We were able to infer DNA replication timing profiles for the centromeres in each of the five cell lines, which showed that centromere replication occurs in mid-to-late S phase. Furthermore, we found that replication timing was more variable between cell lines in the centromere regions than expected, given the distribution of variation in replication timing genome-wide. These results suggest the potential of these, and future, sequence models to enable high-resolution studies of replication in centromeres and other heterochromatic regions.Centromeres serve a critical function in preserving genome integrity across sequential cell divisions, by mediating symmetric chromosome segregation. The repetitive, heterochromatic nature of centromeres is thought to be inhibitory to DNA replication, but has also led to their underrepresentation in human reference genome assemblies. Consequently, centromeres have been excluded from genomic replication timing analyses, leaving their time of replication unresolved. However, the most recent human reference genome, hg38, included models of centromere sequences. To establish the experimental requirements for achieving replication timing profiles for centromeres, we sequenced G Saccharomyces cerevisiae, and to histone hypoacetylation [DNA replication during the S phase of the cell cycle initiates at replication origin loci, which are both spatially dispersed across the genome and asynchronously activated. The resultant spatiotemporal pattern of DNA replication timing is highly reproducible and largely conserved, producing consistent early- and late-replicating regions (reviewed in ). In gentylation  and disttylation  in mouseS. cerevisiae [Schizosaccharomyces pombe [Candida albicans [S. pombe, early centromeric replication has been explained by interactions of heterochromatin protein 1 (HP1) with the replication initiation factors CDC6 [Centromeres are an intriguing potential outlier to the late-replicating heterochromatin paradigm: centromeres replicate early in multiple yeast species, including revisiae , the fises pombe ,22, and albicans . This prors CDC6  and DDK ors CDC6 . Ablatioors CDC6 ,25, thusDrosophila Kc cell line [Drosophila larvae [The time at which centromeres replicate is less clear in higher eukaryotes: centromere replication in early S phase has been reported in the ell line , in mid-ell line , in latea larvae , and evea larvae . Howevera larvae ,31,32, ca larvae ,29,30,32Next-generation sequencing provides a high-resolution assay for replication timing at genome-scale ,33. HoweHere, we report that the centromere sequence models in hg38 enable measuring replication timing of human centromeres. We reveal their timing and variation in five cell lines, and detail the experimental conditions required to obtain this type of information. Our results demonstrate that high-throughput sequencing of human cell lines is both a feasible and a fruitful methodology to clarify a more detailed understanding of the human centromere and its time of replication during the S phase of the cell cycle.2 atmosphere.HEK293T and A2780 cells were cultured in Dulbecco’s modified Eagle medium  supplemented with 10% fetal bovine serum . GM12878, HCC1143, and HCC1954 were grown in Roswell Park Memorial Institute 1640 medium (Corning) supplemented with 15% FBS. All cell lines were obtained from the American Type Culture Collection or the Coriell Institute, and grown at 37 °C in a 5% CO1- and 1 million S-phase cells.Asynchronous populations of ~50 million cells were fixed in 70% ethanol, treated with RNase A (10 mg/mL) for 30 min at 37 °C, and stained with propidium iodide (1 mg/mL) in the dark for 30 min at room temperature. Stained cells were flow-sorted on a FACSAria II  to isolate 1 million GDNA was isolated using the MasterPure™ DNA Purification Kit  and libraries were prepared with the TruSeq DNA PCR-Free Library Prep Kit . Paired-end sequencing was performed for 75 cycles with the Illumina NextSeq 500  or for 150 cycles with the Illumina HiSeq X Ten .Sequence reads were aligned to the human reference genome hg38 using the Burrows–Wheeler Aligner maximal exact matches (BWA-MEM) algorithm (bwa v0.7.13). For HEK293T and A2780, quality-filtered reads were combined from two independent genomic libraries (HEK293T) or two independent sequencing runs (A2780) to enhance read depth. Centromere coordinates were obtained from the UCSC Genome Browser , genome build GRCh38/hg38. To account for repetitive sequences that might be represented as single copies in the reference genome (thus inflating estimates of copy number), reads were binned in 100 Kb windows, and the 99th percentile of windows with the highest read coverage were excluded. Similarly, regions with low mappability were filtered by binning reads in 500 Kb windows and excluding the bottom 0.5% lowest coverage windows. Ploidy was estimated using GenomeSTRiP .1-phase cells were used to define sliding chromosome windows of equal read depth (200 reads), which were then used to bin the reads from the S-phase cells. Outlier read depth values were filtered using a piecewise segmentation model . Contiguously mapped segments between gaps in the reference genome were smoothed with a cubic smoothing spline . Data were then normalized to an autosomal mean of 0 and standard deviation of 1.Replication timing profiles were generated as in . Brieflyhttp://amnonkoren.com/data.Sequence data reported in this study have been submitted to the Sequence Read Archive (SRA) under accession number PRJNA419407. Smoothed replication timing profiles are available at 1 (pre-replicative) and S (replicative) phases of the cell cycle. Pairs of G1- and S-phase fractions were sequenced and aligned to hg38.To assess the feasibility of studying centromere replication by whole-genome sequencing, we generated replication timing profiles for five human cell lines: an apparently healthy lymphoblastoid cell line , an emb1-phase fraction  per megabase to be included in our analysis. For A2780, 13 centromeres were successfully profiled, while 17–18 centromeres passed this threshold in the other four cell lines (We next focused our attention on the centromeres, requiring that any centromere contain on average at least 10 Gll lines A. The idll lines B.The smaller number of successful centromere profiles in A2780 prompted us to consider the effect of sequencing read depth on the ability to infer replication timing profiles: this cell line was sequenced to approximately half the coverage of the others (~80 million filtered read pairs vs. ~140–165 million in the others; Strikingly, while total read depth was an important factor in obtaining sufficient usable centromere sequence reads, paired-end sequencing proved to be even more crucial for the success of our approach. We re-aligned the sequencing data, considering only the first read of each read pair, and found that there was a negligible difference genome-wide in the number of reads passing quality filtering when using single-end sequencing C,D. WithGiven the ability to measure centromeric replication timing, we next compared these profiles among the five cell lines. We first noted a relatively large variability in centromere replication timing among cell lines. The centromere of a given chromosome replicated earlier than the genome average in some cell lines, but later than the genome average in other cell lines . While cWe next analyzed the pattern and timing of centromere replication by aggregating centromeres across all chromosomes within each cell line. Although the centromeres of individual chromosomes did not display consistent replication timing across cell lines, centromeres within a given cell line were notably similar, particularly in the cell lines with later average centromere replication . This trWe also found that the pericentromeric regions replicated progressively later towards the centromeres, indicating that centromeres are embedded within a local area of relatively late replication . This waHow and when human centromeres replicate remains an open question. While most studies in mammalian systems have concluded that centromeres replicate in mid-to-late S phase ,31,32, sUsing the latest human reference genome, hg38, we find that centromeres replicate in mid-to-late S phase, while the neighboring heterochromatin replicates markedly later, in agreement with previous reports that the centromeres replicate earlier than their surroundings ,29,32. IWe measure DNA replication timing without cell synchronization or fractioning S phase, capturing S phase as a continuous process. In addition, this is the first study to our knowledge to assay DNA replication timing of mammalian centromeres without nucleotide analogue incorporation. These methodological advancements enable us to generate high-resolution replication timing data for centromeres in the context of the whole genome. Furthermore, we are able to assay almost all of the centromeres in a chromosome-specific way, rather than using antibodies against centromeric histones , centromThe apparently early replication timing of the centromeres relative to the surrounding pericentromere is compatible with evidence from previous reports that centromeres contain active replication origins. Molecular combing has suggested that replication initiation sites are observed at the same density in centromeric regions compared to other genomic regions, and that α-satellite monomers bind the origin recognition complex in vitro—both of which imply the existence of active replication origins within centromeres . These oWe demonstrate here for the first time that human centromeric replication timing can be inferred by high-throughput sequencing, and establish the technical requirements and the importance of paired-end sequencing for assaying centromere replication. As newer linear centromere reference sequences become available , this ap


The sonic Hedgehog/GLI signaling pathway (HH) is critical for maintaining tissue polarity in development and contributes to tumor stemness. Transcription factors GLI1–3 are the downstream effectors of HH and activate oncogenic targets. To explore the completeness of the expression of HH components in tumor cells, we performed a screen for all HH proteins in a wide spectrum of 56 tumor cell lines of various origin using Western blot analysis. Generally, all HH proteins were expressed. Important factors GLI1 and GLI2 were always expressed, only exceptionally one of them was lowered, suggesting the functionality of HH in all tumors tested. We determined the effect of a GLI inhibitor GANT61 on proliferation in 16 chosen cell lines. More than half of tumor cells were sensitive to GANT61 to various extents. GANT61 killed the sensitive cells through apoptosis. The inhibition of reporter activity containing 12xGLI consensus sites by GANT61 and cyclopamine roughly correlated with cell proliferation influenced by GANT61. Our results recognize the sensitivity of tumor cell types to GANT61 in cell culture and support a critical role for GLI factors in tumor progression through restraining apoptosis. The use of GANT61 in combined targeted therapy of sensitive tumors, such as melanomas, seems to be immensely helpful. The Hedgehog (HH) signaling pathway is a morphogenesis pathway crucial for the growth and patterning of various tissues during embryonic development ,2. The mGlobal significance of the HH pathway for tumor initiation, progression, and metastasis is documented by additional literature. Mounting evidence indicates that HH signaling is required for the maintenance of glioblastoma and its CSC population ,41. GLI2Several studies have implicated a noncanonical activation of the HH route in tumors, thus abrogating the necessity of upstream ligand signaling. Through this mechanism, GLI factors can be activated directly by many different mechanisms upregulated in tumor cells, predominantly operating in RAS/MAPK, Wnt, or AKT pathways ,55,56,57We were interested in studying whether constituents of the HH pathway are invariably present in several tumor cell types or if some components are missing. It would potentially disable the activation of HH pathway in human cancer cell lines. A large screen has been performed and Western blots have shown complete expression of the main HH components in all tumor cells . NoteworVery peculiar was a varying expression of the ligand sonic Hedgehog among the cell lines, irrespective of the tumor type. This nevertheless does not preclude the efficient functioning of the HH pathway, since, in view of the fact that HH is frequently activated noncanonically at the GLI factors level, the production of the ligand itself (acting by an autocrine or paracrine manner in cell lines) is dispensable. Three cell lines were nontransformed and tested for comparison with tumor cells . HeMN-LP (melanocytes) expressed both GLI2 and GLI1, but the two fibroblast cell lines expressed very low GLI2, but retained their GLI1 level. Expression of other components was retained in these normal cell lines. Survivin was present in all tumor cell lines. Our previous results have shown that in IMR90 cells, transfected GLI2 plasmid is capable of evoking the expression of endogenous survivin [We next tested the sensitivity to a GLI inhibitor GANT61 in a panel of 16 tumor cell lines . The tumExpectedly, melanomas were sensitive to GANT61 . We haveTo gain insight into the mechanism underlying the eradication of cells in proliferation assays, we carried out the TUNEL assay that detects apoptosis. Many previous papers indicate that GANT61 kills the cells through apoptosis ,49,57,61To study whether a GLI-responsive promoter-reporter is also affected by HH inhibitors GANT61 and cyclopamine, 12xGLI-luciferase reporter and a reference plasmid were cotransfected in several cell lines that were either variably responsive or nonresponsive to GANT61 in proliferation assays. As shown in The HH signaling pathway, acting through transcription factors GLI1, GLI2, and GLI3, has been identified as critical for the initiation and progression of a number of cancers. Originally, it was believed to be important for only basal cell carcinoma (BCC) and meduloblastoma. Gradually, the pathway becomes a crucial signaling pathway for all frequent cancer types with the GLI family transcription factors being essential in tumor initiation, progression, EMT, CSC, and metastasis, dependent on the tumor cell context. HH signaling is a network rather than as a simple linear pathway because of its cooperation with many other cell signaling pathways and its frequent noncanonical activation. GLI factors have several oncogenic targets . RecentlHere, we have first analyzed the expression of HH cascade components across a panel of 56 tumor types using Western blot analysis. It was found that they are generally expressed . Importantly, either GLI1 or GLI2 were always present in all samples. In three normal control cell lines, the HH proteins were also present. HH signaling is emerging to be essential for the progression of nearly all tumors ,13. The Our results suggest which type of cancer is resistant or sensitive to GANT61 when it is applied directly on cells in culture . Malignal-glutamine, streptomycin, and penicillin . Some cells were cultured in EMEM medium supplemented also with essential amino acids and pyruvate (Sigma). Fresh media were replaced every third day. HH inhibitors GANT61 or cyclopamine were present in media as indicated in Figures and Figure legends. All melanoma cell lines were maintained in RPMI1640 medium with the exception of lines WM35 and WM1552C that were kept in DMEM. NSCLC and SCLC cell lines cells were grown in RPMI1640 medium with the exception of Calu-1 (DMEM). SK-N-SH, SH-N-MC, HT-1080, and T98G cells were maintained in EMEM. The remaining cell lines were grown in DMEM medium.Cells were maintained in appropriate media (DMEM or RPMI1640) supplemented with 10% fetal calf serum , All cell lines were of human origin. Melanoma cell lines DOR, Beu, and Hbl were previously described . Other mCommercially available primary antibodies used were as follows: Sonic Hedgehog, Cell Signaling Technology #2207 ; Patched, Biorbyt #157169 ; SMO, #ab72130 ; SuFu, Cell Signaling #2520; Gli, Abcam #ab134906; Gli2, #sc-271786 ; Gli3, Biorbyt #157158; Survivin, Santa Cruz #sc-17779; BCL-2, BD Pharmingen #556354 ; β-actin, Sigma #A5316. HRP-labelled second antibodies were from Cell Signaling.For Western blot analysis, cells were lysed in RIPA buffer , supplemented with aprotinin, leupeptin, pepstatin (Sigma), COMPLETE, and PhoStop . Total lysates containing 30 μg of protein were separated on SDS-PAGE gels and subsequently transferred onto a PVDF membrane . Membranes were then subjected to probing with antibodies. Western blot signals were detected by using SuperSignal West Pico Chemiluminescent substrate  and exposed on films.To perform proliferation assays, colony outgrowth assays were carried out. Cells were trypsinized and seeded in about 40–50% confluency on 12-well plates (day 0). The next day (day 1), cell lines were treated with 10 μM GANT61 or 20 μM GANT61 , for a maximum of 9 days. The medium was refreshed every third day. The plates were then fixed in 3% paraformaldehyde solution in 1× PBS and stained with 1% crystal violet and photodocumented. Two most important fields  were quantitated using ImageJ software. Two experiments were performed in duplicate. Results of both experiments were similar.A TUNEL  assay was performed according to the manufacturer’s instructions . Results of FITC staining were analyzed on a flow cytometer BriCyte EA . A total of 50,000 cells were analyzed in each sample. The number of apoptotic cells was determined using ImageJ software. Immunofluorescence assays were performed as described previously . BrieflyRenilla luciferase reporter plasmid (as a reference for transfection efficiency) on the 12-well plates in triplicates, the inhibitors GANT61 and cyclopamine were added at concentrations indicated in Luciferase Reporter Gene Assay: Luciferase reporter plasmid with luciferase gene under the transcriptional control of 12xGLI full consensus was obtained from Prof. R. Toftgard . After transfection of the plasmid (1 μg), together with the p values are listed in the corresponding figure legend. In all figures the error bars represent mean + SE. Proliferation assays and TUNEL assay were quantified by ImageJ software .To calculate the statistical significance of the reporter assays, a two-tailed Student test was used. The


Cancer is one of the most frequently occurring diseases and is the second leading cause of death worldwide. In this study, anthraquinone derivatives (Compounds 1–5) were evaluated for their anti-cancer potential against various skin and breast cancer cell lines to assess whether these anthraquinone derivatives may serve as a lead for the augmentation of anti-cancer drug.Rubia philippinensis., whereas ethyl acetate fraction yielded xanthopurpurin (Comp 4) and lucidin-ω-methyl ether (Comp 5). Structures of all the isolated compounds were determined by spectral data analysis. All isolated compounds (Comp 1–5) were assessed for cytotoxicity by the 3--2,5-diphenyltetrazolium bromide (MTT) assay against four different cancer cell lines, i.e. human melanoma (SK-MEL-5), murine melanoma (B16F10), and human breast adenocarcinoma (MCF7 and MDA-MB-231).Anthraquinone derivatives, 2-methyl-1,3,6-trihydroxy-9,10-anthraquinone-3-O-(6′-O-acetyl)-α-rhamnosyl(1 → 2)-β-glucoside (Comp 1), 2-methyl-1,3,6-trihydroxy-9,10-anthraquinone (Comp 2), and alizarin (Comp 3) were isolated from the dichloromethane fraction of the roots of 50 values of 14.65 ± 1.45 and 13.03 ± 0.33 μM, respectively. Encouragingly, IC50 values of 67.89 ± 1.02 and 79.01 ± 0.03 μM against normal kidney epithelial cells (MDCK) were also obtained for compounds 4 and 5, respectively, which indicated very low toxicity and favorable selectivity indices for compounds 4 and 5 in the range of 1.85 to 3.95 and 2.11 to 6.06 against skin cancer cell lines , and breast cancer cell lines (MCF7 and MDA-MB-231), respectively.Significant activity of the compounds 4 and 5 was observed against the breast cancer cell line MDA-MB-231 with ICOur results suggested that the compounds 4 (xanthopurpurin) and 5 (lucidin-ω-methyl ether) showed high selective toxicity towards breast cancer cells at lower concentrations without showing toxicity towards normal cells, thus could be of potential as new lead molecules in cancer treatment.The online version of this article (10.1186/s12906-018-2253-2) contains supplementary material, which is available to authorized users. Soymida fembrifuga (Miliaceae), Tinospora cordifolia (Menispermaceae), Lavandula bipinnata (Lamiaceae), Helicteres isora (Sterculiaceae), Urtica membranacea (Urticaceae), Artemesia monosperma (Asteraceae), and Origanum dayi post (Labiatae) etc., are the source of alternative medicine for cancer therapy in various regions of the globe ; and δH 2.05 , respectively in 1H NMR spectrum. On the other hand, the skeleton of 14 carbon signals along with two ketonic carbonyls  and one methyl functionality (δC 8.1) was representative of 13C NMR data of an anthraquinone. The 1D NMR of compound 2 resemble closely to those of compound 1, except for the absence of signals belonging to sugar units. In comparison with reference values, compound 2 was determined as 2-methyl-1,3,6-trihydroxy-9,10-anthraquinone. Compound 3, 4, and 5 are also anthraquinone derivatives and their structures were elucidated as alizarin, xanthopurpurin, and lucidin-ω-methyl ether, respectively, based on the NMR data analysis. NMR data of all anthraquinone has been provided in Additional file Similar to compound 1, compound 2 also showed resonances of one aromatic singlet proton, one ABX ring system, and one singlet methyl at δ50 values for cancer cell lines treated ranged from 48.68 ± 0.10 to 91.04 ± 1.88 μM for compound 1; 46.75 ± 1.39 to 79.96 ± 1.14 μM for compound 2; 48.64 ± 0.33 to 98.79 ± 2.10 μM for compound 3, 14.65 ± 1.45 to 23.71 ± 1.71 μM for compound 4, and 13.03 ± 0.33 to 42.79 ± 1.32 μM for compound 5. Regarding the normal cell line MDCK cells, the IC50 values were 192.34 ± 0.49, 168.76 ± 0.61, 199.32 ± 1.88, 67.89 ± 1.02 and 79.01 ± 0.03 μM for compounds 1, 2, 3, 4, and 5, respectively. Interestingly, among all the compounds, compounds 4 and 5 showed strong cytotoxicity towards breast cancer cells (MCF7 and MDA-MB-231) than skin cancer cells (SK-MEL-5 and B16F10) with IC50 value of 15.75 ± 1.00 and 24.10 ± 1.06 for MCF7 as well as 14.65 ± 1.45 and 13.03 ± 0.33 for MDA-MB-231, respectively.All compounds were tested for cytotoxicity by MTT assay on cell lines SK-MEL-5, B16F10, MCF7, MDA-MB-231, and MDCK cells as a normal cell line, which showed significant cytotoxicity  than to normal cells -α-rhamnosyl(1 → 2)-β-glucoside (compound 1), 2-methyl-1,3,6-trihydroxy-9,10-anthraquinone (compound 2), alizarin (compound 3), xanthopurpurin (compound 4), and lucidin-ω-methyl ether (compound 5) were isolated from the root of R. philippinensis., and were characterized based on the spectral data analysis [A number of natural compounds have been isolated from different plant sources which have shown enormous biological potential –19. In tanalysis –19.50 values lower than 30 μM against experimental cancer cell lines constitute promising anticancer agents for drug development [50 values greater than 30 μM against all cell lines tested, and were more cytotoxic to normal line to which the cancer cell lines. Moreover, among the testest compounds, anthraquinone derivatives xanthopurpurin (compound 4), and lucidin-ω-methyl ether (compound 5) showed highest selectivity indices in breast cancer cell than skin cancer cells.These anthraquinone derivatives showed significant anticancer potential as confirmed by their cytotoxicity effects against various cancer cell lines, such as cell lines SK-MEL-5, B16F10, MCF7, MDA-MB-231, including normal MDCK cell line. However, according to American National Center Institute, extract/compounds with ICelopment . TherefoMounting evidences have considered that a value greater than or equals to 2.0 is an interesting selectivity index . This vaRubia philippinensis. The results of in vitro studies demonstrate the ability of the compounds 4 (xanthopurpurin) and 5 (lucidin-ω-methyl ether) for high selective toxicity at lower concentrations  against MDCK, SK-MEL-5, B16F10, MCF7, and MDA-MB-231 cell lines. (DOC 899 kb)


In many cancers, somatic mutations confer tumorigenesis and drug-resistance. The recently established clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is a potentially elegant approach to functionally evaluate mutations in cancers. To reproduce mutations by homologous recombination (HR), the HR pathway must be functional, but DNA damage repair is frequently impaired in cancers. Imatinib is a tyrosine kinase inhibitor for BCR-ABL1 in Philadelphia chromosome-positive (Ph+) leukemia, and development of resistance due to kinase domain mutation is an important issue. We attempted to introduce the T315I gatekeeper mutation into three Ph+ myeloid leukemia cell lines with a seemingly functional HR pathway due to resistance to the inhibitor for poly (ADP) ribose polymerase1. Imatinib-resistant sublines were efficiently developed by the CRISPR/Cas9 system after short-term selection with imatinib; resulting sublines acquired the T315I mutation after HR. Thus, the usefulness of CRISPR/Cas9 system for functional analysis of somatic mutations in cancers was demonstrated. Imatinib can achieve durable cytogenetic and molecular remissions not only in CML patient3 but also in patients with Ph+ ALL in combination with conventional chemotherapy5. Despite the remarkable success of imatinib, resistance has been identified due to point mutations in the BCR-ABL1 kinase domain7. Among these mutations, the T315I gatekeeper mutation confers resistance to both imatinib8 and second-generation TKIs such as nilotinib and dasatinib9. Finally, ponatinib was developed as a potent TKI that can inhibit all critical kinase domain mutations including T315I10.Imatinib is a tyrosine kinase inhibitor (TKI) against BCR-ABL1 fusion tyrosine kinase derived from Philadelphia chromosome in chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL)BCR-ABL1 or its mutant cDNAs that were transduced with retrovirus vector13. BCR-ABL1 and its mutants induced spontaneous cell growth of Baf3 in the absence of IL-3. The other commonly used system was imatinib-resistant sublines of human Ph+ leukemia cell lines. A couple of imatinib-resistant sublines with T315I mutation were established after long-term culture of imatinib-sensitive Ph+ leukemic cell lines in the presence of increasing concentrations of imatinib17. However, it has also been reported that long-term culture with increasing concentrations of imatinib induced imatinib resistance due to amplification of the BCR-ABL1 fusion gene and overexpression of P-glycoprotein (P-gp)19. This suggests that imatinib-resistant sublines with T315I (established after long-term selection with imatinib) may acquire additional mechanisms for imatinib resistance. Thus, to directly test the effect of the T315I mutation, establishing a new system that enables the T315I mutation to be introduced into imatinib-sensitive Ph+ leukemia cell lines without long-term imatinib selection is desirable.To investigate the biological significance of T315I mutation and to develop the therapeutic strategy overcoming TKI-resistance, a line of cellular models of T315I-positive leukemia was established. The most common system was murine IL-3-dependent Baf3 cells expressing 22. CRISPR/Cas9 effectively introduces target double-stranded brakes (DSBs) by recognizing a NGG 3-base-pair protospacer adjacent motif (PAM) and causing hybridization between the 20-nucleotide stretch of the sgRNA and the DNA target site, which triggers Cas9 to cleave both DNA strands. DSBs activate two intrinsic repair mechanisms: non-homologous end-joining (NHEJ) and homologous recombination (HR). NHEJ (the predominant pathway for repair of DSBs) can introduce unpredictable insertions and deletions (indels) resulting in knockout alleles through the introduction of frame-shift mutations. HR is achieved in the presence of a single-stranded oligodeoxynucleotides (ssODN) template homologous to the sequences flanking the cleavage site. HR using the CRISPR/Cas9 system could be useful for introducing the T315I mutation into human Ph+ leukemia cell lines; however, to our knowledge, no reports have described success in purely introducing the point mutation of endogenous gene into human leukemia cells by HR using the CRISPR/Cas9 system.The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system consists of a Cas9 endonuclease and a single-guide RNA (sgRNA) that allows sequence-specific gene editing in mammalian cells23. This seems to be true in Ph+ leukemia cells24. Although inactivating mutations in the HR pathway has been rare in leukemia25, BCR-ABL1 reportedly represses genes involved in the HR pathway such as BRCA1 and RAD5128. These observations suggest that DSBs by the CRISPR/Cas9 system might fail to induce HR-mediated gene editing in Ph+ leukemia cells. Poly (ADP) ribose polymerase1 (PARP1) repairs DNA single-stranded breaks (SSBs). Inhibitors for PARP1 have recently been identified and are promising agents for cancer treatment32. PARP1 inhibitors generate DSBs during the replication process by trapping the inactivated PARP1 onto the SSB and/or disrupting the DNA repairment. Subsequently, PARP1 inhibitors induce apoptosis of cancer cells that have deficiency in key components of the HR pathway, which utilizes the sister chromatid as a template for a correct repairment of the DNA sequence. In contrast, cancer cells with intact HR pathways repair DSBs and show resistance to PARP1 inhibitors. Thus, sensitivities of cancer cells to PARP1 inhibitors might be useful to evaluate their status of the HR pathway.To introduce HR-mediated gene editing with the CRISPR/Cas9 system in leukemia cells, the intrinsic HR pathway of leukemia cells must be functionally active. Most cancer cells demonstrate increased genomic instability due to impairment in repair pathways for DNA damageBCR-ABL1 as a result of HR-mediated gene editing.In the present study, using the CRISPR/Cas9 system and ssODN, we attempted to introduce the T315I mutation into three Ph+ myeloid leukemia cell lines that showed resistance to olaparib (a PARP1 inhibitor). After short-term imatinib selection, we obtained imatinib-resistant sublines and confirmed that imatinib-resistant sublines successfully acquired T315I mutation of BRCA1 and RAD5128. To select Ph+ leukemia cell lines with functional HR pathway, we evaluated the sensitivity to olaparib (one of PARP1 inhibitors)32. We determined the IC50 values of olaparib in 20 Ph+ (16 lymphoid and 4 myeloid) leukemia cell lines in comparison with 77 Ph-negative (Ph−) B-cell precursor ALL (BCP-ALL) cell lines using the alamarBlue cell viability assay , suggesting a high on-target activity. Two Cas9 cleavage sites were separated from the target site by 12 and 10 bases in forward and reverse sgRNAs, respectively. As a template for HR, two complementary ssODNs of 102 bases in length were designed ion Fig. , cells wion Fig. , while nuct Fig. . PCR prouct Fig.  confirme34 treatment in HR efficiency. We electroporated recombinant Cas9 protein in combination with reverse sgRNA and anti-sense ssODN  into K562 cells with or without SCR7 treatment. We also electroporated recombinant Cas9 protein in combination with reverse sgRNA and carrier ssODN (which is not template for HR but enables to induce similar transfection efficiency) into K562 cells treated with SCR7. Electroporated cells were cultured in the same way as shown in Fig. Next, we verified the significance of SCR7Next, we introduced the T315I mutation into two other Ph+ myeloid cell lines (TCCS and KOPM28) resistant to olaparib Fig.  in both BCR-ABL1 fusion gene18 and overexpression of P-gp19 were observed in the imatinib-resistant sublines established after long-term culture of imatinib-sensitive Ph+ leukemia cell lines in the presence of increasing concentrations of imatinib. Thus, T315I sublines of three cell lines may acquire these additional mechanisms for imatinib-resistance during imatinib selection. To rule out these possibilities, we evaluated BCR-ABL1 protein expression level in parental cells and imatinib-resistant sublines of three cell lines by Western blotting try Fig. . In the try Fig.  and apoptry Fig.  by imatitry Fig. . T315I-a50 values were approximately 100 nM, which was lower than mean peak concentration (137 nM) of orally administrated ponatinib (45 mg daily) at steady state38. Parental cells of TCCS and KOPM28 were highly sensitive to ponatinib and their IC50 values were less than 0.1 nM. T315I sublines of TCCS and KOPM28 were significantly more resistant to ponatinib than their parental cells; their IC50 values were approximately 0.2 nM and 20 nM, respectively. We also analyzed the sensitivities to chemotherapeutic agents, AraC and DNR  to functionally evaluate the status of the HR pathway. We found that all four Ph+ myeloid leukemia cell lines were highly resistant, suggesting that their HR pathway is functionally not impaired. Indeed, we successfully established imatinib-resistant sublines of three Ph+ myeloid cell lines—K562, TCCS, and KOPM28—which acquired the T315I mutation as a result of HR-mediated gene editing. We previously tried to introduce specific gene mutations of our interested genes in four Ph− ALL cell lines with the same strategy, but we failed to obtain sublines with the desired mutation. Of note, in the present study, all of the four cell lines that we used in the previous attempts turned out to be sensitive to PARP-1 inhibitor, suggesting that their HR pathway was functionally impaired. Thus, although further verifications are required, sensitivity of cancer cell lines to PARP1 inhibitor may be a useful biomarker to predict efficiency of HR-mediated gene editing with the CRISPR/Cas9 system.In the present study, we tried to introduce the T315I mutation of 17, imatinib-resistant sublines with T315I were established from Ph+ leukemia cell lines after 5–10 months’ culturing in the presence of increasing concentrations of imatinib. Long-term culture in the presence of increasing concentrations of imatinib also induced enhancement of BCR-ABL1 expression18 and/or upregulation of cell surface expression of P-gp19 as mechanisms for imatinib-resistance, suggesting that T315I-positive sublines of Ph+ leukemia cell lines may acquire additional mechanism(s) for imatinib-resistance during imatinib selection. Considering these previous reports, to allow an expansion of T315I integrated cells and prevent a spontaneous emergence of imatinib-resistant sublines, we initially cultured cells for 7 days in the absence of imatinib after electroporation. Furthermore, we used 1 μM of imatinib for selection to eliminate emergence of clones that are moderately resistant to imatinib due to mechanism(s) other than an acquisition of T315I mutation. As a result, we successfully expanded T315I acquired sublines of three myeloid Ph+ leukemia cell lines after short-term (7 to 26 days) imatinib selection. Enhancement of BCR-ABL1 expression and upregulation of cell surface expression of P-gp were not induced in imatinib-resistant sublines of three cell lines.To select successfully integrated cells, we used imatinib. In the previous reports39, distance between mutation site and cleavage site39, length of homology arms41, and strand complementarity or orientation of ssODN42. In the present study, all four combinations produced identical distance between mutation site and cleavage site and introduced three silent mutations to block re-cutting of repaired loci. The highest efficiency was observed in the combination of reverse sgRNA and anti-sense ssODN, which was complementary to the transcribed strand and contained the NGG PAM sequence. This was inconsistent with previous reports using different delivery and modality of CRISPR/Cas9 system, since higher efficiency was reported to be obtained by ssODN that was complementary to the non-transcribed strand42 and by ssODN that was complementary to the PAM strand41. Thus, delivery and modality of CRISPR/Cas9 system might affect an optimal combination of sgRNA and ssODN.We prepared forward and reverse sgRNAs in combination with sense and anti-sense ssODNs. Efficiency of HR-mediated gene editing is reported to be affected by structure and composition of sgRNA and ssODN such as incorporation of silent mutation to block re-cutting of repaired lociBCR-ABL1 allele but not the ABL1 allele. We confirmed that all of the imatinib-resistant sublines of two cell lines had both native and recombination alleles of BCR-ABL1, suggesting that these two cell lines may serve as good models of acquired T315I mutation in BCR-ABL1. In contrast to TCCS and KOPM28, K562 has both BCR-ABL1 and ABL1 alleles, and its ABL1 allele also acquired the T315I mutation. Although it is unlikely that the induced ABL1 T315I mutation contributes to imatinib resistance, this may be a confounding issue limiting the use of the K562 subclones for drug testing. Removal of the ABL1 allele by a CRISPR/Cas9-induced deletion in K562 may resolve this problem.TCCS and KOPM28 have the 10. The IC50 value of ponatinib in the kinase assay in vitro was reported to be 0.37 and 2 nM for native ABL1 and T315I mutant, respectively. In Baf3 transfectants, the IC50 value of ponatinib was reported to be 0.5 and 11 nM for native BCR-ABL1 and T315I mutant, respectively10. In the present study, T315I sublines of TCCS and KOPM28 were sensitive to ponatinib, and their IC50 values  were significantly higher than those in parental cells (less than 0.1 nM). In K562, the sensitivities of parental cells and T315I sublines were identical. These observations demonstrated that parental cells and T315I sublines of three cell lines are useful models for the analysis of TKI sensitivities.T315I sublines from the three cell lines showed a marked resistance to dasatinib and nilotinib, as well as imatinib in comparison with their parental cells. Although ponatinib was developed as a potent TKI that can inhibit all critical kinase domain mutations including T315I, its activity against the T315I mutant was reported to be modestly decreased in comparison with the activity against native ABL1 and BCR-ABL1 as followset al.43 recently introduced an oncogenic mutation (R140Q) of the IDH2 gene into K562 cells successfully using the CRISPR/Cas9 system. Since R140Q is located in exon 4 of the IDH2 gene, they inserted exons 4–11 with R140Q as a partial cDNA into exon 4 of the IDH2 gene locus via HR. Thus, the endogenous IDH2 gene promoter regulated the integrated IDH2 R140Q mutation, but the insertion lacks introns and 3′-untranslated regions (UTRs) in the target gene. Recently, it has been reported that introns44 and 3′UTRs46 are involved in transcriptional and post-transcriptional regulation of gene expression. Indeed, 3′UTR of the BCR-ABL1 transcripts were reported to be target of microRNA-203 (miR-203)47, miR-42448 and miR-30e49. In the present study, the introduction of a precise mutation into the BCR-ABL1 gene by harnessing the cells’ HR repair pathway enabled preservation of other regulatory regions in the gene and minimized off-target effects relative to the more disruptive changes induced by NHEJ driven repair.Brabetz BCR. KOPN57bi, KOPN66bi, KOPN72bi, KOPN83bi, YAMN91, Kasumi8, KCB1, SU-Ph2, and SK9 were established from Ph+ ALL with the p190 BCR-ABL1. Seventy-seven Ph- BCP-ALL cell lines listed in Supplemental Table Twenty Ph-positive (Ph+) leukemia cell lines (16 lymphoid and four myeloid) listed in Table 5 cells were plated into a 96-well flat-bottom plate in triplicate and cultured for 68 hours in the presence or absence of seven concentrations of each drug. After a 6 hour additional incubation with alamarBlue, absorbance at 570 nm were monitored by a microplate spectrophotometer using 600 nm as a reference wavelength. Cell survival was calculated by expressing the ratio of the optical density of treated wells to that of untreated wells as a percentage. The concentration of drug required to reduce the viability of treated cells to 50% of untreated cells was calculated, and the median of three independent assays was determined as 50% inhibitory concentration (IC50) for each cell line.To determine the sensitivities to olaparib, imatinib, dasatinib, nilotinib, ponatinib, cytosine arabinoside (AraC), and daunorubicin (DNR), an alamarBlue cell viability assay  was performed. A total of 1–5 × 10http://crispr.mit.edu) as follow; 5′-atcactgagttcatgacctacgg-3′ (forward sgRNA) and 5′-aactcagtgatgatatagaacgg-3′ (reverse sgRNA). 102 base pair template ssODN in a transcribed strand (sense) direction and its complementary template ssODN in a non-transcribed strand (anti-sense) direction were synthesized by Integrated DNA technologies . The sequence of ssODN in the sense direction was as follows; 5′-gggtctgcacccgggagcccccgttctatatcatTaTCgaAttcatgacctacgggaacctcctggactacctgagggagtgcaaccggcaggaggtgaacg-3′. Four mutated nucleotides are capitalized. As a negative control, we used carrier ssODN (Integrated DNA technologies). Recombinant Cas9 nuclease with guide RNA (Integrated DNA technologies) was electroporated into cell lines as a ribonucleoprotein (RNP) complex with template ssODN following manufacturer’s protocol50. Briefly, RNP complex and template ssODN were electroporated into 1 × 105 cells, which were untreated or pretreated with 10 nM of SCR734  for 24 hours before electroporation, using Neon electroporation transfection system (Thermo Fisher Scientific). The electroporated cells were cultured in the absence or presence of 10 nM of SCR7 for 24 hours in one well of 96 well plate. Forty-eight hours after electroporation, the electroporated cells were mixed with untreated cells and plated into 10–12 wells of 24-well plate. After 5 days expansion in the absence of imatinib, the cells were cultured in the presence of 1 μM of imatinib. During imatinib selection, half of the culture medium in each well was exchanged every 2–3 day with fresh medium containing 1 μM of imatinib. When the imatinb-resistant cells were selectively expanded, imatinb-resistant sublines in each well were transferred to culture flask and expanded in the absence of imatinib for further experiments.Two CRISPR guide sequences were designed using the CRISPR design tool  and reverse (5′-cctaggctggggctttttgta-3′) primers that were specific for introns 5 and 6 of the ABL1 gene, respectively. Each PCR product digested with EcoRI was electroporated. Direct sequencing of each PCR product was performed using the forward primer. To detect ABL1 transcripts, cDNA prepared from parental and imatinib-resistant sublines was amplified by RT-PCR using forward primers in exons 1a (5′-gtgggctgcaaatccaagaag-3′) or 1b (5′-aggaatcatcgaggcatggg-3′) and reverse primer in exon 2 (5′-gtccagcgagaaggttttcc-3′). As a control, RT-PCR was also performed using forward primer in exons 6 (5′-ctgctgtacatggccactca-3′) and reverse primer in exon 7 (5′-ctctcgggtgcagtccattt-3′). To distinctively sequence the ABL1 and the BCR-ABL1 transcripts, RT-PCR was performed using forward primers in exon 1b of the ABL1 gene (5′-ccacactgcaatgtttttgtgg-3′) or exon 13 of the BCR gene (5′-agcattccgctgaccatcaa-3′) and reverse primer in exon 6 of the ABL1 gene (5′-tctcccctaccaggcagttt-3′), respectively, and direct sequencing of the RT-PCR products was performed using the reverse primer.To confirm HR in the To evaluate cell surface expression of P-glycoprotein, cells were stained with a fluorescein isothiocyanate (FITC)-conjugated anti-P-glycoprotein antibody  and analyzed by flow cytometry . To analyze phosphorylation status of CRKL, cell cycle, and apoptotic cell death, cells were cultured in the presence or absence of 1 μM of imatinib for 24 hours except for detection of apoptosis in K562 cells (60 hours incubation). To evaluate phosphorylation status of CRKL, the cells were fixed with 10% formaldehyde at 37 °C for 10 min and permeabilized with 90% methanol on ice for 30 min. After washing the cells twice with PBS/0.5% BSA, the cells were stained with anti-phospho CRKL (pTyr207)  and subsequently with Alexa Fluor 488-conjugated goat anti-rabbit IgG (H + L)  and analyzed by flow cytometry. For cell cycle analysis, the cells fixed with 70% ethanol were stained with propidium iodide (PI)  and analyzed by flow cytometry. To detect apoptosis, the cells were stained with FITC-conjugated Annexin-V and 7AAD  and analyzed by flow cytometry.3, 1 mM phenylmethylsulfonyl fluoride, 100 μM sodium vanadate). The lysates were separated on a SDS-polyacrylamide gel under reducing conditions and then transferred to a nitrocellulose membrane. The membrane was incubated with anti-ABL1 antibody  and anti-alpha-Tubulin antibody  at 4 °C overnight, and then with horseradish peroxidase-labeled second antibody  at room temperature for 1 hour. The bands were developed using an enhanced chemiluminescence detection (ECL) kit .Cells were solubilized in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 5 mM EDTA, 0.05% NaNMann-Whitney test was applied for comparison of drug sensitivities, a paired t-test was for comparison in the same subject, and Kaplan-Meier analysis for comparison of efficiencies in the establishment of imatinib-resistant sublines.The authors declare that the data supporting the findings of this study are available within the paper and its supplementary information files.Supplementary Figure S1–7


Epidermal growth factor receptor (EGFR) overexpression is associated with the development of head and neck cancer (HNC) and represents one of the main therapeutic targets for this disease. The use of EGFR inhibitors has limited efficacy due to their primary and acquired resistance, partially because of increased epithelial to mesenchymal transition (EMT). The HDAC inhibitor SAHA has been shown to revert EMT in different tumours, including HNC. In this study, we investigated the cooperative role of SAHA and the EGFR tyrosine kinase inhibitor gefitinib in both HPV-positive and HPV-negative HNC cell lines.A panel of 12 HPV-positive and HPV-negative HNC cell lines were screened for cell viability upon treatment with SAHA, gefitinib and the combination of the two. Epithelial/mesenchymal marker expression, as well as activation of signalling pathway, were assessed upon SAHA treatment. ΔNp63α silencing with shRNA lentiviral particles was used to determine its role in cell proliferation, migration and TGFβ pathway activation.We found that both SAHA and gefitinib have antitumour activity in both HPV-positive and HPV-negative HNC cell lines and that their combination has a synergistic effect in inhibiting cell growth. SAHA treatment reverts EMT and inhibits the expression of the transcription factor ΔNp63α. Suppression of ΔNp63α reduces EGFR protein levels and decreases cell proliferation and TGFβ-dependent migration in both HPV-positive and HPV-negative HNC cell lines.Our results, by giving a clear molecular mechanism at the basis of the antitumour activity of SAHA in HNC cell lines, provide a rationale for the clinical evaluation of SAHA in combination with gefitinib in both HPV-positive and HPV-negative HNC patients. Further knowledge is key to devising additional lines of combinatorial treatment strategies for this disease. A subgroup of HNC, particularly those of the oropharynx, is caused by infection with high-risk types of human papillomavirus (HPV). Although in the past decade the incidence of HNC has been slowly declining in the western world, probably due to a decrease in the prevalence of smoking, oral tongue and particularly oropharyngeal cancers are becoming more prevalent. This may be related to an increase in oropharyngeal HPV infections and recent studies revealed that HPV16 in particular is involved in these tumours. HPV-positive tumours form a distinct group within HNC with different aetiological factors. These tumours are different at the molecular level, changing the clinical outcome and presenting a better prognosis.2 Despite their diversities, HPV-positive and HPV-negative HNC patients are treated using the same therapies, leading the scientific and medical community to reassess the current treatment protocols, in order to develop less toxic strategies while maintaining good oncological outcomes. Recently, the anti-PD-1 antibodies nivolumab and pembrolizumab have been approved as targeted therapies for the treatment of HNC,3 as well as cetuximab. Cetuximab, an anti-epidermal growth factor receptor (EGFR) monoclonal antibody, has been approved for the treatment of HNC in patients with locally advanced tumours in association with radiotherapy and in patients with recurrent or metastatic diseases in combination with cisplatin-based chemotherapy.5 Small-molecule tyrosine kinase inhibitors (TKIs) such as gefitinib, which prevent the binding of ATP to the receptor and thereby inactivate EGFR, have also been investigated for HNC in preclinical and clinical settings, showing tumour growth delay and enhancement of apoptosis4 and single-agent activity in recurrent and metastatic disease.6 Although overexpression of EGFR has been observed in about 90% of HNC specimens and correlates with poor prognosis, advanced disease and reduced survival, cetuximab treatment has yielded only modest clinical outcomes as a monotherapy.7 Taken together, this suggests that primary and acquired resistance mechanisms considerably limit the clinical benefit of cetuximab in HNC,8 which translates in a clear need to understand the molecular mechanisms driving cetuximab resistance to maximise the treatment response by patient selection and to establish new treatment options to overcome resistance.Head and neck cancer (HNC) includes malignant squamous lesions arising in the oral cavity, oropharynx, larynx or hypopharynx, and is the sixth leading cancer by incidence worldwide, with approximately 500,000 new cases annually and only 40–50% 5-year survival rate.9 Acquisition of EMT features has also been associated with chemoresistance acquired after standard chemotherapy.10 Activation of both transforming growth factor β (TGFβ) signalling and receptor tyrosine kinase plays critical roles in EMT either through the downstream SMAD signalling or through a SMAD-independent pathway, such as activation of ERK MAP kinases, Rho GTPases and the PI3 kinase/Akt pathway.11 Cancer cells can increase their production of active TGFβ during development of EGFR-TKI resistance.12 TGFβ triggers EMT and allows the cells to become invasive. EMT seems to also play a role in establishing the resistance to EGFR TKIs in HNC.13Epithelial to mesenchymal transition (EMT) is known to be deeply involved in cancer progression and metastasis. EMT is characterised by the loss of proteins involved in cell junctions such as E-cadherin, and the expression of mesenchymal markers such as vimentin.15 including HNC,16 whereas in other types of cancer the use of HDACi has been shown to increase EMT.19 HDACs are typically overexpressed in cancers, such as HNC,20 altering gene transcription and enhancing cell proliferation. To date, several HDACi have been developed and used in clinical trials for the treatment of cancers, and have been shown to induce differentiation, growth arrest or apoptosis in tumour cells.22 Currently, the US Food and Drug Administration has approved four types of HDACi for cancer therapy, including vorinostat 23 for the treatment of refractory cutaneous T-cell lymphoma (CTCL), romidepsin (Istodax)24 for the treatment of CTCL and peripheral-cell lymphoma (PTCL), belinostat (Beleodaq) for the treatment of PTCL25 and panobinostat (Farydak) for the treatment of multiple myeloma.27 Recently, HDACi have been shown to reduce ΔNp63α protein stability in two cutaneous squamous cell lines.28 The p63 isoform ΔNp63α, member of the p53 family, is a transcription factor essential for terminal differentiation of stratified epithelia.29 ΔNp63α is the predominant p63 isoform expressed in normal squamous epithelia and squamous carcinomas and its expression is upregulated in up to 80% of primary HNC tumours.30 Many studies have highlighted the oncogenic potential of ΔNp63α in HNC promoting squamous epithelial proliferation, migration and inflammation,32 and regulating EMT in primary human keratinocytes and in HNC cell lines in a TGFβ-dependent manner.34 Moreover, HDACi, alone or in combination, have been shown to reduce EGFR expression in HNC cell lines.35In the last years, histone deacetylase inhibitors (HDACi) have been described as valid tools to overcome EMT in many different tumours,In this study we investigated the molecular mechanisms at the basis of the antitumour activity of the HDACi SAHA in HNC cell lines. We demonstrated that SAHA possesses a synergistic inhibitory effect in combination with gefitinib, which is neither dependent on the HPV status nor on the epithelial/mesenchymal phenotype of the cell lines. We then demonstrated that the mechanism by which SAHA controls cell proliferation and reverts EMT is due to its ability to decrease the expression of the transcription factor ΔNp63α. Thus, with our data we uncover new molecular mechanisms underlying the potential efficacy in the clinic of the use of SAHA in combination with gefitinib. Indeed, the use of the two inhibitors together have greater effect in blocking cancer cell proliferation compared to single treatment and might avoid gefitinib resistance in HNC cancer cells, due to the ability of SAHA to revert EMT.36 The UM-SCC-4, UM-SCC-6, UM-SCC-10A, UM-SCC-18, UM-SCC-19, UM-SCC-23 and UM-SCC-47 cell lines were created by Prof. Thomas E. Carey.37 The UD-SCC-2 cell line37 was kindly provided by Prof. Henning Bier: present address LRZ, Munich, Germany. The 93-VU-147T cell line38 was kindly provided by Dr. Martin Rooimans, Free University Medical Centre, Amsterdam, the Netherland. The UM-SCC-104 cell line39 was from Merck spa. The UPCI:SCC-15440 and UPCI:SCC-9041 cell lines were acquired from ATCC®. Cells were grown in Dulbecco’s modified Eagle’s medium supplemented with antibiotics, 2 mM l-glutamine, 10% foetal bovine serum and non-essential amino acids. All cell lines were authenticated by short tandem repeat profiling and tested for mycoplasma contamination every 6 months. Recombinant human TGFβ1 was purchased from PeproTech, SAHA from Alexis Biochemicals and gefitinib  from Sigma Aldrich. Lentiviral plasmid encoding shControl and shp63 were a kind gift from Dr. Leif W.Ellisen . pBABE retroviral plasmid encoding EGFR was a kind gift of Dr. Sara Sigismund .HNC cell lines were obtained from different sources.N-ethylmaleimide (NEM), 0.5 mM NaF and 2 mM sodium Na3VO4, or in E1A buffer . After lysis an equal amount of protein for each sample was resuspended in denaturing sample loading buffer, separated on SDS-polyacrylamide gel electrophoresis gel and immunoblotted with the indicated antibodies. The following antibodies were used: E-Cadherin , Vimentin , p63 , acetyl-histone3 (Lys9) , acetyl-histone4 (Lys8) , phospho-SMAD2 , EGFR , phospho-EGFR (tyr1068) , acetylated-αtubulin , phospho-p70 S6 kinase , phospho-Akt , phospho-p38 , phospho-Erk , HPV16 E7  and GAPDH , and Vinculin  as loading control. Membranes were then incubated with the appropriate horseradish peroxidase secondary antibodies and the signal was acquired with Chemidoc (Bio-Rad).Cells were lysed in either a sodium dodecyl sulphate (SDS) lysis buffer: a 1:3 mixture of buffer I  and buffer II  containing 0.5 mM RNA was extracted from cells with the Quick-RNA MiniPrep kit (Zymo Research). cDNA was generated by reverse transcription-PCR with Reverse Transcriptase (Promega). Relative levels of specific mRNAs were determined with the Fast SYBR Green detection chemistry system (Applied Biosystem). All PCR reactions were performed with a 7500 Fast Real-Time PCR system (Applied Biosystem). Ribosomal phosphoprotein was used as a house-keeper gene for normalisation.® Luminescent Cell Viability Assay and following the manufacturer’s instructions.p63 short hairpin RNA-encoding lentiviral particles were used to transduce HNC cell lines. After puromycin selection, cells were plated in triplicate into 96-well plates at the appropriate density . Cell proliferation was assayed at day 3 and 7 after plating (T0)  or at day 2 and 6 (UM-SCC-104 and UD-SCC-2), using CellTiter-Glo® Luminescent Cell Viability Assay and following the manufacturer’s instructions. Data were analysed using Graphpad Prism software.To assess the half maximal inhibitory concentration (IC50) of SAHA and gefitinib, HNC cell lines were seeded in duplicate at the appropriate density  in 96-well plates. Twenty-four hours later cell lines were treated with vehicle or different concentrations of SAHA (1:3)  and (1:2.5) gefitinib  using serial dilutions, for 72 h. Cell proliferation was assayed using CellTiter-Glo® Luminescent Cell Viability Assay, following the manufacturer’s instructions. The combination index (CI) was calculated by the Chou-Talalay equation, which takes into account both potency (Dm or IC50) and the shape of the dose-effect curve42 using the software CalcuSyn . CI < 1, CI = 1 and CI > 1 indicate synergism, additive effect and antagonism, respectively. The linear correlation coefficient (r) of the median-effect plot is considered the first line of statistics to measure the conformity of the data with the mass-action law principle when the experimental measurement is assumed to be accurate. An r-value = 1 indicates perfect conformity. A poor r-value may be the result of biological variability or experimental deviations. Dose reduction index represents the measure of how much the dose of each drug in a synergistic combination may be reduced at a given effect level compared with the doses of each drug alone.HNC cell lines were seeded at the appropriate density  in duplicate in 96-well plates. At 24 h, cell lines were treated with vehicle or different concentrations of SAHA, gefitinib or the combination of the two drugs in equi-active concentrations using serial dilutions in a 1:1 constant ratio  for 72 h. Cell proliferation was assayed with CellTiter-GloMigration was evaluated by wound-healing assay. Briefly, HNC cells were seeded to 90% of confluence in 35 mm cell culture dishes with grid. Twenty-four hours after plating, the cell monolayer was wounded in two different points with a sterile pipette tip. After washing out the floating cells, cells were rinsed with fresh medium. Migration of wounded cells was observed and photographed at 0 and 24 h with an optic microscope. Photographs were taken at 0 and 24 h after wounding by brightfield and phase contrast microscopy . Quantitative measurements were made by determining the distances between the wound edges in at least three independent wound sites. The migration values were obtained by using ImageJ software , and expressed as % of migration respect to time points 0 h of culture.t test to compare only two samples (Graphpad Prism version 6 software).Statistical differences were evaluated using Dunnet’s multicomparison analysis after one-way analysis of variance to compare multiple samples or unpaired 43 As shown in Table We screened the effect of both SAHA and gefitinib on cell viability in a panel of 12 HNC cell lines, 6 of them deriving from HPV-positive patients  and mutant p53.49 and assessed cellular proliferation and migration. As shown in Fig. We then silenced p63 protein in both HPV-positive and HPV-negative cell lines using a lentiviral vector Fig. 49 and as51 We thus assessed EGFR expression in HPV-positive and HPV-negative HNC cell lines upon p63 knockdown. As shown in Fig. Recently, it has been shown that silencing endogenous ΔNp63α reduces EGFR expression in triple-negative basal-like breast cancer cells and in pancreatic cancer cells promoting cell growth and chemoresistance.8 The HDACi SAHA was already shown to potentially overcome EGFR-TKI resistance in a small panel of HPV-negative HNC cell lines.16 In this study we decided to better investigate the efficacy of SAHA alone or in combination with gefitinib in a larger and heterogeneous panel of HNC cancer cell lines and to characterise the molecular mechanisms underlying its activity. We found that both SAHA and gefitinib were able to inhibit HNC cell proliferation in both HPV-positive and HPV-negative cell lines, with different sensitivity independently from either their HPV status or their epithelial/mesenchymal phenotype. Moreover, the combination of the two drugs had a synergistic effect on both HPV-positive and HPV-negative HNC cell lines. Intriguingly, we found that SAHA treatment was able to increase epithelial marker and partially reduce mesenchymal marker in HNC cell lines, independently of the HPV status. This confirms the role of this drug in reverting EMT also in HPV-positive cell HNC lines, a role that was reinforced by the ability of SAHA to inhibit the activation of the TGFβ pathway, one of the pathways responsible for EMT that is hyperactivated during the acquisition of gefitinib resistance.12HNCs are a biologically heterogeneous group of cancers, originating from different subsites, mucosa of the oral cavity, nasopharynx, oropharynx, hypopharynx and larynx and with different aetiological factors, among them high-risk HPV infection and transformation. Thus, in our study we decided to collect different HNC cell lines, from different subsites, sex and HPV status, to have a better vision of the potential diversity of response to therapy. The cell lines that we obtained were not clearly clustered in subgroups by their expression of epithelial/mesenchymal markers or to the activation of different kinases important in the transduction of signalling pathways, confirming the high heterogeneity of these tumours. Although one of the signatures of these cancer is the overexpression of the EGFR, the inhibition of EGFR as main therapy has very modest efficacy due to intrinsic or acquired resistance.30 They are both involved in the promotion of proliferation and migration of HNC cell lines, cooperating with the TGFβ pathway or independently, activating pathways such as PI3K and MAPK.12 Upon SAHA treatment, we found a remarkable decrease in the expression of both ΔNp63α and EGFR in both HPV-positive and HPV-negative HNC cell lines, together with the inhibition of PI3K and ERK1/2 pathways, and, as mentioned above, decreased activation of the TGFβ pathway. Thus, our results suggest that the inhibition of both EGFR and ΔNp63α expression is responsible for the inhibitory activity in cell proliferation and migration mediated by SAHA. This evidence is reinforced by the remarkable inverse correlation between SAHA sensitivity and SAHA-mediated inhibition of EGFR and ΔNp63α. This behaviour also mirrors the positive correlation between ΔNp63α and EGFR inhibition by SAHA and the less efficacy of SAHA in cells overexpressing EGFR. In particular, ΔNp63α is known to regulate EMT in primary human keratinocytes and in HNC cell lines in a TGFβ-dependent manner34 and its downregulation reduces EGFR expression in triple-negative basal-like breast cancer cells and in pancreatic cancer cells promoting cell growth and chemoresistance.51 Thus, we showed for the first time that p63 silencing reduced EGFR expression in both HPV-positive and HPV-negative cell lines, showing also that SAHA-mediated inhibition of EGFR greatly correlates with ΔNp63α inhibition. Taken together, these data suggest that SAHA-mediated EGFR downregulation is p63-dependent. Moreover, since lack of p63 consistently reduced proliferation and migration of both HPV-positive and HPV-negative cell lines, interfering with the activation of the TGFβ pathway, our results imply a key role  for p63 in the SAHA-dependent regulation of proliferation and migration of these cell lines. In conclusion, we found that not only the HDACi SAHA synergises with gefitinib to decrease HNC cell lines viability, but it is also able to reduce EMT and inhibit TGFβ pathway activation, which are responsible for the induction of gefitinib resistance. A key regulator of this process is the transcription factor ΔNp63α, whose downregulation by SAHA treatment appears to be the major inhibitory activity of this drug, decreasing cell growth, cell migration and TGFβ pathway activation. Thus, this study uncovers a novel molecular mechanism underlying the efficacy of SAHA in the treatment of both HPV-positive and HPV-negative HNC tumours in combination with gefitinib. Gefitinib efficacy in the treatment of these tumours is limited due to the acquired resistance induced by EMT, which we demonstrated can be overcome by the concomitant use of SAHA. These results show that the combination of SAHA with specific inhibitors of EGFR, such as gefitinib, improves the antitumour efficacy of these drugs in both HPV-positive and HPV-negative HNC and should be further explored clinically.EGFR and ΔNp63α are two key markers in HNC since their expression is upregulated in 90% and 80% respectively of HNC primary tumours.Supplementary Figure S1Supplementary Figure S2Supplementary Figure legendsSupplementary Table S1


Although the administration of retinoids represents an important part of treatment for children suffering from high-risk neuroblastomas, approximately 50% of these patients do not respond to this therapy or develop resistance to retinoids during treatment. Our study focused on the comparative analysis of the expression of five genes and corresponding proteins  that have recently been discussed as possible predictive biomarkers of clinical response to retinoid differentiation therapy. Expression of these five candidate biomarkers was evaluated at both the mRNA and protein level in the same subset of 8 neuroblastoma cell lines after treatment with natural or synthetic retinoids. We found that the cell lines that were HMGA2-positive and/or HOXC9-negative have a reduced sensitivity to retinoids. Furthermore, the experiments revealed that the retinoid-sensitive cell lines showed a uniform pattern of change after treatment with both natural and sensitive retinoids: increased DDX39A and decreased PBX1 protein levels. Our results showed that in NBL cells, these putative protein biomarkers are associated with sensitivity or resistance to retinoids, and their endogenous or induced expression can distinguish between these two phenotypes. Neuroblastoma (NBL) is the most common extracranial solid tumor in children, accounting for 8-10% of childhood cancers in the USA and Europe . NBL is In the last decade, differentiation therapy with retinoids in patients with minimal residual disease was proven to be effective, since such a treatment was reported to prevent tumor relapse after myeloablative therapy. Although retinoids as differentiation inducers represent an important part of high-risk NBL treatment, their toxicity and acquired resistance to retinoids limit their use in clinical practice . Therefotrans retinoic acid (ATRA) and 13-cis retinoic acid (13-cis-RA) are the most frequently used retinoids in pediatric oncology, especially in the treatment of acute promyelocytic leukemia and high-risk NBL -2,5-diphenyltetrazolium bromide (MTT) (Sigma) at a final concentration of 455 μg/ml in medium was added to each well. The medium with MTT was replaced by 200 μl of DMSO per well after a 4-h incubation under standard conditions in order to solubilize the MTT product. Subsequently, the absorbance was measured at 570 nm with a reference absorbance at 620 nm wavelength using a Sunrise Absorbance Reader (Tecan). Obtained data were analyzed using one-sample T-test (two-tailed); p<0.05 (*) and p<0.01 (**) were considered statistically significant.HSP90AB1 and GAPDH were used as an endogenous control. Data were analyzed using one-sample T-test (two-tailed); p< 0.05 was considered statistically significant. The primers used in this study are listed in The relative expression levels of genes of interest were studied using RT-PCR, and total RNA for this purpose was extracted using the GenElute™ Mammalian Total RNA Miniprep kit (Sigma-Aldrich). RNA concentration and purity were determined spectrophotometrically, and equal amounts of RNA were reverse transcribed into cDNA using M-MLV reverse transcriptase . RT-PCR was carried out in 20-μL reaction volumes using the GeneQ thermal cycler (BIOER). To ensure that the amplification does not reach a saturation plateau, the suitable number of cycles for analysis of each gene in question was selected on the basis of comparison of RT-PCR products after 25, 30, 35 and 40 cycles of reaction . AccordiWhole cell extracts from all NBL cell lines were loaded onto 10% and 12% polyacrylamide gels and blotted on polyvinylidene difluoride membranes . The membranes were then blocked with 5% nonfat dry milk dissolved in phosphate buffered saline (PBS) containing 0.1% Tween-20 and incubated overnight with the corresponding primary antibody. After a 1-h incubation with a secondary antibody, proteins were visualized with the ECL-Plus detection system . For all experiments, rabbit monoclonal anti-GAPDH served as a loading control. Data were analyzed using one-sample T-test (two-tailed); p< 0.05 was considered statistically significant. Both primary and secondary antibodies used in this study are listed in HMGA2 mRNA levels in most of the cell lines . An overview of all results obtained in this study is given in In general, RA-sensitive cell lines ) responded to the treatment by upregulating the expression of the respective mRNA or protein, whereas downregulation of mRNA levels was observed in RA-resistant cell lines . No change in expression or various responses to particular retinoids was detected for some of the analyzed biomarkers.cis-RA, 13-cis-RA, BEX, 4-HPR) at the same concentration of 1 μM for 7 days , significant downregulation of this gene after treatment with natural retinoids was observed. In contrast, treatment with synthetic retinoids did not show a consistent effect on HMGA2 expression among the analyzed cell lines, and both up- and downregulation were observed , which could indicate ongoing changes towards a more differentiated phenotype mediated by retinoids. Further analysis of protein expression by Western blotting revealed that retinoids modulated the expression of the studied biomarkers at the protein level in an even more significant manner.Our study aimed to analyze the expression of five candidate biomarkers of sensitivity/resistance to retinoids  in a single experimental study using a set of patient-derived and reference NBL cell lines. Our results suggest that on the mRNA level, 7 days of exposure to natural and synthetic retinoids resulted in a change in expression of 3 out of 5 analyzed genes . We consequently concluded that HMGA2 positivity is insufficient in predicting resistance to retinoids in vitro; however, all HMGA2 negative cell lines showed sensitivity to retinoids. Therefore, our data suggest that the absence, rather than presence, of HMGA2 expression could predict the cellular response to retinoids. Furthermore, this study revealed that natural but not synthetic retinoids caused significant downregulation of the HMGA2 gene in all cell lines where HMGA2 was detected. We subsequently performed HMGA2 expression analysis at the protein level, which showed that this protein was completely missing in the NBL cell lines included in this study except for the NBL-36 RA-resistant cell line. Using the HMGA2-positive phenotype as a marker of resistance to retinoids would therefore produce false-negative results, as remaining-RA resistant, HMGA2-negative cell lines would be identified as RA-sensitive. Further study with more cell lines and tumor samples could help to better judge HMGA2 involvement in maintaining resistance to retinoids besides its apparent negative prognostic value in the various tumor types mentioned above.Apart from HMGA1 analysis, we also examined the expression of a second member of the HMGA family – HMGA2. Previously, a study reported that etinoids . All 3 RHOXC9 promoter was epigenetically primed in an active state in ATRA-sensitive reference NBL cell lines and silenced in ATRA-resistant cell lines according to another study, indicating its possible use as a marker of sensitivity to retinoids [HOXC9 mRNA was detected in all RA-sensitive and 2 out of 3 RA-resistant cell lines, indicating that HOXC9 promoter silencing did not generally occur in all cell lines with an RA-resistant phenotype, as suggested in the previously mentioned study. Because we used patient-derived cell lines, whereas reference cell lines were used in the study by Mao and coworkers, a more detailed analysis of the HOXC9 promoter in larger groups of NBL cell lines could help confirm these results. Western blot analysis of HOXC9 protein expression also showed promising results, supporting previous reports on the key role of HOXC9 in NBL differentiation [The transcription factor HOXC9 is discussed as a regulator of differentiation course within a cell  and was etinoids . In the etinoids  and breaetinoids . In our ntiation ,24. In 2cis-RA, was also analyzed in the present study. Previously, published results indicated its upregulation in RA-sensitive cells only, and a study reported that PBX1 downregulation resulted in a more aggressive phenotype [PBX1 mRNA in some RA-sensitive cell lines ), which is in accordance with the study mentioned above. However, we did not observe the same trend in the 3 remaining RA-sensitive cell lines. Compared to research we just mentioned, in which 10 μM 13-cis-RA was administered, all retinoids involved in our project were used at a concentration of 1 μM, which could also have influenced the results. Subsequent analysis of PBX1 expression at the protein level revealed a slight downregulation of this protein in RA-sensitive cell lines. This part of our experiment is different from the study mentioned above, since we did not also confirm the RA-mediated upregulation of PBX1 in RA-sensitive cell lines at the protein level, but PBX1 protein expression is likely downregulated. In addition to NBL, PBX1 was recently extensively studied in other types of cancer, and some of those studies suggested the opposite function of PBX1 in cancer progression that could be in accordance with the results we obtained [PBX1, which was recently suggested to increase sensitivity to 13-henotype . Our expobtained ,26.PBX1 mRNA and/or DDX39A and HOXC9 proteins.Our study aimed to perform an extensive analysis of several candidate biomarkers of RA responsiveness and confirm the potential use of some of these gene products and their corresponding proteins as indicators of resistance or sensitivity to retinoids. Analysis of the expression of these markers in combination might be more useful than analysis of these markers individually. Among all examined biomarkers, endogenous HMGA2 positivity together with HOXC9 negativity could predict a poor response to retinoid treatment. Moreover, cells undergoing RA-mediated differentiation could be identified by upregulation of In summary, our study was the first to evaluate the predictive value of five selected protein biomarkers in the same set of NBL cell lines after treatment with natural or synthetic retinoids. We found that cell lines that were HMGA2-positive and/or HOXC9-negative have a reduced sensitivity to retinoids. Furthermore, our experiments revealed that the retinoid-sensitive cell lines showed a uniform pattern of change after treatment with both natural and sensitive retinoids: increased DDX39A and decreased PBX1 protein levels. Our results proved that in NBL cells, these putative protein biomarkers are clearly associated with retinoid sensitivity or resistance, and the endogenous or induced expression of these markers can distinguish between the two phenotypes.S1 FigHMGA1, Sample 2 for DDX39A, HMGA2, PBX1), NBL-36 , NBL-40 (Sample 1 for DDX39A), SK-N-BE(2) , SH-SY5Y (Sample 2 for HOXC9).For each gene of interest, two different samples, i.e. two different cell lines were chosen to optimize the number of cycles used for semi-quantitative gene expression analysis. Cell lines used in this analysis: NBL-13 (Sample 1 for (EPS)Click here for additional data file.S2 FigDDX39A (A), HMGA1 (B), HMGA2 (C), HOXC9 (D), and PBX1 (E) mRNA expression.Relative band density for  The data are presented as the mean + SD, experiments were repeated in triplicate. The black line crossing the 100% represents the relative band density in untreated cells. * p< 0.05 indicates a significant difference compared to untreated cells. RA sensitive cell lines are illustrated in green and RA-resistant cell lines in red color. ImageJ software was used for visualized bands quantification.(EPS)Click here for additional data file.S3 FigRelative DDX39A (A), HMGA1 (B), HMGA2 (C), HOXC9 (D), and PBX1 (E) protein expression. The data are presented as the mean + SD, experiments were repeated in triplicate. The black line crossing the 100% represents the relative biomarker expression in untreated cells. * p< 0.05 indicates a significant difference compared to untreated cells. RA sensitive cell lines are illustrated in green and RA-resistant cell lines in red color. ImageJ software was used for visualized bands quantification.(EPS)Click here for additional data file.


Rapid changes in the expression of many messenger RNA (mRNA) species follow exposure of cells to ionizing radiation. One of the hypothetical mechanisms of this response may include microRNA (miRNA) regulation, since the amounts of miRNAs in cells also vary upon irradiation. To address this possibility, we designed experiments using cancer-derived cell lines transfected with luciferase reporter gene containing sequences targeted by different miRNA species in its 3′- untranslated region. We focus on the early time-course response (1 h past irradiation) to eliminate secondary mRNA expression waves.Experiments revealed that the irradiation-induced changes in the mRNA expression depend on the miRNAs which interact with mRNA. To identify the strongest interactions, we propose a mathematical model which predicts the mRNA fold expression changes, caused by perturbation of microRNA-mRNA interactions. Model was applied to experimental data including various cell lines, irradiation doses and observation times, both ours and literature-based. Comparison of modelled and experimental mRNA expression levels given miRNA level changes allows estimating how many and which miRNAs play a significant role in transcriptome response to stress conditions in different cell types. As an example, in the human melanoma cell line the comparison suggests that, globally, a major part of the irradiation-induced changes of mRNA expression can be explained by perturbed miRNA-mRNA interactions. A subset of about 30 out of a few hundred miRNAs expressed in these cells appears to account for the changes. These miRNAs play crucial roles in regulatory mechanisms observed after irradiation. In addition, these miRNAs have a higher average content of GC and a higher number of targeted transcripts, and many have been reported to play a role in the development of cancer.Our proposed mathematical modeling approach may be used to identify miRNAs which participate in responses of cells to ionizing radiation, and other stress factors such as extremes of temperature, exposure to toxins, and drugs.The online version of this article (10.1186/s12864-019-5464-0) contains supplementary material, which is available to authorized users. Response of human cells to electromagnetic radiation is of paramount importance in many contexts, including the intertwined fields of carcinogenesis and radiation therapy of cancer. Obtaining insights into the qualitative and quantitative nature of the response is complicated by superposition of potential mechanisms of primary and secondary response. For this reason, we focus on the short-term transcriptional response (mostly up to 1 h past irradiation), and in particular its regulation by other RNA species. Because of this relatively narrow focus, it seems possible to propose that a simple, non-mechanistic, statistical model helps to identify and quantitate the leading components of this regulation and that the modelling outcomes are free of statistical bias.Rapid changes in the expression of many messenger RNAs (mRNAs) follow exposure of cells to irradiation by X-rays, and the change of expression of up- or down-regulated mRNAs is strongly correlated with the distribution of microRNA (miRNA) recognition motifs in their non-coding 3’-UTR sequence . One of p-value < 0.05) of expression of luciferase mRNA occurred after irradiation when the primary transcript contained motifs targeted by miR-21 or miR-24, but in contrast no significant increase occurred in transcripts targeted by let-7 or devoid of miRNA-targeted motifs , recognized by members of let-7 family, miR-21, and miR-24, on ionizing radiation-induced changes in the luciferase mRNA expression. We were mostly interested in early-phase response, before any later effects of irradiation might interfere. Therefore, we focused on the change in luciferase expression between the 0 h (immediately before irradiation), and the 1 h time points. A statistically significant increase  of 6 MV X-ray photons generated by a therapeutic accelerator (Clinac 600) in fresh culture medium (changed 15 min before irradiation).Human melanoma Me45 cell line (established in the Center of Oncology in Gliwice), K562 cell line, and two HCT116 cell lines with different P53 status (ACCT collection) were grown in DMEM/F12 medium in RPMI 1640, both with L-glutamine (Sigma-Aldrich), 10% fetal bovine serum (Gibco), and 80 μg/ml gentamycin at 37 °C in a humidified atmosphere and 5% COFirefly luciferase being a reference and Renilla luciferase with eight tandem repeats of various miRNA target sites in its 3’UTR being the reporter gene. The plasmid contained sequences targeted by either the miRNA let-7, miR-21 or miR-24. The let-7 target sequence had motif TCGAGACTATACAAGGATCTACCTCAG, 71.75% average complementarity to the target sequences of various mature let-7 family members . Resulting C matrix was used to simulate predicted mRNA fold change. Example of single prediction is presented on the left in the form of scatter plot, a result of thousand predictions is presented on the right in the form of histogram, where x axes indicated ρ returned by model. B) The C matrix was permuted . Resulting C matrix was used to simulate predicted fold change. Example of single prediction is presented on the left in the form of scatter plot, a result of thousand predictions is presented on the right in the form of histogram, where x axes indicated ρ returned by model. C) Original C matrix was used to estimate model parameters, after that the C matrix was permuted and used for prediction of mRNA changes. Example of single prediction is presented on the left in the form of scatter plot, a result of thousand predictions is presented on the right in the form of histogram, where x axes indicated ρ returned by model. (TIF 1058 kb)Additional file 5:Table S3. The calculated values for all miRNAs expressed in Me45, K562, HCT116+/+ and HCT116−/− cells together with other characteristics of the miRNAs studied. (XLSX 345 kb)Additional file 6:Figure S3. Proportion of genes represented by one or more transcripts in four cell lines. (TIF 561 kb)Additional file 7:Figure S4. Correlations between the predicted and the experimentally observed fold changes of mRNA levels in all available cell lines. A) AG1522 cell line, 3 h after radiation, dose 2 Gy, ρ = 0.378. B) AG1522cell line, 3 h after radiation, dose 5 Gy, ρ = 0.222. C) MOLT4 (Bay) cell line, 2 h after radiation, dose 4 Gy, ρ = 0.215. D) MOLT4 (DMSO) cell line, 2 h after radiation, dose 4 Gy, ρ = 0.196. E) DU145cell line, 2 h after radiation, dose 10 Gy, ρ = 0.172. F) HCAEC (SD) cell line, 6 h after radiation, dose 10 Gy,, ρ = 0.108. G) HCAECs (MF) cell line, 6 h after radiation, dose 10 Gy, ρ = 0.198. H) MOLT4 cell line, 2 h after radiation, dose 5 Gy, ρ = 0.297. I) PBMC cell line, 2 h after radiation, dose 60 Gy, ρ = 0.179. J) PBMC cell line, 4 h after radiation, dose 60 Gy, ρ = 0.163. K) PBMC cell line, 20 h after radiation, dose 60 Gy, ρ = 0.175. L) SC3 cell line, 2 h after radiation, dose 10 Gy, ρ = 0.160. M) WI38 cell line, 1 h after radiation, dose 2 Gy, ρ = 0.366. N) WI39 cell line, 2 h after radiation, dose 2 Gy, ρ = 0.459 (TIF 812 kb)Additional file 8:Figure S5. Influence of individual miRNAs on the prediction of radiation-induced changes of mRNA levels in K562  HCT116+/+  and HCT116−/−  cells.  Ranking miRNA according to correlation coefficient to lowest.  Using an increasing number of miRNAs added according to decreasing rank.  Using an increasing number of miRNAs added according to increasing rank. (TIF 878 kb)Additional file 9:Table S4. KEGG pathways identified for 30 top-rank miRNAs in Me45 cell line. (XLSX 12 kb)Additional file 10:Figure S6. Influence of the classification criteria on differences between mRNAs with good or poor fit to the model. The plot is an extended version of the data in Table


TERT (Telomerase Reverse Transcriptase) promoter. When hypomethylated, ATRA-inducible TERT repressors can bind the promoter, repressing transcription of TERT, the rate-limiting component of telomerase. Ovarian carcinomas are heterogeneous tumors characterized by several aberrantly methylated genes among which is TERT. We recently found a hypomethylation of TERT promoter in about one third of serous carcinoma, the most lethal histotype. Our aim was to investigate the potential role of ATRA as an anticancer drug in a sub-group of ovarian carcinoma where the TERT promoter was hypomethylated.All-trans retinoic acid (ATRA) is currently being used to treat hematological malignancies, given the ability to inhibit cell proliferation. This effect seems to be related to epigenetic changes of the TERT promoter.The potential antiproliferative and cytotoxic effect of ATRA was investigated in seven serous ovarian carcinoma and one teratocarcinoma cell lines and the results were compared to the methylation status of their TERT promoter, was the best and fastest responder. PA1 and SKOV3, two cell lines with an intermediate methylated promoter, revealed a weaker and delayed response. On the contrary, the other 5 cell lines with a highly methylated promoter did not respond to ATRA, indicative of ATRA-resistant cells.The serous ovarian carcinoma cell line OVCAR3, harboring a hypomethylated TERT promoter and ATRA efficacy in ovarian carcinoma cell lines. Although these results are preliminary, ATRA treatment could become a new powerful, personalized therapy in serous ovarian carcinoma patients, but only in those with tumors harboring a hypomethylated TERT promoter.Our results demonstrate an inverse correlation between the methylation level of  Retinoids are a class of compounds that are structurally associated to vitamin A. Experimental animal models, cellular models, epidemiological data and clinical trials provide a strong rationale for the use of retinoids in cancer therapy and prevention . All-traTERT (Telomerase Reverse Transcriptase). This gene codes for the catalytic subunit of telomerase enzyme liable to elongation of telomeres in up to 95% of malignant tumors [TERT transcription leads to telomerase activity, and is an important factor linked to proliferation, differentiation and senescence. It is not found in the majority of somatic cells, while it is expressed in the proliferative cells such as germ cells, stem cells, and in the majority of tumors [TERT is at its promoter level. The TERT promoter is in a dense CG-rich CpG-island, indicating a role for methylation in the regulation of TERT expression [TERT promoter hypermethylation is very common in many cancers contrarily to normal cells, and a positive correlation was found between TERT methylation and TERT expression, especially in epithelial tumors [TERT promoter. TERT methylation prevents the binding and thus the repressive effect of CTCF, whereas a small hypomethylated region around the transcription start site allows a weak transcription of TERT, despite the hypermethylation of the border regions [The finding that most tumors express telomerase activity has led to an exponential growth of research to discover the role and regulation of this enzyme because the activation of telomerase is a critical step in carcinogenesis and cellular immortalization , 13. Thet tumors . TERT trf tumors , 15. Onepression . TERT prl tumors –19. The  regions , 21.TERT repressors bind to the promoter of TERT, only when this promoter is hypomethylated, blocking transcription of the gene and, consequently, causing malfunction of telomerase. On the one hand, TERT is generally hypomethylated in a great majority of lymphoproliferative tumors [TERT is hypermethylated in a majority of epithelial tumors [TERT [TERT promoter in about one third of serous carcinomas [The study of Azouz et al.  demonstre tumors  where ATl tumors  where ATrs [TERT –28. Nevercinomas , the mosMost of the advanced ovarian cancer patients respond well to first line of chemotherapy based on carboplatin and taxane. Despite this high initial response rate, a majority of patients relapse and die of the disease . The resTERT promoter is hypomethylated. Several ovarian carcinoma cell lines were treated with ATRA, and we determined whether a correlation between the antiproliferative and cytotoxic effects of all-trans retinoid acid and the methylation status of TERT promoter in these cell lines exists.The aim of this study was to predict the role of ATRA as a potential anticancer drug in the sub-group of ovarian carcinomas in which the To evaluate the effective contribution of ATRA treatment on cell growth in ovarian cancer cell lines, we performed cell counts of control and treated cells on days 3, 5 and 7 after treatment. The selected concentration of 1 μM of ATRA was used for this study. This concentration was chosen because it has been shown the strongest support by patients according to the pharmacokinetic studies, which permit to avoid the severe toxicity of the drug . The graThe inhibitory action of ATRA on the cell growth was a rapid and clearly marked process for the OVCAR3 cell line (p<0.05). A substantial diminution of ATRA-treated cell growth was observed on day 3, increasing up to day 7. PA1 and SKOV3 cells showed a cell growth diminution on day 7, whereas they did not show numeric difference on days 3 and 5. There were no changes in growth curves between ATRA-treated and control cells in any of the other ovarian cancer cell lines-A2780, CAOV3, OVMZ6, OVMZ36, OVMZ37 and SKOV3-, and in the HT29 CRC cell line.p<0.05 for OVCAR3), , we observed that the percentage of viable cells decreased at the end of ATRA treatment  no effect on the five ovarian cell lines with a high level of TERT methylation was observed , ovarian teratocarcinoma cell line (PA1) and human colorectal carcinoma (CRC) cell line HT29 were obtained from the Department of Molecular Pathology . A2780, CAOV3, SKOV3 and OVCAR3 were reported to be sensitive to very sensitive to platinum based chemotherapy .Cells were cultured in Roswell Park Memorial Institute Medium (RPMI 1640) + L-glutamine supplemented with 10% Fetal Bovine Serum (FBS), 1% PSF (Penicillin/streptomycin + Fungizone - Amphotericin B) and in Dulbecco’s Modified Eagle Medium + 4.5 g/l D-glucose + Pyruvate (DMEM), supplemented with 10% Fetal Bovine Serum (FBS), 1% PSF. Cells were maintained in a humidified atmosphere at 37 °C and in 5% CO2. Cell lines were stored in liquid nitrogen with 5% DMSO. All products were from ThermoFisher Scientific. All cell lines tested negative for mycoplasma contamination using MycoAlertTM Mycoplasma Detection Kit, Lonza, catalog number LT07–318.5 cells were seeded in six well plates. After 24 h, the cells were treated for 7 days with medium containing ATRA with a final concentration of 1 μM. Cell medium was replaced every 48 h. Cells were collected on day 3, 5 and 7 after ATRA treatment, counted using a Neubauer hematocytometer, and cell pellets were frozen for subsequent analyses. As a control, we used cell lines treated only with ethanol, the solvent for ATRA. Experiments were performed in triplicate.All-Trans Retinoic Acid (ATRA) powder was purchased from Sigma Aldrich. ATRA was dissolved in ethanol at final concentration of 5 mM and stored at − 20 °C. 2 × 10Cell viability was measured using the 3--2,5-diphenyltetrazolium bromide (MTT) assay (Alfa Aesar). Cells were plated in 96-well plates and treated with ATRA as described above. After incubation for the indicated time intervals, 0.5 mg/mL of MTT was added to each well for 4 h. The blue MTT formazan precipitate was then dissolved in 200 μL of an Isopropanol-HCl solution. The absorbance at 540 nm was measured by Synergy™Mx spectrophotometer (BioTek). Gen5™ software (BioTek) was used for the analysis.TERT mRNA expression was normalized with GAPDH as an internal control.RNA extraction was performed by using RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. Subsequently, the reverse transcription-polymerase chain reaction (RT-PCR) was performed with 2 μg of total RNA using SuperScript™ III Reverse Transcriptase (ThermoFisher Scientific). The profile used for the reaction was: 25 °C for 5 min, 50 °C for 50 min and 70 °C for 15 min. Next, the real-time PCR was performed with 2 μL of cDNA using a Platinum® Taq DNA polymerase (ThermoFisher Scientific) and a mix of TERT and GAPDH primers (Table DNA was extracted from cell lines using DNeasy Blood & Tissue Kit as descripted by the manufacturer (Qiagen). Extracted DNAs (500 ng) were bisulfite modified using EpiTect Fast DNA kit according to the manufacturer’s protocol (Qiagen). Briefly, a total of DNA was denaturated with sodium hydroxide and modified with sodium bisulfite. DNA samples were then purified using MiniElute DNA spin columns and elution of converted DNA was performed with 20 μL elution buffer.TERT promoter gene was calculated using the PyroMark Q24 Software (Qiagen) that allows the methylation analysis at each CpG site.The eluted, bisulfite converted DNA was used for pyrosequencing PCR amplification. Bisulfite-PCR-Pyrosequencing was performed in 20 μL reaction mixture containing 3 μL of converted DNA and forward and biotinylated reverse primers (TERT-pyr-Fwd and TERT-pyr-Rev, Table P < 0.05 was considered significant.All assays were performed at least three times. The results were presented as mean ± SD. Statistical analysis was conducted by t-test.