithelium-associated bacterial flora are the major producers of the L- and D-lactate found in vaginal secretions (at concentrations as high as 10–50 mM). Biochemical experiments demonstrated that incubating HeLa cells with L- or D-lactate decreased the activity of class I and II HDACs in living cells as expected. The inhibition of HDACs by lactate was accompanied by the acetylation of histones H3 and H4 and by a decrease in DNA compactness (Fig. 1). Incubating cells with lactate for 24 h evoked a transcriptionally permissive chromatin conformational state and a subsequent significant up-regulation of important genes involved in DNA DSB repair, including LIG4, NBS1, and APTX (Table 1). Indeed, mutation of the genes encoding DNA ligase IV (LIG4), Nibrin (NBS1) or Aprataxin (APTX) results in DDR disorders, specifically LIG4 syndrome, Nijmegen breakage syndrome (NBS) and Ataxia oculomotor apraxia-1, in association with increased radiosensitivity [25–27]. Recent evidence suggests that HATs and HDACs are recruited to DNA DSB sites to create a repair-proficient chromatin state that orchestrates the activity of repair and signalling proteins, thereby promoting DNA repair processes [12–14]. Our study is the first to demonstrate that L- and D-lactate stimulate the activation of DNA-PKcs (Fig. 3), the essential component of NHEJ, although previous studies have demonstrated that histone H3 acetylation induced by trichostatin A  or histone acetylation in response to DSBs  facilitates DNA-PKcs activation. Thus, it is conceivable that such up-regulation of DNA repair mechanisms via a lactate-elicited increase in the activity of DNA-PKcs and in the transcriptional expression of DNA ligase IV, Nibrin and Aprataxin could translate into accelerated processing of DNA damage. Indeed, L- and D-lactate improved the kinetics of γ-H2AX foci formation and resolution and the dynamics of DNA DSB repair after exposure to NCS, DOX or CDDP. Both lactate isomers significantly enhanced DNA repair, although D-lactate, a stronger HDAC inhibitor, was found more effective than L-lactate (Fig. 2). The observations of lactate-induced enhancement of DNA repair were further supported by the protective effects of lactate on cell survival. We showed that lactate increased the survival of three cervical cancer cell lines: HeLa, Ca Ski and C33A; however, the Ca Ski and C33A cell lines were less prone to lactate-induced modulation than the HeLa cell line (Fig. 4). Interestingly, the enhancement of cervical cancer cell survival by L-lactate treatment corresponded to the HCAR1 protein level in the respective cell lines (Fig. 5a, b). Of the three examined cell lines, the HeLa cell line, demonstrating the most abundant expression of HCAR1, showed the most prominent protective effect of lactate on clonogenic survival, as its survival fraction (SF) increased by 2.4-fold after L-lactate treatment, which was higher than that of the Ca Ski and C33A cell lines (both 1.5-fold) (Table 2). Our observations are in line with recent evidence indicating that the HCAR1 levels correlate to the rates of cancer tumour growth and metastasis . In the present study, we demonstrated that cervical cancer cell lines display HCAR1 surface expression and that both lactate isomers induced signalling pathways in a receptor-dependent fashion (Fig. 5a-d). Interestingly, L-lactate preferentially stimulated Gi-mediated pathway, inhibited forskolin-induced intracellular cAMP and slightly activated MAPK pathway, while D-lactate triggered MAPK pathway only. These observation suggest that L- and D-lactate may differ in intrinsic activity towards HCAR1 resulting in differential activation of signal transduction pathways associated with this receptor . In addition, incubating HeLa cells with the HCAR1 agonist 3,5-DHBA improved the kinetics of γ-H2AX foci formation and resolution to a similar but smaller extent than lactate. As expected, uncoupling G-proteins from HCAR1 via pretreatment with PTX decreased L-lactate-, D-lactate- and 3,5-DHBA-stimulated γ-H2AX foci disappearance in cells exposed to NCS (Fig. 6c-e). Furthermore, experiments using HCAR1 shRNA-expressing cells showed that silencing HCAR1 exerted profound effects on DNA repair kinetics, which were observed as considerably diminished γ-H2AX foci disappearance kinetics in the presence or absence of lactate (Fig. 7a, b). Based on the study by Roland and co-workers  HCAR1 is implicated in the regulation of lactate transport mechanisms. Our study using HCAR1 shRNA-expressing HeLa cells revealed that silencing HCAR1 affects the mRNA level of MCT4 (Fig. 7c), which facilitate the cellular uptake of lactate. Complimentary experiments performed using α-CHCA, a classic MCT inhibitor , confirmed that the observed stimulatory effect of lactate on DNA repair and cell survival depends on its intracellular activity (Fig. 7d, e). Taken together, these data demonstrated that lactate transport by MCTs is crucial for its intracellular activity, which leads to chromatin rearrangement and enhanced DNA repair and cell survival. However, although the observed lactate-induced stimulation of DNA repair activity seems to result from its inhibitory activity on HDACs, we cannot rule out the possibility that other HCAR1-dependent responses may affect cellular DNA repair capacity. It is worth noted that D-lactate induced marked Erk phosphorylation via PTX-sensitive pathway compared to slight MAPK signalling activation by L-lactate (Fig. 5d). A detailed study by Li and co-workers  showed that upon HCAR1 activation, the dissociation of the Gβγ subunit from the Gi protein subsequently induces Erk activation via two distinct pathways: a PKC-dependent pathway and an IGF-1R transactivation-dependent pathway . Because IGF-1R and EGFR are known for their cross-talk  and both receptors are involved in HR and NHEJ [28, 29], the involvement of the HCAR1/IGF-1R/EGFR axis in DNA repair requires further investigation.
In the present study, we reported a new potential mechanism underlying the interaction between lower female genital tract microbiota and cervical epithelial cells under physiological conditions. Vaginal and ectocervical microbiota not only protect against pathogen colonisation by acidifying the mucosa using lactic acid but also, according to our results, appear to modulate the activity of the cervical cancer cells in a manner that alters its resistance to chemotherapeutics. Our data indicate a novel mechanism by which lactate modulates the cellular DNA damage repair process in cervical cancer cells; in this mechanism, L- and D-lactate are actively transported across the cell membrane by MCTs to intracellular compartments, leading to the inhibition of class I and II HDACs. This inhibition results in the hyperacetylation of histones H3 and H4, chromatin relaxation, DNA repair gene up-regulation and DNA-PKcs activation in the nucleus. Thus, lactate creates a DNA repair-proficient environment that stimulates DNA repair dynamics and significantly enhances cervical carcinoma cell survival after drug treatment. Furthermore, we also showed that lactate-induced DNA repair enhancement is regulated by the HCAR1/MCT axis, as lactate receptor down-regulation or MCT inhibition notably affects DNA repair efficiency. We suggest that the enhancement of DNA repair machinery activity by lactate may account for the increased resistance of malignant cervical tumours to standard clinical therapy (such as cisplatin and ionising radiation). Thus, targeting lactate-mediated signalling in cervical cancer environment, e.g. by locally delivered MCTs inhibitors and/or HCAR1 antagonist, might improve efficacy of anticancer therapy.
Materials and methods
All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated. Doxorubicin, forskolin, α-CHCA and U0126 were dissolved in anhydrous DMSO and added to cells at a final DMSO concentration of 0.5 % (v/v). Control cells were incubated in 0.5 % DMSO alone.
The HeLa, Ca Ski and C33A human cervical cancer cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and were authenticated by short tandem repeat profiling (LGC Standards, UK) at the end of the study. HeLa cells were cultured in DMEM, and Ca Ski and C33A cells were cultured in RPMI medium (Life Technologies, Carlsbad, CA, USA) supplemented with 10 % foetal bovine serum (PAA Laboratories GmbH, Pasching, Austria) and antibiotics (Life Technologies) at 37 °C in a humidified atmosphere containing 5 % CO2. The cells were routinely tested for mycoplasma contamination and were passaged every 3 days using TrypLE Express (Life Technologies).
The inhibitory effects of lactate and butyrate on cellular HDAC activity were measured using an HDAC-Glo I/II Assay Kit (Promega, Madison, WI, USA) for class I/II HDACs according to the manufacturer’s protocol. Briefly, HeLa cells grown on 96-well plates were treated with sodium L-lactate, sodium D-lactate, sodium butyrate or sodium chloride for 2 h. Then, HDAC-Glo™ I/II Reagent was added, and the luminescence was directly measured using a chemiluminescence plate reader after a 30-min incubation at room temperature.
Western blot analysis
The histone acetylation status of HeLa cells was evaluated after 24 h incubation in lactate or butyrate. The harvested cells were washed twice with ice-cold phosphate-buffered saline (PBS) supplemented with either lactate (10 or 20 mM) or butyrate (5 mM) and centrifuged at 400 g for 8 min at 4 °C. Then, the cells were suspended in Triton extraction buffer (TEB: PBS containing 0.5 % Triton X-100 (v/v), 2 mM PMSF, and 0.02 % (v/v) NaN3) at a density of 1 × 106 cells/100 μl and lysed on ice for 10 min. After centrifugation at 6500 g for 10 min, the pellet was washed with a half volume of TEB, suspended in 0.2 N HCl at a density of 3 × 106 cells/70 μl, and incubated at 4 °C overnight for acidic extraction. The next day, the samples were centrifuged, and the extracts were neutralised with a 1/50 volume of 10 N NaOH, followed by SDS-PAGE (NuPAGE gradient gel 4–12 %, Life Technologies) and transfer to a nitrocellulose membrane. After 1 h of blocking (SuperBlock Blocking Buffer, Thermo Fisher Scientific, Inc., Waltham, MA, USA) and overnight incubation at 4 °C in anti-acetyl-H3 and -H4 primary antibodies (#39140 and #39967, Active Motif, Carlsbad, CA, USA), the membrane was washed with TBS and incubated in an HRP-conjugated secondary antibody (Dako, Ely, UK) for 1 h at RT. Antibody binding was visualised via chemiluminescence using SuperSignal West Pico Substrate (Thermo Fisher Scientific, Inc.). The membrane was stripped and re-probed with anti-H3 and -H4 primary antibodies (#39763 and #61300, Active Motif) for 1 h at RT as described above. The experimental design for evaluating the HCAR1 protein level and Erk phosphorylation was as described above with minor changes. For analysis of the HCAR1 protein level, cells grown at 70 % confluence were harvested using a cell scraper, centrifuged and lysed with RIPA buffer supplemented with a protease inhibitor cocktail (Roche Diagnostics GmbH). For Western blot analysis of Erk phosphorylation, HeLa cells that were serum-starved (DMEM, 0.5 % FBS) overnight were stimulated with lactate for 5 min in the presence or absence of 100 ng/ml PTX (overnight pretreatment) or 10 μM of the MAPK inhibitor U0126 (2 h pretreatment). The cells were harvested using a cell scraper, centrifuged and lysed with PhosphoSafe Extraction Reagent (Novagen, EMD Chemicals, CA, USA) containing a protease inhibitor cocktail (Roche). The primary antibodies used were anti-GPR81 (ab106942, Abcam, Cambridge, UK), anti-p-Erk1/2, anti-Erk and anti-β-actin conjugated to HRP (sc-101760, sc-94 and sc-1616, respectively, Santa Cruz Biotechnology, Inc.). The membrane handling and incubation conditions were as described above. Chemiluminescence signals were captured and quantified using a G:BOX gel imager (Syngene, Cambridge, UK).
Quantification of DNA compactness
Cells grown on a 96-well plate were washed with ice-cold PBS, fixed in 4 % formaldehyde for 20 min and washed twice with PBS. After the final PBS wash, the cells were stained with 1 μg/ml of Hoechst 33,342 solution in PBS for 10 min and subjected to analysis using an ArrayScan VTI HCS Reader (Thermo Fisher Scientific, Inc.) equipped with a 40× objective. To visually assess changes in chromatin structure after lactate or butyrate treatment, representative images were subjected to Sobel edge detection and thresholding using ImageJ software. The process of chromatin expansion decreases the number of distinct spaces within the nucleus, and these changes can be visualised via the detection of the Sobel edges as shown by Irianto and co-workers . Quantitative analysis of chromatin compactness was performed by calculating the CV for the Hoechst intensity of all pixels within individual nuclei according to the method described by Contrepois and co-workers . The Target Activation Bioapplication software (Thermo Fisher Scientific, Inc.) was set up to analyse 100 cells per well and to calculate the mean fluorescence and standard deviation of Hoechst staining within each nucleus. The CV parameter was obtained by dividing the standard deviation by the mean fluorescence. Each experiment was performed in six replicates.
DNA DSB repair assay
DNA DSB repair was measured via a neutral comet assay as previously described  using a Comet Assay Kit (Trevigen, Gaithersburg, MD, USA). Comets were stained with SYBR Green I, visualised using a Nikon D-Eclipse Ti fluorescence microscope (5× objective) and analysed using CASP software .
Colony formation assay
Actively growing cells in flasks were incubated in the presence or absence of lactate for 24 h before treatment with chemotherapeutics for the next 24 h. Then, the cells were harvested via trypsinisation, seeded in 10-cm-diameter Petri dishes at densities ranging from 500 to 400,000 per dish in triplicate in drug-free medium and then allowed to form colonies. After 14 days, the colonies were fixed in Carnoy’s solution and stained with crystal violet. Images of the dishes were captured using a G:BOX imager and processed using ImageJ software, which was set up to count colonies containing > 50 cells. The SIF was calculated by dividing the SF of cells treated with a cytotoxic agent and lactate by the SF of cells treated with a cytotoxic agent alone. The SF values used to determine the SIF were calculated using the linear quadratic model [SF = exp(−aD-bD2)] in GraphPad Prism software according to the least-squares fit.
γ-H2AX, phospho-DNA-PKcs and HCAR1 immunocytochemistry
Cells grown on a 96-well plate were washed with ice-cold PBS and fixed with ice-cold methanol:acetone (1:1) for 20 min at −20 °C. After the blocking procedure (1 % BSA in PBST, 1 h), the cells were stained with a rabbit antibody against γ-H2AX (ab2893, Abcam, Cambridge, UK) at 4 °C overnight in a humidified chamber. Primary antibody binding was visualised using an Alexa Fluor 594-conjugated goat anti-rabbit antibody (Life Technologies) followed by nuclear staining with 1 μg/ml Hoechst 33342 for 20 min. The plate was analysed using an ArrayScan VTI HCS Reader equipped with a 40× objective. Images of 20 fields per well were routinely acquired, and 150 cells/well were analysed using Spot Detector Bioapplication V3 software. Each experiment was performed in triplicate. The experimental design for evaluating DNA-PKcs phosphorylation was as described above with minor changes. The cells were fixed (4 % formaldehyde, 20 min), permeabilised (0.25 % Triton X-100 in PBS, 10 min) and blocked (3 % BSA in PBST, 30 min) before incubation in an anti-pS2056-DNA-PKcs antibody (ab18192, Abcam) as the primary antibody overnight at 4 °C. Images were acquired using an ArrayScan VTI HCS Reader equipped with a 20× objective and analysed using Spot Detector Bioapplication V3 software (p-DNA-PKcs, 250 cells/well). Each experiment was performed in six replicates. For cell surface HCAR1 staining, cells grown on Lab-Tek chamber slides (Nunc, Thermo Fisher Scientific, Inc.) were washed with HBSS and incubated in an anti-FKSG80 antibody (sc-32647, Santa Cruz Biotechnology, Inc.) in HBSS at 4 °C for 60 min. After fixation with 4 % formaldehyde and blocking with 5 % normal donkey serum in PBS for 30 min, the cells were incubated in an Alexa Fluor 488-conjugated anti-goat secondary antibody (Invitrogen) at RT for 1 h and stained with Hoechst 33342. Images were acquired using a confocal microscope (Nikon D-Eclipse C-1 Plus) equipped with a 63× objective.
Total RNA was extracted from cells using TRIzol reagent (Sigma-Aldrich) according to the manufacturer’s instructions. Then, total RNA (5 μg) was reverse-transcribed using a Maxima First Strand cDNA Synthesis Kit for RT-qPCR (Thermo Fisher Scientific, Inc.). PCR was performed using LightCycler 480 SYBR Green I Master Mix (Roche Diagnostics GmbH, Mannheim, Germany) and a Roche LightCycler 480 Instrument (Roche Diagnostics GmbH). Relative gene expression was normalised to the housekeeping genes hydroxymethylbilane synthase (HMBS) and hypoxanthine phosphoribosyl transferase (HPRT) and were calculated using the ΔΔCt method. In total, 5 reference genes (GAPDH, ACTB, HPRT, HBMS, TBP) were tested and Normfinder  was used to identify the most stably expressed housekeeping genes (stability values: GAPDH: 0.153, ACTB: 0.240, HPRT: 0.091, HBMS: 0.095, TBP: 0.115).The study of mRNA expression included the following genes: LIG3, XRCC1, PNKP, PARP1, PARP2, RAD51, BRCA1, BRCA2, RAD50, MRE11A, NBS1, XRCC6, XRCC5, PRKDC, LIG4, XRCC4, DCLRE1C, WRN, NHEJ1, ATM, ATR, TP53, APTX, PARD3, MDC1, MCT1, MCT2, MCT4, and HCAR1. The primer sequences are listed in Additional file 10.
cAMP accumulation assay
The day before the experiment, the culture medium was replaced with serum-free medium. All experiments were conducted in the presence of the phosphodiesterase inhibitor IBMX at 500 μM. For the cAMP accumulation studies, HeLa cells were treated with 10–20 mM L- or D-lactate in the presence of forskolin (10 μM) to stimulate cAMP synthesis. The reaction was incubated at 37 °C for 30 min and then terminated via two cold PBS washes, immediately followed by cell harvesting using a cell scraper. The centrifuged cells were resuspended in lysis buffer, and the measurement of the intracellular cAMP levels was performed using a cAMP assay kit (R&D Systems). The data are presented as the means ± SEM of at least three separate experiments.
Short hairpin RNA
shRNA (Dharmacon, Lafayette, CO, USA) was used to silence HCAR1 according to the protocol provided by the manufacturer. Briefly, 24 h before transfection, HeLa cells were seeded on 24-well plates at a density of 5 × 104 cells/well. Next, the cells were transfected with 1 μg of HCAR1 shRNA plasmid DNA using 2 μl of TurboFect Transfection Reagent (Life Technologies) in serum-free DMEM. Control cells were treated with non-targeted shRNA (Dharmacon). The next day, the medium was replaced with DMEM containing 10 % FBS and puromycin (7.5 μg/ml) (Life Technologies), and the selection of cells expressing the transgene was continued for 3 weeks, and the selection medium was changed every 3 days. Resultant puromycin-resistant and GFP-positive cells were evaluated for HCAR1 expression via real-time PCR and Western blot.
The experiments were repeated at least three times, each of which was conducted in three or six replicates (except for Western blot analysis). The data are presented as the means ± SEM. GraphPad Prism software was used to analyse and plot the data. Statistical significance was evaluated using Student’s t-test or one-way ANOVA followed by Tukey’s test.
This project was supported by The Polish National Science Centre under grant number DEC-2011/03/B/NZ4/00046.
- Hunt TK, Aslam RS, Beckert S, Wagner S, Ghani QP, Hussain MZ, et al. Aerobically derived lactate stimulates revascularization and tissue repair via redox mechanisms. Antioxid Redox Signal. 2007;9:1115–24.PubMed CentralPubMedView ArticleGoogle Scholar
- Vegran F, Boidot R, Michiels C, Sonveaux P, Feron O. Lactate influx through the endothelial cell monocarboxylate transporter MCT1 supports an NF-kappaB/IL-8 pathway that drives tumor angiogenesis. Cancer Res. 2011;71:2550–60.PubMedView ArticleGoogle Scholar
- Sonveaux P, Copetti T, De Saedeleer CJ, Végran F, Verrax J, Kennedy KM, et al. Targeting the lactate transporter MCT1 in endothelial cells inhibits lactate-induced HIF-1 activation and tumor angiogenesis. PLoS One. 2012;7:e33418.PubMed CentralPubMedView ArticleGoogle Scholar
- Liu C, Wu J, Zhu J, Kuei C, Yu J, Shelton J, et al. Lactate inhibits lipolysis in fat cells through activation of an orphan G-protein-coupled receptor, GPR81. J Biol Chem. 2009;284:2811–22.PubMedView ArticleGoogle Scholar
- Bozzo L, Puyal J, Chatton JY. Lactate modulates the activity of primary cortical neurons through a receptor-mediated pathway. PLoS One. 2013;8:e71721.PubMed CentralPubMedView ArticleGoogle Scholar
- Roland CL, Arumugam T, Deng D, Liu SH, Philip B, Gomez S, et al. Cell surface lactate receptor GPR81 is crucial for cancer cell survival. Cancer Res. 2014;74:5301–10.PubMedView ArticleGoogle Scholar
- Stäubert C, Broom O, Nordström A. Hydroxycarboxylic acid receptors are essential for breast cancer cells to control their lipid/fatty acid metabolism. Oncotarget. 2015;14. [Epub ahead of print].Google Scholar
- Cheeti S, Warrier BK, Lee CH. The role of monocarboxylate transporters in uptake of lactic acid in HeLa cells. Int J Pharm. 2006;325:48–54.PubMedView ArticleGoogle Scholar
- Latham T, Mackay L, Sproul D, Karim M, Culley J, Harrison DJ, et al. Lactate, a product of glycolytic metabolism, inhibits histone deacetylase activity and promotes changes in gene expression. Nucleic Acids Res. 2012;40:4794–803.PubMed CentralPubMedView ArticleGoogle Scholar
- Gong F, Miller KM. Mammalian DNA repair: HATs and HDACs make their mark through histone acetylation. Mutat Res. 2013;750:23–30.PubMedView ArticleGoogle Scholar
- Price BD, D’Andrea AD. Chromatin remodeling at DNA double-strand breaks. Cell. 2013;152:1344–54.PubMed CentralPubMedView ArticleGoogle Scholar
- Tamburini BA, Tyler JK. Localized histone acetylation and deacetylation triggered by the homologous recombination pathway of double-strand DNA repair. Mol Cell Biol. 2005;25:4903–13.PubMed CentralPubMedView ArticleGoogle Scholar
- Miller KM, Tjeertes JV, Coates J, Legube G, Polo SE, Britton S, et al. Human HDAC1 and HDAC2 function in the DNA-damage response to promote DNA nonhomologous end-joining. Nat Struct Mol Biol. 2010;17:1144–51.PubMed CentralPubMedView ArticleGoogle Scholar
- Ogiwara H, Ui A, Otsuka A, Satoh H, Yokomi I, Nakajima S, et al. Histone acetylation by CBP and p300 at double-strand break sites facilitates SWI/SNF chromatin remodeling and the recruitment of non-homologous end joining factors. Oncogene. 2011;30:2135–46.PubMedView ArticleGoogle Scholar
- Jiang X, Sun Y, Chen S, Roy K, Price BD. The FATC domains of PIKK proteins are functionally equivalent and participate in the Tip60-dependent activation of DNA-PKcs and ATM. J Biol Chem. 2006;281:15741–6.PubMedView ArticleGoogle Scholar
- Kaidi A, Jackson SP. KAT5 tyrosine phosphorylation couples chromatin sensing to ATM signalling. Nature. 2013;498:70–4.PubMedView ArticleGoogle Scholar
- Domingue PA, Sadhu K, Costerton JW, Bartlett K, Chow AW. The human vagina: normal flora considered as an in situ tissue-associated, adherent biofilm. Genitourin Med. 1991;67:226–31.PubMed CentralPubMedGoogle Scholar
- Boskey ER, Cone RA, Whaley KJ, Moench TR. Origins of vaginal acidity: high D/L lactate ratio is consistent with bacteria being the primary source. Hum Reprod. 2001;16:1809–13.PubMedView ArticleGoogle Scholar
- Bannister AJ, Kouzarides T. Regulation of chromatin by histone modifications. Cell Res. 2011;21:381–95.PubMed CentralPubMedView ArticleGoogle Scholar
- Bouquet F, Ousset M, Biard D, Fallone F, Dauvillier S, Frit P, et al. A DNA-dependent stress response involving DNA-PK occurs in hypoxic cells and contributes to cellular adaptation to hypoxia. J Cell Sci. 2011;124:1943–51.PubMedView ArticleGoogle Scholar
- de Laval B, Pawlikowska P, Petit-Cocault L, Bilhou-Nabera C, Aubin-Houzelstein G, Souyri M, et al. Thrombopoietin-increased DNA-PK-dependent DNA repair limits hematopoietic stem and progenitor cell mutagenesis in response to DNA damage. Cell Stem Cell. 2013;12:37–48.PubMedView ArticleGoogle Scholar
- Li G, Wang HQ, Wang LH, Chen RP, Liu JP. Distinct pathways of ERK1/2 activation by hydroxy-carboxylic acid receptor-1. PLoS One. 2014;9:e93041.PubMed CentralPubMedView ArticleGoogle Scholar
- Urban JD, Clarke WP, von Zastrow M, Nichols DE, Kobilka B, Weinstein H, et al. Functional selectivity and classical concepts of quantitative pharmacology. J Pharmacol Exp Ther. 2007;320:1–13.PubMedView ArticleGoogle Scholar
- Sonveaux P, Vegran F, Schroeder T, Wergin MC, Verrax J, Rabbani ZN, et al. Targeting lactate-fueled respiration selectively kills hypoxic tumor cells in mice. J Clin Invest. 2008;118:3930–42.PubMed CentralPubMedGoogle Scholar
- O’Driscoll M, Cerosaletti KM, Girard PM, Dai Y, Stumm M, Kysela B, et al. DNA ligase IV mutations identified in patients exhibiting developmental delay and immunodeficiency. Mol Cell. 2001;8:1175–85.PubMedView ArticleGoogle Scholar
- Chrzanowska KH, Gregorek H, Dembowska-Bagińska B, Kalina MA, Digweed M. Nijmegen breakage syndrome (NBS). Orphanet J Rare Dis. 2012;7:13.PubMed CentralPubMedView ArticleGoogle Scholar
- Rass U, Ahel I, West SC. Actions of aprataxin in multiple DNA repair pathways. J Biol Chem. 2007;30:9469–74.View ArticleGoogle Scholar
- Wang Y, Yuan JL, Zhang YT, Ma JJ, Xu P, Shi CH, et al. Inhibition of both EGFR and IGF1R sensitized prostate cancer cells to radiation by synergistic suppression of DNA homologous recombination repair. PLoS One. 2013;8:68784.View ArticleGoogle Scholar
- Myllynen L, Rieckmann T, Dahm-Daphi J, Kasten-Pisula U, Petersen C, Dikomey E, et al. In tumor cells regulation of DNA double strand break repair through EGF receptor involves both NHEJ and HR and is independent of p53 and K-Ras status. Radiother Oncol. 2011;101:147–51.PubMedView ArticleGoogle Scholar
- Irianto J, Lee DA, Knight MM. Quantification of chromatin condensation level by image processing. Med Eng Phys. 2014;36:412–7.PubMedView ArticleGoogle Scholar
- Contrepois K, Thuret JY, Courbeyrette R, Fenaille F, Mann C. Deacetylation of H4-K16Ac and heterochromatin assembly in senescence. Epigenetics Chromatin. 2012;5:15.PubMed CentralPubMedView ArticleGoogle Scholar
- Ciszewski WM, Tavecchio M, Dastych J, Curtin NJ. DNA-PK inhibition by NU7441 sensitizes breast cancer cells to ionizing radiation and doxorubicin. Breast Cancer Res Treat. 2014;143:47–55.PubMedView ArticleGoogle Scholar
- Końca K, Lankoff A, Banasik A, Lisowska H, Kuszewski T, Góźdź S, et al. A cross-platform public domain PC image-analysis program for the comet assay. Mutat Res. 2003;534:15–20.PubMedView ArticleGoogle Scholar
- Andersen CL, Jensen JL, Ørntoft TF. Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res. 2004;64:5245–50.PubMedView ArticleGoogle Scholar
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