Increasing evidence shows that static magnetic fields (SMFs) can affect cell proliferation but mixed results have been reported. Here we systematically examined the effects of 1 T (Tesla) SMF, which is close to the SMF intensity that patients are exposed to MRI (magnetic resonance imaging) scanners in hospitals, for its effect on 15 different cell lines, including 12 human and 3 rodent cell lines. Our results show that 1 T SMF does not have apparent impact on cell cycle or cell death. However, at higher cell density, it reduced cell numbers in six out of seven solid human cancer cell lines. We found that both cell type and cell density had evident impacts on SMF effects. Moreover, the EGFR-Akt-mTOR pathway, which varies significantly between different cell types and densities, contributes to the differential effects of SMF. In addition, SMF also increases the efficacy of Akt inhibitors on cancer cell growth inhibition. Therefore 1 T SMF affects cell proliferation in a cell type- and cell density-dependent manner, and the inhibition effect of 1 T SMF on multiple cancer cells at higher cell density may indicate its clinical potential in late stage cancer therapy. However, the seemingly inconsistent observations are mostly due to the different magnetic field parameters and multiple experimental variables. It is obvious that magnetic fields of different types (static or time-varying magnetic fields), field intensity  or frequencies  can lead to diverse and sometimes completely opposite results [Although there are numerous reports of  results –4.Besides various parameters of the magnetic fields, different biological samples in individual studies often have distinct genetic background, which makes them respond to the magnetic fields differentially. For example, Aldinucci et al. found that 4.75 T SMF significantly inhibited Jurkat leukemia cell proliferation but did not affect normal lymphocytes , but othIn comparison to Dynamic/Time-varying Magnetic Fields, static magnetic field (SMF) is more suitable to study the biological effects and their underlying mechanisms because they have less variable parameters. Electromagnetic fields from power lines, microwave ovens and cell phones are all dynamic/time-varying magnetic fields, whose effects on human bodies are still debated and causing widespread public health concerns. In contrast, SMF is characterized by steady, time-independent field strengths, and the reported biological effects of SMFs are mostly negligible or even beneficial. The core component of the MRI (magnetic resonance imaging) machines in most hospitals is a strong SMF with field intensities ranging between 0.1-3 T, in combination with pulsed radiofrequency magnetic fields. The SMF intensities in the 0.1-3 T range are currently considered to be safe to human bodies because no severe health consequences have been reported. The discomforts in patients such as dizziness are all temporary, which disappear after the MRI examination. However, mixed experimental reports from the laboratories are in the literature, which seem to be controversial. Some studies show that SMFs in this range do not affect cell growth or cell cycle , 10, whiHere in this study, we chose 1 T SMF to test its effect on 15 different cell lines side-by-side, including 12 human cell lines (7 solid cancer and 5 non-cancer cell lines) and 3 rodent cell lines. We found that 1 T SMF not only affected cell proliferation in a cell type-dependent manner, but also cell density-dependent manner. We revealed that cell growth of most human solid cancer cell lines we tested, but not non-cancer cell lines, can be inhibited by 1 T SMF at higher cell densities, in which the EGFR-Akt-mTOR pathway may play essential roles.5 cells/ml, cultured with or without 1 T SMF for 2 days and examined them side-by-side  can effectively inhibit human nasopharyngeal cancer CNE-2Z cell proliferation . However5 cells/ml for cell seeding, depending on which concentrations could reach maximum confluence at the end of experiments. We found that there was no significant cell morphology change by 1 T SMF treatment  and the mTOR pathway could be affected by SMFs , 11. SpeNext we compared the cancer vs. non-cancer cells from human lungs. We examined different densities of A549 lung cancer cells and HSAEC2-KT normal lung cells side-by-side to see whether the EGFR-Akt-mTOR pathway is differentially regulated by cell type and cell density Figure . Not sur5 cells/ml, the cell number of CHO-EGFR cells were increased by 1 T SMF , the cell number of CHO-EGFR cells are reduced by 1 T SMF . Their treatment time goes far beyond the MRI machines. However, our results showed that the cell growth for one of the seven cancer cell lines, the bladder cancer EJ1 cell, was not reduced by SMF at all. In addition, we have only tested 7 human solid cancer cell lines, whether other cancer cells in suspension, such as various types of leukemia cells, can be affected in the same pattern is still unknown. Therefore, although at higher cell density, 1 T SMF reduced the cell number in majority of cancer cell lines we tested, further investigations are strongly needed to explore the clinical potential of SMFs on cancer growth inhibition. It is very possible that only some cancer cell types can be inhibited by SMFs, which is likely relevant to their different genetic background. In addition, the tendency of SMFs to promote some cancer cell growth at lower cell density should also raise caution about the potential oncogenic effect at early cancer stages for this prolonged SMF exposure. More investigations are needed to address this issue.One limitation of our study was that we did not address the potential issue of metabolism, nutritional competition for cells at high density, which is apparently more stringent than cells at lower density. Theoretically, SMFs may affect cell metabolism, for example, utilization of carbon and nitrogen sources, oxygen consumption, respiration etc. However, we recently started to examine two types of leukemia cells in suspension and found that 1 T SMF inhibited both of them at lower cell concentration but not at higher cell concentrations. Therefore we think it is less likely that the reasons mentioned above such as oxygen consumption or nutrient competition are the major factors. In the meantime, it has been shown that moderate intensity SMFs could affect intracellular signaling pathways , 33. AltIt was known that the biological effects of magnetic fields can be influenced by the magnetic field types, strength, frequency, treatment time and other parameters, which all contribute to the mixed results of biological effects of magnetic field in the literature. Here by systematically testing 15 different cell lines at 4 different cell densities, we revealed that both cell types and densities are key factors that influence the effects of SMF. In addition, EGFR-Akt-mTOR likely contributes to the cell types and densities induced variations. The fact that most cancer cell lines can be inhibited at high density indicates the clinical potential of SMF in solid tumors or end-stage tumors.We tested 15 cell lines in total, which are all adherent cell lines. For the 12 human cell lines, we chose 7 cancer cell lines  and 5 non-cancer cell lines . The three rodent cell lines are Chinese Hamster Ovary cell line CHO and mouse embryo fibroblast cell line NIH-3T3, as well as CHO cells that overexpresses EGFR-FLAG (CHO-EGFR cells). All cells were from ATCC except for CHO-EGFR, which was constructed as previously described [5 cells/ml are equivalent to the density of 2 x 104 cells/cm2. On the second day, they were placed in regular full-sized CO2 cell incubator  that has accurate control of temperature (37°C), humidity and CO2 (5%)  . The shaThe magnet surface dimension is 5 cm x 5 cm and the diameter of the cell culture plates we used was 3.5 cm . TherefoAs mentioned above, cells were plated at different concentrations in 35 mm cell culture plates. After SMF exposure, bright field images were taken before the cells were harvested by trypsinization. An aliquot of the cells were counted by hemocytometer and the rest cells were used for flow cytometry analysis (cell death and cell cycle). Experiments were repeated for at least three independent times by two researchers. The results were gathered together for analysis.4 cells per sample. Data were analyzed using ModFit LT. Experiments were done for at least two times and representative results were shown in the figures.Cells were trypsinized and washed with PBS. Then they were fixed in 70% ice-cold ethanol overnight at 4 °C before they were washed with PBS, and incubated in PI (propidium iodide) solution (BD Pharmingen) for 30 min at room temperature in the dark. Samples were then analyzed on a BD Flow Cytometry . For each condition, we collected 1 x 106 cells/ml. Then 100 μl of them was transferred to a 5 ml culture tube and 5 μl of FITC-Annexin V + 5 μl of PI (FITC-Annexin V Apoptosis Detection Kit was from BD PharmingenTM) were added to the tube, mixed, and incubated in the dark for 15 min at room temperature. Then, 400 μl of binding buffer was added to the stained cells before they were analyzed by flow cytometry within 1 h. Approximately 1 x 104 cells were collected by flow cytometer. Experiments were done for at least two times and representative results were shown in the figures.Cells were trypsinized and washed twice with ice-cold PBS before they were resuspended in binding buffer at 10TM XRS+ System and Beijing Tanon Fine-do X6. ImageJ software was used to quantify the protein relative level shown by Western blots.Cells grown in tissue culture plates were lysed directly in plates by 200 or 100 μl of M-PER lysis buffer (Pierce) supplemented with protease and phosphatase inhibitor cocktail at 4°C for 20 min. The whole cell lysate was mixed with 2 x SDS loading buffer, boiled, and subjected to Western blotting. The PVDF membrane was blocked with 5 % NFDM (non-fat dry milk) at room temperature for around 1 h. Corresponding primary antibodies were diluted in AbDil-Tween  at 1:1000 dilution. Primary antibodies used include phospho-specific antibodies, EGFR, Akt, S6K, 4EBP1, and the HRP-linked anti-rabbit and anti-mouse IgG antibodies which were from Cell Signaling Technology. The mouse monoclonal antibodies for beta-tubulin and beta-actin were from Beijing TransGen Biotech. All primary antibodies were diluted in AbDil-Tween at 1:1000 dilution (TBS supplemented with 2% BSA and 0.1% Tween-20) and HRP conjugated secondary antibodies were diluted in TBS with 0.1 % Tween-20 and 5 % NFDM at 1:5000 dilution. Western blotting results were obtained by Bio-Rad ChemiDoc5 cells seeded at the beginning of the experiment.To ensure the reproducibility of the experiments, most experiments in this work were repeated by two independent researchers on different days. Each researcher did their experiment independently, according to the same protocol. For quantifications presented in the figures, experiments were repeated for at least three independent times (n=3). In each experiment, one plate of sham control and one plate of magnetic exposure were examined. Each plate had 1-10 x 10For quantifications in this manuscript, mean values are shown in all figures, and standard deviations are shown as error bars. All images shown in figures are representative results from multiple experiments. Comparisons between different treatments were analyzed by a two-tailed Student t test. P values are labeled in figures for where data were compared.We calculated the coefficient of drug interaction (CDI = AB/(A x B)) of SMF in combination with Akt inhibitors for their effects on cell numbers in Figure


Int. J. Mol. Sci. 2017, 18, 928). SIPS is a growth-arrested state in which the cells acquire flattened and enlarged morphology, remain viable, secrete growth-promoting factors, and can give rise to tumor-repopulating progeny. The impact of SIPS on radiosensitivity measured by short-term assays remains largely unknown. We report that in four p53 wild-type human solid tumor-derived cell lines : (i) the conventional short-term growth inhibition assay (3 days post-irradiation) generates radiosensitivity data comparable to that measured by the laborious and time-consuming colony formation assay; (ii) radiation dose-response curves obtained by multiwell plate colorimetric/fluorimetric assays are markedly skewed towards radioresistance, presumably reflecting the emergence of highly enlarged, growth-arrested and viable cells; and (iii) radiation exposure  does not trigger apoptosis or loss of viability over a period of 3 days post-irradiation. Irrespective of the cell-based assay employed, caution should be exercised to avoid misinterpreting radiosensitivity data in terms of loss of viability and, hence, cell death.In most p53 wild-type human cell types, radiosensitivity evaluated by the colony formation assay predominantly reflects stress-induced premature senescence (SIPS) and not cell death ( Activation of the p53 signaling pathway by ionizing radiation was originally proposed to result either in cell cycle checkpoint activation to facilitate DNA repair and promote survival, or in apoptotic cell death . Based SIPS is a sustained growth arrested state characterized by the acquisition of flattened and enlarged cell morphology in cells that retain viability for a long time (months) post-treatment and acquire the ability to secrete factors that can promote proliferation and invasiveness in cell culture models and tumor development in vivo ,11. In aWAF1  protein is the founding member of the CIP/KIP family of cyclin-dependent kinase (CDK) inhibitors [The p21hibitors ,17. It ihibitors ,19. Conshibitors ,6,7,20.The purpose of the present study was to determine the impact of SIPS on radiosensitivity when evaluated by short-term tests in the commonly-used p53 wild-type cancer cell lines HCT116 (colon carcinoma), MCF7 (breast carcinoma), SKNSH (neuroblastoma), and A172 . These cell lines are known to respond to moderate doses of ionizing radiation  by exhibiting sustained nuclear accumulation of p21, coupled with growth arrest through SIPS. We demonstrate that radiosensitivity as measured by multiwell plate colorimetric/fluorimetric assays is markedly skewed towards radioresistance when compared to that measured by growth inhibition (3 days post-irradiation) and colony formation (10 days post-irradiation) assays, and that the response measured by these cell-based assays reflects growth inhibition (SIPS) and not apoptosis or loss of viability.The colony formation assay  for assessment of radiosensitivity in cultured human cells was developed by Puck and Marcus over half a century ago . It has 50) were comparable when measured by colony formation and growth inhibition assays.As alluded to earlier, in most cell types, activation of the p53 pathway in response to moderate doses of ionizing radiation  results in activation of early cell cycle checkpoints, suppression of apoptosis and sustained growth arrest through SIPS ,3,4. ConInterestingly, we have recently reported that these two assays also indicate a comparable degree of radiosensitivity for solid tumor-derived cell lines that lack p21 or wild-type p53 activity . RadioseAlthough the colony formation assay is considered to be the “gold standard” for genotoxicity assessment, it has several drawbacks. Aside from being time consuming and labor intensive, preparation of good quality single-cell suspensions and evaluation of borderline macroscopic colonies are not trivial for some cell types, requiring considerable expertise. In addition, some solid tumor-derived cell lines  cannot be reliably evaluated by this assay because of their poor cloning efficiencies, and some cell lines  do not yield a good single-cell suspension by conventional approaches  .The growth inhibition assay circumvents these limitations. As pointed out previously , in addiShort-term cell metabolic activity assays are widely used to assess radiosensitivity and chemosensitivity with solid tumor-derived cell lines ,25,26,27In a typical assay, the cells are plated at “optimal” densities in the wells of a microtiter plate, exposed to a genotoxic agent, and incubated for 3 days. XTT (or MTT) is added to each well, and cultures are incubated for a few hours to allow the viable cells to covert the yellow tetrazolium salt (XTT or MTT) to its colored formazan derivative; XTT formazan is orange in color whereas MTT formazan is purple. The number of cells inoculated per well and the duration of cell incubation with XTT or MTT need to be optimized for each cell line.MTT is readily taken up by the cells, but its formazan metabolite is water insoluble, requiring a solubilization step before data evaluation. XTT, on the other hand, requires an electron coupling step to facilitate its uptake by the cells, but the XTT metabolite is water soluble.After these treatments, either with (MTT) or without (XTT) the metabolite solubilization step, the medium overlaying the control (sham-treated) cultures turns purple (MTT) or orange (XTT), as a result of conversion of the yellow tetrazolium salt to a colored metabolite by viable cells. Genotoxic treatment results in a decrease in color intensity of the medium, which is proportional to the number of viable cells/well at the time of incubation with XTT or MTT . The resThe CellTiter-Blue assay is also widely used for genotoxicity assessment. This assay is based on the ability of living cells to convert the redox dye resazurin (blue) into the resorufin product (pink). Resazurin has little intrinsic fluorescence until it is converted to its metabolite, which is highly fluorescent (579 nm excitation/584 nm emission). Thus, the CellTiter-Blue assay not only eliminates the need for electron coupling or metabolite solubilization steps required for tetrazolium-based assays, but it also enables fluorimetric evaluation of the data, which is considered to be more sensitive than absorbance measurements.The properties of MTT make it particularly useful for microscopic assessment of individual cells in the absence of solvent treatment. To this end, cultures are incubated with medium containing MTT to allow viable cells to covert MTT to its purple, water-insoluble formazan. Under these conditions, MTT metabolites appear as intercellular formazan granules as well as needle-like formazan crystals . We usedSeveral pitfalls of multiwell plate colorimetric assays have been extensively reviewed, including the impact of cell density and possible interference of test compounds with optical density measurements ,31,32,33Thus, although many suppliers refer to these colorimetric assays in terms of “Cell Viability” or “Cytotoxicity,” all such assays that are performed in a multiwell plate format lack specificity and can lead to incorrect interpretations. As illustrated in 50 values measured by growth inhibition and multiwell plate assays (both measured 3 days post-irradiation) amounted to ~3 Gy and >8 Gy, respectively.Assuming that radiation exposure would minimally alter the size of cancer cells, radiosensitivity as measured by multiwell plate assays would be expected to be comparable to that measured by growth inhibition and colony formation assays. This is not always the case, however; cancer cells undergoing SIPS following radiation exposure exhibit a markedly enlarged morphology. Sohn et al. , for exaIn most human cell types, activation of p53 signaling in response to genotoxic stress has been shown to supress (rather than promote) apoptosis ,19. SuppApoptosis in our previous work was evaluated by microscopic examination of Hoechst 33342-stained cells for morphological criteria (nuclear condensation and fragmentation). We sought to verify these observations by flow cytometric determination of Annexin V-positive  cells. The results are presented in As a positive control, we also determined the apoptotic response of selected cancer cell lines  following incubation with high concentrations of the chemotherapeutic drug cisplatin or the nuclear export inhibitor leptomcyin B. Treatment with cisplatin (60 μM) or leptomycin B (2 μM) for 3 days resulted in apoptosis in a high proportion of HCT116 cells when evaluated by Annexin V/flow cytometry  and morpv/v) fetal bovine serum as described in [Mycoplasma contamination.The four cancer cell lines used in the present study were purchased from the American Type Culture Collection . Cells were cultured as monolayers in DMEM/F12 nutrient medium supplemented with 5% , the CellTiter-Blue reagent  and the tetrazolium dyes MTT and XTT  were used as recommended by the manufacturers.60Co γ-rays was performed in a Gammacell 220 unit as described [Exposure to escribed .Radiosensitivity evaluation by growth inhibition, colony formation and 96-well plate assays was performed as described . Flow cyIn the current study, we have demonstrated that the conventional growth inhibition assay generates radiosensitivity data comparable to that obtained by the slower and more technically challenging colony formation assay for p53 wild-type cancer cell lines. On the other hand, the response measured by multiwell plate colorimetric/fluorimetric assays is markedly skewed towards radioresistance, which we assume to reflect the emergence of highly enlarged, growth-arrested and viable cells post-irradiation . In addition, we have confirmed that exposure to moderate  doses of ionizing radiation does not induce apoptosis (as judged by the Annexin V/flow cytometry approach) or loss of viability in the p53 wild-type cancer cell lines that we examined. These observations, in concert with those recently reported by us for p53 null or mutant p53-expressing cancer cell lines , give crShort-term multiwell plate assays are indispensable for high throughput studies, e.g., screening compound libraries for genotoxicity (proliferation block and/or cell death) towards the US NCI panel of cancer cell lines. However, it is becoming increasingly evident that the effect measured by such assays primarily reflects growth inhibition and not loss of viability ,19,23. O


In contrast, the commercially available line HepaRG, postulated to be a surrogate for hepatocytes and an ideal tool for mutagenicity tests, did not detect IQ and was relatively insensitive towards ROS. All other lines failed to detect two or more compounds. HCC1.2 cells have a high and unstable chromosome number and mutated p53, these features distract from its use in routine screening. HepG2 was frequently employed in earlier studies, but pronounced inter-laboratory variations were observed. HuH6 was never used in genotoxicity experiments and is highly promising, it has a stable karyotype and we demonstrated that the results of genotoxicity experiments are reproducible.Cell lines which are currently used in genotoxicity tests lack enzymes which activate/detoxify mutagens. Therefore, rodent-derived liver preparations are used which reflect their metabolism in humans only partly; as a consequence misleading results are often obtained. Previous findings suggest that certain liver cell lines express phase I/II enzymes and detect promutagens without activation; however, their use is hampered by different shortcomings. The aim of this study was the identification of a suitable cell line. The sensitivity of twelve hepatic cell lines was investigated in single cell gel electrophoresis assays. Furthermore, characteristics of these lines were studied which are relevant for their use in genotoxicity assays . Three lines  detected representatives of five classes of promutagens, namely, IQ and PhIP (HAAs), B(a)P (PAH), NDMA (nitrosamine), and AFBThe online version of this article (10.1007/s00204-017-2109-4) contains supplementary material, which is available to authorized users. One of the fundamental problems of in vitro tests is the inadequate representation of drug metabolizing enzymes in cell lines which are currently used in routine screening of chemicals. Therefore, exogenous liver-derived enzyme homogenates are prepared from rodents which have been treated with enzyme inducers Kirkland . The homIn the early 1990s, Darroudi and Natarajan  showed t2O2) was included in all experimental series as a positive control, this compound is directly active and causes damage of the DNA via formation of reactive oxygen species (ROS).The use of the different human-derived liver cells in mutagenicity tests was not based on targeted comparative screening of currently available cell lines and we hypothesized that other lines which are currently available may be equally or even more suitable for the detection of mutagens. Therefore, we performed a comprehensive study in which we compared the sensitivity of a panel of cell lines towards representatives of different classes of genotoxic carcinogens which require enzymatic activation in genotoxicity experiments. In addition to HepG2, Hep3B, HCC1.2 and HepaRG, eight lines were included which have never been used in genotoxicty assays before. The test compounds which were used are listed in Table The DNA damaging properties of the different compounds were monitored in single cell gel electrophoresis (SCGE) assays which are based on the determination of DNA damage in an electric field and are increasingly used in genetic toxicology  the morphology of the cells which provides information about their origin and similarity to primary liver cells, (2) their karyotype allowing to draw conclusions concerning their chromosomal stability, (3) measurements of the mitotic activities providing information about the duration of the repair phase which is needed for the design of tests which require cell division  and (4) the 1 , benzo(a)pyrene (B(a)P, CAS-No. 50-32-8), hydrogen peroxide  and N-nitrosodimethylamine  were purchased from Sigma-Aldrich . 2-amino-3-methyl-3H-imidazoquinolone  came from Toronto Research Chemicals  and 2-amino-1-methyl-6-phenylimidazopyridine  from Santa Cruz Biotechnology, Inc. . Anti-GADPH antibody (ab9485), anti-mutant-p53 antibody (ab32049) and anti-p53 antibody (ab1101) were purchased from Abcam plc . Anti-rabbit IgG HRP conjugate (W4018), anti-mouse IgG HRP (W4028) conjugate came from Promega Corporation .Citric acid (CAS-No. 77-92-9) and di-natriumhydrogenphosphate-dihydrate (CAS-No. 10028-24-7) for McIlvaine buffer were obtained from Merck . Quinacrine dihydrochloride (CAS-No. 69-05-6) and Thymol (CAS-No. 89-83-8) for quinacrine staining solution, aflatoxin BLow melting point agarose (LMPA) and normal melting point agarose (NMPA) were acquired from Gibco . Inorganic salts, dimethyl sulfoxide (DMSO), propidium iodide, hydrogen peroxide, Triton X-100, Trizma base, fetal bovine serum (FBS), Dulbecco’s Phosphate Buffered Saline (DPBS), Dulbecco’s modified Eagle Medium (DMEM), Eagle’s Minimum Essential Medium (EMEM), Minimal essential Medium Eagle (MEME), Roswell Park Memorial Institute Medium (RPMI-1640) were purchased from Sigma-Aldrich . William’s E medium came from Thermo Fisher Scientific . Trypsin–EDTA was ordered from Life Technologies .The origin of the cell lines is listed in Table 2. All lines except HepaRG™ were grown in TC-treated flasks and dishes  as described in previous publications  was diluted to obtain a final concentration of 0.2 mg/ml collagen. Subsequently, the growth areas of the flasks or dishes were covered with 0.1 ml/cmThe identity of the cell lines (except of HepaRG™) was verified by short tandem repeat (STR) analyses  in 5.0 ml medium. DMSO was added drop-wise , then the suspensions were distributed equally to cryovials , which were placed in a polystyrene box at − 80 °C for 24 h (to ensure slow freezing) and transferred to liquid nitrogen.® Cell Counter and Analyzer System  which was used as described by the manufacturer . Cell lines were grown in Petri dishes  in 5.0 ml medium; ca. 5 × 105 cells were seeded in each dish at the start of the experiments. After 24 h intervals, the cells were detached with trypsin–EDTA  and 50 µl of these suspensions were transferred to CASY-cups . For each experimental point, three plates were evaluated. On the basis of the results, means and standard deviations (SDs) were calculated. Nonlinear fits with exponential growth equations (least square fits) were calculated to determine the doubling times .The proliferation kinetics and size of the cells were monitored by use of a CASYCell lines were cultured and prepared according to standard cytogenetic techniques; at least 20 metaphases per cell line were karyotyped. Slides, which had been stained with quinacrine solution , were incubated in McIlvaine solution , covered with a cover slip and analyzed using Applied Spectral Imaging Case Data Manager .p53 gene in the different cell lines was analyzed using real-time quantitative PCR (RT-qPCR) according to the description of Straser et al. , all other compounds in DMSO. The final DMSO concentrations in the media did not exceed 1.0%. The exposure time in experiments with AFB1, B(a)P and NDMA was 24 h and in assays with PhIP and IQ 48 h. To terminate the treatment, the cells were washed twice with PBS (1.0 ml), trypsinized  and then resuspended in 400 µl culture medium containing FBS. Subsequently, the suspensions were transferred to Eppendorf tubes and centrifuged . Cell pellets were resuspended in cold PBS. H2O2 treatment was conducted with cells embedded on the slides by exposure to different concentrations (10–50 µM) of cold H2O2 solution in PBS for 5 min, followed by washing with cold PBS (5 min).Briefly, cells were sub-cultured in 1.0 ml medium in 24–well tissue-culture plates . After 24 h, the media were changed and the cells exposed to different concentrations of the test compounds or to the solvent controls (PBS or DMSO). H® Cell Counter and Analyzer System   Doi .The proliferation kinetics of the different cells are shown graphically in Figures S2a and b. On the basis of the experimental data, the doubling times were calculated Table . The “faThe results of the karyotyping experiments are summarized in column four of Table Western Blot and RT-qPCR analyses  also in assays with HCC1.2 while relatively strong differences of the responses were observed with HepG2. Experiments with HepaRG were not included as the reproducibility of comet experiments was addressed already in a study by Le Hegarat et al. (Induction factors (IF) represent the ratios of %DNA in tail induced in chemically treated cells and respective untreated controls. It can be seen that the values obtained in two independent experiments with different mutagens are in general similar in experiments with HuH6 and for certain model compounds P, five NDMA, four PhIP, and three IQ. Three hepatic lines  were sensitive towards representatives of all groups, HepRG to four compounds, HuH7 to two; only four lines were not responsive at all.The results of the SCGE experiments confirm our assumption that several human-derived hepatoma cell lines detect the genotoxic properties of different groups of DNA reactive carcinogens without addition of exogenous enzyme activation mix. Seven out of twelve lines were sensitive towards AFB1 and B(a)P, which were positive at concentrations between 5 and 10 µM while higher doses of IQ and NDMA (in most cases ≥ 100 mM) were required to cause significant effects. It is notable that HCC1.2 was clearly more sensitive towards these latter compounds as the other lines.The sensitivity of the different hepatic lines was in general similar. The most pronounced effects were observed in general with AFBp53 and an epithelial morphology, its chromosome number is in a narrow range and twice as high as in primary hepatocytes; the doubling time is similar to that of most other lines.The most important finding of the present study is the identification of a hepatic line (HuH6) which is apparently more suitable for the detection of genotoxins as the other liver-derived lines. This line was isolated more than 40 years ago from a hepatic tumor of a 1-year-old boy Doi . HuH6 cep53. These findings indicate that they are not suitable for routine testing of chemicals; as mentioned above, it was stressed by Fowler et al. P, PhIP, and NDMA) and their sensitivity was in most cases identical or lower as that of the other cell types . With the heterocyclic aromatic amine IQ, negative results were obtained under all experimental conditions. These observations are in agreement with previous findings of Lehegerad et al. (N-acetyl transferase (NAT) . It is well documented that ROS play a crucial role in the induction of DNA damage by a large number of chemicals including peroxides and PAHs and also by radionuclides  IARC . The latAT) IARC  which arAT) IARC . In thisAT) IARC ; data foAT) IARC  are not AT) IARC . AnotherAn ideal cell type for the detection of genotoxic carcinogens would be primary human hepatocytes. However, it is difficult to obtain these cells  and they are not available for routine testing. We found only two papers concerning comet assays with primary liver cells. The first by Monteith and Vanstone  describeTaken together, the results of the present study show that several human-derived liver cell lines detect promutagens without addition of rodent-derived liver homogenate. Furthermore, they indicate that HuH6 is the most promising line which may be useful for routine testing of chemicals. Results of experiments with HepaRG cells indicate that differentiated cells express higher levels of drug metabolizing enzymes Below is the link to the electronic supplementary material.


VEGFR1/2, expression of PDGFR α/β in particular has been documented as characteristic of metastatic disease correlating with poor prognosis. Therefore, we have been suggested that the clinically approved multi‐kinase angiogenesis inhibitor Axitinib, which specifically targets these kinases, might constitute a promising option for medulloblastoma treatment. Indeed, our results delineate anti‐neoplastic activity of Axitinib in medulloblastoma cell lines modelling the most aggressive c‐myc‐amplified Non‐WNT/Non‐SHH and SHH‐TP53‐mutated tumours. Exposure of medulloblastoma cell lines to Axitinib results in marked inhibition of proliferation and profound induction of cell death. The differential efficacy of Axitinib is in line with target expression of medulloblastoma cells identifying VEGFR 1/2, PDGFR α/β and c‐kit as potential markers for drug application. The high specificity of Axitinib and the consequential low impact on the haematopoietic and immune system render this drug ideal multi‐modal treatment approaches. In this context, we demonstrate that the clinically available PI3K inhibitor GDC‐0941 enhances the anti‐neoplastic efficacy of Axitinib against c‐myc‐amplified medulloblastoma. Our findings provide a rational to further evaluate Axitinib alone and in combination with other therapeutic agents for the treatment of most aggressive medulloblastoma subtypes.Aberrant receptor kinase signalling and tumour neovascularization are hallmarks of medulloblastoma development and are both considered valuable therapeutic targets. In addition to  Medulloblastoma is the most common malignant brain tumour of childhood. Despite current intensive therapy including a combination of surgery, chemotherapy and radiation, still one‐third of the patients succumb to this disease via vascular endothelial growth factor receptor VEGFR 1/2, platelet‐derived growth factor receptor α/β (PDGFR α/β) and c‐kit is critical for medulloblastoma development and progression Signalling Axitinib has been approved by the Food and Drug Administration for the treatment of advanced renal cell carcinoma While the anti‐angiogenic capacity of Axitinib has been extensively delineated, only few reports show that the anti‐tumour activity of Axitinib is also mediated by inhibition of its target kinases VEGFR1‐3, PDGFR α/β and c‐kit expressed by the tumour cells themselves c‐myc‐amplified Non‐WNT/Non‐SHH and SHH‐TP53‐mutated medulloblastoma variants associated with a very poor prognosis in vitro and in vivo the anti‐neoplastic potential of the phosphoinositid‐3‐kinase (PI3K) inhibitor GDC‐0941 for medulloblastoma therapy c‐myc‐amplified medulloblastoma. Thus, our data provide a rational for further assessment of Axitinib alone and in combination with other therapeutic approaches such as PI3K inhibitors for medulloblastoma treatment.Here, we report that Axitinib displays anti‐proliferative, anti‐clonogenic and pro‐apoptotic activity in cell lines modelling the most aggressive Axitinib and GDC‐0941 were obtained from LC Laboratories . The primary antibodies pSTAT3 , STAT3 (124H6), pAKT (Ser473), AKT (11E7) and GAPDH were purchased from Cell Signalling while secondary antibodies were purchased from Dianova . Carboxyfluoreszein‐succinimidyl ester (CFSE) was purchased from Invitrogen , while Hoechst 33258 was provided by Sigma‐Aldrich .l‐glutamine supplemented with 1 mM sodium pyruvate (PAA), 1% penicilline/streptomycine (Invitrogen) and 10% foetal bovine serum .The human medulloblastoma cell lines Daoy (HTB 186) and D283 Med (HTB‐185) were obtained from American Type Culture Collection (ATCC). The medulloblastoma cell line MEB‐Med‐8A was generated by Prof. T. Pietsch. The medulloblastoma cell lines Daoy, D283 Med and MEB‐Med‐8A were maintained in complete medium, namely Dulbecco's modified Eagle's medium , with 5), D283 Med (5 × 105) and Daoy (3 × 105) cells were seeded in 6‐well cell culture dishes in complete medium. After overnight cultivation, the cells were treated with the respective drug concentration for 24 and 48 hrs. Dimethylsulfoxid (DMSO) served as control. Thereafter, dead and living cells were harvested and resuspended in an equal volume of PBS. Dead cells were identified by trypan blue stain according to the supplier's instructions. Results of microscopic cell count by means of a haemocytometer were confirmed by a flow cytometric analysis. Dead cells were excluded from analysis by Hoechst33258 stain. For each sample, the total number of living cells measured within 100 sec. was determined.MEB‐Med‐8A (7 × 105) and D283 Med (5 × 105) and Daoy (3 × 105) cells were seeded in 6‐well cell culture dishes in complete medium. After overnight cultivation, the cells were treated with respective concentrations of Axitinib and GDC‐0941 for 48 hrs. Thereafter, floating and attached cells were collected, stained with Hoechst33258 and analysed by flow cytometry. Proliferation was traced by CFSE staining and normalized to the control DMSO, while dead cell was determined by Hoechst33258 staining.Medulloblastoma cells were stained with CFSE according to the supplier's instructions. MEB‐Med‐8A  and MEB‐Med‐8A (1000 cells/well) were plated in 6‐well cell culture dishes. The cells were allowed to adhere and spread properly for 12 hrs at 37°C. Thereafter, the cells were exposed to the stated concentration of Axitinib. After 48 hrs of exposure, the cells were washed with standard medium to remove any trace of the inhibitor and cultured for another week. Colony numbers and colony size were assessed by IMAGEJ. Particles smaller than 40 pixelg, and the protein concentration was determined by means of the Bradford assay. During the entire procedure, cells and lysates, respectively, were kept at 4°C. A total protein concentration of 25 μg derived from medulloblastoma cell lines was separated by SDS‐polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes . The membranes were blocked for 1 hr at RT in 1× Tris‐buffered saline containing 0.1% tween‐20 (TBST) supplemented with 5% BSA. Thereafter, the membranes were incubated with the primary antibodies (1/1000) overnight at 4°C and subsequently with the respective secondary antibody  for 1 hr at room temperature. Immunoreactivity was detected by chemiluminescence and quantified by means of a ChemiDoc XRS Imaging System (Bio‐Rad).The medulloblastoma cell lines Daoy, MEB‐Med‐8A, D283 Med and D341 were exposed to different Axitinib concentrations for 1 and 48 hrs, respectively. Thereafter, the cells were harvested and subsequently lysed in RIPA buffer (Sigma‐Aldrich) containing protease and phosphatase inhibitors ). The lysates were centrifuged 10 min. at 10,000 × t‐test was applied to determine statistical significance of differences between groups. P < 0.05 (*) was considered as statistically significant. Values stated within text and figures represent mean ± standard deviation.The two‐sided Student's c‐myc‐amplified Non‐WNT/Non‐SHH medulloblastoma while the third analysed cell line, Daoy, shows markers of a subgroup of SHH tumours characterized by a mutation of tumoursuppressor protein 53 in vitro concentrations of 0.5 and 1 μM Axitinib corresponding to plasma levels observed in patients  Vandetanib We previously showed that the clinically available PI3K inhibitor GDC‐0941 exerts significant anti‐neoplastic efficacy against medulloblastoma c‐myc‐amplified Non‐WNT/Non‐SHH medulloblastoma lines MEB‐Med‐8A and D283 Med, Axitinib reduces viable cell number in a dose‐dependent manner starting at the lowest concentration of 0.5 μM, while in the SHH‐TP53‐mutated cell line Daoy, a significant decrease was only observed at 2 μM, the highest concentration applied. In both the c‐myc‐amplified Non‐WNT/Non‐SHH medulloblastoma cell lines, an additive cytoreductive effect of GDC‐0941 is noticeable when combined with 1 and 2 μM Axitinib.In the % in MEB‐Med‐8A and to 14 ± 3% in D283 Med. In MEB‐Med‐8A, cell loss was complete after combined exposure to 2 μM Axitinib and 1 μM GDC‐0941. Yet, in D283 Med, there was no further loss in viable cell number. Daoy, in contrast, is considerably more resistant to Axitinib. Viable Daoy cells are 90 ± 22 and 91 ± 19% at concentrations of at 0.5 μM and 1 μM Axitinib. Notably, cytoreductive activity is documented at 2 μM Axitinib with 30 ± 2.5% cell surviving, which is significantly lower than after exposure to 1 μM GDC‐0941 alone but without any additional effect by the drug combination.After treatment of MEB‐Med‐8A and D283 Med with 1 μM Axitinib, a decline to 39 ± 5% and 36 ± 5% residual viable cells is observed, respectively, while after exposure to 1 μM GDC‐0941, 19 ± 1.6% and 47 ± 5% cells survive. Compared to either drug alone, the combination of 1 μM Axitinib with 1 μM GDC‐0941 significantly decreases the number of viable cells to 3.6 ± 1We further dissected the anti‐neoplastic effect of Axitinib with regards to its pro‐apoptotic and anti‐proliferative capacity based on the combined CFSE‐Hoechst33258 stain. There was a substantial cell death effect upon escalation of Axitinib from 1 to 2 μM. After treatment with 2 μM Axitinib, 75 ± 4% of dead cells were detected in MEB‐Med‐8A and 36 ± 3% in Daoy. In MEB‐Med‐8A and Daoy, this exceeds the cell death observed with 1 μM GDC‐0941 resulting in 40 ± 3.5% and 9 ± 4% cell death, respectively. Compared to single drug application, the Axitinib‐GDC‐0941 combination significantly enhances cytotoxicity in MEB‐Med‐8A for all Axitinib concentrations, while in Daoy, dose escalation of Axitinib had the major cytotoxic effect. In D283 Med, increasing the Axitinib dose has no effect on cell death, which remains below 20% as is the case with 1 μM GDC‐0941 alone. However, in combination with GDC‐0941, cytotoxicity is twice as pronounced reaching 43 ± 10% and 37 ± 8% when coadministered with 1 and 2 μM Axitinib, respectively. Moreover, AnnexinV/7ADD staining reveals that Axitinib alone or in combination with GDC‐0941 exerts a pro‐apoptotic effect on all three medulloblastoma cell lines Fig. .With regards to the anti‐proliferative activity, in MEB‐Med‐8A and Daoy, the most prominent effect is achieved with 2 μM Axitinib alone compared to all other experimental conditions with 57 ± 3.5% and 43 ± 1.5% of growth inhibition, respectively. At lower Axitinib concentrations, the combination with GDC‐0941 augmented the anti‐proliferative effect in MEB‐Med‐8A and D283 Med compared to single Axitinib application at the respective concentration. However, there was no additional effect compared to GDC‐0941 alone in MEB‐Med‐8A and Daoy.To date, targeted therapies have become an integral element of cancer treatment c‐myc‐amplified Non‐WNT/Non‐SHH medulloblastoma variant While Axitinib is currently under clinical investigation in glioblastoma, this is a first report documenting profound anti‐neoplastic activity in paediatric medulloblastoma cell lines The profound loss of cell viability in all three MB lines can be attributed to marked pro‐apoptotic and anti‐proliferative activity. Axitinib arrests proliferation in G2/M phase Fig.  and intein vitro experiments proof anti‐neoplastic efficacy against different medulloblastoma cell lines. Morover, in vivo, drug accumulation or retention as a result of newly formed dysfunctional tumour vessels might lead to spatial Axitinib concentration in the brain tumour tissue that exceeds the patient plasma concentration in vivo, in addition to direct anti‐tumour effects, modulation of the tumour microenvironment contributes to MKI efficacy. Thus, a recent report on a phase II clinical study in recurrent glioblastoma concludes that Axitinib improves response and progression‐free survival In clinical pharmacokinetic studies, there is a high degree of variability in plasma levels even in healthy volunteers. However, although it is well documented that Axitinib is primarily metabolized by CYP3A4/5 and to a lesser extent by CYP1A2, CYP2C19 and UGT1A1, in two meta‐analyses, no association between plasma levels and polymorphisms of these enzymes was found. in vivo xenograft model that the oral administration of the broad‐spectrum MKI Sorafenib and Pazopanib attenuate tumour growth and prolong survival in vitro cytotoxicity of Axitinib shown here further in vivo studies evaluating Axitinib in orthotopic medulloblastoma mouse models are warranted. Pazopanib and Sorafenib also target VEGFR1‐3, PDGFR α/β and c‐kit among other kinases and affect medulloblastoma proliferation and cell survival by inhibition of STAT3 and, to a lesser extend, AKT phosphorylation In medulloblastoma, we have previously shown in an orthotopic c‐myc amplification compared to treatment with either drug alone. Combination of Axitinib with the AKT inhibitor GDC‐0941 results in near to complete abrogation of viable cells in MEB‐Med‐8A and D283 Med. Corresponding to the enhanced cytotoxicity of the Axitinib‐GDC‐0941 combination, AKT and STAT3 signalling are markedly impaired. While there is a cumulative suppressive effect of Axitinib and GDC‐0941 on STAT3 activity, as expected the specific PI3K inhibitor dominates the inhibitory effect on AKT. The significance of PI3K/AKT signalling has not only been delineated for c‐myc‐amplified medulloblastoma but also for cerebellar cells overexpressing c‐myc resulting in a similar molecular profile and PI3K dependency c‐myc‐amplified MEB‐Med‐8A and D283 Med medulloblastoma cells, in the SHH‐TP53‐mutated medulloblastoma line Daoy, at the highest dose, the anti‐neoplastic effect of Axitinib exceeds the cytotoxic activity of GDC‐0941 and combination of the two drugs has no additional effect. Of note, across the different medulloblastoma entities, not only the activity of STAT3 and AKT but also protein levels are diminished by the applied inhibitors, which is most likely because of the role of STAT3 and AKT as key regulators of protein expression With acceptable tolerability including limited haemotological side effects, Axitinib is particularly well suited for combination therapy c‐myc‐amplified Non‐WNT/Non‐SHH and SHH‐TP53‐mutated tumours. In this setting, marked anti‐proliferative property of Axitinib is complemented by profound cytotoxic capacity. We further show that Axitinib anti‐neoplastic efficacy correlates with the target expression profile on the tumour cells. As the availability of anti‐cancer agents with partially redundant target profiles such as Axitinib, Vandetanib, Sorafenib, Sunitinib and Pazopanib is steadily increasing, biomarkers supporting the choice of MKI are of increasing importance. With regards to multi‐modal treatment approaches, PI3K/AKT pathway inhibition by means of GDC‐0941 enhances the efficacy of Axitinib against c‐myc‐amplified medulloblastoma. Our data suggest that Axitinib single treatment or in combination with PI3K inhibitors such as GDC‐0941 might prove a promising strategy to clinically target the most aggressive c‐myc‐amplified and SHH‐TP53‐mutated medulloblastoma subtypes.In conclusion, our study demonstrates that Axitinib, a highly specific clinically available inhibitor of angiogenesis, displays marked cytotoxic activity against paediatric medulloblastoma cell lines modelling most aggressive The authors declare that they have no conflict of interest.Fig. S1 Expression of the cancer stem cell markers CD24, CD44 and CD133 is absent in MEB‐Med‐8A.Click here for additional data file.Fig. S2 Axitinib induces a G2/M‐phase cell cycle arrest in medulloblastoma.Click here for additional data file.Fig. S3 Determination of pro‐apoptotic effects of Axitinib in medulloblastoma cells.Click here for additional data file.Table S1 Relative expression of Axitinib target proteins in medulloblastoma cell lines Click here for additional data file.


Cervical cancer is a major cause of cancer-related mortality in women in the developing world. Cancer Stem cells (CSC) have been implicated in treatment resistance and metastases development; hence understanding their significance is important.Primary culture from tissue biopsies of invasive cervical cancer and serial passaging was performed for establishing cell lines. Variable Number Tandem Repeat (VNTR) assay was performed for comparison of cell lines with their parental tissue. Tumorsphere and Aldefluor assays enabled isolation of cancer stem cells (CSC); immunofluorescence and flow cytometry were performed for their surface phenotypic expression in cell lines and in 28 tissue samples. Quantitative real-time PCR for stemness and epithelial-mesenchymal transition (EMT) markers, MTT cytotoxicity assay, cell cycle analysis and cell kinetic studies were performed.+ phenotype. In tissue samples of untreated invasive cervical cancer, CD133+ CSCs ranged from 1.3–23% of the total population which increased 2.8-fold in radiation-resistant cases. Comparison of CD133+ with CD133− bulk population cells revealed increased tumorsphere formation and upregulation of stemness and epithelial-mesenchymal transition (EMT) markers with no significant difference in cisplatin sensitivity.Four low-passage novel cell lines designated RSBS-9, − 14 and − 23 from squamous cell carcinoma and RSBS-43 from adenocarcinoma of the uterine cervix were established. All were HPV16+. VNTR assay confirmed their uniqueness and derivation from respective parental tissue. CSC isolated from these cell lines showed CD133+ phenotype and are increased in relapsed cases and hence should be targeted for achieving remission.Low-passage cell lines developed would serve as models for studying tumor biology. Cancer Stem Cells in cervical cancer display CD133The online version of this article (10.1186/s12885-018-4237-5) contains supplementary material, which is available to authorized users. Cancer of cervix is the fourth most common cancer among women and the third most common cause of cancer related mortality worldwide . Develop+) [CSC have been demonstrated in several solid cancers. In cancer cervix, cancer stem-like cells were first identified to exist by Feng et al.  and subs+) , 6, Anne+)  and CD49+)  have all+) –8. HenceThe study was approved by the Institute’s Ethics Committee (vide letter No.IC-CCRT/07/DTM-3226 dated 16.11.2009). All patients were enrolled after obtaining a voluntary written informed consent and the study was conducted as per the Helsinki declaration (2000). Tissue samples obtained from untreated patients of invasive cervical cancer comprising of 3 radical hysterectomies in FIGO stage IIA and 30 cervical biopsies from FIGO stage IIb/III were subjected to primary culture. The sample was collected directly in Dulbecco’s Modified Eagle Medium/ Nutrient Mixture F-12 Ham  (Invitrogen). The rest of the specimen was subjected to formalin fixation and paraffin-embedded sections were evaluated light microscopically for histopathological diagnosis.2) and 95% air. 17 cases showed contamination and in 16 cases successful primary cultures could be established. In 7 of these 16 cases, long-term primary cultures got established; and 4 were pursued further. The morphology of the adherent cells was observed under an indirect microscope by phase-contrast and morphology documented.The specimen collected was immediately mechanically disaggregated with a sterile surgical blade, washed 3 times with sterile phosphate buffered saline (PBS), digested by collagenase II by incubation for 1 h at 37 °C , filtered through a 70 μm mesh to get rid of stromal fragments and obtain a cell suspension. The tumour cells were placed in 6 well plates (Corning) containing DMEM/ F12 supplemented with 10% fetal bovine serum , penicillin (100 U/mL), and streptomycin (100 μg/mL) and placed in a humidified incubator at 37 °C containing 5% carbon dioxide  equipped with XR81M-B Camera .Approximately 5 × 10Karyotyping was performed on metaphase spreads of RSBS-9, RSBS-14, RSBS-23 and RSBS-43 cells at passage 32 by standard G-banding.Cultured cells were also placed under anchorage-independent/stem-cell conditions in Ultra Low Attachment plates  in serum-free DMEM/F12 supplemented with 5 μg/mL insulin (Sigma), 20 ng/mL human recombinant epidermal growth factor , 10 ng/mL basic fibroblast growth factor , and 0.4% bovine serum albumin . Floating aggregates or tumorspheres were observed microscopically and their numbers per well documented. Efficiency of tumorsphere formation ability was calculated based on manual counting of number of tumorspheres after plating equal numbers of sorted cells wherever required.high and ALDHlow cells  as per manufacturer’s instructions detailed in Additional file 6 cells derived from tumorspheres and corresponding adherent cells were screened for surface markers by flow Cytometry using fluorescent  labelled antibodies against CD117, CD90, CD44, CD49f and CD71. Isolation of the CD133+ cells was performed using a FACS Aria flow cytometer  and analyzed further  and in 6 patients who had been treated with chemoradiation therapy 46 Gy/23 fraction external beam radiation administered concurrently with 40 mg/m2 cisplatin weekly dose followed by intracavitary brachytherapy and who had subsequently relapsed. The levels of CD133, CD49f and CD44 was evaluated in each case after gating for CD45 negative cells  was performed on the biopsies of patients with untreated invasive cervical cancer  were used to plot the growth curve by using the MTT 3--2,5-diphenyl tetrazolium bromide assay detailed in Additional file Cell cycle analysis was performed after labelling the cellular deoxyribonucleic acid (DNA) with propidium iodide (PI) . The cells were then subjected to cell cycle analysis by FACS (BD FACS Aria II). Each experiment was analysed in duplicate and four independent experiments were performed  were used to prove and validate that the cervical cancer cell lines were derived from primary cervical cancer biopsies. Amplification of VNTR loci D1S80 (range of amplification product 387 to 723 bps) was achieved using the primers  for variable duration . Cytotoxicity was determined by MTT assay All experiments were performed twice in triplicates to determine the ICOCT-4, SNAIL, SLUG, VIMENTIN, ABCG2, N-CDHERIN, NANOG, TIWST and E-CADHERIN using gene specific primers  per the manufacturer’s instructions. 1 μg RNA was reverse-transcribed into cDNA using specific primers and per the manufacturer’s instructions . 1 μL of cDNA was used as template for qRT-PCR for Successful long-term primary cultures could be established in 7/33 or 21.2% cases; four of these were pursued and 4 permanent cell lines were derived. They were designated as RSBS-9, RSBS-14, RSBS-23 and RSBS-43 respectively with the ages of the patients being 49, 34, 45 years and 63 years respectively. All 4 cell lines were derived from cervical biopsy specimen and from patients with FIGO stage III disease. All the cell lines established were regularly checked for mycoplasma contamination.The histology of the primary tumour corresponding to the RSBS-9 cell line was a moderately differentiated keratinizing squamous cell carcinoma, for RSBS-14 and RSBS-23 cell lines were non-keratinizing squamous cell carcinoma, moderately and poorly differentiated respectively. RSBS-43 cell line was derived from a moderately differentiated adenocarcinoma. The parental tissue biopsies and the respective adherent cell lines derived are shown in Fig. Electron microscopy of the cell lines derived from squamous cell carcinoma  showed high nucleo-cytoplasmic ratio with keratin filaments in the cytoplasm and cell junctions in the form of hemi-desmosomes. RSBS-43 cell line derived from adenocarcinoma cervix showed a few irregular microvilli, prominent nucleolus and prominent rough endoplasmic reticulum and occasional cell junctions. Representative images from RSBS-14 and RSBS-43 are shown in Fig. Karyotyping was done on all the four cell lines at passage 32. All 4 cell lines displayed aneuploidy. The number of chromosomes ranged from 47 to 53 in the RSBS-9, from 44 to 47 in RSBS-14, from 47 to 57 in RSBS-23 and from 47 to 53 in the RSBS-43 cell lines. The modal chromosome number was 53, 47, 56 and 51 in the RSBS-9, 14, − 23 and − 43 respectively [Additional file The VNTR assay uses multiple primer sets read as informative or non-informative. This established that the cell lines were indeed derived from the corresponding tissue sample and are unique Fig. .Fig. 3VaHPV DNA typing for HPV-16, − 18, − 31, and − 45, the most common HPV types in cervical cancer, revealed positivity only for HPV-16 sequence  [Additional file Cell cycle analysis showed 9–15% cells in the S-phase and 0.05–7.6% in subG1 phase [Additional file Cisplatin is commonly used in cervical cancer as part of the chemotherapy protocol and hence cisplatin sensitivity was determined in these cell lines [Additional file Tumorsphere formation could be demonstrated from cells derived from the primary cultures obtained from biopsy specimen of invasive cervical carcinoma, from the four novel cell lines established and from HeLa and SiHa cell lines. In these cases, tumor cells were dissociated and grown under anchorage-independent or ‘stem-cell’ conditions Floating aggregates of cells formed small spheroids on day 7 and representative images (RSBS-14 and RSBS-43) are shown in Additional file high whereas rest were ALDHlow in Hela, SiHa and four novel cell lines. A representative plot is shown in Additional file high and ALDHlow cells. Equal numbers of cells  of ALDHhigh and ALDHlow cells were cultured and tumorspheres was assessed at day 10. ALDHhigh positive cells showed higher capacity for spheroid formation [Additional file Adherent cultures from the four novel cell lines as well as SiHa and HeLa cell lines were tested by the ALDEFLUOR assay which identifies cancer stem cell population based on the high aldehyde dehydrogenase activity (ALDH) activity of cells in the presence or absence of the ALDH inhibitor DEAB. Further, these cells were sorted by flow cytometry based on high / low ALDH activity. Compared with control cells treated by the DEAB inhibitor, we observed 2.9% to 6% cells to be ALDHNext, we wished to identify the surface phenotypic expression of putative stem cell markers in the tumorspheres enriched for cancer stem cells. Initially,the expression of known stem cell markers including CD44, CD49f, CD133 and CD71, CD90, CD105 was tested by flow cytometry in the tumorspheres and adherent bulk cells in each of the 4 cell lines RSBS-9, − 14, − 23 and − 43 and also HeLa and SiHa cell lines. CD71, CD90 and CD105 did not show any differential expression and so were not pursued further. There was a differential expression of CD44, CD49f and CD133. CD44 levels were higher in adherent cells as compared to tumorshpere cells. Adherent cells showed CD49f expression in the range of 45.7–89% whereas in tumorspheres it ranged from 61.1–90.7%. Similarly CD133 positivity ranged from 1.4 to 19% percentage in adherent cells whereas it was 12.5–63.6% in corresponding tumorspheres. A representative plot in RSBS-9 cell line is shown [Additional file + is a surface phenotypic marker of cervical cancer stem cells.Direct immunofluorescence was carried out on adherent cells and tumorspheres derived from all four novel cell lines and their corresponding tissue sections. Similar results were observed in all cell lines tested and a representative image panel from RSBS-14 cell line is shown in Fig. n = 22), CD133 showed 1.3–50.6% positivity (median 10.4%); CD49f showed 3.2–91.8% positivity (median 58.4%), CD44 showed positivity in 1.2–58.1% (median 14.3%). In 6 cases who had received chemoradiation as per standard protocol but had relapsed cases within 1 year and hence represent radiation resistance or treatment failure, there was a trend towards downregulation of CD44 (median 6.2%) and upregulation of CD133 (median 28.2%). CD49f expression was very variable and no trend was observed.Expression of CD44, CD49f and CD133 in patient samples by flow cytometry are shown in Fig. + cancer stem cells were sorted from adherent cultures of RSBS 9, − 14, − 23 and − 43 cells tumorosphere formation assessed. CD133+ cells formed more and larger spheroids as compared to CD133− cells [Fig. + cancer stem cells to cisplatin determined by MTT assay. There was no appreciable difference in the CD133+ versus the CD133− cells in the response to cisplatin in all 4 cell lines tested [Fig. CD133lls Fig. . Furtherted Fig. .Fig. 6a OCT4, NANOG) and EMT markers  in CD133+ sorted cells in relation to unsorted cells and after normalization to β-ACTIN housekeeping gene was evaluated and results shown in Fig. + RSBS-9 cells showed marked upregulation of OCT-4, NANOG, ABCG-2, SLUG, SNAIL, TWIST, N CADHERIN, and VIMENTIN; however, E-CADHERIN was also upregulated. CD133+RSBS-14 cells showed upregulation of OCT-4 and NANOG, upregulated E-CADHERIN with little change in the other markers. CD133+ RSBS-23 cells showed upregulation of VIMENTIN, TWIST, SNAIL, SLUG AND N-CADHERIN, nil expression of E-CADHERIN with no change in the other markers. CD133+ RSBS-43 cells showed upregulation of OCT-4, TWIST and SNAIL and downregulation of E-CADHERIN with no appreciable change in the other transcripts. Taken together, our results indicate a variable albeit an overall upregulation of stemness and EMT markers in the CD133+ sorted cancer stem cells in all the four cell lines evaluated.Relative expression of stemness markers Additional file 2:Figure S1. Representative karyotypes with modal chromosome numbers of RSBS-9 (A), RSBS-14 (B), RSBS-23 (C) and RSBS-43 (D) respectively. (TIFF 127 kb)Additional file 3:Figure S2. A. PCR for HPV-16 in cell lines and corresponding parental tissues showing positivity for all cell lines and the parental tissues. B. Cell cycle analysis. C. Cell growth curve and estimation of doubling time showing similar doubling time of 48 h approximately. D. Cisplatin sensitivity assay showing variable chemosensitivity in the cell lines. (TIFF 192 kb)Additional file 4:Figure S3. A. Representative figure of tumorspheres from RSBS-14 and RSBS-43 cell lines on day 7. B. Aldefluor assay: Representative plots of RSBS-14 cell line showing 3.2% cells with high ALDH levels. Cell sorted into ALDH low (−) and high (+) levels and tumorsphere assay performed showing spheres in ALDHhigh sorted cells. (TIFF 223 kb)Additional file 5:Figure S4. CD133 and CD49f expression in adherent (ADH) vs Tumorspheres (SPH) in RSBS-9 cell line by Flow Cytometric Immunophenotyping. Representative plots of CD49f and CD133 expression and data represented in histograms. Both markers showed increased expression in tumorspheres as compared to adherent cells. (TIFF 123 kb)Additional file 6:Figure S5. CD49f expression in adherent cells and tumorspheres. Representative images panel of RSBS-14 cell line showing similar and moderate levels in adherent cells and in tumorspheres. (TIFF 2512 kb)


The objective of this study was to evaluate the effect of platinum-based drugs on nuclear-factor erythroid2 like 2 (NRF2) signaling in non-small cell lung cancer cell lines with or without Kelch-like ECH-associated protein 1 (KEAP1) mutations and to determine the role of NRF2 and KEAP1 on platinum-based drug treatment.We used real-time PCR to assess relative mRNA expression and used western blotting and immunofluorescence assays to assess protein expression. Small interfering RNA and shuttle plasmids were used to modulate the expression of NRF2, wild-type KEAP1, and mutant KEAP1. Drug sensitivity to platinum-based drugs was evaluated with Cell Count Kit-8.We found that platinum-based therapies modified the NRF2 signaling pathway differently in KEAP1-mutated non-small cell lung cancer (NSCLC) cell lines compared with wild-type KEAP1 cell lines. The reactive degree of NRF2 signaling also varies between nedaplatin and cisplatin. The modification of NRF2 or KEAP1 expression in NSCLC cell lines disrupted downstream gene expression and cell sensitivity to platinum-based drugs. Finally, gene expression data retrieved from The Cancer Genome Atlas (TCGA) consortium indicated that KEAP1 mutation significantly affects NRF2 signaling activity in patients with NSCLC.Our findings suggest that NRF2 signaling plays an indispensable role in NSCLC cell sensitivity to platinum-based treatments and provides a rationale for using NRF2 as a specific biomarker for predicting which patients will be most likely to benefit from platinum-based treatment. Non-small cell lung cancer (NSCLC) is associated with considerably high mortality worldwide . SurgeryNuclear-factor (NF)-E2-related factor 2 (NRF2) is a core transcription factor involved in the cellular response to oxidative stress . Under bIn urethane-induced lung carcinogenesis model, NRF2 activation suppresses tumorigenesis, but somatic activation of NRF2 in cancer cells enhanced growth of tumors . StudiesUnless otherwise stated, all chemicals were purchased from Sigma-Aldrich . Antibodies against NRF2 (ab62352) were purchased from Abcam; antibodies against HO1 (#5853) and NQO1 (#3187) were purchased from Cell Signaling Technology; and antibodies against KEAP1 (10503-2-AP), GCLM  (14241-1-AP), and β-actin were purchased from Proteintech.The A549 (human lung adenocarcinoma), H292 (human mucoepidermoid carcinoma), H460 (human large cell lung cancer), and SKMES-1 (human squamous lung cancer) cell lines were obtained from the Cell Resource Center, Peking Union Medical College. The cells were cultured in RPMI-1640, with the exception of SKMES-1 cells, which were cultured in MEM. All media were supplemented with 10 % fetal bovine serum . No antibiotics were added to the media.G333C-forward, TGATCTACACCGCGGGCTGCTACTTCCGACAGTCG333C-reverse, AGCCCGCGGTGTAGATCAGGCGGCCCACCD236H-forward, AGCCCGCGGTGTAGATCAGGCGGCCCACCD236H-reverse, GGTCCCGGCTGATGAGGGTCACCAGTTGGThe small interfering RNAs used to silence NRF2 were:Sense, GGUUGAGACUACCAUGGUUTTAntisense, AACCAUGGUAGUCUCAACCTTThe wild-type KEAP1 coding sequence (CDS) was cloned into the pEnter shuttle plasmid. The Fast Mutagenesis system  was used to introduce the G333C and D236H mutations. The primers used were:Transfection was conducted with Lipofectamine 2000 (Invitrogen) according to a previously published method .2O. Cycling conditions were as follows: 95 °C for 10 min and 40 cycles of 95 °C for 15 s and 60 °C for 60 s. Cycle thresholds were calculated as the expression fold-changes according to the 2−△△CT algorithm. The primers used for real-time PCR are:NRF2, forward, TCCAGTCAGAAACCAGTGGATReverse, GAATGTCTGCGCCAAAAGCTGHO1, forward, AAGATTGCCCAGAAAGCCCTGGACReverse, AACTGTCGCCACCAGAAAGCTGAGNQO1, forward, GAAGAGCACTGATCGTACTGGCReverse, GGATACTGAAAGTTCGCAGGGGCLM, forward, TGTCTTGGAATGCACTGTATCTCReverse, CCCAGTAAGGCTGTAAATGCTCTotal messenger RNA (mRNA) was extracted from cells using TRIzol according to the manufacturer’s protocol . Seven hundred fifty-nanogram total mRNA was used to synthesize first strand complementary DNA (cDNA) using the cDNA synthesis kit (ThermoFisher Scientific). Quantification reactions were performed with the ABI 7900 HT platform. Each reaction consisted of 5 μl SYBR Green/ROX qPCR Master Mix (ThermoFisher Scientific), 1 μl cDNA, 1 μl 10 μM forward and reverse primer mix, and 3 μl ddHFragment 1, forward AGAGGTGGTGGTGTTGCTTATReverse TGGAGATGGAGGCCGTGTAFragment 2, forward CAGGTCAAGTACCAGGATGReverse GATGAGGGTCACCAGTTGFragment 3, forward ATCGGCATCGCCAACTTCReverse AGGTAGCTGAGCGACTGTFragment 4, forward CAGAAGTGCGAGATCCTGReverse GCTCTGGCTCATACCTCTFragment 5, forward GCCCTGGACTGTTACAACReverse GTCTCTGTTTCCACATCGTAFragment 6, forward GCTGTCCTCAATCGTCTCReverse AGTTCTGCTGGTCAATCTGNRF2 exon2, forward TCGTGATGGACTTGGAGCTGReverse AGCATCTGATTTGGGAATGTGTotal RNA was extracted and used to synthesize the first chain cDNA according to the procedure described in the real-time PCR analysis description. cDNA was used as a template to synthesize six segments of the KEAP1 gene using the following cycling conditions: initial denaturing step at 94 °C for 5 min followed by 30 cycles at 94 °C for 40 s, 60 °C for 40 s, and 72 °C for 55 s. Products were separated on 1 % agarose gels, and bands were visualized with ethidium bromide. The products were sequenced using the ABI3730 XL DNA Analyzer  and analyzed with Chromas 2.4.3 software. Primers used to amplify the KEAP1 gene fragments were:Sections were spliced together after manual inspection and compared with the reference sequence with BLAST to identify potential mutations.Cytoplasm and nuclear protein were extracted by using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (ThermoFisher Scientific). For total protein extraction, we used standard protocols for protein isolation and antibody detection, as previously described , 25. CelImmunofluorescence was performed as previously reported with some modifications . Cells wv/v) CCK-8 was added to each well. Absorbance at 490 nm was measured after a 2-h incubation at 37 °C. Cell survival was calculated as the ratio between the treated group and control groups. Ratios were graphed in line charts with Graphpad Prism 6 software.Cellular proliferation was evaluated using a Cell Count Kit-8 (CCK-8) with some modifications . Lung cahttp://www.compgenome.org/TCGA-Assembler/) in an R program. KEAP1 mutation status was defined as previously described [Expression data from The Cancer Genome Atlas (TCGA) squamous lung cancer patients was retrieved using the package TCGA-Assembler  Fig.  and thatp < 0.01, **p < 0.05) and H460 , nedaplatin induced a weak but significant increase in three downstream gene expression. Cisplatin treatment, however, significantly enhanced HO1, NQO1, and GCLM expression in both A549 and H460 cell lines after 24 h of treatment.To profile the expression of NRF2 signaling pathway components in response to platinum treatment, the four NSCLC cell lines  were treated with 30 μM nedaplatin or 30 μM cisplatin for 24 h. Total RNA from each group was extracted, and HO1, NQO1, and GCLM gene expression levels were assessed with real-time PCR. In the wild-type KEAP1 cell lines H292 Fig.  and SKMETo evaluate protein levels, the four cell lines were treated with 30 μM nedaplatin or 30 μM cisplatin for 3 or 24 h. Total proteins were extracted, and HO1, NQO1, GCLM protein expression was evaluated by using western blot analysis. In the wild-type KEAP1 H292 and SKMES-1 cell lines Fig. , a mild The results of immunofluorescence analysis of HO1 protein expression were consistent with the results of the western blot analysis. Over a 24-h treatment period, 30 μM nedaplatin or cisplatin induced a mild increase in HO1 protein expression in H292 and SKMES-1 cells Fig. . In the p < 0.001, **p < 0.05) and H460 , constitutive expression of wild-type KEAP1 over a 72-h period reduced the expression of the NRF2 downstream target genes HO1, NQO1, and GCLM at the mRNA and protein levels. Overexpression of wild-type KEAP1 sensitized A549 cells  and H460 cells  to nedaplatin and cisplatin over a 24-h treatment period.To investigate the impact of KEAP1 mutations status on NRF2 signaling, the KEAP1 CDS was cloned into the pEnter vector and transfected into the NSCLC cell lines. In the mutant KEAP1 cell lines A549  and SKMES-1 cells  transfected with the mutant KEAP1 construct. An identical trend was observed at the protein level  and SKMES-1  treated with 60 μM cisplatin or nedaplatin.Next, we introduced the G333C and D236H mutations into the KEAP1 pEnter construct. The mRNA expression levels of the NRF2-associated genes HO1, NQO1, and GCLM were significantly elevated in H292 cells  and SKMES-1 , siRNA transfection over a 72-h period reduced the expression of NRF2 and its downstream targets HO1, NQO1, and GCLM at the mRNA and protein levels. Pretreatment with siRNA targeting NRF2 increased sensitivity to nedaplatin and cisplatin in H292 cells  and SKMES-1  over a 24-h treatment period.To assess the effects of NRF2 expression on NSCLC cells exposed to platinum-based drugs, we designed a small interfering RNA (siRNA) construct targeting NRF2 mRNA to inhibit NRF2 activity. In the wild-type KEAP1 cell lines H292  and SKMES-1 , administration of the NRF2 activator over a 24-h period significantly increased the expression of the NRF2 downstream genes HO1, NQO1, and GCLM at the mRNA and protein levels. Pretreatment with 60 μm t-BHQ conferred chemo-resistance to nedaplatin and cisplatin in H292 cells  and in SKMES-1 cells  over a 24-h treatment period.In addition, NSCLC cell lines were treated with t-BHQ, a specific activator of NRF2. In the wild-type KEAP1 NSCLC cell lines H292  and KEAP1  were observed between the mutated patients with mutant KEAP1 cases and with wild-type KEAP1. However, expression levels of the NRF2 downstream target genes HO1 , NQO1 , and GCLM  were significantly enhanced in patients with mutant KEAP1 cases compared with those with wild-type KEAP1.A total of 100 individuals diagnosed with NSCLC were selected from the TCGA group for analysis. KEAP1 mutation status was defined as previously described . We founA large genomic study has demonstrated that multiple oncogenic and tumor suppressor pathways are involved in the initiation and progression of lung cancer . In addiFunctional KEAP1 represses NRF2 activity by recruiting the NRF2 protein to the actin cytoskeleton . SequencAfter exposure to cisplatin, the KEAP1 mutant cell lines A549 and H460 exhibited considerably increased levels of NRF2 signaling, whereas when the KEAP1 wild-type cell lines H292 and SKMES-1 exhibited no changes or only mild changes. Cisplatin is a weak inducer of ARE in the MCF-7 human breast cancer cell line (without KEAP1 mutation ) and indHowever, nedaplatin induces only weak activation or no activation of NRF2 signaling. The antineoplastic activity of nedaplatin, a cisplatin derivate developed in 1983 , might bAn early study by Devling et al.  has revet test p value 0.0116). This effect was also observed in Fig. t test p value 0.0218). We speculated that under siRNA intervention or activator treatment, some other pathways such as NF-kappa B and BACH1 are compensatorily involved in the process [NRF2 activity is involved in chemosensitivity in breast cancer  and ovar process . Our worTo confirm the significance of KEAP1 mutation in vivo, we evaluated public gene expression data from the publically available TCGA consortium. As expected, KEAP1 mutation did not significantly change alter the mRNA expression of NRF2 and KEAP1. However, mRNA levels of NRF2 downstream target genes were significantly higher in patients with KEAP1 mutations compared with patients with wild-type KEAP1, suggesting that KEAP1 and NRF2 interact primarily at the protein level and that KEAP1 mutations strongly affect NRF2 signaling Fig. . The assIn summary, the mRNA and protein expression profile of NRF2 and components of the NRF2-associated genes in four NSCLC cell lines were characterized and their responses to platinum-based therapies were analyzed. A causative effect of KEAP1-NRF2 signaling on platinum-based treatment was defined by engineered expressing either wild-type or mutant KEAP1 and knocking-down or activating NRF2. It is reasonable to hypothesize that KEAP1/NRF2 plays a key role in the cellular response to platinum chemotherapy in NSCLC and that KEAP1 could be explored as a specific biomarker for predicting a patient’s response in personalized therapy.


Bacterial L-asparaginase (ASNase), hydrolyzing L-asparagine (Asn), is an important drug for treating patients with acute lymphoblastic leukaemia (ALL) and natural killer (NK) cell lymphoma. Although different native or pegylated ASNase-based chemotherapy are efficient, disease relapse is frequently observed, especially in adult patients. The neo-synthesis of Asn by asparagine synthetase (AsnS) following ASNase treatment, which involves the amino acid response and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathways, is believed to be the basis of ASNase-resistance mechanisms. However, AsnS expression has not emerged as an accurate predictive factor for ASNase susceptibility. The aim of this study was to identify possible ASNase sensitivity/resistance-related genes or pathways using a new asparaginase, namely a pegylated r-crisantaspase, with a focus on classic Asn-compensatory responses and cell death under conditions of Asn/L-glutamine limitation. We show that, for B-ALL cell lines, changes in the expression of apoptosis-regulatory genes  are associated with ASNase susceptibility. The response of malignant NK cell lines to ASNase may depend on Asn-compensatory mechanisms and other cellular processes such as cleavage of BCL2A1, a prosurvival member of the Bcl-2 protein family. These results suggest that according to cellular context, factors other than AsnS can influence ASNase susceptibility. Bacterial L-asparaginase (ASNase) is an important component of anticancer regimens for acute lymphoblastic leukemia (ALL)134268910121314AsnS mRNA and protein expression; 2) a decrease in Asn efflux through Na+-independent exchange system; 3) a production of aspartic acid, the substrate for Asn synthesis by AsnS, via transamination; 4) a higher Gln synthetase (GlnS) activity through post-transcriptional regulation; 5) an activation of the Gln transporter91011161713141920Despite the high survival rate of patients treated with chemotherapeutic drugs including ASNase, about 20% of children and more than 50% of adults with ALL relapse, with leukemic cells becoming resistant to treatmentAsnS transcription for further neo-synthesis of Asn10222324252627293032Moreover, at the level of signaling transduction, the amino acid response (AAR) pathway, a well-known pathway that senses and responses to a deficiency of amino acids, as well as the survival-related mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) and mammalian target of rapamycin complex 1 (mTORC1) pathways are believed to be involved in ASNase resistance through supplying Asn and limiting its waste. Indeed, upon amino acid depletion, uncharged tRNAs activate general control nonderepressible 2 kinase (GCN2) of AAR pathway, which phosphorylates subsequently eukaryotic initiation factor 2 alpha (elF2α), leading to translational up-regulation of a transcription factor ATF4. Enriched ATF4 proteins promote activation of AsnS at baseline or after exposure to ASNase353637AsnS in ALL patient samples are generally so low that AsnS expression cannot be used as a predictive factor for ASNase sensitivity or resistanceAsnS has not been identified as a discriminating gene for ASNase sensitivity in the study of gene-expression patterns in sensitive versus resistant children ALL samplesAlthough AsnS expression levels are believed to be a key factor in these mechanisms of resistance to ASNase, several studies using clinical samples from children and adult ALL fail to show any correlations between the sensitivity to ASNase and the mRNA expression of In the present work, we sought to identify discriminating factors for ASNase susceptibility using two pairs of B-ALL and malignant NK cell lines with high or low sensitivity to ASNase. We demonstrate that the classical resistance-mechanisms involving the MEK/ERK pathway as well as increase in GlnS expression are associated with resistance to ASNase in the KHYG1 NK leukemia cell line. Furthermore, we show that ASNase induced cell death by apoptosis and that the induction of apoptosis after exposure to ASNase is delayed in resistant cell lines. Analysis of gene-expression indicates that activation of the NFκB pathway is associated with higher sensitivity of the RS B-ALL cell line, while cleavage of the prosurvival Bcl-2 family member BCL2A1 was observed in the sensitive MEC04 NK T-cell lymphoma cell line. These data point to different mechanisms involved in ASNase-susceptibility according to disease type.50 < 5 × 10−5), whereas Nalm-6 and KHYG1 were much less sensitive to ASNases (EC50 > 1 × 10−2)In our previous study, we have defined using MTT assay the susceptibility of different leukemic cell lines  and Nalm-6), non-Hodgkin lymphoma cell lines as well as malignant NK cell lines (including the NK T-cell lymphoma cell line MEC04 and the NK cell leukemia cell line KHYG1), to ASNase treatmentWe first analyzed the classic compensatory AAR and MER/ERK pathways in the four cell lines . WithoutThese results indicate that the increased activity of the MEK/ERK pathway and the moderate up-regulation of AsnS and GlnS expression, favoring the neo-synthesis of Asn to compensate for Asn limitation, are associated to the ASNase-resistance of KHYG1 cells.To determine whether differences in mTORC1 activity exist between sensitive and resistant cell lines, the phosphorylation of 4E-BP1 on T70 was analyzed . For malThese observations show that in response to ASNase treatment the expression of phosphorylated 4E-BP1 was reduced in both the sensitive and the resistant cell lines and that the activity of mTORC1 pathway does not represent a discriminating factor for ASNase susceptibility in these cell lines.−5/ml). In contrast, the induction of apoptosis (~23%) and late apoptosis, (~18%) in resistant Nalm-6 cells was observed only 48 h after ASNase treatment and only at high doses of ASNase (e.g. ≥ 5 × 10−1 U/ml)  through which ASNase-treated cells were dying, and the difference in the induction of cell death between sensitive and resistant cell lines . For B-A−1 U/ml) .We obser−1 U/ml) . In all These results indicate that B-ALL and malignant NK cells predominantly die by apoptosis in response to ASNase treatment and that, expectedly, the induction of apoptosis is delayed and less marked in resistant versus sensitive cell lines.In order to determine which apoptosis-related factors may influence the susceptibility of cells to ASNase, the mRNA expression of 93 apoptosis-related genes was analyzed using TaqMan® Human Apoptosis Array in cells treated or not with ASNase. According to their prominent function in the regulation or execution of apoptosis, the studied genes were classified as pro-apoptotic n = 72) or anti-apoptotic (n = 21) and were divided into four sub-groups: Bcl-2 family, caspases, NFκB and TNF/Fas pathways. We compared the baseline mRNA expression of these genes in sensitive versus resistant cell lines from the same disease type as well as between treated versus non-treated cells. The fold change of each gene at baseline (ratio sensitive/resistant) or 6 h after ASNase treatment (ratio treated/non-treated) were summarized in  or anti-CASP4, PYCARD, RELB, NFκB2, BCL3, TNFSF10, FAS; 2 anti-apoptotic: BCL-2, BCL2A1) were more expressed at baseline and 10 NFκB target genes  were increased in ASNase-treated cells. Among these genes, four  were both more expressed at baseline and induced after ASNase treatment  were more expressed in sensitive RS cells at baseline and 9 genes (7 pro and 2 anti-apoptotic) were less expressed, compared to resistant Nalm-6 cells. In ASNase-treated sensitive RS cells, the expression of 21 genes (16 pro- and 5 anti-apoptotic ) were induced compared to non-treated cells, whereas in ASNase-treated resistant Nalm-6 cells, only 5 pro-apoptotic genes were induced. These data indicate that the expression pattern of apoptosis genes is consistent with the phenotype of ASNase sensitivity/resistance of the analyzed cell lines. Moreover, in sensitive RS cells, 9 NFκB target genes  were less expressed in sensitive MEC04 cells at baseline, and 5 genes (4 pro- and 1 anti-apoptotic) were more expressed, compared to resistant KHYG1 cells. In response to ASNase, only the expression of 1 pro-apoptotic gene was increased in sensitive MEC04 cells, whereas 2 pro-apoptotic genes were decreased. In resistant KHYG1 cells, 4 genes (2 pro-apoptotic and 2 anti-apoptotic) were induced, and 5 pro-apoptotic genes displayed reduced expression levels after ASNase treatment . ContrarBCL2A1 , was ~80 times more expressed at the transcript level in sensitive MEC04 cells, compared to resistant KHYG1 cells. Because the prosurvival BCL2A1 protein was recently reported to be converted into a pro-apoptotic factor after proteolytic cleavageBCL2A1 could represent a discriminating factor for ASNase susceptibility in these cell lines. As shown in We noticed that a specific anti-apoptotic member of the Bcl-2 family, ASNase is an important drug for ALL treatment. Although this molecule is efficient, some patients, especially adults, relapse, and refractory ALL cases are also reported. It is therefore essential to identify possible ASNase resistance determinants in leukemic cells. In this work, we sought to exploit the differential ASNase susceptibility of two pairs of B-ALL and malignant NK cell lines, in order to identify ASNase sensitivity/resistance-related genes or pathways, with a focus on classic compensatory responses and cell death under conditions of Asn/Gln limitation.AsnS at baseline and after ASNase exposure, albeit showing high GlnS expression. Given that the phosphorylation of elF-2α was not induced (and even decreased) after ASNase exposure, AAR pathway was inactivated in both cell lines, which could be an explanation for the absence of induction of AsnS protein expression following ASNase treatment. Those observations are in line with previous findings showing absence of AsnS mRNA expression before and after ASNase treatment in sensitive RS cells36AsnS gene is silenced and the promoter region of AsnS gene is highly methylated42AsnS expression might be associated with specific epigenetic processes such as AsnS gene silencing.The survival of both highly ASNase-sensitive RS and MEC04 cell lines seems strictly dependent on extracellular Asn, because these two cell lines had undetectable protein expression of AsnS by binding to its promoter, resulting in an increase in the protein expression of AsnS. The authors propose that tumor-derived p53 mutants induce oncogenesis by transactivating growth-promoting genes such as AsnS and hTERT genes45In resistant KHYG1 cells, the high baseline expression of AsnS as well as the increase in both AsnS and GlnS expression following ASNase treatment could compensate for the deficiency in Asn and Gln by promoting the synthesis of both amino acids, explaining the low susceptibility of this cell line to ASNase. Yet, intriguingly, in ASNase-treated cells, the phosphorylation of Erk1/2 was greatly induced with a slight increase in AsnS expression, while phosphorylation of elF-2α was reduced to undetectable levels. These data are in contradiction with previous published data suggesting the existence of a positive regulatory loop between the AAR and MEK/ERK pathways as well as the involvement of the activated AAR pathway in the activation of the MEK/ERK pathway and in the induction of AsnS expression in response to amino acid restriction2745We demonstrated that ASNase-induced cell death was mediated by apoptosis, in agreement with previous reports4710In conclusion, our data provide evidence that, for B-ALL cell lines, changes in the expression of apoptosis-regulatory genes, especially NFκB-related genes, rather than AsnS-regulatory pathways could be crucial for the susceptibility of cells to ASNase. Regarding malignant NK cell lines, AsnS-regulatory pathways appear to be the main determinant implicated in the susceptibility of cells to ASNase, BCL2LA1 cleavage representing another potential mechanism. Anomalies making cells resistant to ASNase could be multiple and variable from one patient sample to another according to the type of disease and cellular context. Thus, our study provided a piece of puzzle for the understanding of ASNase-resistance mechanisms and for further development of more adaptive and personalized combinatory treatment for ALL (e.g. including inhibitors of Erk1/2 or modulators of the NFκB pathway).2. Malignant NK cell lines, MEC04  and KHYG1 (aggressive NK cell leukemia), were cultured under the same condition in the presence of 10 ng/ml interleukin-2 (IL-2) .The B-ALL cell lines RS (ATCC CRL-1873) and Nalm-6 (DSMZ ACC128) were cultured in RPMI 1640 medium , containing 10% fetal calf serum (FCS), 2 mM L-glutamine, 100 U penicillin and 100 μg streptomycin, at 37°C in a humidified atmosphere with 5% COWestern blot was carried out as previously described6 cells/ml in round-bottom 96-well plates and treated with increasing doses of pegylated r-crisantaspase−5 U/ml to 5 U/ml for 24, 48 and 72 h. Non-treated cells were used as control. All samples were then subjected to Annexin V/PI apoptosis assay. Cells were incubated in 100 μl of Annexin V-binding buffer containing 10 μg/ml of PI (Sigma-Aldrich) and 2.5 μl Annexin V  for 15 min at room temperature before analysis by flow cytometry .Cells were seeded at 0.2 × 106 cells/ml and treated with or without 5 U/ml of pegylated r-crisantaspase for 6 h. Total RNA was extracted using Trizol solution and RNA quality was assessed by Bioanalyser . The reverse transcription was performed using High Capacity RNA-to-cDNA Master Mix  with 1 μg of total RNA in a volume of 20 μl. The product of reverse transcription diluted in TaqMan® Universal PCR Master Mix (Life Technologies) was then loaded into TaqMan® Human Apoptosis Array (Life Technologies) and quantitative PCR was performed with the Applied Biosystems 7900 Sequence Detection system (Life Technologies) according to the manufacturer's instructions. Relative quantification (RQ) was obtained using the equation RQ = 2−ΔΔCt after normalization of the Ct data to that of the reference gene, where Ct is the threshold cycle of fluorescence detection. Results were the combined data of two independent experiments. The reference gene for baseline expression analysis was 18S. The mean and standard deviation (S.D.) were Ct = 9.8 ± 0.4 for non-treated B-ALL samples and Ct = 10.2 ± 0.5 for non-treated malignant NK samples. 18S, GADPH and β-actin were the reference genes for normalizing data of ASNase-treated samples to that of non-treated samples. RS cells: 18S Ct = 8.7 ± 0.1; GADPH Ct = 17.5 ± 0.1; β-actin Ct = 17.9 ± 0.1. Nalm-6 cells: 18S Ct = 8.4 ± 0.2; GADPH Ct = 17.9 ± 0.1; β-actin Ct = 18.4 ± 0.1. MEC04: 18S Ct = 8.6 ± 0.2; GADPH Ct = 16.7 ± 0.2; β-actin Ct = 18.9 ± 0.2. KHYG1: 18S Ct = 9.9 ± 0.3; GADPH Ct = 17.4 ± 0.1; β-actin Ct = 17.3 ± 0.2.Cells were seeded at 0.2 × 10