AKTIP is a shelterin-interacting protein required for replication of telomeric DNA. Here, we show that AKTIP biochemically interacts with A- and B-type lamins and affects lamin A, but not lamin C or B, expression. In interphase cells, AKTIP localizes at the nuclear rim and in discrete regions of the nucleoplasm just like lamins. Double immunostaining revealed that AKTIP partially co-localizes with lamin B1 and lamin A/C in interphase cells, and that proper AKTIP localization requires functional lamin A. In mitotic cells, AKTIP is enriched at the spindle poles and at the midbody of late telophase cells similar to lamin B1. AKTIP-depleted cells show senescence-associated markers and recapitulate several aspects of the progeroid phenotype. Collectively, our results indicate that AKTIP is a new player in lamin-related processes, including those that govern nuclear architecture, telomere homeostasis and cellular senescence. A- anLMNA, LMNB1 and LMNB2. Lamins A and C are alternatively spliced isoforms of the LMNA gene. Lamins B1 and B2 are the products of the LMNB1 and LMNB2 genes. Lamins A, B1 and B2 are initially synthesized as precursors, known as prelamins, which are processed into mature lamins through a sequential series of post-translational modifications . G. Gd) [233.2.a).Previous work has shown that A- and B-type lamins form two prominent nuclear meshworks with points of co-localization during interphase . During figure 3b). Double staining of HPFs for AKTIP and either lamin A or lamin A/C showed a partial co-localization of AKTIP with A-type lamins at the nuclear periphery (b). We also observed partial colocalization of AKTIP intranuclear signals with lamin A/C signals (b).We next investigated whether AKTIP co-localizes with A- and B-type lamins in interphase nuclei. The analysis of HPFs stained for both AKTIP and lamin B1 showed that the two proteins have multiple co-localization points, both at the nuclear rim and in the nucleoplasm b. Doubleeriphery b. We als signals b.c–e and electronic supplementary material, figure S4). In all cell types, antibody staining detected specific AKTIP-enriched mitotic structures, which were absent in AKTIP-depleted cells (shAKTIP) . The AKTIP signal was associated with structures at the cell poles that overlapped the spindle microtubules and the centrosomes. In addition, AKTIP concentrated to the midbody, a transient structure that connects the two daughter cells at the end of cytokinesis. The midbody contains microtubules that overlap in a narrow region at the centre of the structure. This region often appears dark after tubulin immunostaining (dark zone), because it is tightly associated with proteins that block antibody binding to tubulin [figure 3d), suggesting a possible role of AKTIP in cytokinesis.To determine AKTIP localization during mitosis, we used a fixative containing 0.25% Triton X-100. This fixative results in a high level of protein extraction allowing a clear visualization of AKTIP, but it does not fully preserve spindle microtubules. We analysed AKTIP localization in BJ-hELT (BJ fibroblasts expressing hTERT and SV40 Large T) and HeLa cells and HPFs (figure 3 tubulin . Remarkae). By contrast, lamin B1 accumulated at the centrosomes and partially co-localized with AKTIP, which was enriched at both the centrosomes and the spindle .To ask whether AKTIP co-localizes with lamins during mitosis, we co-immunostained HPFs for AKTIP and either lamin A or B. As expected from previous work , lamin Afigure 33.3.LMNA gene, leading to a lamin A form deleted of 50 aa and permanently farnesylated [LMNA mutation that replaces an arginine with a histidine at position 527, are characterized by accumulation of prelamin A and progeroid traits [a,b). In unextracted LV-progerin cells expressing both endogenous lamin A and progerin, and more effectively in unextracted HGPS cells, which are heterozygous for the LMNA mutation, the AKTIP signal in the cytoplasm was reduced and the nuclear rim staining was weaker and more discontinuous than in controls (a); the defective AKTIP localization at the nuclear rim of these cells was more evident in preparations extracted with Triton X-100 (b). In MADA cells, homozygous for the LMNA mutation, AKTIP was almost undetectable .Given the connection between AKTIP and lamins, we investigated the functional relationships between these proteins. We first asked whether mutations in lamin genes affect AKTIP localization. We analysed AKTIP localization in HPFs from patients affected by HGPS or MAD type A (MADA). The HGPS mutation exposes a cryptic splice site in the esylated ,36. MADAd traits . In addicontrols a; the deon X-100 b. In MADtectable a,b.Figua–d). In addition, Q-PCR-mediated mRNA quantification in HeLa cells at 7 days after shAKTIP infection showed a significant decrease in the lamin A mRNA level compared to control (e). However, AKTIP deficiency did not affect the level of lamin C mRNA, which is the main alternative splicing product of the LMNA gene. Consistent with these results, lamin A immunostaining in AKTIP-depleted HeLa cells showed a substantial reduction of the lamin A signal, which, however, maintained its normal association with the nuclear rim (f).We next investigated whether loss of AKTIP affects lamin expression. We reduced the AKTIP level by RNAi in early passage HPFs and HeLa cells . Western blotting performed on extracts from both cell types collected at 7 days post-infection showed that AKTIP depletion results in a substantial decrease in lamin A compared with control cells, without affecting lamin C and lamin B1 levels a–d. In a control e. Howevelear rim f.FigureWe have previously shown that AKTIP is required for telomere replication and that its depletion generates telomere-associated DNA repair foci . Depleti3.4.LMNA mutations. Cell labelling with the empirical senescence marker SA-βgal [a) exhibit a significant increase in senescent cells compared with controls . We next analysed K9 trimethylation of histone H3 (trimethyl H3K9), an epigenetic marker of cell senescence that is reduced in prematurely ageing lamin-defective cells [a) displayed higher frequencies of nuclei with diffuse trimethyl H3K9 distribution than passage-matched controls . In addition, Western blotting showed that in AKTIP-depleted early passage (p6) HPFs, the trimethyl H3K9 level was reduced with respect to controls and was comparable to that observed in naturally aged HPFs at p22 (e).Lamin mutations cause defects in nuclear structure, variations in heterochromatic marks and cell senescence . These f SA-βgal  revealedcontrols a,b. We nve cells . Early pve cells ,37,39. Econtrols c,d. In as at p22 e.Figurea) and examined 7 days post-infection cells immunostained with the C20 antibody, which specifically reacts with prelamin A. shAKTIP HPFs at p22 displayed a higher frequency of prelamin A-positive cells than passage-matched control HPFs . We also examined AKTIP-deficient HPFs for distorted nuclei, which have been described in lamin mutant cells [c,d).Previous studies on naturally aged cells have shown that they accumulate unprocessed lamin A (prelamin A) . This acrol HPFs a,b. We ant cells ,14,39. WTaken together, these data indicate that AKTIP depletion results in phenotypic traits that are characteristically associated with lamin A mutations.4.We have recently shown that AKTIP interacts with the PCNA and RPA70 components of the DNA replication machinery and with the shelterin subunits TRF1 and TRF2. Consistent with these results, we found that AKTIP is required for both general DNA replication and telomere replication . Our MS We focused on the relationships between AKTIP and lamins and found that these proteins exhibit similar localization patterns in interphase nuclei, with AKTIP enriched at the nuclear rim, showing partial co-localization with both the A- and B-lamin meshworks. We also found that AKTIP is enriched in structures that partially co-localize with the centrosomes and the spindle of mitotic cells. In addition, AKTIP forms a ring around the midbody during late cytokinesis. Previous studies in different cell types have shown that lamin B is also enriched at the centrosomes and spindle poles ,41,42 asLMNA RNA or by controlling both prelamin A degradation and LMNA gene expression [Indeed, we found that AKTIP knockdown results in a specific reduction of lamin A at both the mRNA and the protein level. A specific downregulation of A-type lamins has been also found in other systems. For example, in mouse brains both lamin A and progerin expression is downregulated by miR-9, a brain-specific microRNA; miR-9 targets prelamin A 3′-UTR but not the lamin C transcript . Lamin Apression . It is apression . The mosWe also found a modest increase (from 3 to 9%) in the frequency of prelamin A-positive cells in late passage samples of AKTIP-depleted cells. This finding is in apparent contrast with the downregulation of lamin A observed in in early passage AKTIP-depleted cells. However, it should be considered that prelamin A accumulation reflects a slowdown in prelamin A processing, a phenomenon that characterizes the senescent phenotype associated with physiological and premature ageing . It is tWe showed that AKTIP is mislocalized in LV-progerin and HGPS cells and in cells from MADA patients that accumulate prelamin A ,14,39. TOur MS analyses, in addition to lamins, identified matrin 3, importin 7 and several mitochondrial proteins as AKTIP partners. The functional relationships between AKTIP and these interacting partners deserve further investigation. In this respect, it should be noted that previous studies have shown that proteins involved in telomere maintenance such as the ATM kinase and the shelterin subunit TIN2 localize to mitochondria and regulate mitochondrial activity ,51.Altogether, our results identify AKTIP as a new lamin interactor, expanding the connection between telomeres and lamins and, more in general, between telomeres and nuclear envelope. In addition, the interplay between AKTIP and both lamin A and its aberrant forms responsible for HGPS and MADA suggests a possible involvement of AKTIP in the pathogenesis of these diseases.


MYC, but not the mature B-cell lymphoma associated oncogenes BCL2 and BCL6. Instead, U-2946 cells expressed the antiapoptotic BCL2 family member MCL1 which was highly amplified genomically (14n). MCL1 amplification is recurrent in DLBCL, especially in the activated B cell (ABC) variant. Results of microarray expression cluster analysis placed U-2946 together with ABC-, but apart from germinal center (GC)-type DLBCL cell lines. The 1q21.3 region including MCL1 was focally coamplified with a short region of 17p11.2 . The MCL1 inhibitor A-1210477 triggered apoptosis in U-2946 (MCL1pos/BCL2neg) cells. In contrast to BCL2pos DLBCL cell lines, U-2946 did not respond to the BCL2 inhibitor ABT-263. In conclusion, the novel characteristics of cell line U-2946 renders it a unique model system to test the function of small molecule inhibitors, especially when constructing a panel of DLBCL cell lines expressing broad combinations of antiapoptotic BCL2-family members.Diffuse large B cell lymphoma (DLBCL) is the most common form of non-Hodgkin lymphoma worldwide. We describe the establishment and molecular characteristics of the DLBCL cell line U-2946. This cell line was derived from a 52-year-old male with DLBCL. U-2946 cells carried the chromosomal translocation t and strongly expressed  Continuous cell lines have become indispensable tools in leukemia/lymphoma research. Self -renewing and transportable in the frozen state, cell lines provide unlimited supplies of cellular tumor material worldwide. Descendent from single cells of a patient´s tumor, they retain oncogenomic aberrations and gene expression patterns characteristic of the primary tumor. Tumor congruence is a key reason why cell lines are used as models to study the function of aberrantly expressed genes or to test the efficiency of novel anti-cancer drugs ,2.BCL2, BCL6 and MYC [BCL2 and MCL1 are recurrently overexpressed, as result of chromosomal translocations, amplification or other mechanisms [MCL1. Several DLBCL cell lines have been used to validate novel drugs targeting antiapoptotic BCL2 family members [Diffuse large B cell lymphoma (DLBCL) is the most common form of lymphoma in adults. Various genes are recurrently affected by mutations and chromosomal aberrations therein, including the germinal center oncogenes  and MYC . Express and MYC . DLBCL-d and MYC –9. In co and MYC . Cell li and MYC ,7,10. On and MYC . In DLBCchanisms –14. We d members –18. We pwww.dsmz.de) or were supplied by the originators for research purpose. Detailed references and cultivation protocols have been described previously [BCL2 family inhibitors ABT-263 and A-1210477 were obtained from Selleckchem .Authenticated stocks of cell line U-2946 were grown in RPMI 1640  containing 10% fetal bovine serum (FBS) . Cell lines applied in this study are all held by the DSMZ—German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany (eviously . The BCLin situ hybridization (FISH) were performed as described previously [https://genome-euro.ucsc.edu/) guided by the results of combined conventional cytogenetic, SKY and Cytoscan copy number array data (see below). For colocalization experiments commercial chromosome painting probes were mixed 1:1 with labelled BACs. Probe preparation and labelling were as described previously [Cells were harvested and fixed as described previously . Spectraeviously . Tilepateviously . Brieflyeviously .CytoScan HD Array  hybridization analysis was performed to identify numerical aberrations. DNA was prepared using the Qiagen Gentra Puregene Kit . Data were analyzed using the Chromosome Analysis Suite software version 2.0.1.2 (Affymetrix).Immunophenotyping was conducted on a FACSCalibur . Antibodies (Abs) against CD3 (Leu4), CD5 (UCHT2), CD10 (HI10a), CD19 (Leu12), CD20 (Leu16), CD34 (clone 581), CD37 (M-B371), CD38 (HIT2), CD138 (MI15) and HLA-DR (Tu39) were purchased from Becton Dickinson. Ab CD13 (My7) was from Beckman Coulter . Optimal dilutions of the Abs were determined with positive and negative control cell lines. FITC-labeled Abs against IgG, IgM, Ig kappa and Ig lambda were obtained from Southern Biotech/Biozol .Rearrangement of the immunoglobulin (Ig) heavy chain was determined by genomic PCR using primers described by van Dongen et al. . The PCRFive hundred ng total RNA were used for biotin labelling according to the 3´ IVT Express Kit (Affymetrix); 7.5 μg of biotinylated cDNA were fragmented and placed in a hybridization cocktail containing four biotinylated hybridization controls . Samples were hybridized to an identical lot of Affymetrix GeneChip HG-U133 Plus 2.0 for 16 h at 45°C. Steps for washing and SA-PE staining were processed on the fluidics station 450 using the recommended FS450 protocol (Affymetrix). Image analysis was performed on GCS3000 Scanner and GCOS1.2 Software Suite (Affymetrix). Data processing and analysis  were performed via R/Bioconductor using limma and affy packages –25.BCL2 (Hs00153350_m1), BCL6 (Hs00153368_m1), MAP2K3 (Hs00177127_m1), MCL1 Exon 1/2 (Hs03043899_m1), MYC exon 1/2 (Hs00905030_m1) and MYC exon 2/3 (Hs00153408_m1) expression levels with TBP as endogenous control. Relative expression levels were calculated using the ΔΔCt method.Quantitative PCR was performed on a 7500 Applied Biosystems real-time PCR system . RNA was prepared using the RNeasy Mini kit (Qiagen). Reverse transcription was performed using the SuperScript II reverse transcriptase kit . TaqMan probes (Applied Biosystems) were used to quantify human BCL2, MAP2K3 and MCL1 were determined by genomic PCR applying the SYBR green assay system (Applied Biosystems) with ABL1 as internal control and NC-NC as diploid reference cell line. DNA was prepared using the High pure PCR template preparation kit . The applied primers are listed in Ploidy levels of Samples were prepared as described previously . Anti BC4 cells (in 100 μl) were seeded in triplicate in 96-well flat-bottom microtiter cell culture plates. Inhibitors were added as 2x concentrated solution in a 100 μl volume. For the last 3 h of the incubation period, 1 μCi 3H-thymidine  was added to each well.Assays of 3H-thymidine incorporation were executed as follows: 1.25 x 10Apoptotic cells were detected and quantified with the annexin-V/propidium iodide method using the TACS Annexin-V-FITC kit  according to the manufacturer’s instructions. Binding of fluorescein isothiocyanate-labeled annexin-V and propidium iodide staining of the cells were determined by flow cytometry on the FACSCalibur (Becton Dickinson).The patient was a previously healthy man, age 52 years who sought medical care due to abdominal pain and tiredness. Computer tomography examination revealed an abdominal tumor. In July 1996 he underwent a right-sided hemicolectomy due to a tumor of the ascending colon. The tumor, 15 cm in diameter, removed macroscopically radically. Pathological examination showed DLBCL. Diagnostic work-up revealed lymphomatous engagement in the right testicle and in paratesticular tissue with a diameter of >10 cm, in lymph nodes and in the bone marrow (stage IVB). International Prognostic Index was retrospectively assigned to 3 ). At diagnosis he also had a palsy of the left N. abducens but examination of cerebrospinal fluid showed no signs of lymphoma and a CT of the brain was normal.9/l. The patient gave informed consent to use the pleural effusion for research including the possible establishment of a cell line. He died of progressive lymphoma three months after diagnosis. The overall picture of the lymphoma was an extremely aggressive disease with fast progression and very limited sensitivity to chemotherapy.He was treated with VACOP-B : a weeklMYC translocation was detected by in situ hybridization.Large blast cells with the appearance of both immunoblasts and centroblasts grew in a diffuse and infiltrative pattern in the intestine. Several mitotic figures and apoptotic cells were present. Immunohistochemical stainings were performed and the lymphoma cells were positive for CD20, CD79a, CD10, BCL-6, MYC, p53 and negative for BCL-2, CD5, CD30, cyclin D1, SOX11 with a high proliferation index of Ki67. Partial expression of MUM1 and FOXP1 was detected. According to the WHO classification and update ,30 the l2 humidified environment. Cells were split every 72 h with an optimal density of 0.3 x 105 cells/ml and a doubling time of 48 h. After one month, the cell line U-2946 was considered established and grew in continuous culture in RPMI 1640 with 10% FBS for another 2 months. U-2946 cells tested EBV-negative excluding a lymphoblastoid origin. May-Grünwald-Giemsa stain of the cell line is shown in Tumor cells from pleural effusion were obtained by Ficoll-Isopaque  gradient separation and cultured in RPMI 1640 containing 10% of FBS, glutamine and antibiotics (100 IU/ml penicillin and 50 μg/ml streptomycin) at 37°C in 5% COWe tested the Ig rearrangement status of cell line U-2946 using previously described primers . The celIGH and MYC loci t, effecting copy number increase of the terminal 8q region just including PVT1, but excluding MYC.The consensus karyotype, performed using classical  and specMYC loci  was resoMCL1, ENSA), while that at 1p11 extending some 461 kbp covered four loci . Coamplification of gene pairs normally arises when a genomically remote gene pair, ectopically juxtaposed or fused by chromosome rearrangement is locally subjected to linear copy number amplification, e.g. following selection. Such copy number aberrations are hallmarks of oncogenes and are thought to arise by conferral of proliferation or antiapoptopic advantage.Cytogenetic analysis also revealed a novel rearrangement, focal co-amplification of 1q21 and 17p11.2 replacing a deleted segment of chromosome 17p . The ca.Expression array analysis allows distinction between two forms of DLBCL, the GC and the ABC forms . Based oBCL2, BCL6 and MYC. These aberrations can occur in the same tumors, which are then called “double-hit” or “triple-hit” lymphoma according to their aberration scores. Cell lines representing double-hit and triple-hit lymphomas have recently been reviewed in detail [BCL2), 3q27 (BCL6) were not detected in U-2946 cells. As shown in MYC on 8q24 was fused to IGH on 14q32. In accordance with the genomic findings, cell line U-2946 was BCL2neg/BCL6low/MYCpos . Accordingly qRT-PCR showed MYC transcript in exon 2 but not in exon 1 suggesting (i) that the genomic break was located in intron 1/2, and (ii) that translation started with the alternative ATG start codon in exon 2 and not with the “internal ribosome entry site” (IRES)-mediated CTG codon in exon 1  were upregulated in U-2946; another 7 genes (15%) lay below the average of B-lymphoma cell lines . High deMAP2K3 (MEK3) was amplified in cell line U-2946  is consistent with the ABC character proposed here for cell line U-2946  or BCL2, BCL-XL and BCL-W (ABT-263). The MCL1 inhibitor  substantially reduced 3H-thymidine uptake and triggered apoptosis in the MCL1pos cell line U-2946, while MCL1neg cell lines were not affected  were responsive to low doses of the BCL2 inhibitor , the MCL1 inhibitor (7.5 μM A-1219477) did not have any effect on U-2932 cells, suggesting that BCL2 was dominant over MCL1 in the U-2932 cell system  had no effect. Other DLBCL cell lines expressed BCL2 and responded to ABT-263. U-2932 was one of these BCL2pos cell lines. This cell line also expressed MCL1, but did not respond to the MCL1 inhibitor. Even with low doses of the BCL2 inhibitor, A-1210477 was inefficient which suggests that the effect of BCL2 is dominant over MCL1, at least in this cell system.Translocations and/or numerical aberrations affecting the germinal center oncogenes BC cases ,14. MCL1proteins ,15–18. Rproteins . Cell lipos/BCL2neg cell line U-2946 promises to be a valuable model for inhibitor studies, useful especially in combination with cell lines expressing other antiapoptotic BCL2 family members. Cell line U-2946 will be available from the public repository DSMZ (www.dsmz.de).We conclude that the MCL1BCL2 family member MCL1. The MCL1 chromosomal region (1q21.3) was involved in a translocation to chromosome 17. The amplified MCL1 gene is highly expressed in the cell line (MCL1pos/BCL2neg). Inhibitor experiments revealed the importance of MCL1 for growth and survival of U-2946 cells. Cell line U-2946 will prove to be an important model system for the functional analysis of small BCL2-family inhibitors, especially in a panel of certified DLBCL cell lines showing other MCL1 and BCL2 expression profiles.We describe the establishment and characteristics of the DLBCL cell line U-2946. According to gene expression data, the cell line clusters together with ABC DLBCL cell lines. The cell line carries an amplification of the antiapoptotic S1 FigA-E) Immunohistochemical stainings of lymphoma cells. Positive: BCL6 (A), CD10 (B), CD20 (C), CD79a, FOXP1, MUM1 , MYC (D), p53 and negative: BCL2 (E), CD3, CD5, CD30, cyclin D1. (F) May-Grünwald-Giemsa stain of cell line U-2946.(TIF)Click here for additional data file.S2 FigCells were stained with Abs against the antigens indicated and analyzed by flow cytometry.(TIF)Click here for additional data file.S3 FigThe 47 genes with strongest expression differences between U-2946 and median of 55 B-lymphoma cell lines in relation to variance –highest differences on the left. Data base on expression array analyses. Red dots, cell line U-2946.(TIF)Click here for additional data file.S4 FigBCL2, MAP2K3 and MCL1. The bars indicate means with standard deviation (n = 3). NC-NC as diploid reference cell line for genomic PCR.Quantitative genomic PCR (upper) and qRT-PCR (lower) detecting correlation between amplification and RNA expression in (TIF)Click here for additional data file.S5 FigMCL1 inhibitor A-1210477 (7.5 μM) inhibits growth of the MCL1pos/BCL2neg cell line U-2946, but has no effect on the MCL1pos/BCL2pos cell line U-2932 –neither alone nor together with suboptimal doses of the BCL2 inhibitor ABT-263 (50 nM). The bars indicate means with standard deviation (n = 3).3H-thymidine uptake after 48 h. The (TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 TableOutliers are listed according to chromosomal position. Note the perfect correlation between genomic imbalances and expression in cell line U-2946, 6/6 hemiploid genes (black and bold) being repressed, 5/5 amplified genes (red and bold) being overexpressed. Up, higher than average; down, lower than average.(XLSX)Click here for additional data file.S3 TableAmplified genes on 1q21.3 are listed from centromere to telomere, genes on 17p11.2 from telomere to centromere. Bold: strongly expressed genes as assessed by expression array analysis.(XLSX)Click here for additional data file.


NOD1 has also been suggested to be a tumor suppressor gene in a model of estrogen receptor-dependent breast cancer. Contrarily, NOD2 polymorphisms are associated with higher risk of breast cancer, with no tumor suppressor role being reported. To better delineate this issue, we investigated NOD1 and NOD2 expression in a panel of breast cancer cell lines, as well as their potential impact in breast tumorigenesis based on in vitro assays.Immune-related pathways have been frequently associated to tumorigenesis. NOD1 and NOD2 are innate immune receptors responsible for sensing a subset of bacterial-derived components, and to further translate these pathogenic signals through pro-inflammatory and survival pathways. NOD1 and NOD2 have been further associated with tumorigenesis, particularly in gastrointestinal cancers. NOD1 expression and the lowest NOD2 expression in our panel. Therefore, we investigated whether NOD1 and/or NOD2 might act as a tumor suppressor in this cell model. Our studies indicate that overexpression of either NOD1 or NOD2 reduces cell proliferation and increases clonogenic potential in vitro. Elucidation of NOD1 and NOD2 effects on tumor cell viability and proliferation may unveil potential targets for future therapeutic intervention.The highly invasive Hs578T breast cell line presented the second highest  Breast cancer is the malignancy with the highest incidence in women worldwide, accounting for 29% of all diagnosed cancers in females . DespiteBreast cancer is classified according to immunohistological detection of protein markers, including receptors for estrogen (ER), progesterone (PR), androgen (AR), and the amplified HER2  receptor . Approxid-glutamyl-meso-diaminopimelic acid) and MDP (muramyl dipeptide), respectively , where Δt is the duration of cell proliferation  in hours, and N0 and Nt are the respective numbers of cells at the beginning and end of this period , 20.2 cells from each population were cultured in 9.5 cm2 wells (6-well plate) for 12 days. Colonies were counted after fixation  and staining . Statistical analysis was performed using two-tailed, unpaired, student’s t-test  (Graphpad Prism V6).2 × 104 cells from each population were seeded in 1.9 cm2 wells (24-well plate) previously covered with 0.5 mL DMEM growth medium containing 0.6% agar. After seeding, a layer of 0.5 mL 0.3% agar DMEM was added and allowed to gellify before addition of 0.5 mL DMEM per well. Cultures were maintained for 14 days. Colonies were fixed in 4% formaldehyde and counted. Statistical analysis was performed using two-tailed, unpaired, student’s t-test  (Graphpad Prism V6).1 × 10NOD1 and NOD2 genes in a panel of breast cancer-derived cell lines, including estrogen-positive (MCF-7 and ZR-75-1) and estrogen-negative  cell lines. We found that NOD1 and NOD2 expression varies among these cell lines, with no clear pattern discriminating estrogen receptor-positive or negative groups. Interestingly, the Hs578T cell line presented the highest expression of NOD1 and the lowest expression of NOD2 in our panel. Due to its specific characteristics, namely, its tumorigenic potential and TNBC origin [NOD1 or NOD2 in Hs578T cells for further functional studies.Figure C origin , we deciGFP, displayed NOD1 and NOD2 expression levels similar to those of the wild-type  Hs578T cell line  and GFP-only control cells . Control populations (wild-type and GFP-transduced cells) displayed statistically identical proliferative rates, with doubling times of 24.9 and 23.9 h, respectively. Intriguingly, the Hs578T/NOD2 population displayed the lowest growth rate, even when compared to Hs578T/NOD1 cells   when compared to the controls. This effect was further enhanced by treatment with 5 μg/mL γ-tri-DAP, a NOD1-specific agonist . Hs578T/NOD2 displayed a non-statistical tendency for increased number of colonies in the absence of any treatment, and a statistically significant increase in colony number upon treatment with 5 μg/mL MDP, a NOD2-specific agonist   [Our in vitro colony formation assays indicated an increased viability and cell growth rate in adherin) , ICAM1 (adherin)  and VCAMadherin) .Given the incidence and high mortality rate of TNBC, decoding sub-pathways through which NOD receptors modulate cell proliferation may offer potential new targets for future therapeutic interventions.NOD1 and NOD2 amounts are usually evaluated by RT-qPCR [A more conclusive assessment of NOD1/2 levels could benefit from western blot detection. However, due to their typically limited protein levels in a vast number of cell models, endogenous  RT-qPCR , 25, 26. RT-qPCR .NOD1 and/or NOD2 would provide a valuable functional corroboration model to complement our overexpression results. Also, determining the sensitivity to apoptotic induction in our overexpressing populations could allow a better understanding of the pathways participating in the cellular effects observed.Additionally, generating Hs578T populations with null expression of


BRCA1/2 mutant tumors , such as disruptive gains in PTEN or FANCD2 and/or losses of ATM, BAP1, BARD1 or CHEK2, were susceptible to talazoparib-induced reduction of cell viability . Consistent with their high sensitivity to talazoparib, MG63 and ZK-58 cells scored positive in a DNA-based measure of genomic instability (i.e. homologous recombination deficiency (HRD)-loss of heterozygosity (LOH) score). In contrast, U2OS cells that carry a heterozygous BRCA2 mutation and therefore most likely have one intact BRCA2 allele left proved to be resistant to talazoparib. Furthermore, we identified TMZ as the most potent chemotherapeutic drug together with talazoparib to synergistically reduce cell viability, as confirmed by calculation of combination index (CI) values, and to suppress long-term clonogenic survival. Mechanistically, talazoparib and TMZ cooperated to induce apoptotic cell death, as demonstrated by activation of BAX and BAK, loss of mitochondrial membrane potential (MMP), caspase activation, DNA fragmentation and caspase-dependent cell death. Genetic silencing of BAX and BAK or pharmacological inhibition of caspases by zVAD.fmk significantly rescued OS cells from talazoparib/TMZ-induced apoptosis. These findings have important implications for the development of novel treatment strategies using PARP inhibitors alone or together with chemotherapy in a subset of OS with features of BRCAness.We recently discovered mutation signatures reminiscent of BRCA deficiency in the vast majority of a set of primary osteosarcomas (OS). In the current study, we therefore investigated the sensitivity of a panel of OS cell lines to the poly(ADP)-ribose polymerase (PARP) inhibitor talazoparib alone and in combination with several chemotherapeutic drugs (i.e. temozolomide (TMZ), SN-38, doxorubicin, cisplatin, methotrexate (MTX), etoposide/carboplatin). Here, we identified an association between homologous recombination (HR) repair deficiency and the response of OS cell lines to talazoparib. All OS cell lines with molecular features characteristic of  BRCA1/2 mutations or to mutations in HR pathway genes, such as FANCD2 [BRCA1 or BRCA2 as well as BRCAness have previously been reported to confer sensitivity towards PARP inhibitors, since ineffective HR repair induces a dependency on base excision repair to cope with DNA damage [OS is the most common primary bone cancer in children and young adults . There is FANCD2 . Loss-ofA damage . Thus, bA damage , 6.Currently, several PARP inhibitors  are being investigated in clinical trials . In 2014L and MCL-1), the proapoptotic multidomain BCL-2 proteins BAK and BAX and the proapoptotic BH3-only domain proteins [Programmed cell death such as apoptosis plays an important role in the response of tumors to cytotoxic therapies, as most cytotoxic therapies exert their antitumor activity by engaging programmed cell death . Apoptosproteins .In the current study, we aimed i) to investigate the vulnerability of a panel of OS cell lines to the PARP inhibitor talazoparib, ii) to explore the antitumor activity of talazoparib in combination with chemotherapeutics and to identify the most potent synergistic drug combinations and ii) to elucidate the molecular mechanisms of synergy with a specific focus on cell death pathways.50 values in the nanomolar range  , whereas U2OS cells remained largely resistant   towards treatment with the PARP inhibitor talazoparib. According to the observed response pattern among the OS cell lines, we defined three subgroups. MG63 and ZK-58 cells displayed the highest sensitivity towards talazoparib with IC) Figure . SaOS-2,) Figure .BAP1, FANCA and FANCD2  in four of the tested OS cell lines  that we defined as highly synergistic , intermediately synergistic  and weakly synergistic . In contrast, we found no consistent synergistic interaction of talazoparib with the tested anticancer drugs in U2OS cells, as CI values showed no synergism (CI >0.9) and/or Fa values were below 0.3 with the exception of a few drug combinations Figure .For TMZ in combination with talazoparib we detected a highly synergistic drug interaction in all four responding OS cell lines , as indicated by CI values < 0.2 and Fa values > 0.7 Figure . For SN-Since we identified MG63 and ZK-58 cells to be most sensitive to treatment with talazoparib alone Figure  and in cTo better understand the molecular mechanisms of cell death underlying the synergistic antitumor activity of talazoparib/TMZ combination treatment, we monitored the kinetics of cell death. Talazoparib acted in concert with TMZ to increase DNA fragmentation in a time-dependent manner in both cell lines with a significant increase in cell death upon talazoparib/TMZ cotreatment at 24 hours in ZK-58 cells and at 48 hours in MG63 cells Figure . To moniTo explore whether the addition of talazoparib would potentiate the cytotoxicity of TMZ also in non-malignant cells, we tested the effect of TMZ in the presence and absence of talazoparib on human peripheral blood lymphocytes (PBLs) that were freshly isolated from two different donors. We used PBLs, as hematological toxicity has been described for talazoparib . NeitherNext, we asked whether talazoparib and TMZ cooperate to activate BAK and BAX, two multidomain proapoptotic BCL-2 proteins that control MOMP . To addrBRCA1/2 mutations, including disruptive gains in PTEN and FANCD2 and/or losses of ATM, BAP1, BARD1, FANCA or CHEK2, although none of the tested OS cell lines showed a bi-allelic BRCA1/2 mutation [BRCA2 mutation [BRCA2 allele left.Using exome sequencing, we recently identified mutation signatures characteristic of BRCA deficiency in OS , indicatmutation . In contmutation  and most50 values scored positive in HRD-LOH score, a DNA-based measure of genomic instability [50 values for talazoparib (i.e. SaOS-2 and MNNG-HOS cells) were borderline, respectively positive by HRD-LOH score. Testing of talazoparib monotherapy in the pediatric preclinical testing program has previously shown minimal in vivo activity against OS; however, the BRCAness status of OS was not reported in that study [Importantly, the susceptibility of OS cell lines to talazoparib broadly correlated with their degree of genomic instability, as MG63 and ZK-58 cells that exhibited high sensitivity to talazoparib with nanomolar ICtability . In contat study .PTEN, ATM, BAP1, BARD1, CHEK2, FANCA and FANCD2 have been shown to exert pivotal functions in HR repair [PTEN function has previously been described to cause genomic instability [PTEN-deficient cells were found to be sensitive to the PARP inhibitor olaparib [ATM-deficient lymphoid tumor cells have shown vulnerability to olaparib in vitro and in vivo [R repair . Loss oftability , and PTEolaparib . Also, A in vivo . Further in vivo . CHEK2-m in vivo . Ubiquit in vivo  and the  in vivo . Thus, lFurthermore, among the tested chemotherapeutic drugs we identified TMZ to be most potent for combination treatment with talazoparib in OS, which was confirmed by highly synergistic CI values and by a large fraction of cells affected. In addition, talazoparib and TMZ acted together not only to reduce cell viability, but also to trigger cell death and to suppress long-term clonogenic survival. We and other groups previously demonstrated that the DNA-damaging agent TMZ is particularly suitable for PARP inhibitor-based combination treatment, yielding highly synergistic drug interaction in Ewing sarcoma cells –30. DepeOur molecular studies revealed that the synergistic cytotoxicity of talazoparib and TMZ in OS cells involves the induction of apoptotic cell death. First, we showed that talazoparib/TMZ cotreatment triggered DNA fragmentation and loss of MMP as characteristic markers of apoptotic cell death. Second, talazoparib/TMZ-stimulated activation of BAK and BAX contributed to the induction of apoptosis, since genetic silencing of BAK and BAX significantly reduced talazoparib/TMZ-induced cell death. Third, talazoparib and TMZ acted together to activate caspases, which were required for apoptosis, as the caspase inhibitor zVAD.fmk significantly rescued talazoparib/TMZ-mediated cell death. While our data showing that zVAD.fmk as well as BAK/BAX silencing significantly reduced talazoparib/TMZ-induced cell death demonstrated that caspases and BAK/BAX activation contributed to talazoparib/TMZ-induced apoptosis, additional mechanisms are also likely to be involved, since the protection by zVAD.fmk or BAK/BAX knockdown was only partial.BRCA1/2 mutations or the signature of BRCAness such as ovarian cancer, prostate cancer, pancreatic cancer, glioblastoma, and soft-tissue sarcomas [BRCA1/2-mutated ovarian cancer and castration-resistant prostate cancer [BRCA deficiency [in vivo model of OS. Since the addition of talazoparib did not increase the cytotoxicity of TMZ on non-malignant PBLs, there might be a therapeutic window for the incorporation of talazoparib into treatment protocols for OS. Selective synthetic lethality of the talazoparib/TMZ combination treatment in OS cells might be explained by the fact that cancer cells but not normal cells harbor HR repair deficiency. The doses of talazoparib and TMZ that caused synergistic induction of cell death in our study correspond to clinically achievable plasma concentrations of those drugs , underlOS cell lines  were kindly provided by M. Nathrath  and authenticated by STR profiles. Cells were maintained in RPMI 1640 medium , supplemented with 10% fetal calf serum (FCS), 1% penicillin/streptomycin, 1 mM sodium pyruvate . Cell lines were regularly tested for mycoplasma contamination to ensure that experiments were performed only with mycoplasma-free cell lines. Talazoparib was obtained from Selleckchem . TMZ, SN-38, cisplatin, doxorubicin and etoposide were purchased from Sigma-Aldrich , MTX and carboplatin were purchased from Medac , zVAD.fmk from Bachem . Chemicals were purchased from Sigma-Aldrich or Carl Roth  unless otherwise indicated.Apoptosis was determined by flow cytometric analysis  of DNA fragmentation of propidium iodide (PI)-stained nuclei as described previously . Cell viWestern blot analysis was performed as described previously  using thFor determining caspase activity cells were treated as indicated and caspase activity was assessed by caspase-3/7 staining according to the manufacturer's instructions for CellEvent Caspase-3/7 Green Detection Reagent  using ImageXpress Micro XLS system .Cells were reversely transfected with a combination of targeting siRNAs  using Lipofectamine RNAiMAX reagent  and Opti-MEM medium . After six hours of incubation with transfection solution, the medium was changed and cells recovered for 48 hours before drug treatment.BAK and BAX activation was determined by immunoprecipitation of active conformation by specific antibodies. Briefly, cells were lysed in CHAPS lysis buffer . 500 μg protein was incubated overnight at 4°C with 8 μg mouse anti-BAX antibody  or 0.5 μg mouse anti-BAK antibody  and 10 μl pan mouse IgG Dynabeads , washed with CHAPS lysis buffer and analyzed by Western blotting using rabbit anti-BAX NT antibody  or rabbit anti-BAK antibody (BD Biosciences). Loss of MMP was assessed by JC-1 staining according to the manufacturer's instructions (ThermoFisher scientific).For PBL isolation, blood was drawn from two independent donors and leukocytes were isolated using density centrifugation. Afterwards, leukocytes were treated with indicated concentrations of talazoparib and/or TMZ and cell death was assessed directly after PI staining as described previously .Affymetrix CytoscanHD array data processed with Nexus 8.0 software such that only segments with copy-number changes larger than 100 Kb and with at least 21 probes were considered for analysis using a public algorithm. In brief, HRD-LOH score requires at least 15 sub-chromosomal LOH segments of a size exceeding 15 Mb, which do not affect the whole chromosome and are at least 10 Mb apart. Borderline cases were tumors with 14 LOH events, while negative cases had 13 or less LOH events.Statistical significance was assessed by Student's t-Test  using Microsoft Excel . Drug interactions by CI values and Fa were analyzed by the Chou-Talalay method described by Chou , 35 usin


Nullomer peptides are the smallest sequences absent from databases of natural proteins. We first began compiling a list of absent 5-amino acid strings in 2006 (1). We report here the effects of Nullomer-derived peptides 9R, 9S1R and 124R on the NCI-60 panel, derived from human cancers of 9 organs , and four normal cell lines .NCI-60 cancer cell panel and four normal cell lines were cultured in vitro in RPMI1640 supplemented with 10% Hyclone fetal bovine serum and exposed for 48 h to 5 μM, 25 μM and 50 μM of peptides 9R, 9S1R and 124R. Viability was assessed by CCK-8 assay. For peptide ATP depletion effects, one cell line representing each organ in the NCI-60 panel, and four normal cell lines were exposed to 50 μM of peptides 9R, 9S1R and 124R for 3 h. The ATP content was assessed in whole cells, and their supernatants.Peptides 9S1R and 9R are respectively lethal to 95 and 81.6% of the 60 cancer cell lines tested. Control peptide 124R has no effect on the growth of these cells. Especially interesting the fact that peptides 9R and 9S1R are capable of killing drug-resistant and hormone-resistant cell lines, and even cancer stem cells. Peptides 9R and 9S1R have a broader activity spectrum than many cancer drugs in current use, can completely deplete cellular ATP within 3 h, and are less toxic to 3 of the 4 normal cell lines tested than they are to several cancers.Nullomer peptides 9R and 9S1R have a large broad lethal effect on cancer cell lines derived from nine organs represented in the NCI-60 panel. This broad activity crosses many of the categorical divisions used in the general classification of cancers: solid vs liquid cancers, drug sensitive vs drug resistant, hormone sensitive vs hormone resistant, cytokine sensitive vs cytokine non sensitive, slow growing vs rapid growing, differentiated vs dedifferentiated cancers. Furthermore peptides 9R and 9S1R are lethal to cancer stem cells and breast canrcinosarcoma.The online version of this article (doi:10.1186/s12885-017-3514-z) contains supplementary material, which is available to authorized users. Nullomers are the shortest string of monomers absent from a species or species group. In 2007 Hampikian and Andersen  publisheThe current treatment of cancer cannot address many aspects of human tumors driven by their cellular heterogeneity. For example, some cells in a tumor may be designated drug resistant  cancer c2. Passaging of cells was done at 80% confluence after a PBS wash, cells were treated with Trypsin-EDTA 0.25% (Invitrogen).The NCI-60 panel of cancer cells was purchased from Development Therapeutics of the NCI. Cells were thawed in a water bath at 37 °C, and collected by centrifugation at room temperature. For maintenance, the cell were grown in RPMI 1640 supplemented with 10% Hyclone fetal bovine serum (FBS), and 1× penicillin streptomycin  at 37 °C and 5% CONormal cell lines were purchased from the American Type Culture Collection . Human endothelial cell line HUVEC (ATCC CRL-1730) was grown in the ATCC formulated F-12 K medium with 0.1 mg/ml Heparin, 0.05 mg/ml endothelial cell growth supplement and 10% FBS. Human skin fibroblast BJ (ATCC CRL-2522) was grown in the ATCC formulated Eagle’s Minimum Essential Medium supplemented with 10% FBS. Human prostate epithelial cell line RWPE-1 (ATCC CRL-11609) was grown in the Keratinocyte Serum Free Medium  supplemented with 0.05 mg/ml bovine pituitary extract (BPE) and 5 ng/ml human recombinant epidermal growth factor (EGF). Human colon epithelial cell line FHC (ATCC CRL-1831) was grown in the ATCC formulated DMEM-F12 supplemented with 10 mM HEPES , 10 ng/ml cholera toxin, 0.005 mg/ml insulin, 0.005 mg/ml transferrin, 100 ng/ml hydrocortisone and 10% FBS. Only early cell passages were used for all experiments. No human subjects  has been used in the research reported in this manuscript. All cancer ell lines were obtained from the NCI depository, and all normal cells from the ATCC.Peptides 9R, 9S1R and 124R were made by Pierce Biotechnology . HPLC purified peptides (purity >98%) were delivered in 0.5 mg/tube (lyophilized) and stored at −20 °C. Peptides were solubilized in 1 M Trehalose to produce 50 mM stock solutions. Only freshly prepared solutions were used to treat cells. Trehalose final concentration in all reactions was 1 mM . Sodium Azide (Sigma Aldrich) was solubilized in water at 100 mM.2 incubator. After the 4 h incubation, plates were monitored by a plate reader  at an absorbance of 450 nm. Plate readings were exported to Microsoft Excel and GraphPadPrism software. All the wells were analyzed in triplicates. The statistical analysis was done with GraphPadPrism*. The survival index (SI) is the CCK-8 reading ratio of peptide-treated to untreated cells , multiplied by 100 to yield an index of the percent surviving. A dose response using several concentrations of the peptides  was used for all cancer and normal cell lines. The SI was used for screening in this study at the 50 μM concentration of each peptide.Three thousand–Five thousand normal or cancer cells/well were seeded in 96-well plates. After 24 h incubation, peptides 9R, 9S1R and 124R were added to the wells, with untreated cells as a control. After 48 h exposure to 5 μM, 25 μM, or 50 μM peptides, cell viability was quantified by the addition of 10 μl of cell counting kit  to each well, incubated for 4 h at 37 °C in a 5% COCancer cell lines (one cell line for each organ represented in the NCI-60 panel) and normal cell lines (3000-5000/well) were seeded in 96 well microplates (white plates from Nunc to block luminescence bleeding between the wells), and allowed to attach for 24 h. Then cells were incubated for 3 h with peptides , or 100 mM sodium azide as a control. After 3 h, 10 μl of a single reagent from Cell Titer Glow™ (Promega) was added to cells. For the cellular supernatant ATP content, the supernatant of each well was transferred to a new microplate and 10 μl of a single reagent from Cell Titer Glow™ (Promega) was added to each supernatant. Complete reagent mixing in 96 wells plates required gentle orbital shaking for 2–10 min. The plate reading was taken by a SynergyMx plate reader. Plate readings were exported to Microsoft Excel and Graph-PadPrism software. All the wells were analyzed in triplicates. The statistical analysis was done with the GraphPadPRISM*.In a previous work  peptide The three peptides were prepared as previously published: first solubilized in 1 M Trehalose and then diluted to three doses, 50 μM, 25 μM, and 5 μM final concentration. Cells were incubated for 48 h, and then their viability was assessed by CCK-8 assay . Cell liAs shown in ). Comparison of Nullomer derived peptides (9R and 9S1R) sensitivity with current anticancer drugs approved by the FDA. (XLSX 13 kb)Additional file 4:Results 2.p<0.05,**p <0.01, ***p<0.001. (PDF 276 kb)Effect of 50 μM, 25 μM and 5 μM Nullomer peptides on normal cell growth. Cells (3000-5000 cells/well) were seeded in 96-well plates. After 24 h incubation, peptides 9R, 9S1R and 124R were added to the wells, with untreated cells as control. After 48 h exposure to the peptides, cell viability was quantified by the addition of 10 μl of cell counting kit  to each which was then incubated for 4 h at 37 °C in a 5% CO2 incubator. After the incubation, the plates were monitored by a microplate reader (BioTek) at an absorbance of 450 nm. (A) Endothelial cell line, (B) Skin fibroblast, (C) Prostate cell line, (D) Intestinal cell line. Results are as mean ± SE (standard error) of three different experiments. NS, not significant. *Additional file 5:Table 3ATP depletion of cancer and normal cell lines after treatment with 9R, 9S1R and 124R. ATP content: expressed as % of control. Three-hour incubations with 50uM peptide. (XLSX 9 kb)


Meningioma tumors arise in arachnoid membranes, and are the most reported central nervous system (CNS) tumors worldwide. Up to 20% of grade I meningioma tumors reoccur and currently predictive cancer stem cells (CSCs) markers for aggressive and drug resistant meningiomas are scarce.Meningioma tissues and primary cell lines were investigated using whole transcriptome microarray analysis, immunofluorescence staining of CSCs markers , and drug treatment with cisplatin or etoposide.Reelin (RELN), Calbindin 1 (CALB1) and Anterior Gradient 2 Homolog (AGR2). Immunofluorescence staining for four tissues confirmed stemness variation in situ. Biological characterization of fifteen meningioma primary cell lines concordantly separated cells into two functionally distinct sub-groups. Pleomorphic cell lines (NG type) grew significantly faster than monomorphic cell lines (G type), had a higher number of cells that express Ki67, and were able to migrate aggressively in vitro. In addition, NG type cell lines had a lower expression of nuclear Caspase-3, and had a significantly higher number of CSCs co-positive for CD133+ Sox2+ or AGR2+ BMI1+. Importantly, these cells were more tolerant to cisplatin and etoposide treatment, showed a lower level of nuclear Caspase-3 in treated cells and harbored drug resistant CSCs.Unsupervised hierarchical clustering of six meningioma samples separated tissues into two groups. Analysis identified stem cells related pathways to be differential between the two groups and indicated the de-regulation of the stem cell associated genes Collectively, analyses of tissues and primary cell lines revealed stem cell associated genes as potential targets for aggressive and drug resistant meningiomas.The online version of this article (doi:10.1186/s12935-017-0441-7) contains supplementary material, which is available to authorized users. They acplicated . Genomicr (KLF4) –11.Deregulated molecular pathways identified based on bulk tumor analyses, have been utilized to predict prognosis and help determine appropriate targeted therapy . HoweverA crucial component for the development of targeted therapy is the availability of live tumor models . Early pPreviously we published the gene expression profiles for meningioma patients tissues collected for our cohort . For thiTumor tissue RNA were isolated and processed for microarray and gene expression analysis as previously described , and datg) for 3 min at room temperature, re-suspended in DMEM-F12 (Gibco), 10% FBS (HyClone), 100 U/mL penicillin, and 100 µg/mL streptomycin, and maintained in standard humidified incubators at 5% CO2. After 24 h, non-adherent cells were removed by washing with Phosphate Buffered Saline  and the adherent cells were cultured until they reached confluence, then split (1:2) every 12–30 days dependent on the cell line. For each of tumors Jed62_MN and Jed79_MN, tissues were divided into four representative portions and each was placed into a 25 cm2 tissue culture flasks containing a type of media  with 10% FBS, or DMEM high glucose concentrations of 4500 mg/L (Gibco) with 5% FBS, or DMEM low glucose concentrations of 1000 mg/L (Gibco) with 10% FBS).Meningioma specimens collected between February 2013 and December 2015 were obtained within 30 min of tumor removal and distributed into both RNAlater solution (Life Technologies) for RNA/DNA extraction and cell culture using mechanical and enzymatic dissociation methods . For celCell cultures were observed for a period of 4 weeks with an inverted microscope (Leica DMI6000) to determine their morphologies. Twenty images of cells were taken at 10× magnifications each week using a digital microscope camera (Leica DFC425). The average number of counted cells per cell line exceeded 500 per week. The weekly morphology percentages were calculated as the total number of cells displaying a particular morphology, divided by the total number of cells counted. Morphological profiles for cells were confirmed by immunofluorescence staining with mouse anti-Vimentin . All cell lines and images were reviewed independently at least twice to confirm counts.T test or Chiχ2 values were retrieved using Statistical Package for the Social Sciences (SPSS) Graduate Pack 21.0.For cell lines, cells were seeded on chamber slides and fixed with 4% Paraformaldehyde (PFA). For in situ staining, fresh frozen tissues were cut at 4 μM sections, then fixed with 4% Paraformaldehyde (PFA). The antibodies used for standard immunostaining were rabbit anti-Caspase-3 , mouse anti-CD133 , rabbit anti-Sox2 , mouse anti Sox2 , mouse anti Nestin , rabbit anti-Ki67 , mouse anti-Vimentin , rabbit anti- Frizzled 9 , mouse anti-BMI1 proto-oncogene, polycomb ring finger (BMI1) antibody , and rabbit Anti-Anterior Gradient 2 antibody . For secondary goat antibodies, 488 anti-mouse  and 555 anti-Rabbit  were used. Pictures were taken at 20× magnifications using Leica DMI6000 microscope and Leica DFC425 camera. Photos were edited in Photoshop 7.0 and signal levels were compared to negative controls of secondary only. 50 values for cisplatin and etoposide, preliminary growth inhibition experiments were completed for three cell lines that had fast growth capability ,  or etoposide (in DMSO) at increasing concentrations of drug. Twelve days following treatment, cells were fixed with 4% paraformaldehyde (PFA) and stained with crystal violet. 10% acetic acid was added to cells and absorbance was measured at 590 nm. The survival fractions  were calculated. All assays were performed in triplicate.To determine ICSpalt-Like Transcription Factor 4 (SALL4) (fold change: 3.570) and Wingless-type MMTV Integration Site Family, member 6 (WNT6) (fold change: 2.340) were deregulated. For individual genes, the top four up-regulated molecules in group 1 tumors were Reelin (RELN) , Calbindin 1 (CALB1) , Killer Cell Lectin-Like Receptor Subfamily C, Member 4 (KLRC4) , and Anterior Gradient 2 Homolog (AGR2) . The top four down-regulated molecules in group 1 tumors were Cytochrome P450, Family 4 Subfamily B Polypeptide 1 (CYP4B1) , F-Box Protein 32 (FBXO32) , Mesenteric Estrogen-Dependent Adipogenesis (MEDAG) , and Serpin Peptidase Inhibitor, Clade A , Member 3 (SERPINA3) . Expression for the stem cell related markers Nestin, CD133, Sox2, AGR2 and the known oncogenic BMI1 protein, investigated using in situ tissue immunofluorescence for four additional tumors , support the observed gene expression trends, Additional file Table Consistent with differences observed in the analyzed meningioma tissues, newly initiated primary cell lines appeared to separate into two main groups with differences detect for morphological profiles, proliferation, and invasion in vitro. Following cell lines initiations, distinct cell shapes were observed in all cells lines, Fig. 2: 9.681, P < 0.01; Ki67+ Nestin-, χ2: 9.953, P < 0.01), Fig. 2: 12.156, P < 0.001). The expression of the recently associated stem cell protein AGR2, which was detected earlier to be deferentially expressed in our tissues, was also analyzed in NG type cell lines. Three NG type cell lines were co-stained to detect AGR2 with the oncogenic stem cell associated protein BMI1, Fig. Since stem cell pathways appeared to be differential in tissues, primary G type and NG type cell lines were also investigated for differences in stemness. Immunofluorescence was used to co-stain cells for stem cell markers CD133 Sox2, Nestin Ki67, or Vimentin Frizzled 9 in four cell lines . Interestingly, CSCs stained co-positive for all combinations were present in all cell lines, Fig. To address whether the sub-groups had different drug response, four NG and four G type cell lines were treated with cisplatin (50 µM) or etoposide (100 µM). NG type cell lines survived treatment significantly better than G type cell lines , Fig. To test whether meningioma CSCs survived treatment with cisplatin or etoposide, 12 days following treatment, treated Jed49_MN cells (NG type) were stained with CD133 and Sox2 or Nestin and Ki67. Cells co-positive for CD133+ Sox2+ or Nestin+ Ki67+ were detected following treatment with cisplatin or etoposide, Fig. We examined multiple bio-parameters for meningiomas through analysis of both eight tissue samples and 15 primary cell lines. Whole transcriptome microarray analysis for six tumor samples identified two groups of meningiomas with differential stem cell related pathways and a number of novel stem cell related biomarkers, including AGR2. Biological characterization of primary meningioma cell lines also sub-grouped cell lines into two main types; G type with predominantly glial like cells that grow slowly and were less viable, and NG type that had a mix of neuronal like and glial like cells, grow faster and showed invasive properties. In addition, NG type cells had a significantly higher percentage of cancer stem cells that express CD133+ Sox2+ or AGR2+ BMI1+, and were more tolerant to treatment with the chemotherapeutic agents cisplatin and etoposide. Importantly, drug treatment of NG type cells resulted in the enrichment of CD133+ Sox2+ or AGR2+ BMI1+ cells.Gene expression profiling has been employed to grade, identify and characterize various sub-types within tumors. In this study, unsupervised hierarchical clustering of tumor samples divided tumors into two groups that were primarily different in their stem cell pathways. In addition, three of the top differentially up-regulated genes Reelin, Calbindin 1 and Anterior Gradient 2 Homolog, and two of the most down differentially regulated genes Cytochrome P450 and F-Box Protein 32 are associated with stemness –41. UnfoMost importantly, tumors that formed group 2, with low stem cell gene expression, generated G type cell lines that also had a lower expression of stem cell related markers, while tumors that showed high stem cell gene expression (including Jed45_MN) generated NG type cell lines that showed similar characteristics. This indicated that the generated primary cell lines do represent the essence of their corresponding tumors’ tissues, as previously shown with glioblastoma multiform tumors . Thus fuClear biological features, including morphology which is influenced by the repository of biochemical interactions flowing through the cell signaling system , separatPrevious work has shown the presence of cancer stem cells in meningioma cell lines –31, but Importantly, NG type cell lines showed a significantly higher tolerance of cisplatin and etoposide treatment compared with G type cell lines. Although etoposide has been traditionally thought of as a cell-cycle specific drug, recent data suggests a strong binding of the drug to chromatin and implicates a binding affinity for histone proteins, in particular histone 1, thus contributing to DNA damage regardless of the cell cycle status , 55. NotIn conclusion, analysis of patterns for cellular architecture, drug tolerance and associated resistance makers for primary cell lines in combination with genomic analysis of corresponding tissues of meningiomas, led to the functional sub grouping of meningiomas and the identification of novel stem cells related markers that appear to be associated with drug resistance and are likely to play a role in tumor aggressiveness. Importantly, this work highlights the need to use stem cells molecular markers and early in vitro analysis to classify functional variants of meningioma and predicts tumor aggressiveness and drug resistance.Additional file 1: Figure S1. Growth inhibition assays for A) Cisplatin and B) Etoposide for NG cell lines Jed38_MN, Jed45_MN and Jed49_MN.Additional file 2: Figure S2. H&E slides for studied variants including meningothelial  showing lobules of uniform eosinopholic cells with central nuclei and intranuclear inclusions forming abundant whorls with no evidence of atypia, necrosis or mitosis; fibroblastic  showing spindle cells with indistinct cell boundaries running in fascicle; transitional  with ratios of meningothelial to fibroblastic patterns ranging from 20:80 for Jed04_MN, 50:50 for Jed09_MN, 30:70 for Jed36_MN, and 40:60 for Jed38_MN; rhabdoid (Jed45_MN) showing hypercellular sheets with rhabdoid morphology  and necrosis; psammomatous (Jed43_MN) composed of whorled clusters of spindle cells with numerous psammoma bodies; and angiomatous (Jed12_MN) showing neoplastic growth in the form of nests and whorled of bland- looking polygonal cells with vascular component exceeding 50% of total tumor area and with no evidence of atypia, necrosis or mitosis. All images were taken at 20×.Additional file 3: Table S1. Differentially expressed genes between group 1  versus group 2 .Additional file 4: Table S2. Predicted differential pathways generated by Ingenuity Pathway Analysis Software. The transcriptome comparison was carried out for group 1  versus group 2 .Additional file 5: Figure S3. CSCs markers expression in situ. A) Images for immunofluorescence co-staining of stem cell markers CD133+Sox2+  or Nestin+Ki67+  and AGR2+ BMI1+  in low grade (Jed62_MN) and high grade (Jed45_MN) tumors. B) Mean percentages of co-positive cells. Error bars represent count errors between three independent regions within each tissue. All images were taken at 20×.Additional file 6: Figure S4. AGR2 co-expression with CSCs markers in situ. A) Images for immunofluorescence co-staining of stem cell markers Nestin+AGR2+, CD133+AGR2+, and Sox2+AGR2+  in low grade (Jed62_MN and Jed40_MN) and high grade (Jed49_MN and Jed45_MN) tumors. B) Mean percentages of co-positive cells. Error bars represent count errors between three independent regions within each tissue. All images were taken at 20×.Additional file 7: Figure S5. Average percentages of morphologies counts for Jed62_MN and Jed79_MN. Each tumor was divided into four portions that were grown in either DMEM-F12 +10% FBS (Blue line), or DMEM high glucose concentrations of 4500 mg/L (Gibco) +10% FBS (Grey line), or DMEM high glucose concentrations of 4500 mg/L (Gibco) + 5% FBS (Yellow line), or DMEM low glucose concentrations of 1000 mg/L (Gibco) + 10% FBS (Orange line). Horizontal accesses represent morphologies described in Figure Additional file 8: Figure S6. The expression of AGR2 in G type cell lines. A) Immunofluorescence images for two G Type cell lines (Jed39_MN and Jed40_MN). Images show cells co-stained positively for Vimentin+AGR2+ . B) Average percentages of cells positive for AGR2. Error bars represent errors between three independent counts.


Resistance formation after initial therapy response (acquired resistance) is common in high-risk neuroblastoma patients. YM155 is a drug candidate that was introduced as a survivin suppressant. This mechanism was later challenged, and DNA damage induction and Mcl-1 depletion were suggested instead. Here we investigated the efficacy and mechanism of action of YM155 in neuroblastoma cells with acquired drug resistance. The efficacy of YM155 was determined in neuroblastoma cell lines and their sublines with acquired resistance to clinically relevant drugs. Survivin levels, Mcl-1 levels, and DNA damage formation were determined in response to YM155. RNAi-mediated depletion of survivin, Mcl-1, and p53 was performed to investigate their roles during YM155 treatment. Clinical YM155 concentrations affected the viability of drug-resistant neuroblastoma cells through survivin depletion and p53 activation. MDM2 inhibitor-induced p53 activation further enhanced YM155 activity. Loss of p53 function generally affected anti-neuroblastoma approaches targeting survivin. Upregulation of ABCB1 (causes YM155 efflux) and downregulation of SLC35F2 (causes YM155 uptake) mediated YM155-specific resistance. YM155-adapted cells displayed increased ABCB1 levels, decreased SLC35F2 levels, and a p53 mutation. YM155-adapted neuroblastoma cells were also characterized by decreased sensitivity to RNAi-mediated survivin depletion, further confirming survivin as a critical YM155 target in neuroblastoma. In conclusion, YM155 targets survivin in neuroblastoma. Furthermore, survivin is a promising therapeutic target for p53 wild-type neuroblastomas after resistance acquisition (neuroblastomas are rarely p53-mutated), potentially in combination with p53 activators. In addition, we show that the adaptation of cancer cells to molecular-targeted anticancer drugs is an effective strategy to elucidate a drug's mechanism of action. YM155 (sepantronium bromide) was introduced as a transcriptional suppressor of survivin expression that displayed activity against a broad range of cancer types in preclinical models.3 However, further studies suggested that the YM155-induced inhibition of survivin expression may be a secondary effect downstream of YM155-induced DNA damage5 or associated with Myeloid Cell Leukemia 1 (Mcl-1) depletion.6Survivin, a member of the inhibitor of apoptosis protein (IAP) family, comprises a nodal protein implicated in a multitude of cellular pathways, including apoptosis and mitosis regulation, and is frequently found highly expressed in cancer cells, making it a potential target for anticancer therapies.rCDDP1000), doxorubicin (UKF-NB-6rDOX20), or vincristine (UKF-NB-3rVCR10 and UKF-NB-6rVCR10). Neuroblastoma is the most frequent solid extracranial pediatric cancer entity. About half of the patients are diagnosed with high-risk disease associated with overall survival rates below 50%, despite myeloablative therapy and differentiation therapy using retinoids.8 Although many neuroblastomas respond initially well to therapy, acquired drug resistance represents a major obstacle in clinical practice.8 Survivin had been previously shown to be a potential drug target in neuroblastoma.13 However, survivin had not been investigated as a therapeutic target in the acquired resistance setting in neuroblastoma prior to this study. Our principal findings are that survivin is a promising drug target in p53 wild-type neuroblastoma cells with acquired drug resistance and that YM155 impairs neuroblastoma cell viability in clinically achievable concentrations via survivin depletion. The drug-resistant cell lines displayed decreased sensitivity to YM155, with upregulation of the ATP-binding cassette (ABC) transporter ATP Binding Cassette Subfamily B Member 1  and downregulation of Solute Carrier Family 35 Member F2  as the major drug-specific resistance mechanisms and loss of p53 function as resistance mechanism that affects all approaches targeting survivin in neuroblastoma. In accordance with these findings, neuroblastoma cells adapted to YM155 displayed decreased levels of SLC35F2, increased levels of ABCB1, a p53 mutation, decreased levels of survivin, and decreased sensitivity to RNAi-mediated survivin depletion.Here we investigated the mechanism of action of YM155 in a panel consisting of the neuroblastoma cell lines UKF-NB-3 and UKF-NB-6 and their sublines with acquired resistance to cisplatin (UKF-NB-3rCDDP1000) or vincristine (UKF-NB-3rVCR10), as well as the UKF-NB-6 sublines resistant to doxorubicin (UKF-NB-6rDOX20) or vincristine (UKF-NB-6rVCR10), displayed substantially reduced YM155 sensitivity compared to the parental cell lines, resulting in IC50 values ranging from 5.32 nM (UKF-NB-3rCDDP1000) to 49.3 nM (UKF-NB-6rVCR10)  . There w6rVCR10) .14 Among the investigated cell lines, UKF-NB-3rVCR10, UKF-NB-6rDOX20, and UKF-NB-6rVCR10 display detectable ABCB1 activity  polymerase (PARP) cleavage  inhibition.of YM155  and p53 of YM155 , suggest4The anticancer mechanism of action of YM155 is under dispute. The compound was introduced as a suppressor of survivin expression. However, this mechanism was challenged and some studies suggested that the YM155-induced inhibition of survivin expression may be a secondary effect downstream of YM155-induced DNA damage.γ-histone H2A (γH2AX) levels indicating DNA double-strand breaks became only detectable after 24 h at YM155 concentrations of 10 nM or higher (γH2AX levels and Checkpoint Kinase 2 (Chk2) phosphorylation in UKF-NB-3 and UKF-NB-6 cells  not detected after 24 h treatment of p53-depleted UKF-NB-3 cells with YM155 concentrations of up to 25 nM , and that p53 is involved in mediating anti-neuroblastoma effects as a consequence of RNAi- or YM155-induced reduction of cellular survivin levels.rYM15520nM). UKF-NB-3rYM15520nM cells displayed a 497-fold increased resistance to YM155 (IC50: 303±8 nM) compared to parental UKF-NB-3 cells (IC50: 0.61±0.08 nM)  sensitized UKF-NB-3rYM15520nM cells to YM155 (IC50: 90.5±3.1 nM) but not to the level of parental UKF-NB-3 cells  . Moreove SLC35F2 , and har-3 cells .rYM15520nM cells also displayed reduced survivin levels (UKF-NB-3n levels  and redun levels . This furCDDP1000), doxorubicin (UKF-NB-6rDOX20), and vincristine (UKF-NB-3rVCR10 and UKF-NB-6rVCR10). The YM155 IC50 values ranged from 0.49 nM (UKF-NB-3) to 49.3 nM (UKF-NB-6rVCR10). This is in the range of therapeutically achievable plasma levels that were documented to reach about 50 nM.22 Notably, animal models indicated that the YM155 concentrations in tumor tissues are likely to be higher than those found in the plasma.23 Previously, YM155 IC50 values were reported to range from 3.2 to 11 nM in melanoma cells24 and from 2 to 50 nM in multiple myeloma cells.25 In a screen of 124 cell lines from 17 cancer entities, the YM155 IC50s ranged from 0.35 to >1000 nM.26 In a panel of 23 neuroblastoma cell lines, the YM155 IC50s ranged from 0.5 to >10 000 nM.10 This shows that the sensitivity to YM155-mediated survivin depletion may substantially vary even in cells from the same cancer type. Hence, it will be critical to understand which cancer cells are particularly sensitive to YM155 and survivin depletion in order to design tailored therapies.Here we investigated the efficacy of the survivin suppressant YM155 in a panel of neuroblastoma cell lines consisting of the V-Myc Avian Myelocytomatosis Viral Oncogene Neuroblastoma Derived Homolog (MYCN)-amplified neuroblastoma cell lines UKF-NB-3 und UKF-NB-6 and their sublines with acquired resistance to cisplatin , UKF-NB-6rDOX20 (from 11.80 to 1.35 nM), and UKF-NB-6rVCR10 (from 49.30 to 3.60 nM), but not to the level of the parental cell lines that displayed IC50 values of 0.49 nM (UKF-NB-3) and 0.65 nM (UKF-NB-6). Hence, additional resistance mechanisms appear to contribute to the decreased YM155 sensitivity that was observed in the drug-resistant neuroblastoma sublines in comparison to the respective parental chemosensitive cells.The parental cell lines UKF-NB-3 and UKF-NB-6 displayed a similar response to YM155 treatment. The YM155 IC50s were comparable , and YM155 induced survivin depletion and p53 induction in both cell lines. All investigated drug-resistant sublines of UKF-NB-3 and UKF-NB-6 showed substantially reduced YM155 sensitivity relative to the parental cell lines. Three mechanisms may explain the decreased YM155 sensitivity in the drug-resistant cancer cell lines (at least to a large degree): upregulation of ABCB1, downregulation of SLC35F2, and loss of p53 function. Upregulation of the ABC transporter ABCB1 had already been shown to affect the efficacy of YM155.15 The drug-resistant neuroblastoma cell lines UKF-NB-3rCDDP1000, UKF-NB-6rDOX20, and UKF-NB-6rVCR20 were characterized by reduced SLC35F2 expression relative to the respective parental cells. Hence, these decreased SLC35F2 levels are likely to contribute to the increased YM155 resistance observed in these cell lines when compared to the respective parental cell lines. This notion is further supported by our finding that SLC35F2 depletion increases the resistance of ABCB1-expressing neuroblastoma cells. Since SLC35F2 and ABCB1 affect intracellular YM155 concentrations, these represent drug-specific resistance mechanisms that may not be shared by other therapeutic strategies that target survivin.YM155 is transported by SLC35F2 into cells and lack of SLC35F2 has been associated with decreased cellular YM155 sensitivity.rVCR10 was the only drug-resistant cell line that did not display decreased SLC35F2 levels. However, ABCB1 inhibition did not resensitize UKF-NB-3rVCR10 cells to YM155 to the level of the parental UKF-NB-3 cell line. In contrast to the other investigated cell lines, UKF-NB-3rVCR10 harbors a C135F loss-of-function p53 mutation.18 Hence, loss of p53 function may impair the YM155 sensitivity of neuroblastoma cells. Indeed, p53 depletion resulted in decreased YM155 sensitivity of UKF-NB-3 and UKF-NB-6 cells. Moreover, the nutlin-3-resistant UKF-NB-3 subline UKF-NB-3rNutlin10μM that harbors a G245C loss-of-function p53 mutation18 displayed reduced sensitivity to YM155 relative to UKF-NB-3. Other reports had suggested that YM155 exerts its anticancer effects independently of the cellular p53 status.28 However, the relevance of the p53 status for cancer cell sensitivity to YM155 and survivin depletion may depend on the cellular context.UKF-NB-326 However, other studies suggested that YM155 may primarily induce DNA damage and that the reduction of survivin levels may rather be a consequence of DNA damage.4 In our study, however, survivin downregulation preceded the occurrence of DNA damage in response to YM155 treatment, suggesting that survivin depletion happens prior to DNA damage formation. In accordance, RNAi-mediated survivin depletion also resulted in the induction of DNA damage, demonstrating that DNA damage formation can be a consequence of the reduction of cellular survivin levels. Notably, we and others had previously shown that survivin depletion resulted in increased yH2AX foci detection following irradiation30 that is functionally related to a direct physical interrelationship of survivin with components of DNA damage repair pathways.32The anticancer mechanism of action of YM155 has been under dispute and may depend on the cellular background. YM155 was introduced as a compound that suppresses the expression of survivin.6 However, YM155 reduced cellular Mcl-1 levels at substantially higher concentrations than survivin, and Mcl-1 depletion did not affect neuroblastoma cell viability. The finding that Mcl-1 depletion does not impair neuroblastoma cell viability is in accordance with previous results demonstrating that Mcl-1 depletion sensitizes neuroblastoma cells to paclitaxel, but does not exert significant effects on neuroblastoma cell viability on its own.33 Hence, the anti-neuroblastoma effects of YM155 do not appear to be caused by Mcl-1 depletion. Finally, survivin depletion-mediated X-linked inhibitor of apoptosis (XIAP) degradation had been shown to contribute to the effects of YM155 against breast cancer cells.34 However, survivin depletion did not decrease XIAP levels in our models  and the resistance-associated changes were investigated. We detected a R248W p53 mutation as target-specific resistance mechanism, and decreased SLC35F2 and increased ABCB1 as drug-specific resistance mechanisms. UKF-NB-3rYM15520nM cells also displayed decreased survivin levels and sensitivity to RNAi-mediated survivin depletion relative to UKF-NB-3, further confirming that YM155 primarily exerts its anti-neuroblastoma effects through survivin depletion.Next, UKF-NB-3 cells were adapted to growth in the presence of YM155  was purchased from Selleck Chemicals via BIOZOL Diagnostica GmbH , vincristine and cisplatin from TEVA GmbH , doxorubicin from Cell Pharm , and verapamil from Sigma-Aldrich .39 UKF-NB-3 and UKF-NB-6 sublines with acquired resistance to cisplatin (UKF-NB-3rCDDP1000), doxorubicin (UKF-NB-6rDOX20), vincristine (UKF-NB-3rVCR10 and UKF-NB-6rVCR10), or nutlin-3 (UKF-NB-3rNutlin10μM) were established by continuous exposure to increasing drug concentrations as described previously39 and derived from the Resistant Cancer Cell Line collection (www.kent.ac.uk/stms/cmp/RCCL/RCCLabout.html).The MYCN-amplified neuroblastoma cell lines UKF-NB-3 and UKF-NB-6 were established from stage 4 neuroblastoma patients.All cells were propagated in Iscove's modified Dulbecco's medium supplemented with 10% fetal calf serum, 100 IU/ml penicillin, and 100 mg/ml streptomycin at 37°C. Cells were routinely tested for mycoplasma contamination and authenticated by short tandem repeat profiling.41, using the Lentiviral Gene Ontology (LeGO) vector technology (www.lentigo-vectors.de).42p53-depleted neuroblastoma cells or neuroblastoma cells showing high expression of ABCB1 were established as described previouslySLC35F2-depleted cells were established using the same technology. The shRNA sequences (5′ to 3′) were sh1 SLC35F2, CTCTTTCTGTTTGGCTATA; sh2 SLC35F2, GAGGAATACATCGTGAAGA; sh3 SLC35F2, CAGGGAGTGATGTATTGAT.41Cell viability was determined by the 3--2,5-diphenyltetrazolium bromide (MTT) dye reduction assay after 120 h incubation, modified as described previously.6 cells were resuspended in 200 μl resuspension buffer R including 2.5 μM siRNA. The electroporation was performed using two 20 ms pulses of 1400 V. Subsequently, the cells were transferred into cell culture plates or flasks, containing prewarmed cell culture medium.Transient depletion of SLC35F2 was achieved using synthetic siRNA oligonucleotides (ON-TARGETplus SMARTpool) from Dharmacon . Non-targeting siRNA (ON-TARGETplus SMARTpool) was used as negative control. Cells were transfected by electroporation using the NEON Transfection System  according to the manufacturer protocol. Cells were grown to 60–80% confluence, trypsinized, and 1.2 × 10β-actin , SLC35F2 , ABCB1, Chk2, phosphorylated Chk2 (Thr68), Baculoviral IAP repeat containing 3 (cIAP-2), histone H2AX, phosphorylated histone H2AX (Ser139) (γH2AX), myeloid cell leukemia sequence-1 (Mcl-1), p21, cleaved PARP, XIAP , p53 , survivin and Baculoviral IAP repeat containing 2 (cIAP-1) . Protein bands were visualized by laser-induced fluorescence using infrared scanner for protein quantification .Cells were lysed using Triton-X-100 sample buffer, and proteins were separated by SDS-PAGE. Detection was done by using specific antibodies against A DNA damage kit that determines apurinic/apyrimidinic sites as indicators of DNA lesions by using an Aldehyde Reactive Probe  was used following the manufacturer's instructions.All coding exonic and flanking intronic regions of the human TP53 gene were amplified from genomic DNA with Platinum Taq DNA polymerase (Life Technologies) by multiplex PCR, using two primer pools with 12 non-overlapping primer pairs each, yielding ~180 bp amplicons. Each sample was tagged with a unique 8-nucleotide barcode combination using twelve differently barcoded forward and eight differently barcoded reverse primer pools. Barcoded PCR products from up to 96 samples were pooled, purified, and an indexed sequencing library was prepared using the NEBNext ChIP-Seq Library Prep Master Mix Set for Illumina in combination with NEBNext Multiplex Oligos for Illumina (New England Biolabs). The quality of sequencing libraries was verified on a Bioanalyzer DNA High Sensitivity chip (Agilent) and quantified by digital PCR. 2 × 250 bp paired-end sequencing was carried out on an Illumina MiSeq (Illumina) according to the manufacturer's recommendations at a mean coverage of 300 ×.43 To identify putative mutations, variant calling was performed using SAMtools in combination with VarScan2 (v2.3.9).44 Initially, SAMtools was used to create pileups with a base quality filter of 15. Duplicates, orphan reads, unmapped, and secondary reads were excluded. Subsequently, Varscan2 was applied to screen for SNPs and InDels separately, using a low-stringency setting with minimal variant frequency of 0.1, a minimum coverage of 20, and a minimum of 10 supporting reads per variant to account for cellular and clonal heterogeneity. Minimum average quality was set to 20 and a strand filter was applied to minimize miscalls due to poor sequencing quality or amplification bias. The resulting list of putative variants was compared against the IARC TP53 (R17) database to check for known p53 cancer mutations.Read pairs were demultiplexed according to the forward and reverse primers and subsequently aligned using the Burrows-Wheeler Aligner against the Homo sapiens Ensembl reference (rev. 79). Overlapping mate pairs were combined and trimmed to the amplified region. Coverage for each amplicon was calculated via SAMtools (v1.1).t-test. Three and more groups were compared by ANOVA followed by the Student–Newman–Keuls test. P values lower than 0.05 were considered to be significant.Results are expressed as mean±S.D. of at least three experiments. Comparisons between two groups were performed using Student's


Elucidating the poorly defined mechanisms by which inflammatory lesions are spatially restricted in vivo is of critical importance in understanding skin disease. Chemokines are the principal regulators of leukocyte migration and are essential in the initiation and maintenance of inflammation. The membrane-bound psoriasis-associated atypical chemokine receptor 2 (ACKR2) binds, internalizes and degrades most proinflammatory CC-chemokines. Here we investigate the role of ACKR2 in limiting the spread of cutaneous psoriasiform inflammation to sites that are remote from the primary lesion. Circulating factors capable of regulating ACKR2 function at remote sites were identified and examined using a combination of clinical samples, relevant primary human cell cultures, in vitro migration assays, and the imiquimod-induced model of psoriasiform skin inflammation. Localized inflammation and IFN-γ together up-regulate ACKR2 in remote tissues, protecting them from the spread of inflammation. ACKR2 controls inflammatory T-cell chemotaxis and positioning within the skin, preventing an epidermal influx that is associated with lesion development. Our results have important implications for our understanding of how spatial restriction is imposed on the spread of inflammatory lesions and highlight systemic ACKR2 induction as a therapeutic strategy in the treatment and prevention of psoriasis and potentially a broad range of other immune-mediated diseases. Chemokines, the primary in vivo regulators of leukocyte migration, are central to inflammatory pathogenesis . They biRecently, we showed that ACKR2 expression is particularly elevated in uninvolved skin of psoriatic patients . We hypoTo establish a mouse model appropriate for assessing the mechanism of differential ACKR2 regulation and the role of ACKR2 in regulating localized inflammation, we determined the role of ACKR2 in the well-characterized imiquimod (IMQ) mouse model of psoriasis . At rest+ T-cell localization in the IMQ model. There are no basal differences in total cutaneous or epidermal T-cell numbers in ACKR2-deficient, compared with WT, mice  could regulate human T-cell migration. For this purpose, we established an in vitro three-dimensional cell migration assay. The T-cell chemoattractant chemokine CCL20 has been associated with psoriasis . However–/– mice treated with or without IMQ  for 24 hours at 4 °C. For long-term storage, RNAlater-treated tissues were stored at –80 °C. Tissues for histology and immunohistochemistry were fixed in 10% neutral buffered formaldehyde (Sigma-Aldrich).RNA was extracted and purified on RNeasy Micro columns with on-column DNase digestion as per manufacturer’s instructions . Quantitative PCR was performed as described in the + staining, paraffin-embedded fixed skin was cut to 2-μm thickness. Heat-induced antigen retrieval was performed in pH 6 sodium citrate buffer for 100 seconds at 125 °C under pressure. Polyclonal rabbit anti-human CD3 (1:100 dilution) and polyclonal rabbit anti-Ki67 (1:400) , or negative controls, were used and counterstained with hematoxylin.For hematoxylin and eosin, Ki67, and CD3Details are provided in the Details are provided in the Details are provided in the Full-thickness mouse skin was submerged in 0.025-mmol/L EDTA  at 37 °C, after which the epidermal layer was curetted off, leaving the dermis intact. Both skin components were immediately lysed in Qiazol, using a Qiagen TissueLyser LT .t test, Mann-Whitney U tests, one-way analysis of variance, two-way analysis of variance, and correlation tests were performed in Prism Version 7.0 , with multiple comparisons tests as appropriate. Vector data were assessed by the Rayleigh test for vector data in chemotaxis plugin in ImageJ (available at http://imagej.nih.gov/ij/). P < 0.05 was deemed significant. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.005, ∗∗∗∗P < 0.0001. All data are n ≥ 3 and given as mean ± standard error of the mean unless otherwise stated.Student The authors state no conflict of interest.