Despite significant therapeutic progress in multiple myeloma, drug resistance is uniformly inevitable and new treatments are needed. Our aim was to identify novel, efficacious small-molecule combinations for use in drug resistant multiple myeloma.A panel of 116 small molecule inhibitors was used to screen resistant myeloma cell lines for potential therapeutic targets. Agents found to have enhanced activity in the bortezomib or melphalan resistant myeloma cell lines were investigated further in combination. Synergistic combinations of interest were evaluated in primary patient cells.The overall single-agent drug sensitivity profiles were dramatically different between melphalan and bortezomib resistant cells, however, the bromodomain inhibitor, CPI203, was observed to have enhanced activity in both the bortezomib and melphalan resistant lines compared to their wild-type counterparts. The combination of bortezomib and CPI203 was found to be synergistic in both the bortezomib and melphalan resistant cell lines as well as in a primary multiple myeloma sample from a patient refractory to recent proteasome inhibitor treatment. The CPI203-bortezomib combination led to enhanced apoptosis and anti-proliferative effects. Finally, in contrast to prior reports of synergy between bortezomib and other epigenetic modifying agents, which implicated MYC downregulation or NOXA induction, our analyses suggest that CPI203-bortezomib synergy is independent of these events.Our preclinical data supports a role for the clinical investigation of the bromodomain inhibitor CPI203 combined with bortezomib or alkylating agents in resistant multiple myeloma. Multiple myeloma (MM) is a hematologic malignancy characterized by bone marrow infiltration of neoplastic plasma cells. Despite significant therapeutic progress, MM remains incurable. Currently the most effective treatment strategies for MM rely on combination regimens including novel agents (proteasome inhibitors or immunomodulators) and standard chemotherapies . However, therapeutic options become limited over the course of the disease resulting from the development of drug resistance, treatment related toxicity, and intolerance . While tWith a rapidly increasing number of targeted agents available in the preclinical setting, an effective and high throughput approach for identifying active agents followed by rationally designed treatment is desirable. We have developed a panel of molecularly targeted small-molecule inhibitors for direct interrogation of primary leukemia patient samples that collectively informs pathway dependence, and identifies candidate therapeutic options . In our BET  family proteins include BRD2, BRD3, BRD4, and BRDT and are the so called “readers of the histone modification code”. Bromodomain (BRD)-containing proteins selectively bind to the acetyl lysine marks placed by histone modifying enzymes  . TargeteMYC pathway activation can be involved in MM pathogenesis through a variety of mechanisms. Transcriptional profiling identifies MYC pathway activation in up to 67% of MM patient samples . In earlReport of a myeloma mouse model involving a MYC transgene showed activity with (+)-JQ1 alone and in combination with bortezomib or a deacetylase inhibitor. In particular it was noted that activity with these agents persisted even after the model was serially passaged to induce BTZ resistance . In our Using a panel of 116 targeted agents, we performed cell viability testing on BTZ (ANBL6 BR and 8226.BR) and melphalan (8226/LR5) resistant cell lines, in addition to the well characterized “parent” MM cell lines U266, ANBL6 WT, and RPMI8226 from which the resistant lines were derived.Concentration of inhibitor required to suppress cell growth and viability by 50% (IC50) was obtained for each individual agent across the various cell lines. In order to prioritize on-target effects across the large panel, we compared the IC50 values from the tested MM cell lines to the pooled results of 151 patient bone marrow aspirate samples tested on the same panel that we have previously published . Table 1One can postulate that the biological alterations that occur upon acquiring resistance to one drug may lead to an increased sensitivity to another drug. Given our aim to identify novel combinations for drug resistant disease, we directly compared the differences in the IC50 values between our resistant lines  with their wild type parent lines (RPMI8226 and ANBL6 WT). Dividing the wild type IC50 values by the resistant cell line IC50 values represents the relative fold magnitude increase sensitivity the resistant lines have to a particular agent. Figure The activity of CPI203 was of particular interest due to the impressive enhancement of activity in both the BTZ and melphalan resistant cell lines compared to their wild type counterparts. We therefore investigated CPI203 in combination with BTZ. The combination of CPI203-BTZ showed synergy in the MTS cell viability assay in both ANBL6 WT and ANBL6 BR, as well as in 8226/LR5, but not the wild type RPMI8226 based on calculated CI values . At low concentrations of CPI203 , there was no enhancement of apoptosis either as single agents or when added to BTZ. At a concentration of 1600nM, CPI203 exerted increased apoptosis as a single agent as well as in combination with BTZ Figure .To better understand the effects of the combination on cell growth kinetics, ANBL6 WT and BR cells were exposed to different concentrations of BTZ and CPI203, and serial cell counts were assessed based on guava ViaCount over the course of 72 hours Figure . BTZ trend generation proteasome inhibitor). Cytogenetic analysis of the MM sample showed high risk genetic features including: chromosome 17p deletion, gain of 1q, t, and monosomy 13. A significant synergistic effect was shown at low nanomolar concentrations of BTZ and CPI203 at 48 hours, even overcoming the cytoprotective effects of the native bone marrow microenvironment , P-glycoprotein expression (efflux pump), microenvironmental upregulation of IL-6, IGF-1, or tumor upregulation of HSP90, PI3K, MYC, and IGF-1 , 32-36. Additionally, a recent report suggests that higher MYC expression promotes survival through activation of the PERK/eIF2alpha/ATF4 dependent aggresome pathway, a resistance mechanism known to be important in the setting of proteasome inhibition . EvidencOur data show that treatment with CPI203 sensitized both BTZ resistant and sensitive cells to BTZ despite the CPI203 mediated downregulation of MYC and NOXA expression. The mechanism for the synergistic effect in the BTZ resistance setting is unlike those previously described in the literature. In contrast to previous reports , 40 showThe synergistic mechanisms of the BTZ combinations that have been studied also seem to vary. The effect of combining a deacetylase inhibitor (ie panobinostat or vorinostat) with BTZ in MM has been described , 40, 41.in vitro settings. Collectively our findings provide support for the clinical investigation of combined BET and proteasome inhibition in drug resistant MM.Our data suggests that the previously reported reliance on MYC expression for NOXA-mediated apoptosis is more complicated, and likely involves other mediators. Accumulating data on role of MYC in BTZ resistance supports the notion that there is an intricate balance of MYC expression levels, and the influence of other mediators may tip the balance toward either a proliferative or apoptotic phenotype. Based on our data, we may simply hypothesize that CPI203 downregulates MYC enough to preferentially reduce proliferation and aggresome mediated survival, yet permitting enough NOXA expression for BTZ potentiated apoptosis. However, given the far-reaching transcriptional effects of BET and the pleotropic effects of proteasome inhibition, the synergistic relationship observed is likely far more complex. While the precise mechanism of the synergistic relationship is not clearly understood at this time, the combination of BTZ + CPI203 shows compelling responses that are consistent across multiple The characteristics and sources of the human MM cell lines used are depicted in Table 5 cells/mL and incubated for 48 hours in R10 media supplemented with 1ng/mL IL-6, then tested for cell viability using the CellTiter 96 Aqueous One Solution Cell proliferation assay (Promega).Primary cells from a patient with relapsed-refractory MM was collected by bone marrow aspiration with informed consent of the patient under a protocol approved by the institutional review board at Oregon Health and Science University. The bone marrow aspirate underwent independent clinical pathologic review and was composed of 90% myeloma cells. Red cell lysis of the bone marrow sample with Ammonium-Chloride-Potassium (ACK) buffer was performed. Given the significant myeloma cell population, and to preserve the marrow microenvironment, CD138 selection of tumor cells was omitted. The primary bone marrow cells were seeded at a concentration of 3.0 × 104 cells/mL in 50 μL of media per well and incubated for 72 hours. All cell lines were initially screened using a panel of small molecule inhibitors as previously described [Cell lines were seeded in 96-well plates at a concentration of 3.0 × 10Cells were stained for Annexin V-PE/7-AAD as per manufacturer instructions  after mechanical resuspension. The relative percentage of cells undergoing apoptosis was then quantified by flow cytometric analysis . All apoptotic measurements were performed in duplicate.Cell lysis buffer  with phenylmethanesulfonyl fluoride (PMSF) (Sigma), aprotinin (Sigma), and protease inhibitor cocktail (Sigma) was used to make cell lysates. Proteins were separated by electrophoresis on Tris-HCL precast gels (Bio-Rad Laboratories). Proteins were transferred to PVDF membranes  with pore size 0.45um for MYC and 0.22um for NOXA blots then probed with antibodies against MYC , NOXA , actin (Millipore), and GAPDH (Ambion). Chemiluminescence detection was performed using Clarity Western ECL substrate (Bio-Rad) or SuperSignal West Femto (Thermo Scientific) and viewed using ChemiDoc MP imaging system with Image Lab software .Data presented as means of duplicate or triplicate samples or assays. The student t-test was used to determine statistical difference between the groups. Combination index (CI) analyses were performed using Calcusyn software . A CI value of 1 indicates additive effect, a CI of >1 indicates antagonistic effect, and a CI of <1 indicates synergy. Based on synergy growth curves, calculated CI values are reported at the effective doses of the combination resulting in 50%, 75%, and 90% inhibition  .


Chk1 inhibitors are currently in clinical trials as putative potentiators of cytotoxic chemotherapy drugs. Chk1 inhibitors may exhibit single agent anti-tumor activity in cancers with underlying DNA repair, DNA damage response or DNA replication defects.Here we describe the cellular effects of the pharmacological inhibition of the checkpoint kinase Chk1 by the novel inhibitor V158411 in triple-negative breast cancer and ovarian cancer. Cytotoxicity, the effect on DNA damage response and cell cycle along with the ability to potentiate gemcitabine and cisplatin cytotoxicity in cultured cells was investigated. Western blotting of proteins involved in DNA repair, checkpoint activation, cell cycle and apoptosis was used to identify potential predictive biomarkers of Chk1 inhibitor sensitivity.The Chk1 inhibitors V158411, PF-477736 and AZD7762 potently inhibited the proliferation of triple-negative breast cancer cells as well as ovarian cancer cells, and these cell lines were sensitive compared to ER positive breast and other solid cancer cells lines. Inhibition of Chk1 in these sensitive cell lines induced DNA damage and caspase-3/7 dependent apoptosis. Western blot profiling identified pChk1 (S296) as a predictive biomarker of Chk1 inhibitor sensitivity in ovarian and triple-negative breast cancer and pH2AX (S139) in luminal breast cancer.This finding suggests that Chk1 inhibitors either as single agents or in combination chemotherapy represents a viable therapeutic option for the treatment of triple-negative breast cancer. pChk1 (S296) tumor expression levels could serve as a useful biomarker to stratify patients who might benefit from Chk1 inhibitor therapy. Breast cancer is the most common cancer affecting women and the second most common cause of death due to cancer . This hiBreast tumors arising from hereditary BRCA mutation carriers are mostly triple-negative and share a striking resemblance to sporadic basal-like and triple-negative breast cancers suggesting that TNBC might bear defects in DNA repair thereby endowing a degree of “BRCAness” on these cancers . BRCA muThe DNA damage signaling response pathway (DDR) is activated by DNA breaks induced by a variety of endogenous and external insults including therapies currently used for the treatment of cancer such as ionizing radiation and cytotoxic chemotherapeutic agents such as gemcitabine, irinotecan and cisplatin. Activation of the DDR results in a number of cellular responses including checkpoint activation and cell cycle arrest, initiation of DNA repair, regulation of transcription and apoptosis , 16. TheIt has also been postulated that DNA damage response checkpoints and especially Chk1 kinase activity may be critical for the normal replication of cancer cell lines with specific underlying defects in DNA repair or DNA damage response pathways, or DNA replication defects. For example, the Fanconi Anaemia (FA) repair pathway is responsible for repairing crosslinked DNA and maintaining chromosomal stability . FA defiin vitro with IC50s of 3.5 and 2.5nM respectively [50 of 48 nM and Chk2 on Ser516 with an IC50 of 904 nM indicating a 19-fold cellular selectivity for Chk1 over Chk2. V158411 potentiated cytotoxic chemotherapy in p53 defective cancer cells in vitro and in vivo. We therefore evaluated the single agent cytotoxic potential of V158411 against a panel of solid cancer cell lines including those derived from breast and ovarian cancer. We further profiled the panel of cell lines to understand and identify potential biomarkers predictive of response to Chk1 inhibition. The data provides a preclinical rationale to support the clinical testing of Chk1 inhibitors as single agents and in combination with cytotoxic chemotherapy in patients with triple-negative breast cancer.V158411 is a novel, potent, selective inhibitor of recombinant Chk1 and Chk2 kinases ectively . In p53 All cells were obtained from the American Type Culture Collection and cultured in DMEM, RPMI or McCoys 5a containing 10% FCS (Invitrogen). The cytotoxicity of V158411 was determined following exposure of cells in 96 well plates to a 10-point titration for 72 hours. Cell proliferation was determined using sulphorhodamine B (Sigma) staining following protein precipitation with 10% TCA. For cell counts, cells were seeded in 6 well plates and counted following trypsinisation after 72 hours using a haemocytometer with trypan blue staining.2O; cisplatin (Selleckchem), 3.33 mM in 1% NaCl in H2O; oxaliplatin (Tocris), 5 mM in H2O; carboplatin (Tocris), 25 mM in H2O; PF-477736 (Selleckchem), 20 mM in DMSO and AZD7762 (Axon Medchem), 20 mM in DMSO.V158411 was synthesized according to the method described in  and prep50 of V158411 for 24 or 48 hours. Caspase-3/7 activity was determined using a homogenous caspase-3/7 luminescence kit (Promega).Cells were seeded in 96 well plates and treated with 10-times the GIL, GAPDH and pH2AX (S139) from Cell Signaling Technologies; pChk1 (S296), FANCF and FANCD2 from Abcam, and Bcl-2 and Mcl-1 from Santa Cruz. Treated and untreated cells were washed once with PBS and lysed in RIPA buffer containing protease and phosphatase inhibitor cocktails (Roche). Protein concentration was determined using BCA kit (Pierce). Equal amounts of lysate were separated by SDS-PAGE and western blot analysis conducted using the antibodies indicated above.Anti-pHistone H3 (S10) was obtained from Millipore; Chk1, pChk1 (S317), pChk1 (S345), pChk2, pChk2 (T68), pCdc25c (S216), 53BP1, Cdc2, pCdc2 (Y15), Cyclin B1, D1 and E, PARP, pERK1/2, ERK 1/2, AKT, pAKT (S473), Bcl-XCells were seeded in 6 well plates and subsequently treated with the indicated concentrations of V158411 for 24 or 48 hours. All cells were harvested, fixed in 70% ethanol and stained with propidium iodide/RNase A. Cell cycle profiles were examined by flow cytometry using a FACSArray cytometer (BD) and FACSDiva software (BD).3 cells per well were seeded in 96 well plates and incubated overnight. Cells were treated with a 10-point titration of gemcitabine or cisplatin in the presence of a fixed concentration of V158411 for 72 hours. The effect on cell proliferation was determined using a CellTiter 96 AQueous One Solution Cell Proliferation Assay .5x10None of the research in this manuscript involved human subjects, human material, or human data, or used regulated vertebrates or invertebrates.Sporadic basal-like or triple-negative breast cancers strongly resemble breast cancers originating from hereditary BRCA defective patients and may harbor BRCA-mimetic defects in DNA repair pathways. To test the hypothesis that triple-negative breast cancers are sensitive to Chk1 inhibitors in the absence of cytotoxic chemotherapy agents, a panel of sporadic triple-negative and luminal breast cancer cell lines  as well 2 = 0.899, Figure 2 = 0.653 and 0.635 respectively) and may be due to the reduced kinase selectivity of AZD7762 compared to V158411 and PF-477736. Sensitivity to V158411 was independent of p53 or kRas mutational status . Inhibition of the estrogen receptor with 4-hydroxytamoxifen did not increase the sensitivity of the ER positive BT474 and MCF7 cell lines to Chk1 inhibition . This inhibition of Chk1 correlated with an activation of ATR and increased DNA strand breakage as measured by increased Chk1 phosphorylation on serine 317 and 345 and increased pH2AX (S139)  polymerase. Recent reports have demonstrated the sensitivity of TNBC to PARP inhibitors as well as DNA damaging cytotoxic agents such as gemcitabine and cisplatin . TNBCs hin vitro and in vivo[This hypothesis was substantiated in a screen of 26 solid cancer cell lines where TNBC cancer cell lines were among the most sensitive to growth inhibition by the novel, selective Chk1 inhibitor V158411. Even though V158411 is an extremely selective inhibitor of Chk1, it is difficult to ascertain the absolute selectivity of any small molecule kinase inhibitor. To confirm this observation, two structurally unrelated Chk1 kinase inhibitors PF-477736  potently in vivo, 43. In In light of these results, we attempted to understand the mechanism by which single agent Chk1 inhibitors induced TNBC and ovarian cancer cell death. The clearest marker of response to Chk1 inhibition in all the sensitive cell lines was the time dependent increase in phosphorylation of H2AX on serine 139. Phosphorylation on this site is generally associated with an increase in DNA double strand breaks . CoupledIn all breast and ovarian cancer cell lines, including those relatively resistant to V158411 single agent cytotoxicity, reduction in total Chk1 protein levels following V158411 treatment was evident. This was especially notable at the higher concentrations of V158411 and appeared to correlate with an increase in pH2AX (S139) in the V158411 sensitive cell lines. V158411 treatment of leukemia and lymphoma cell lines induced Chk1 degradation that was proteasome dependent  whilst det al.[et al., [kRas mutational status, both of which have previously been implicated in Chk1’s mechanism of action [The precise mechanism for the sensitivity of the TNBC and ovarian cancer cell lines compared to other solid cancer cell lines remains to be fully understood. The resistance of the two ER-positive breast cancer cell lines BT474 and MCF-7 could not be overcome with 4-hydroxytamoxfien suggesting that estrogen receptor signaling did not contribute to the relative sensitivities in the breast cancer cell lines. Chk1 expression has been demonstrated to be elevated in histological grade 3 TNBC primary tumors compared to other grade 3 breast cancers . Sensitiet al. who iden[et al.,  identifif action . The outBRCA1[The underlying mechanism for the sensitivity of these cancer cell types to single Chk1 inhibitor therapy is not yet clear and the phosphorylation events identified as potential predictive markers of sensitivity (pChk1 S296), (S317) and pH2AX (S139)) are most likely symptomatic rather than the cause of the underlying sensitivity. This observation suggests that the molecular defects in these cell lines occur in pathways for which Chk1 can mutually compensate to protect genomic integrity and therefore Chk1 inhibition is lethal. An example of this so far discovered is the Fanconi’s Anemia (FA) DNA repair pathway. Cells defective in FA were sensitive to Chk1 siRNA and the small molecule Go6976 due to an accumulation of unrepairable DNA double strand breaks [6, (S317)BRCA1 and thisCells under replicative stress due to oncogene amplification or activation of oncogenic signaling pathways are addicted to Chk1 kinase activity for the completion of a normal S-phase , 36. TheTriple-negative status was not a sufficient prognostic marker for response to Chk1 inhibition as other cell line types such as the HER2 positive SKBr3 breast cancer cell line and SKOV-3 ovarian cancer cell line were among the most sensitive cell lines to growth inhibition by V158411. For this treatment to be better focused in the clinic to those patients most likely to benefit then additional biomarkers prognostic of sensitivity would be extremely beneficial. High expression of pChk1 (S296) in untreated tumors and the induction of pH2AX (S139) post Chk1 inhibitor therapy appear to be the most sensitive and predictive markers for response to Chk1 inhibitor therapy. However, phospho-protein profiling of tumor biopsies by immunohistochemistry or western blotting is not without its technical challenges. Alternative methods of identifying potential clinical responders, such as gene signature profiles, would aid the use of Chk1 inhibitors as monotherapy.From this study and others, it is becoming clear that Chk1 inhibitors may have clinical utility as a single agent as well as in combination with cytotoxic chemotherapy agents in a variety of human cancer types. Chk1 inhibitors either as single agents or in combination with cytotoxic chemotherapy are a potentially viable therapeutic option for the treatment of triple-negative breast cancer in the clinic. High tumor expression of pChk1 (S296) could serve as a useful biomarker to select those patients who would most likely benefit from Chk1 inhibitor therapy.Clinical testing of Chk1 inhibitors is currently focused on their ability to potentiate the anti-tumor efficacy of cytotoxic chemotherapy drugs and anti-metabolite therapies. Recent studies have demonstrated single agent activity of Chk1 inhibitors in cancer cells harboring defects in DNA damage repair or response pathways or high levels of replicative stress. V158411 either as a single agent or in combination with cytotoxic chemotherapy are potentially viable therapeutic options for the treatment of triple-negative breast cancer in the clinic. High tumor expression of pChk1 (S296) could serve as a useful biomarker to select those patients who would most likely benefit from Chk1 inhibitor therapy. The BRCAness of triple-negative breast cancer may underlie the sensitivity of this cancer type to Chk1 inhibition.


Notch signalling regulates cell fate in most tissues, promoting precursor cell proliferation in some, but differentiation in others. Accordingly, downregulation or overactivity variously contributes to cancer development. So far, little is known about Notch pathway activity and function in the normal urothelium and in urothelial carcinoma (UC). We have therefore investigated expression of Notch pathway components in UC tissues and cell lines and studied the function of one receptor, NOTCH1, in detail.Expression of canonical Notch pathway components were studied in UC and normal bladder tissues by immunohistochemistry and quantitative RT-PCR and in UC cell lines and normal cultured urothelial cells by qRT-PCR, immunocytochemistry and Western blotting. Pathway activity was measured by reporter gene assays. Its influence on cell proliferation was investigated by γ-secretase inhibition. Effects of NOTCH1 restoration were followed by measuring cell cycle distribution, proliferation, clonogenicity and nuclear morphology.NOTCH1 and its ligand, DLL1, were expressed at plasma membranes and in the cytoplasm of cells in the upper normal urothelium layer, but became downregulated in UC tissues, especially in high-stage tumours. In addition, the proteins were often delocalized intracellularly. According differences were observed in UC cell lines compared to normal urothelial cells. Canonical Notch pathway activity in reporter assays was repressed in UC cell lines compared to normal cells and a mammary carcinoma cell line, but was induced by transfected NOTCH1. Inhibitors of Notch signalling acting at the γ-secretase step did not affect UC cell proliferation at concentrations efficacious against a cell line with known Notch activity. Surprisingly, overexpression of NOTCH1 into UC cell lines did not significantly affect short-term cell proliferation, but induced nuclear abnormalities and diminished clonogenicity.Our data indicate that canonical Notch signalling is suppressed in urothelial carcinoma mainly through downregulation of NOTCH1. These findings can be explained by proposing that canonical Notch signalling may promote differentiation in the urothelium, like in many squamous epithelia, and its suppression may therefore be advantageous for tumour progression. As an important corollary, inhibition of canonical Notch signalling is unlikely to be efficacious and might be counter-productive in the treatment of urothelial carcinoma.The online version of this article (doi:10.1186/1471-2407-14-628) contains supplementary material, which is available to authorized users. Bladder cancer is the fifth most common malignancy in most Western industrialized countries with urothelial carcinoma (UC) representing the major histological subtype. UC can be further classified into papillary and invasive carcinomas . While mNOTCH genes [The Notch signalling pathway is a promising target for cancer therapy , 9. In mCH genes , 13 is oCH genes –16. TherCH genes .Hairy and Enhancer of Split (HES) and Hairy and Enhancer of Split related (HEY) target genes. Through such effectors Notch determines cell fate [Notch signalling constitutes a short-range communication system between adjacent cells. Canonical Notch signalling is activated by ligand-receptor interactions releasing the receptor intracellular domain (NICD) by two proteolytic steps, including an essential cleavage by γ-secretase. The NICD translocates into the nucleus where it binds to C Promoter Binding Factor-1 (CBF-1), recruits co-activators (MAML1 and KDM5A) and typically activates the transcription of ell fate .To date, little information exists on the Notch signalling pathway in normal urothelium or urothelial cancers. Decreased expression of several Notch receptors and ligands in immunohistochemistry has been reported especially in papillary UC . We haveThe benign and tumour bladder samples used for gene expression studies were a subset of those described previously  comprisiAll UC cell lines  and HEK293 cells were cultured in DMEM GlutaMax , supplemented with 10% fetal calf serum . They weHuman N1ICD-pIRES2 was a kind gift from Prof. Giebel, Essen; human full-length wildtype NOTCH1 in a pcDNA3-HA/FLAG vector was kindly provided by Prof. Di Fiore, IFOM, Italy .Cells were treated with 0.25 - 20 μM γ-secretase inhibitors DAPT , L-685,458  or Compound E  or, as a control, compound W  for 48 h when viability was analysed by MTT assay.For reporter gene assays, cells seeded in 6-well plates were transiently transfected with XtremeGene9 (Roche) with 1 μg of either pJH26A (Notch responsive reporter) or pJH28A (Notch non-responsive reporter), kindly provided by Prof. D. Hayward , or in o1C Fc secondary antibody , washed, and then incubated for 2 h with 10 μg/ml DLL1 extracellular domain (aa 1–545) fused at its C-terminus to the Fc portion of human IgG  or human IgG , followed by a further wash. After a further 24 h, cells were harvested and assayed for luciferase activities by the Dual-Luciferase reporter assay (Promega). Basal activity in cells exposed to immobilized IgG was set as 1 for each cell line.The ligand-dependent activation assay was carried out as described . Briefly5 cells were seeded in 6 well plates and transfected with pIRES2-N1ICD or the corresponding pIRES2 empty vector, or with pcDNA4/to-Notch1 full-length (N1-FL) or the corresponding pcDNA4/to-lacZ plasmid. After 24 h cells were seeded in triplicates into 10 cm plates and selection for stable integration of the plasmids was started the next day. Cells transfected with pIRES2 plasmids were treated with 1.0 - 1.8 mg/ml Geneticin Selective Antibiotic G418-Sulfat (Invitrogen) and cells transfected with pcDNA4/to-plasmids were treated with 100–150 μg/ml Zeocin Selection Reagent (Invitrogen) every two days for 2 weeks. After a one week recovery period with lower antibiotic concentrations, the remaining colonies were fixed with 100% methanol and stained with Giemsa solution . Colonies were then counted and statistical comparisions were made by Student’s T-Test.For the colony forming assay 10TBP was used as a reference gene. Statistical comparisons were made by Mann–Whitney U test with SPSS 21.Total RNA was isolated from sub-confluent cell lines using the RNeasy Mini Kit . RNA was reverse transcribed using 200 U SuperScriptII reverse transcriptase , with 300 ng oligo-dT and 25 ng random hexamer primers. Real Time-PCR assays were performed with the ABI7900HT System using the QuantiTect SYBR Green PCR Kit (Qiagen) and premade or self-designed primers , JAG1 , DLL1  and NOTCH2  and subsequently with goat-anti-mouse Alexa Fluor488 antibody. Nuclei were stained with 1 μg/ml DAPI .Subconfluently grown cells were lysed in RIPA buffer with protease and phosphatase inhibitors. Western blot analysis after SDS-page was performed using primary antibodies against NOTCH1 , JAG1 , DLL1 , NOTCH2  and α-tubulin  as a control. Secondary antibodies labelled with HRP and the ECL advanced or ECL Select kits  were used for detection.Immunohistochemistry was performed by standard techniques with antigen retrieval by citrate buffer pH 9. Antibodies used were NOTCH1 (Epitomics), JAG1 (Santa Cruz) and DLL1 (Santa Cruz). Detection was achieved via a biotin-conjugated polyvalent antibody with an avidin-biotin-peroxidase reagent  and diamino-benzidine staining. Sections were counter-stained with hemalum. The staining results of the reference tissues for antibody specificity are provided in Additional file NOTCH1 and NOTCH2 was significantly decreased in almost all urothelial cancers . Among the ligands, DELTA1 (DLL1) expression was strongly and significantly decreased in cancer tissues, whereas JAGGED1 (JAG1) and JAGGED2 (JAG2) mRNA levels were not significantly changed , whereas NOTCH2 expression was only slightly diminished. Expression of DLL1 was strongly reduced, but that of JAG1 and JAG2 less so , providing a specificity control. Indeed, in HEK293 cells which are sensitive to inhibition of Notch signalling, the former three inhibitors, but not Compound W, inhibited proliferation in a dose-dependent manner Figure . In contWhereas pharmacological Notch signalling inhibition did not diminish proliferation in urothelial cancer cell lines, overexpression of N1-FL or N1ICD diminished clonogenicity of urothelial carcinoma cell lines Figure A. In shohttp://cancergenome.nih.gov/) indicate only few mutations in Notch pathway components in urothelial carcinoma  which occur in up to 30% of invasive UC. However, neither mutations nor gene loss can fully account for the high prevalence of NOTCH1 downregulation. As both, NOTCH1 and DLL1, are predominantly localized in more differentiated cells in the normal urothelium, their decreased expression in tumours may conceivably reflect to some extent the loss of this differentiated cell population.Taken together, our results from tissues and cell lines suggest that canonical Notch signalling towards the nucleus is inactive in urothelial carcinoma, especially in the invasive subtype. NOTCH1 and DLL1 were most consistently diminished in both cell lines and tissues. Downregulation of these factors is in accord with a previous report , whereasIn addition to downregulation, we frequently observed delocalization of NOTCH1 and DLL1 to the cytoplasm in tumour cells. Delocalization of NOTCH proteins to the cytoplasm in some cells reflects lack of activation by external ligands in trans but enables cis-inhibition by ligands expressed in the same cell compartment , 30. CanWe note that NOTCH2 expression was less strongly changed than that of NOTCH1 and that active N2ICD could be detected in the nuclei of some UC cell lines. Interestingly, it appeared more strongly expressed in mesenchymal UC cell lines albeit localized to the cytoplasm. Increased expression of NOTCH2 in UC cell lines with a mesenchymal phenotype as compared to lines with an epithelial phenotype has recently been reported by others , 34. TheAcross the cancer tissues, expression of HES1 mRNA was significantly increased compared to normal bladder, despite the significant downregulation of NOTCH1, NOTCH2 and DLL1. Although no upregulation of HES1 was observed in the UC cell lines compared to normal urothelial cells, this finding is somewhat puzzling, since HES1 is a common target gene of the canonical Notch pathway. However, target genes of the pathway vary strongly between tissues and even HES1 is not a universal target . MoreoveIn normal urothelial cells and urothelial carcinoma cell lines, overexpression of full-length NOTCH1 or its active intracellular domain induced canonical reporters but was not compatible with long-term proliferation of UC cells. Inhibition of proliferation did not appear to occur by straightforward inhibition of cell cycling or by a major induction of apoptosis. Rather, NOTCH1 transfected cells often showed misshaped or abnormally large nuclei or became obviously binuclear. Intriguingly, in normal urothelium NOTCH1 is localized in the upper layers together with its likely ligand DLL1, where they might contribute to terminal differentiation. In particular, umbrella cells of the urothelium are usually polyploid . In squaCollectively, our findings indicate that canonical Notch signalling is lost in urothelial carcinoma mainly via inactivation of NOTCH1, which in normal urothelium may promote specific steps of urothelial differentiation. As it has been shown that inhibition of the γ-secretase leads to hyperproliferation in tissues, where Notch signalling promotes differentiation , inhibitFigure S2. Immunocytochemical staining of NOTCH1, NOTCH2, DLL1 and JAG1 in UC cell lines. Table S1. Self-designed primer assays for qPCR analysis. Table S2. QuantiTect primer assays from Qiagen for qPCR analysis. Table S3. Mutations in Notch receptors, ligands and ubiquitin ligase in urothelial cancer based on the TCGA study. Table S4. Statistical analysis of changes in N1ICD transfected cell lines. (DOCX 9 MB)Additional file 1: Figure S1: Immunohistochemical staining of reference tissues.


Porichthys notatus) exhibit similar increases in the steroid-dependent frequency sensitivity of the saccule, the main auditory division of the inner ear. This form of auditory plasticity enhances detection of the higher frequency components of the multi-harmonic, long-duration advertisement calls produced repetitively by males during summer nights of peak vocal and spawning activity. The molecular basis of this seasonal auditory plasticity has not been fully resolved. Here, we utilize an unbiased transcriptomic RNA sequencing approach to identify differentially expressed transcripts within the saccule’s hair cell epithelium of reproductive summer and non-reproductive winter fish.Successful animal communication depends on a receiver’s ability to detect a sender’s signal. Exemplars of adaptive sender-receiver coupling include acoustic communication, often important in the context of seasonal reproduction. During the reproductive summer season, both male and female midshipman fish  term enrichment analyses of the differentially expressed transcripts showed that the reproductive saccular epithelium was transcriptionally, translationally, and metabolically more active than the non-reproductive epithelium. Furthermore, the expression of a specific suite of candidate genes, including ion channels and components of steroid-signaling pathways, was upregulated in the reproductive compared to the non-reproductive saccular epithelium. We found reported auditory functions for 14 candidate genes upregulated in the reproductive midshipman saccular epithelium, 8 of which are enriched in mouse hair cells, validating their hair cell-specific functions across vertebrates.We identified a suite of differentially expressed genes belonging to neurotransmission and steroid-signaling pathways, consistent with previous work showing the importance of these characters in regulating hair cell auditory sensitivity in midshipman fish and, more broadly, vertebrates. The results were also consistent with auditory hair cells being generally more physiologically active when animals are in a reproductive state, a time of enhanced sensory-motor coupling between the auditory periphery and the upper harmonics of vocalizations. Together with several new candidate genes, our results identify discrete patterns of gene expression linked to frequency- and steroid-dependent plasticity of hair cell auditory sensitivity.The online version of this article (doi:10.1186/s12864-015-1940-3) contains supplementary material, which is available to authorized users. Understanding how genes are regulated within neural networks to produce and modify behavior is a major goal in neuroscience and behavioral genetics. One strategy for achieving this objective is to use model systems for identifying changing patterns of gene expression under different behavioral states. Exemplars include circadian rhythms in flies and mice e.g., –4, alterMidshipman hearing is an excellent model of neural plasticity for several reasons. First, midshipman exhibit reproductive state-dependent behavioral responses to playback of advertisement calls . Second,To date, we have successfully employed hypothesis-driven approaches to identify neuro-molecular mechanisms of seasonal variation in peripheral auditory function, namely in the hair cell epithelium and eighth nerve afferents of the saccule, the main auditory division of the inner ear in many teleost fish including midshipman , 15. ThiThough our hypothesis-driven approach has been fruitful, it provides a limited view of the cascade of events underlying steroid-dependent, seasonal auditory plasticity. Advances in next-generation sequencing and high-throughput analyses can provide a global view of gene expression. Here, we use transcriptome sequencing to uncover seasonal and reproductive state-dependent differences in transcript abundances within the auditory saccular epithelium. We identified a suite of candidate genes and pathways with known auditory function in midshipman fish, and vertebrates in general, that likely underlie seasonal and reproductive state-dependent variation in hearing. The results have been reported, in part, in abstract form .Midshipman have two male reproductive morphs; we used type I males here because they are the most abundant during collections and have the most dynamic vocal repertoire , 20. GivWe sequenced and annotated transcriptome libraries produced from the SE, along with libraries derived from the vocal motor nuclei (VMN) and the hindbrain region surrounding the VMN. The VMN and hindbrain samples were used for a comprehensive companion study that identified daily and seasonal variation in gene expression patterns in the VMN, the final node of the vocal control network that sends a command signal to the vocal muscles . In thatWe obtained approximately 200 million total paired-end reads; over 20 million reads were produced from each pooled SE sample Table . Using ttpt1), which functions to prevent cell death .


Anaplastic pancreatic cancer (APC) cell lines have been scarcely established.The morphology, gene expressions, karyotyping and epithelial-mesenchymal transition markers of newly established APC cell lines OCUP-A1 and OCUP-A2 were analyzed. Their abilities of proliferation under normoxia and hypoxia, migration and invasion were compared to 4 commercially available pancreatic ductal adenocarcinoma (PDA) cell lines. Their induction of angiogenesis, stem-like cell population and subcutaneous tumor growth in nude mice were estimated, comparing 2 PDA cell lines examined here.OCUP-A1 and OCUP-A2 cells continuously grew with spindle and polygonal shapes, respectively. Gene analysis revealed 9 gene mutations including KRAS and TP53. Karyotyping clarified numerical structural abnormalities in both cells. Loss of E-cadherin and expression of vimentin in both cell lines were observed. The doubling time of both cell lines was approximately 20 h. Proliferation, migration and invasion abilities were not notable compared to other PDA cell lines. However stem-like cell population of both cell lines was superior to a part of PDA cell lines. Moreover OCUP-A1 showed stronger hypoxia tolerance and induction of angiogenesis than other PDA cell lines. The tumorigenicity in vivo of OCUP-A2 was stronger than conventional PDA cell lines.The OCUP-A1 and OCUP-A2 cell lines of rare malignancies might be useful for investigating the biology of pancreatic cancer. Although the number of treatment strategies has been increasing for pancreatic ductal adenocarcinoma (PDA), it still has poor prognosis. The 5-year survival rate is <5 %, and the outcomes have not changed for almost 50 years , 2. AnapA man in his 60s with abdominal pain was suspected as having pancreatic cancer, as seen on CT. The values of carcinoembryonic antigen (CEA), cancer antigen (CA) 19–9, SPan-1 and DUPAN-2 were 3.1 ng/mL, 4 U/mL, >3 U/mL , and >25 U/mL , respectively. The patient underwent distal pancreatectomy with resection of the portal vein which accompanied a tumor embolism. The pathological stage was T3N1M0, UICC stage IIB. He died due to recurrence and rapid progression of the cancer at two months after surgery.A man in his 30s with left upper quadrant pain was diagnosed by CT as having a malignant pancreatic neoplasm with liver metastasis. The values of CEA, CA19-9, SPan-1 were 0.7 ng/mL, 394 U/mL and 60 U/mL, respectively. Although the diagnosis was unresectable pancreatic cancer, the patient underwent distal pancreatectomy in order to control the bleeding in his stomach caused by the invading primary tumor. The pathological classification of the cancer was T4NXM1, UICC stage IV. After surgery, the patient was treated with gemcitabine. Although his activities of daily living had improved at the beginning of chemotherapy, he died five months after surgery due to progression of the cancer.The histological diagnosis in both cases was determined as the pleomorphic type of anaplastic pancreatic cancer because tissue imaging with hematoxylin and eosin (H&E) staining was composed of a large portion of pleomorphic cells and a portion of spindle cells and poorly differentiated adenocarcinoma in the pancreas.−1 penicillin , 100 μgmL−1 streptomycin (Sigma), and 0.5 mM sodium pyruvate (Sigma). Then, the pellets were plated into a 100-mm culture dish  and cultivated at 37 °C in a humidified atmosphere with 5 % CO2. The cell lines were trypsinized every 7–10 days, and maintained in complete culture medium. PDA cell lines  and breast cancer cell line (MCF-7) were purchased from American Type Culture Collection  for use in various assays. As above, these cell lines were also cultured and passed. The morphological findings on OCUP-A1 and OCUP-A2 were investigated by phase-contrast microscopy.Samples of the patients’ ascites were removed in CELLSTAR® tubes  and centrifuged at 1500 rpm for 5 min. The pellets were suspended in Dulbecco’s Modified Eagle’s Medium  containing 10 % heat-inactivated fetal bovine serum , 100 IUmLShort tandem repeat genotyping was performed using genomic DNA extracted from OCUP-A1 and OCUP-A2. This analysis was performed by Promega . This experiment was conducted using the PowerPlex® 16 System  according to the manufacturer’s instructions.G-banding was performed by SRL, Inc.  via the manufacturer’s procedures. The cells of OCUP-A1 and OCUP-A2 were cultured until exponential proliferation was reached for this analysis. Then, the cells were treated with colcemid (0.02 μg/mL) for 60 min and hypotonic treatment by potassium chloride (0.075 mol/L) for 20 min at 37 °C. The cells were fixed by Carnoy’s solution (methanol: acetic acid = 3: 1 by volume) for 10 min. The slides were air-dried and analyzed via G-banding. G-banding was carried out by trypsinizing the slides and stained by Giemsa staining using laboratory procedures. The slides were analyzed with the iCas system , and the images of the metaphases were captured. Using approximately 50 cells in both cell lines, the number of chromosomes was counted. Ten cells that resembled the mode number were karyotyped in detail.Genomic DNA samples of OCUP-A1 and OCUP-A2 were extracted using the QIAamp® DNA Mini Kit  according to the manufacturer’s protocol. The NanoDrop system  quantified the extracted DNA and estimated the quality before sequencing.The Ion AmpliSeq™ Cancer Hotspot Panel v2  was used as multigene panels for sequencing. The amplicon library for exploring hotspot mutations of 50 cancer-related genes was generated using 10 ng of DNA from each sample. The 50 genes were as follows: ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CDKN2A, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, EZH2, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, GNA11, GNAS, GNAQ, HNF1A, HRAS, IDH1, JAK2, JAK3, IDH2, KDR/VEGFR2, KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, SRC, STK11, TP53, and VHL. Multiplex polymerase chain reaction amplification was conducted with 10 ng of DNA samples to make the sequence library according to the Ion AmpliSeq™ Library Preparation kit . The quality of the library was estimated by the Agilent 2100 Bioanalyzer . After emulsion PCR, sequencing was run on the Ion PGM™ system (Life Technologies). The Torrent Suite™ software program v 4.0.2 (Life Technologies) was used to analyze the data, including alignment of the sequences to the reference genome (human genome build 19) and base calling. Variants were detected by the Torrent Variant Caller plug-in v 4.0-r76860 (Life Technologies) and visualized by the Integrative Genomics Viewer . These procedures were consigned to TaKaRa Bio, Inc. . For a reliable sequence variant, a sequencing coverage of 250× and a variant frequency of at least 10 % in the background of the wild type were used as minimum requirements in the current study, as well as in the previous study .5 cells per well that were disseminated and incubated at 37 °C in a humidified atmosphere with 5 % CO2. When the cell number count was performed, monolayer cells were trypsinized. The cell number was counted every 24 until 96 h after the beginning of culture. The Bio-Rad TM10™ automated cell counter  was used. The doubling time was determined from the growth curve obtained by the count.For estimation of proliferation, the doubling times of OCUP-A1, OCUP-A2 and 4 PDA cell lines were measured. A 6-well plate was used to culture these cell lines. There were 1.5 × 10The migratory and invasive abilities were estimated using the IncuCyte ZOOM . These assays were performed according to the manufacturer’s protocol. Before assay, each pancreatic cancer cell line was cultured in a 96-well plate, and incubated until semi-confluent in 5 % CO2 at 37 °C. Then, the 96-pin WoundMaker™ (Essen BioScience) created a wound in each well of the 96-well plate. After washing by phosphate buffered saline (PBS), DMEM + 2%FBS was added into each well, and the plate was placed into the IncuCyte. The scratched area was scanned with the IncuCyte™ Live-Cell Imaging System and software (Essen BioScience) every 3 h. With the invasion assay, each pancreatic cancer cell line was cultured on Matrigel®  in a 96-well plate then incubated until semi-confluent in 5 % CO2 at 37 °C. After washing with PBS, DMEM + 2%FBS and Matrigel® of the same quantity as the medium were added. Similarly to the migration assay, the scratched area was scanned with the IncuCyte™ Live-Cell Imaging System and software (Essen BioScience) every 3 h.3 cells/well) were seeded into a 96-well plate in DMEM. After 24 h, a different concentration of anti-cancer drugs was added to each well. Furthermore, after incubation for 72 h at 37 °C, 10 μL of MTT (5 mg/mL in PBS) were added to each well and the plates were incubated at 37 °C for 3 h, and then 200 mL of dimethyl sulfoxide (Wako) was added. The formazan product of MTT was measured as the absorbance at 550 nm using a microtiter plate leader . The percentage of cell viability was determined as the ratio of the absorbance of the sample to control. The IC50 value was measured as the drug concentration showing 50 % cell growth inhibition compared with the control cell proliferation in the 6 cell lines. The anti-cancer drugs used were 5-fluorouracil , gemcitabine , irinotecan , oxaliplatin , and paclitaxel .To estimate the effect of anti-cancer drugs on the viability of the 6 pancreatic cancer cell lines, a 3-  - 2, 5- diphenyltetrazolium bromide  colorimetric assay was performed. The cancer cells . These 4 tumor markers are generally measured for PDA patients. CEA was estimated by immunoassay . CA19-9 was investigated by chemiluminescent enzyme immunoassay . SPan-1 was measured by immunoradiometric assay . DUPAN-2 and VEGF were investigated by enzyme-linked immunosorbent assay (ELISA) . These analyses were conducted according to each manufacturer’s method by the Mitsubishi Chemical Medience Corporation, Tokyo, Japan.There were 106 cells of each cell line were cultured in 10 mL of DMEM + 5%FBS for 4 days. The supernatant was added to co-cultured green fluorescent protein-labeled human umbilical vein endothelial cells (HUVECs) and normal human dermal fibroblasts in a 96-well plate, and then HUVEC tube formation was monitored using IncuCyte ZOOM (Essen BioScience). As negative and positive control of angiogenesis, 5%DMEM and VEGF (4 ng/mL) were used instead of the supernatant of each cell line, respectively. The medium was replaced on days 5, 7, and 9 after cell plating. Phase-contrast and fluorescent images of tube formation were automatically captured, and tube length and branch points were also automatically measured every 6 h for 10 days using IncuCyte ZOOM (Essen BioScience). Kinetic plots of the angiogenesis metrics  could be formed using the IncuCyte software.Angiogenesis affected by cancer cells was investigated using the CellPlayer™ Angiogenesis PrimeKit (Essen BioScience). This assay was performed according to the manufacturer’s method. Before starting the assay, 103 cells/well) in DMEM + 10%FBS were seeded into a 96-well plate and incubated at 37 °C in a humidified atmosphere with 5 % CO2 and 1 or 21 % O2. Using IncuCyte ZOOM (Essen Bio-Science), two images per well were captured in both phase-contrast and fluorescence every 3 h during 48 h. The proportions of the fluorescent areas to all areas in the images were measured by the software under both hypoxia and normoxia. The fluorescent areas were regarded as indicating cell growth. Then, the cell viability under hypoxia at 48 h was calculated considering the cell viability under normoxia at 48 h as control in each cell line.For investigating the proliferation of OCUP-A1 and OCUP-A2 under low oxygen condition, the growth of each cell line was compared between normoxia and hypoxia. Cancer cells  and vimentin  of OCUP-A1 and OCUP-A2 were examined by Western blotting. Approximately 30 μg of protein extracts were separated through 10 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride membrane using the Trans- Blot® Turbo™ Transfer System . Then, using the SNAP i.d.® 2.0 system , the membranes underwent application of Tris-buffered saline-Tween (TBS-T) solution containing each primary antibody against anti-rabbit E-cadherin , anti-rabbit vimentin , and anti-mouse β-actin  for 10 min after blocking with ECL blocking agent  and incubated with HRP-conjugated anti-mouse or anti-rabbit secondary antibody  for 10 min. Protein bands were visualized with the Luminescent Image Analyzer LAS 4000-plus .Stem-like cells were measured by SP analysis. The cells were incubated in pre-warmed DMEM supplemented with 5 % FBS containing 5 μg/mL Hoechst 33342  in the absence or presence of 30 μg/mL verapamil at 37 °C for 60 min. Then, 1 μg/mL propidium iodide was added and then filtered through a 40-μm cell strainer  and maintained at 4 °C. The analysis was performed using FACS AriaII (Becton Dickinson). Hoechst 33342 was excited with the UV laser at 350 nm, and fluorescence emission was measured with 405/BP30 (Hoechst blue) and 570/BP20 (Hoechst red) optical filters.7 viable cells of each cell line  that were suspended by 200 μL of DMEM + 10%FBS and were inoculated subcutaneously into the back of four-week-old BALB/c nude mice. In each cell line, five mice were injected subcutaneously. In all cases, the injected cells developed a subcutaneous tumor. During approximately one month, the diameter of the subcutaneous tumor in each mouse was measured every 3–5 days. The volume of the tumor was calculated with the formula:There were 10The mice were sacrificed with an overdose of sevoflurane. Part of each subcutaneous tumor was fixed in 10 % formalin and embedded in paraffin section for staining and immunohistochemistry.Tissues from primary APC were obtained from the 2 study patients who underwent surgery at our institution. Then, the xenografts of OCUP-A1 and OCUP-A2 were also used for immunohistochemistry. Formalin-fixed paraffin-embedded specimens were made from each tissue sample. In each sample, the representative block of APC and the cell lines were chosen. Each block was sliced at 4 μm for H&E staining and immunohistochemistry for E-cadherin and vimentin. Immunohistochemistry was performed according to the protocol of our institution. We used antibodies against E-cadherin  and vimentin  for immunohistochemical labeling. The expression of each protein was scored as 0 (no labeling in cancer cells), 1+ (mild labeling), 2+ (moderate labeling), and 3+ (strong labeling). The scoring was performed by two surgeons and a pathologist.Results of statistical analyses were expressed as the means ± standard error from at least three independent experiments. Significance of difference was analyzed with Student’s t tests or Tukey’s honest significant difference (HSD) test using JMP 10 . P-values less than 0.05 were regarded as statistically significant.We succeeded in establishing the cell lines of OCUP- A1 and OCUP- A2. Although both cell lines had little adhesion in a non-confluent state, they formed monolayer sheets in confluence. The cells of OCUP- A1 were mainly spindle-shaped, and appeared as if they were extended for communication among the cancer cells Fig. . In addiAll DNA extracted from the 2 established cell lines showed identical STRs, which did not corresponding to the cells of the database of the Japanese Collection of Research Bioresources (JCRB) .The chromosome number of OCUP- A1 ranged from 55 to 106. The range of chromosome numbers of OCUP- A2 was 74 to 79. Figure A sufficient library from each sample led to subsequent successful sequencing. In DNA samples of OCUP-A1 and OCUP-A2, a mean 100× coverage of 98.8 % and 97.3 % with a mean read length of 105 bp was obtained, respectively.Thirteen gene mutations of the targeted genes were identified in both cell lines Table . 9 gene Table Figure 50 values of each cell line are shown for each anti-carcinogenic agent in Table The ICThe values of tumor markers in the supernatant of the cell lines are shown in Table 2, respectively. However, negative control (FBS included in the supernatant) hardly caused cord extension . The culture supernatant of OCUP-A1 and OCUP-A2 significantly contributed to longer cord extension than Panc-1. The value of VEGF in the supernatant of each cell line were shown in Fig. As seen in Fig. Figure The protein bands of OCUP-A1 and OCUP-A2 were compared with that of the breast cancer cell line MCF7, which was known to expresses E-cadherin Fig. . Both OCFigure In all cell lines, xenografts were successfully made from all mice. Figure Immunohistochemical stainings of the primary tumor and the xenograft of OCUP-A1 and OCUP-A2 were negative for E-cadherin, but almost positive for vimentin Fig. . The expDue to the rarity, the characteristics of APC are not well documented. The present study intended to clarify the biological behaviors of APC using two novel APC cell lines established in our institute, compared to conventional PDA. It may create an important role for future pancreatic therapy.Conventional chromosomal analysis has drawn attention to significant implications of common chromosomal aberrations in pancreatic cancer . The chrRegarding gene mutation analysis, there were common and different features between APC and PDA. Mutations of KRAS and TP53 were identified in OCUP-A1 and OCUP-A2. Although these mutations have been reported most frequently in ordinary PDA , 16 and Several assays were performed for investigating the aggressiveness of APC using the experimental model in the present study. The results demonstrated that APC cell lines did not have prominent abilities of proliferation, migration and invasion compared to other PDA cell lines in vitro. However, the tumorigenicity in vivo of OCUP-A2 was stronger than that of MiaPaca-2 and Panc-1 and that of OCUP-A1 was also stronger than that of Panc-1. These results were consistent with its clinical characteristics that APC often develops huge tumor. The average size in most series of APC has been reported to be 9 or 10 cm. We hypothesized that the high proliferation ability in vivo and in clinical observation might arise from the tumor microenvironment and increment of stem-like cells. Therefore, we performed assays for the angiogenesis, hypoxic tolerance and SP cells. The cord formation of HUVEC and production of VEGF showed that angiogenesis of OCUP-A1 was superior to that of other PDA cell lines resulting from the higher production of VEGF. Previous studies have suggested that VEGF and its receptors (VEGFR) contribute to an autocrine/paracrine mitogenic loop via mitogen-activated protein kinase (MAPK) signaling on the proliferation of cancer cells, including pancreatic cancer, besides the stimulation of angiogenesis –20. The α [The high malignancy of APC might also be related to hypoxia. The present study indicated that OCUP-A1 had hypoxia tolerance. Hypoxia tolerance could be formed through a gene mutation such as a p53 mutation, which relates to apoptosis, and the interaction between oncogenic signaling such as RAS and hypoxia-induced transcription factor (HIF) 1α . It has α . Anotherα . As a reα . Future SP cells in APC cell lines were more enriched than in MiaPaCa-2. Previous studies on SP analysis showed that SP cells related to tumorigenic potential and drug resistance, such as cancer stem cells in various type of tumors . EspeciaTo sum up, these results suggested the biological aggressiveness of APC might be related with induction of angiogenesis, acquisition of tolerance to hypoxia, and increment of stem-like cells.Chemosensitivity analysis showed that OCUP-A1 and OCUP-A2 were comparatively sensitive to several anti-cancer drugs including gemcitabine. OCUP-A2 was also the most sensitive to some of anti-cancer drugs among the examined cell lines. Strobel et al. reported that adjuvant or palliative chemotherapy did not contribute to improvement of prognosis in APC . HoweverRegarding EMT, the absence of E-cadherin and the expression of vimentin were observed in APC cell lines, xenografts and primary specimens from western blotting or immunohistochemistry. This is a feature of APC pathology , 28. In The present study was limited in two respects. One limitation was that these cell lines were samples derived from ascites. Therefore, OCUP-A1 and OCUP-A2 might lose the properties possessed in the primary site. Another limitation was the monoclonality of the cell lines OCUP-A1 and OCUP-A2 that could not reflect heterogenic properties of the pleomorphic type of APC. To resolve these limitations, a new APC cell line derived from a primary site needs to be established and then divided into several cell types using limiting dilution.The present study succeeded in establishing two APC cell lines. Angiogenesis, hypoxia tolerance, the presence of SP cells and EMT might be associated with the biology of APC. These cell lines could be useful for investigating the progression of pancreatic cancer. Effective treatment for APC could potentially be developed through further investigations using these cell lines.This study was carried out in compliance with the Helsinki declaration and was approved by the ethics committee of Osaka City University. The consent was obtained from the two patients with APC to use ascites specimens and resected tissue samples of the two patients in writing. Also, the experiment about animals was approved by the Osaka City University Ethical Committee for animal experiments.The consent to publish the individual data was obtained from the two patients in this study in writing.


Callithrix jacchus). For the first time we document derivation of NHP ESCs derived from morula stages. We show that quantitative chromosome-wise transcriptome analyses precisely reflect trisomies present in both morula-derived ESC lines. We also demonstrate that the female ESC lines exhibit different states of X-inactivation which is impressively reflected by the abundance of the lncRNA X inactive-specific transcript (XIST). The novel marmoset ESC lines will promote basic primate embryo and ESC studies as well as preclinical testing of ESC-based regenerative approaches in NHP.Embryonic stem cells (ESCs) are useful for the study of embryonic development. However, since research on naturally conceived human embryos is limited, non-human primate (NHP) embryos and NHP ESCs represent an excellent alternative to the corresponding human entities. Though, ESC lines derived from naturally conceived NHP embryos are still very rare. Here, we report the generation and characterization of four novel ESC lines derived from natural preimplantation embryos of the common marmoset monkey ( Embryonic stem (ES) cells are generally derived from the inner cell mass of preimplantation embryos at the blastocyst stage123456in vitro differentiation capability human ES cells hold great promise for curative cell replacement approachesDue to their Macaca mulatta) and cynomolgus (Macaca fascicularis) macaques belong to the group of Old World monkeys and are the most frequently used NHP species in biomedical researchRhesus  is a New World monkey endemic to Brazil. It has several significant practical and biological advantages compared to macaques. The marmoset monkey is small, easy to handle and free of zoonoses. These characteristics contribute to relatively low housing costs. Additionally, common marmosets have a significantly shorter generation time as well as a higher fecundity than macaques. Therefore, the common marmoset is the NHP model of choice for specific applications and purposes and nicely complements the macaque models. This is reflected by many studies performed in the fields of stem cell research21232426et al.The common marmoset monkey  were obtained from the self-sustaining breeding colony of the German Primate Center  and kept as described previouslyMarmoset monkeys  in the age range of 3.9 to 7.3 years were used as embryo donors. All females were kept pairwise with fertile males, and their reproductive cycles were basically monitored as described previouslyGamma-irradiated MEFs served as feeder cells and were obtained as described previouslyex vivo processing of the embryos was done at 37 °C. The ZP was removed using pronase  dissolved in KO-DMEM (Gibco). Embryos were first washed in a 100 μL drop of pronase solution, then transferred into another drop of pronase solution and kept there for 1–3 min until degradation of the ZP was observed. ZP-free embryos were immediately washed sequentially in four drops of ESM to remove the pronase and finally transferred onto MEFs in a 35 mm diameter well with ESM. Embryos were allowed to attach without any disturbances for three days before cultures were checked. If primary outgrowths were observed, the culture was continued for 2 to 3 weeks until further passaging. All pluripotent cells were cultured under hypoxic conditions  in ESM, and medium was changed every two to three days. Passaging of primary outgrowths and of resulting ES cells is described below.After recovery, preimplantation embryos were transferred to and kept in embryonic stem cell medium (ESM)For further passaging of the primary outgrowths and ES cells, StemPro Accutase  was used. Briefly, cells of one well in a six-well plate were washed with PBS and incubated with 1 mL Accutase at 37 °C for 4 min. The cell suspension was transferred to 5 mL of pre-warmed ESM and the remaining feeder layer was washed with 3 mL ESM. Cells were pelleted , resuspended in ESM and seeded onto fresh MEFs. Medium was changed every two to three days.Oligonucleotides (Sigma) used for detection of mRNA coding for pluripotency associated genes are listed in Immunofluorescence stainings were performed as described previouslyFor detection of Alkaline Phosphatase (AP), Alkaline Phosphatase Live Stain  was used. Briefly, growth medium was removed and the culture was washed with pre-warmed DMEM/F-12 two times for 2–3 minutes. Then a 1X AP Live Stain working solution was applied directly on to the cell culture and incubated for 20–30 minutes. The AP Live Stain was removed and pre-warmed DMEM/F-12 was applied to the culture prior to the visualization of fluorescent-labeled colonies under fluorescent microscopy using a standard FITC filter. Images were captured immediately.5 mouse embryonic feeder cells were combined with 8–9 × 105 ES cells in a final volume of 70 μL PBS. 60–75 μL Matrigel  were added to this cell suspension and injected subcutaneously into the inguinal region of male immunodeficient SCID/beige mice. Teratomas were retrieved 10–17 weeks, in one case 24 weeks after injection. Teratomas were immediately fixed after recovery in Bouin’s solution. After paraffin embedding, they were sectioned at 5 μm. Sections were then Hematoxylin and Eosin stained or processed for immunohistochemistry as described previouslyFor teratoma formation, 1–2 × 10et al.Karyotyping was performed by the Cytogenetic Laboratory in the Department of Human Genetics at the Universitätsklinikum Hamburg-Eppendorf (Germany) according to standard procedures. Briefly, for each cell line chromosome preparation was done from two or three 35 mm wells with ES colonies. ES cells from the wells were pooled before analysis. Then the cells were arrested with 0.2 μg/ml colcemid for 3 h and dissociated with 0.25% trypsin EDTA. For hypotonic treatment, cells were subjected to 55 mM KCl and fixed with methanol/acetic acid (3:1). For each cell line, 15 metaphases from GTG banded chromosome spreads were analysed under a light microscope at a 1000× magnification and at least four metaphases were karyotyped using a cytogenetic image analysis system . Karyotyping was done according to the chromosome assigning of Neusser Callithrix jacchus (Ensembl genome assembly 3.2.1) using the STAR alignment software (version 2.3.0e)www.bioconductor.org) using the DESeq2 package (version 1.8.1)Callithrix jacchus entries from Ensembl v78 (www.ensembl.org) via the biomaRt package (version 2.24.0)Two independent biological replicates were analyzed for each cell line. For sequencing, the RNA-samples were prepared with the “TruSeq RNA Sample Prep Kit v2” according to the manufacturer’s protocol (Illumina). Single read (50 bp) sequencing was conducted using a HiSeq 2000 (Illumina). Sequences were aligned to the genome reference sequence of We established four embryonic stem cell lines, designated DPZcjESC1-4. The embryos were obtained from different donors . Three o26730SOX2, OCT4, KLF4, cMYC, NANOG, all being transcription factors, and LIN28, an RNA binding protein. All factors have been used for reprogramming of marmoset monkey somatic cells to a pluripotent state30SALL4 was also detected. , and GDF3. Analyses of the transcriptome data revealed similar relative expression levels of all markers analyzed in the individual samples of both the morula and the blastocyst-derived cell lines  which is updated every five yearsIrrespective of the ambiguity associated with the assignment of marmoset chromosomes 19 and 20, we subjected all four ES cell lines to karyotype analysis. While the cell lines DPZcjESC3 and DPZcjESC4 had a normal female  and male  karyotype respectively, DPZcjESC1  displayed a trisomy for chromosomes 18 and 19  and DPZcjESC2  a trisomy for chromosome 19  .−12 (paired t-test), blue arrows in −6, (paired t-test), orange arrow in Irrespective of their karyotype, all generated ES cell lines were indistinguishable from each other in all assays performed so far. Therefore we were wondering whether the additional copies of two chromosomes in DPZcjESC1 and of one chromosome in DPZcjESC2 might be subjected to regulatory mechanisms which lead to at least partial inactivation of the respective chromosome. This phenomenon of dosage compensation is known for the X chromosome (see below). Therefore we compared the relative expression per chromosome  between the five ES cell lines. We found that the additional chromosomes are not subject to chromosome-wide regulatory mechanisms in the sense of dosage compensation since trisomies are clearly reflected by the deep sequencing data. Indeed, the relative expression of DPZcjESC1 was approximately 50% higher for two chromosomes, identified as 19 and 20 (p ~8 × 10We conclude that (i) two of our four new ES cell lines show trisomies, (ii) there is no evident compensatory gene expression (down-) regulation due to the presence of an additional copy of a chromosome, (iii) the transcriptome of the ES cell lines reflects their karyotype, (iv) it needs to be determined which chromosomes are affected (by clarification of numbering of the marmoset monkey chromosomes).−6, paired t-test) (X-inactive specific transcript (XIST) is a large long non-coding RNA known to be an important effector in the process of X inactivationXIST. Indeed, analysis of the transcriptome data revealed an evident correlation between XIST-expression and transcription activity from the X chromosome. While the relative amount of detected transcripts from the X chromosome was comparable in all ES cell lines generated in this study (XIST (<3 reads per million) was detected in the two male ES cell lines (DPZcjESC1 and 4) whereas both female lines (DPZcjESC2 and 3) expressed XIST at high levels (945 and 851 reads per million)   . X-inactmillion) . In sharmillion) .XIST. However, X-inactivation seems not to be an essential inherent characteristic of female ES cells.We conclude that X-inactivation can occur in female ES cells of the common marmoset monkey and that X-inactivation correlates with expression of REX1 (ZFP42), NROB1, FGF4, FGF5, and T (Brachyury)  in human pluripotent stem cells. However, mouse ES cells with a stable knockdown of NROB1 were predisposed to differentiation but still maintained pluripotency55REX1 expression was robust in all samples tested, and the average expression was comparable between the novel ESC lines, the previously established ESC line cjes001NROB1 expression was hardly detectable in any of the cell lines except for DPZcjESC4. Moderate FGF4 expression was detected in all ES cell lines and the iPS cell line but not in the fibroblasts. In contrast to FGF4, FGF5 expression was not detected in any of the novel ES and iPS cell lines, while very weak FGF5 expression was detected in the established ESC line cjes001 and in neonatal skin fibroblasts. Expression of Brachyury (T) was detected at different levels in all cell types except for the marmoset fibroblasts. In summary, quantitative analysis of these marker transcripts indicates a state of primed pluripotency of the novel marmoset monkey ES cells under the conditions used in this study.Our novel ESC lines exhibit morphological characteristics of primed pluripotency, as the colonies are flat  and not achyury) . Since tCallithrix jacchus). Although ESCs are usually derived from the inner cell mass of a blastocyst, it was previously shown that it is also possible to derive ESCs from embryos of earlier stages in mouse and human23562356We report the derivation of four novel embryonic stem cell lines from natural preimplantation embryos of the common marmoset monkey  and also whole transcriptome analyses by deep sequencing proved the absence of global differences between the cell lines of different embryonic origin. In-depth analyses of the transcriptome data revealed differentially expressed genes. However, to prove that potential differences are due to the embryonic origin and not to other parameters, more (euploid) cell lines are needed.Two of the four novel ES cell lines generated in this study, one male and one female line, have a normal karyotype while karyotype analyses revealed trisomies in the male cell line DPZcjESC1 and in the female cell line DPZcjESC2. According to karyotype analysis, chromosomes 18 and 19 are affected in DPZcjESC1, while global transcriptome data generated by deep sequencing and subsequent transcript mapping indicated trisomies of chromosomes 19 and 20. Analogously, in DPZcjESC2 there are three copies of either chromosome 19 or 20, depending on the method of analysis. Since we were not able to unambiguously bring in line the banded chromosomes 19 and 20 from two different publications32et al.606162in vitro fertilization (IVF) are a mosaic of euploid and aneuploid blastomeresin vitro manipulation or that early human embryos undergo a unique, transient phase of chromosomal instabilityIrrespective of the karyotypic characteristics of the four lines , we did not observe any obvious differences between the euploid and aneuploid cell lines. Therefore, it still remains to be investigated in detail whether all cell lines have the same characteristics. Moreover, despite the unresolved issue of cytogenetic nomenclature, it would be very interesting to determine which human chromosomes correspond to the marmoset trisomic chromosomes in the novel ES cell lines in order to find out which human trisomies may be  represented by the DPZcjESC lines 1 and 2. However, ignoring the difficulties of chromosome assignment and nomenclature described above, according to Sherlock XIST-expression and transcriptional activity of X chromosomal genes. This allows the conclusion that X-inactivation in genotypically female ES cells of the common marmoset monkey occurs in a XIST-dependent manner. However, X inactivation does not seem to be an essential inherent characteristic for pluripotency in vitro67Transcriptome analysis of the ES cell lines revealed an evident inverse correlation between OCT4A and NANOG dramatically decreased within a few days to less than 2% compared with the levels of cells cultured on MEFs (data not shown). We conclude that culture protocols established for human pluripotent cell are not directly applicable to these marmoset ES cell lines and that adaptations of the protocols for the feeder-free culture of human cells are necessary to enable feeder-free marmoset monkey ES cell culture.Quantitative analysis of the expression of marker genes for naïve or primed pluripotency of the common marmoset monkey ES cells suggests that the novel NHP ES cells are in a primed state of pluripotency . We atteThe four novel ES cell lines derived in this study extend the available repertoire of marmoset monkey ES cell lines. Since ES cells derived from natural embryos are considered as the gold standard for pluripotent stem cells, their availability is crucial to identify fundamental (epigenetic) differences between primate ES and induced pluripotent stem (iPS) cells. Furthermore, pluripotency of the novel ESC lines 3 and 4, although apparently in a primed state under culture conditions currently established, may be worth testing in chimera formation assays.Hierarchical clustering showed that the novel ES cell lines’ transcriptomes are very similar, but that they – together with the transcriptomes of an iPS cell line we previously generatedFinally, two of our novel cell lines are female and two are male allowing the investigation of possible sex-specific differences between the lines. Interestingly, except for one ES cell line, which unfortunately is not available any more26In the context of the increasing interest in use of the common marmoset as a non-human primate model species that closely resembles human physiology .


This data paper shows functional activities of the three cell lines in vitro and in vivo. Phase-contrast images show the morphology of these cells, metabolic and luciferase activity has been determined. Survival data of mice peritoneally injected with SK-OV-3 Luc or SK-OV-3 Luc IP2 is available with H&E histology of the peritoneal implants. Tumor growth curves and bioluminescent images of mice inoculated with a different number of SK-OV-3 Luc IP2 cells are also included.This data paper contains information about the  Specifications tableValue of the data•in vivo selection of SK-OV-3.This data shows intraperitoneal tumor formation with low cell inoculation after •This method can be applied to other cancer cell lines to increase metastasis take rate even with lower inoculation numbers.•in vivo model for peritoneal implants, providing researchers with a references for their in vivo studies.These data provides growth curves, survival data and histology about the SK-OV-3 Luc (IP2) 1in vivo model for the development of peritoneal ovarian tumor implants. Usually inoculation numbers of 1–2×106 SK-OV-3 (Luc) are used 6 SK-OV-3 Luc cells gives a 100% tumor take with an average survival time of over 2 months  of SK-OV-3 Luc IP2 is sufficient to form peritoneal implants this model has been applied successfully Intraperitoneal injection (IP) of SK-OV-3 (Luc) cells is an established 2 months  [3]. To 22.12 in air.SK-OV-3 is a human ovarian cancer cell line (ATCC number: HTB-77). SK-OV-3 Luc (Luciferase positive SK-OV-3 cells) were prepared by pFL4.76 plasmid transfection and selection . SK-OV-3 Luc (IP1/2) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics (penicillin/streptomycin), and incubated at 37 °C with 10% CO2.26 SK-OV-3-Luc cells were inoculated intraperitoneally in immunodeficient female Swiss/nu mice  to isolate populations that form peritoneal implants. Tumor implants were collected in saline. The implants were cut into small pieces (1 mm) and dissociated by de gentleMACS Dissociator  in the presences of 1 mg/ml collagenase from Clostridium histolyticum (Sigma-Aldrich) in PBSD+. The mixture was cleared through a cell strainer (70 µm) and the homogenized single cell suspension was expanded in culture by seeding in DMEM 10% FBS. After 24 h, non-adhered cells were removed and the medium was refreshed; the resulting culture was maintained as the parental cell line. Each subsequent intraperitoneal metastatic generation is designated IP1, IP2. in vivo selection. Animal experiments were conducted accordance with the local ethics committee . 1×102.3SRT-profiling was conducted on the SK-OV-3, SK-OV-3 Luc and SK-OV-3 Luc IP2 cell lines to confirm the identity of the cell lines. 9 DNA sites were profiled and the alleles were identical between the three cell lines and to the profile published by ATCC  [6].2.44 SK-OV-3 Luc or SK-OV-3 Luc IP1 cells per well, were seeded in a 96-well plate. After 96 h, MTT analysis was performed. Briefly, the culture medium was replaced by 100 μl culture medium containing 1 mg/ml MTT. Following 2 h incubation at 37 °C, MTT-containing medium was removed and 150 μl of dimethylsulfoxide (DMSO) was added to dissolve formazan crystals. Absorbance was measured at 570 nm and background measured at 650 nm, with a plate reader . n=3) of 6 independent metabolic activity analysis 96 h after seeding 4×104 SK-OV-3 Luc or SK-OV-3 Luc IP1.A single cell suspension of 4×102.5A single cell suspension of SK-OV-3 Luc, SK-OV-3 Luc IP1 and SK-OV-3-Luc-IP2 cells were seeded in a 96-well plate in different cell numbers . Cells were allowed to adhere for 2 h, just before luciferase activity was measured with IVIS (PerkinElmer), 150 µg/ml D-Luciferin, firefly (Perkin-Elmer) was added. 2.66 SK-OV-3-Luc or SK-OV-3-Luc-IP2 cells. Mice were monitored and from the first visual signs of advanced carcinomatosis  the mice had reached their end-point and were sacrificed. Peritoneal organs were embedded in paraffin before standard hematoxylin and eosin (H&E) staining was conducted. Animal experiments were conducted in accordance with the local ethics committee . Immune deficient female Swiss/nu mice were inoculated intraperitoneally with 1×102.76, 0.5×106, 0.25×106 and 0.1×106 SK-OV-3 Luc IP2 cancer cells. Tumor growth was monitored by weekly bioluminescence imaging (BLI). Immune deficient female Swiss/nu mice were inoculated intraperitoneally with 1×10


In total we found six clusters of drugs of which five consisted of drugs with related mode of action and one cluster with two drugs not previously connected. In total, 25 correlated and four anti-correlated drug sensitivities were revealed of which only one drug, Sirolimus, showed significantly lower IC50 values in the luminal/ERBB2 breast cancer subtype. We found expected interactions but also discovered new relationships between drugs which might have implications for cancer treatment regimens.With substantial numbers of breast tumors showing or acquiring treatment resistance, it is of utmost importance to develop new agents for the treatment of the disease, to know their effectiveness against breast cancer and to understand their relationships with other drugs to best assign the right drug to the right patient. To achieve this goal drug screenings on breast cancer cell lines are a promising approach. In this study a large-scale drug screening of 37 compounds was performed on a panel of 42 breast cancer cell lines representing the main breast cancer subtypes. Clustering, correlation and pathway analyses were used for data analysis. We found that compounds with a related mechanism of action had correlated ICThe online version of this article (doi:10.1186/s40064-015-1406-8) contains supplementary material, which is available to authorized users. Life expectancy and survival of breast cancer patients have increased significantly over the last decades, due to—amongst other factors—an increasing number of effective drug therapies , between Quisinostat, Panobinostat, Vorinostat and Belinostat , between Docetaxel and Paclitaxel , between JNJ-707 and JNJ-493  and between MI-219 and Nutlin-3 ; all correlations are listed additionally in Table nes Fig. —a mechannes Fig. —showed nUnanticipated but highly significant correlations were observed between particularly Doxorubicin and Azacitidine , between Decitabine and 5-Fluorouracil (r = 0.58) and Serdemetan (r = 0.56); and between Serdemetan and Tipifarnib (r = 0.52). Additional weaker, but expected correlations were found for Vandetanib with Erlotinib and Gefitinib . Decitabine was correlated with Lapatinib (r = 0.52) and Veliparib with Serdemetan and Decitabine . Furthermore, we also detected a remote relation between various tyrosine kinase inhibitors like JNJ-493 with the multi-receptor tyrosine kinase inhibitor Sunitinib  , except for Bortezomib (2.00−5 to 6.00−11 M), Sirolimus and 17-AAG (both: 2.00−6 to 6.00−12 M). Drug diluent was used as negative control treatment. DMSO was used as drug solvent and diluent for all drugs except for Methotrexate  and Cyclophosphamide .Forty-five breast cancer cell lines with confirmed identity  Voigt : After t50 values. For drug correlation analysis, IC50 values per compound of each cell line were correlated with each other in Excel 2007  using Pearson correlation. Cell lines with missing data for several drugs were discarded from correlation and cluster analysis, as were drugs that did not show differential IC50 values. The programs Cluster 3.0  and Java Treeview version 1.1.6r2 (Saldanha Cell lines were clustered in a hierarchical fashion based on their ICSaldanha  were use50- versus cell lines with a low IC50-value per drug. Cell lines were grouped in the high or low group by individual evaluation per drug instead of pre-selecting a fixed IC50 value for all drugs. This method was chosen to test the drug response extremes rather than testing values with little difference, which are present in gradual IC50 distributions. For this distribution type we used the cell lines at the distribution extremes and removed the intermediate values to reduce noise. A few drugs showed an IC50 profile that precluded a sensible grouping; e.g. Paclitaxel had only two cell lines with a high IC50 value while the others had a very low IC50; too few cell lines in a group renders the pathway analysis useless. For this reason, we excluded JNJ-208, Sirolimus, Docetaxel and Paclitaxel from the pathway analysis. For all other drugs, we were able to use meaningful group sizes of at least five samples each (Additional file For pathway analysis we analyzed gene expression levels of cell lines with a high ICFor all cell lines, previously published mRNA expression data of our laboratory (Riaz et al. 50 profiles and a Chi Square or Fisher’s Exact test for two-group IC50 profiles. Previously published ER protein expression data (Riaz et al. All drugs were tested for association with breast cancer cell line subtypes. First, cell lines were grouped into luminal, basal, ERBB2-overexpressing and normal-like on the basis of intrinsic subtypes as previously reported (Hollestelle et al.


On the other hand, there was a clear dose-dependent increase in protein β-sheet structure related peaks shift in resistant cell lines after exposure to cisplatin. This would probably indicate an easier protein interaction site for cisplatin in the resistant cell lines, which would probably inhibit cisplatin from binding to DNA, as the cytotoxic target. As a conclusion, FTIR biospectroscopy has proven its potency to identify the interactions, as well as the false engagement cellular sites for cisplatin in sensitive and resistant cell lines.Fourier Transformed Infrared Spectroscopy (FTIR) has extensively been used for biological applications. Cisplatin is one the most useful antineoplastic chemotherapy drugs for a variety of different human cancers. One of the clinical problems in its application, which would consequently affect the therapeutic outcome of its application, is the occurrence of resistance to this agent. In this project three different pairs of sensitive and resistant cell lines of human ovarian A2780 and its resistant pair of A2780-CP, human ovarian OV2008 and its resistant pair of C13, and finally human lung carcinoma of HTB56 and its resistant pair of HTB56-CP were grown in the laboratory under the standard procedure. Saline was exposed to control cells, whereas 1, 5 and 10 µg/mL of cisplatin was exposed to experimental cells, for 1 h. Cells were then collected and lyophilized from which spectra were taken. According to our results, we could not trigger a well-recognized cells biomolecular band at 1015 cm Cancer is one of the life-threatening diseases, killing many people every year. Cisplatin is one of the most effective anticancer drugs for the treatment of ovarian, breast, head and neck, and childhood leukemia . One of Among the different methods for the investigation on the cellular mechanisms is biospectroscopy . Using dIn this study, we have tried to apply FTIR spectroscopy to investigate three paired human tumor cell lines, sensitive and resistance to cisplatin. The idea is to compare the spectral patterns of sensitive and resistant cell lines to each other and look for the most likely related peaks appeared, disappeared and/or altered in all three pairs of resistant vs. sensitive cell lines, and/or following the exposure to cisplatin. We are presenting data, for the first time, to support the application of FTIR spectroscopy for pharmacological investigations, as well as clues to clarify the mechanism of resistance to cisplatin in these cell lines.Cell lines(ATCC® HTB-56™) and its cisplatin resistant variant of HTB56-CP were obtained from the Pharmacology Lab, Ottawa Regional Cancer Centre, Ottawa, Ont., Canada. The cells were cultured following the standard methodology of the American Type Culture Collection . Cells were cultured in DMEM/F12 medium  supplied with 10% heat-inactivated fetal bovine serum in 37 ºC humidified incubator with 5% CO2. All cellular experiments were done in triplicate. All experiments were performed on the exponentially growing cells, after a minimum of three passages from the initial seed of frozen stock. Human ovarian carcinoma cell lines of OV2008 and A2780 , and theChemicals Cisplatin was the clinical formulation of 1 mg/mL in 0.9% saline supplied by Horner Laboratories . This formulation was diluted in additional 0.9% saline to the appropriate concentrations and incubated for 1 h at 37 °C before use.Drug Treatment of cellsCells were grown in 6-well plates separately and then were exposed to 1, 5, and 10 μg/mL of cisplatin in serum-free media to prevent cisplatin denaturation through the protein binding, based on the published method . ControlFreeze-drying protocol-2 millibar pressure. Temperature was then increased gradually up to the room temperature of 25 °C in 22 h under the same vacuum condition. Vial lead was then sealed using 7x10-3 millibar pressure. These vials containing freeze-dried cells were kept in refrigerator before the FTIR experiments. Cells lyophilization was performed using a Usifroio model SMH-50 instrument. Readily collected cells were transformed into specific lyophilization 10 mL vials and the initial freezing was performed at -40 °C in 2.5 h. Primary drying procedure was then conducted on these cells during 4.5 h at the same temperature at 5x10FTIR analysisAt the time of spectroscopy, vials containing freeze-dried cells were opened and 5 mg of cell powder were mixed with 95 mg of potassium bromide rapidly. A small amount of this mixed powder was put in the sample holder of a Perkin Elmer instrument model Spectrum one provided with a TGF detector, and analyzed using the DRIFT (Diffuse Reflectance Infra-red Fourier Transform) method. Pure potassium bromide was used as the control for background spectra. -1 resolution, were applied in the range of 600-4000 cm-1. Spectral mathematical manipulations  and statistical parameters (such as peak frequency shifts and intensity ratios of various infrared bands) were calculated using Perkin Elmer software 1B version 3.02. For each spectrum, a total of 512 scans, at 4 cmStatistical analysis-1) and absorbance ratios based on the peak height and area under the curves. Results, expressed as mean ± S.D in tables and graphs were created using nonlinear regression analysis in GraphPad PRISM® Software. One-way analysis of variance (ANOVA) followed by Dunnett's post hoc test was used to assess significant differences (p < 0.05) between spectra resulted from different cells exposed to different concentrations of cisplatin. All the statistical analysis was performed using GraphPad PRISM® version 5 Software .Different spectral parameters were used for the comparison purposes between the control and cisplatin exposed cells, including wave numbers  is one of the most promising techniques to distinguish biological variations among different samples. Simplicity, prompt response, sensitivity and subject independence properties hallmark this technique among other conventional diagnostic methods. Although it is in the beginning and discovering steps, its advantages on cancer detection have widely been examined  and new 2O/D2O substitute techniques (data not shown).We have aimed to try this technique for the estimation of cancer cells sensitivity to cisplatin cytotoxicity as a model for its application in chemotherapy outcome prediction use. Cisplatin is one of the most widely used cancer chemotherapeutic agents in clinics, and many sensitive or resistance cell lines to this agent are recognized and well established. Three pairs of the same origin, but cisplatin sensitive and resistant variant cell lines, were selected in this study and exposed to the clinically relevant doses of cisplatin for a double period of clinically relevant half life of this drug. Cells were then collected, lyophilized and examined under FTIR instrument to look for a well discriminative peak to be consistent with sensitivity or resistance of the cell line to cisplatin. Lyophilization has been introduced to the collected cells to set up a repeatable unique method of cell preparation and long term preservation to be useful for biospectroscopy, which will also eliminate the large proportion of cellular and intracellular water with its related large interfering peak which mask many spectroscopic peaks from various cellular biomolecules in FTIR spectroscopy. A good and acceptable reproducibility in cell spectra have also been acquired using the lyophilized cells instead of other sample drying or Het al 2 symmetric stretching in aliphatic compounds, it has not yet been well recognized in biological structures of cell lines , cellular phospholipids (indicating with peak at 1080 cm-1) and a not yet known biomolecule indicated at 1015 cm-1, instead of cisplatin target site on the α-helical structure of DNA. More investigations are ongoing to further explore this finding.As a conclusion, our work has further proved the application of FTIR spectroscopy in biological discoveries. Our unique protocol of FTIR biospectroscopy on three sensitive and resistance cell lines to cisplatin and after exposure to the different concentrations of this agent has presented some candidate biomolecular sites for the action, as well as resistance to this drug in these cell lines, to be further investigated in detail in future works. Here, we have presented clues to support a new hypothesis that resistance to cisplatin might well be related to the engagement of this agent to β-sheet structures of proteins (indicating with peak at 1625 cm