Among its several activities, Nef is essential for maintaining HIV-1 maximal infectivity. In cell culture, such activity can account for as much as 98% of the HIV-1 virion infectivity and requires Nef to be expressed in virus producing cells rather than in target cells. However, the cause of the defective infectivity of Nef-negative HIV-1 and the mechanism by which Nef is able to restore such a defect remain elusive. The purpose of this research is to establish whether Nef contributes to HIV-1 infectivity by counteracting a cellular inhibitor or by promoting a cellular activity required for optimal infectivity.Nef is an HIV-1 accessory protein with a fundamental role for virus replication Several human cell lines of different histological origin were used as HIV producer cells in order to study the variability of the requirement of Nef for infectivity. Single cycle infectivity was measured on a HeLa-based reporter cell line after normalization based on reverse transcriptase of the inocula. To determine the dominance of the Nef requirement for optimal infectivity, virus is produced from heterokaryons generated from cells responsive or not-responsive to Nef.We analyzed the ability of Nef to enhance HIV infectivity produced in more than 50 cell lines. We found that the requirement of Nef is highly variable. We identified a group of cell lines (Nef-responsive) in which the lentiviral protein is most required (10-50 fold) and a group of cell lines in which Nef is not at all or only weakly required . All Nef-responsive cell lines belong to the lymphoid lineage, in line with the lymphotropic nature of HIV, while most cell lines belonging to the Nef-unresponsive group include mostly non-lymphoid cells. However, we identified two Nef-unresponsive cell lines of lymphoid origin, providing useful tools for further investigation to study the dominance of the Nef requirement for infectivity. We have established a heterokaryon assay which allows HIV-1 production only upon fusion of producer cells expressing complementary parts of the virus. We are now in the process of coupling producer cell lines with opposite Nef-responsiveness in order to verify the possibility that Nef counteracts a dominant cellular inhibitor of retroviral infectivity predominantly expressed in lymphocytes.Our findings show that the Nef requirement for virus infectivity is extremely variable depending on the type of producer cells and is strongest in lymphoid cells. From the study of cell lines with marked differences for Nef-responsiveness, we aim to identify cellular factors which determine the Nef requirement for HIV infectivity.


Here, we describe the successful establishment and characterization of three patient-derived MMR-D cell lines  along with their corresponding xenografts.Colorectal cancer (CRC) represents a morphologic and molecular heterogenic disease. This heterogeneity substantially impairs drug effectiveness and prognosis. The subtype of mismatch repair deficient (MMR-D) CRCs, accounting for about 15% of all cases, shows particular differential responses up to resistance towards currently approved cytostatic drugs. Pre-clinical in vitro and in vivo.MMR-D cell lines  were established from a total of ten clinicopathological well-defined MMR-D cases . Cells were comprehensively characterized by phenotype, morphology, growth kinetics, invasiveness, and molecular profile. Additionally, response to clinically relevant chemotherapeutics was examined wt, B-rafmut, HROC87: K-raswt, B-rafmut), whereas the HROC113 cell line  was HNPCC-associated. A diploid DNA-status could be verified by flow cytometry and SNP Array analysis. All cell lines were characterized as epithelial (EpCAM+) tumor cells, showing surface tumor marker expression (CEACAM+). MHC-class II was inducible by Interferon-γ stimulation. Growth kinetics as well as invasive potential was quite heterogeneous between individual lines. Besides, MMR-D cell lines exhibited distinct responsiveness towards chemotherapeutics, even when comparing in vitro and in vivo sensitivity.Two MMR-D lines showing CIMP-H derived from sporadic CRC (HROC24: K-rasThese newly established and well-characterized, low-passage MMR-D cell lines provide a useful tool for future investigations on the biological characteristics of MMR-D CRCs, both of sporadic and hereditary origin. Additionally, matched patient-derived immune cells allow for comparative genetic studies. Developing preclinical cancer models has substantially contributed to a more detailed understanding of colon carcinoma (CRC) initiation and progression V600E mutations and loss of PTEN expression.MMR-D accounts for 15% of all CRCs in vitro and in vivo models. Cell line establishment has been described to be successful either directly from fresh tumor tissue or from so-called xenopatients by engrafting tumor fragments in immunocompromised mice before the in vitro culture step. For both cases, maintenance of the original tumor’s cell differentiation, morphology and molecular signature is initially warranted Mandatory for identifying accurate novel molecular markers and for testing innovative treatment regimen is the availability of suitable in vitro and in vivo chemosensitivity towards selected antineoplastic drugs provides a ready basis for preclinical evaluation of innovative treatment regimens. Such patient-derived cell line sets are especially ideal tools to optimize development of individualized therapeutic strategies in the near future.In this study, we describe a feasible and straightforward method for establishing new MMR-D cell lines, along with their corresponding xenopatients. Subsequent detailed analysis of tumor biology, genetic, as well as 2. All cell culture reagents were obtained from PAA , antibiotics and antifungal agents were provided by the university hospital’s pharmacy.Primary CRC resection specimens were received fresh from surgery, with informed written patient consent (n = 10). All procedures were approved by the Ethics Committee of the Medical faculty, University of Rostock  in accordance with generally accepted guidelines for the use of human material. Tumor samples were cut into small pieces. For cryopreservation and subsequent xenografting, pieces (3×3×3 mm) were frozen  at −80°C. Other pieces were stored in liquid nitrogen for molecular analysis. Cell culture was started from single cell suspensions, seeded on collagen-coated plates in Quantum tumor medium  and incubated at 37°C in a humidified atmosphere of 5% C02 culture flask was performed when tumor cell growth was observed. Continually growing cell cultures were further passaged and regularly stocked in low passages.Medium was changed regularly. Initial passage into a 25 cmin vivo engraftment, six-week-old female NMRI nu/nu mice were used as recipients. Mice were bred in the university’s animal facility and maintained in specified pathogen-free conditions. All experimental procedures were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the University of Rostock . All surgery was performed under Ketamin/Xylazin anesthesia (dose: 90/25 mg/kg bw), and all efforts were made to minimize suffering. Subcutaneous (s.c.) tumor implantation was performed as described 3) were removed and underwent in vitro culture protocols as described above.For Histopathological examination of primary tumors was done according to standard protocols for clinicopathological CRC staging V600E genes were done as described. Finally, DNA-methylation in CIMP-sensitive promoters was traced by the MethyLight technology with a modified marker panel originally published by Molecular classification was done according to B-lymphoid cell lines (B-LCLs) were generated from purified peripheral blood leukocytes by Epstein-Barr virus (EBV)-transformation as described 2 flasks and daily counted for seven days. Ploidy and cell cycle analysis was conducted by flow cytometry  as described Population doubling times were determined by viable cells seeded into replicate 25 cmCell surface marker expression on established tumor cell lines was traced by flow cytometry with and without IFN-γ pre-treatment using a panel of Abs . Primers were: forward: 5′-GGC GAA TGG GTG AGT AAC ACG-3′; reverse: 5′-CGG ATA ACG CTT GCG ACC TAT G-3′ yielding an approximately 500 bp product . Potential polyomavirus infection was tested from gDNA according to Mycoplasma contamination was tested by the 16S-rRNA-gene-based polymerase chain reaction (PCR) amplification method from cell lysates. Amplification was carried out in a total volume of 25 µl (2 mM MgCl3 or 1×104 cells/well). Two days after plating, triplicate wells were treated with increasing drug concentrations . This procedure was repeated after three days of treatment ( = two cycles). Cellular metabolic activity in treated versus control wells was estimated by MTT assay as described above. Drug effects were determined at the level of 50% inhibition (IC50) compared to controls.Cells were seeded into 96-well microtiter plates  were implanted s.c. into nude mice under anaesthesia (Ketamin/Xylazin 90/25 mg/kg bw) and allowed to grow until tumor establishment. Mice with established tumors received therapeutic applications of selected drugs . Tumor-carrying mice receiving PBS (n = 7 per group) served as controls. Tumor growth was controlled regularly and volume was estimated according to the formula: V = width2 * length * 0.52. All mice  were sacrificed at day 21 or when they became moribund before the tumor volume reached 2000 mm3. Tumors were removed for further histological examinations.Thereafter, response to selected therapeutics was tested in vitro data and mean ± SEM for in vivo data. Statistics were done on in vivo experiments. After proving the assumption of normality, differences between saline and treated animals were determined by using the unpaired Student’s t-test. If normality failed, the nonparametric Mann–Whitney U-Test was applied. The tests were performed by using Sigma-Stat 3.0 . The criterion for significance was set to p<0.05.Values are reported as the mean ± SD for In this study, ten cases of clinicopathological well-defined MMR-D tumors were collected from a series of 120 CRC cases. Samples were obtained from resection specimens without prior therapy. Clinicopathological patient’s characteristics are summarized in in vitro and in vivo approaches were combined: parts of surgical tumor specimens were either directly processed for in vitro cell line establishment or frozen native for subsequent xenopatient generation  originated from a sporadic CRC patient and HROC113P was derived from a HNPCC patient with a germline MLH1 mutation.In a parallel series of experiments, xenografting was performed on 8/10 tumors by subcutaneous implantation into immunocompromised recipients. Preservation of morphology was confirmed by comparing histology of xenografts with the original tumor .in vitro approach .Comprehensive molecular classification on freshly established MMR-D cell lines was paralleled by examinations on original tumor material as well as on corresponding xenografts (HROC24 and HROC87). This analysis revealed no difference in any of the samples and hence data presented in The MMR-D status was evidenced by using the advanced Bethesda panel. All three cell lines exhibited instability in the six markers analyzed. Mutations in tumor-associated genes varied among cell lines. HROC24P and HROC87X displayed high-degree of CIMP (methylation in 6/6 markers), including MLH1 promoter methylation. The HROC113P cell line showed characteristic molecular features associated with the HNPCC syndrome like mutations in codon 12 of the K-ras gene, but wildtype B-raf. Moreover, methylation –except for MGMT– was absent. MMR-D tumors are by definition devoid of or have very low levels of CIN Determining morphology revealed tight adherence to the bottom of the cell culture flasks. All cell lines were characterized as epithelial-like cells without contaminating fibroblasts. Initially, HROC24P cells proliferated as tightly packed multi-cellular islands , two different cellular clones were observed. After serial passages, numbers of large cells gradually decreased and were entirely replaced by the dominant smaller cell clone . Highest levels were observed for the neutrophil-attracting chemokine IL8. Values of the Th2 cytokine IL4 were identical between all three lines, ranging from 110 to 230 pg/ml . A similar pattern was seen for IL10. IL6 production was quite low in the MMR-D lines and none of them secreted detectable levels of the immunostimulatory IFN-γ.in vitro system was used. Exponentially growing cells were treated for a total of six days. In order to mimic the in vivo situation, cells received two chemotherapeutic cycles and IC50 levels were calculated  were sensitive towards irinotecan-mediated growth inhibition; with doses comparable to or even lower than plasma concentrations in patients . InteresThereafter, sensitivity towards several additional cytostatic drugs was tested. The microtubule-stabilizing compound paclitaxel showed effectiveness comparable to the standard drugs. Again, highest efficacy was observed against HROC24P and HROC87X cells, while it was less potent towards HROC113P cells. A somewhat unexpected finding was the high responsiveness towards gemcitabine. All three cell lines showed substantial response, even at very low doses. Inhibition of cell proliferation was achieved at concentrations well below plasma levels under standard therapy.in vitro chemosensitivity patterns to cytotoxic drugs were very individual with the HNPCC-derived HROC113 tending to be more resistant than their sporadic counterparts.In summary, in vivo. Similar to the in vitro growth behavior, HROC24P tumors displayed fastest growth, HROC87X tumors grew slowest and HROC113P tumors showed an intermediate growth rate. No distant metastases were detected neither at necropsy, nor following histologic examination of the inner organs (data not shown).All three cell lines engrafted well and gave rise to growth in vivo  were directly established from surgical resection specimens, whereas one cell line (HROC87) was obtained subsequent to xenografting. In addition, a paired xenograft cell line from the HROC24 tumor was obtained, too. By combining xenografting and direct in vitro cell culture, even more individual patient-derived cell lines, reflecting the original tumors’ molecular signature, will probably become available in the future. Moreover, preselection based on criteria like node infiltration, advanced stage, and elevated CEA in serum may increase the rate of successful tumor engraftment rate into immunocompromised mice In this study, we picked up the idea of establishing and characterizing patient-derived individual MMR-D tumor models for testing treatment modalities. Firstly, to ameliorate the success rate for CRC cell line establishment, an experimental strategy –based on the combination of +, CEACAM+) with heterogeneous adhesion  and co-stimulatory marker (CD80) expression as well as different cytokine secretion patterns. MHC class I was only expressed by HROC113 cells. Whereas MHC-class II expression was normally absent but inducible by Interferon-γ stimulation. These findings are of interest for development of immune-based therapeutic concepts. Of note, paired immune cells, i.e. antigen-presenting B-LCL and (effector) T cells, offer the chance to work in complete autologous settings. For the MMR-D models described here, limited amounts of immune cells are available to us.The three newly established MMR-D cell lines exhibited variations in terms of morphology and growth kinetic. Confirming their origin, they were recognized as epithelial tumor cells (EpCAMin vitro passages (<50), thereby reflecting the original tumors’ biology and not a genotypic and phenotypic heterogeneity that could arise following long-term culture V600E mutations and high-degree promoter methylations in CpG islands of sporadic tumors ; and a K-ras mutation but CIMPneg in the HNPCC-associated HROC113P. Which molecular markers predict patients’ clinicopathological outcome best is currently a matter of debate. Bae and colleagues recently claimed that CIMP status might be crucial for MMR-D CRCs response to cytotoxic chemotherapeutics neg counterparts. Also, BRAFV600E mutations, which have never been found in CIMPneg tumors, seem to be another independent prognosticator of poor outcome and thus an important confounder to the prognostic role of CIMP Comparing molecular profiles of MMR-D cell lines with original tumor material as well as matched xenopatients revealed virtually no differences. Somehow unexpected, tumor-associated mutations, MMR-D-status, DNA-methylation in CIMP-specific promoters and LOH profiles remained identical between these samples. It is noteworthy that cells were exclusively studied at early in vitro and in vivo response, especially of the HNPCC-associated HROC113P cells. MMR-DD tumors were described to be more sensitive towards microtubule-stabilizing agents than their CIN counterparts in vitro, but failed in vivo.Additionally, it led us to hypothesize that even molecular closely matched cancer cell lines may respond different towards certain chemotherapeutics. Indeed, chemosensitivity varied between our cell lines.MMR-D. Of particular interest was the difference in terms of Of particular interest was the sensitivity towards gemcitabine, the standard drug for treatment of pancreatic carcinoma – but not of CRC in vitro and subsequently verified in xenopatients. This will help to identify patients likely to benefit from optimal care, while sparing those unlikely to benefit. Thus, unnecessary toxicity and costs are avoided.These experiments shall be combined with systems biology-based approaches for predicting patient outcome and responsiveness to conventional and novel, targeted treatment strategies http://www.northgermantumorbank-crc.de).Our comprehensive biobanking strategy for CRC includes the collection of primary material  together with established xenografts, patient as well as xenopatient-derived cell lines and matched B-LCLs. Recently, we broadened this approach by collecting tumor samples and correlating clinical data from multiple clinical centers in the context of a third-party funded biobanking initiative (This strategy may thus be ideal to address the question of whether cell lines, xenopatients or both are suited models for predicting treatment and response. If so, this strategy will pave the way towards truly personalized therapy.


The amyloid precursor protein (APP) as well as its homologues, APP-like protein 1 and 2 (APLP1 and APLP2), are cleaved by α-, β-, and γ-secretases, resulting in the release of their intracellular domains (ICDs). We have shown that the APP intracellular domain (AICD) is transported to the nucleus by Fe65 where they jointly bind the histone acetyltransferase Tip60 and localize to spherical nuclear complexes (AFT complexes), which are thought to be sites of transcription. We have now analyzed the subcellular localization and turnover of the APP family members. Similarly to AICD, the ICD of APLP2 localizes to spherical nuclear complexes together with Fe65 and Tip60. In contrast, the ICD of APLP1, despite binding to Fe65, does not translocate to the nucleus. In addition, APLP1 predominantly localizes to the plasma membrane, whereas APP and APLP2 are detected in vesicular structures. APLP1 also demonstrates a much slower turnover of the full-length protein compared to APP and APLP2. We further show that the ICDs of all APP family members are degraded by the proteasome and that the N-terminal amino acids of ICDs determine ICD degradation rate. Together, our results suggest that different nuclear signaling capabilities of APP family members are due to different rates of full-length protein processing and ICD proteasomal degradation. Our results provide evidence in support of a common nuclear signaling function for APP and APLP2 that is absent in APLP1, but suggest that APLP1 has a regulatory role in the nuclear translocation of APP family ICDs due to the sequestration of Fe65. The amyloid precursor protein (APP) is a type I transmembrane glycoprotein, encoded by a single gene on chromosome 21q21, which is causally involved in Alzheimer’s disease (AD) Drosophila, and APL-1 in C. elegans. APP and APLP2 are ubiquitously expressed, with high expression in the brain APLP2 is located on chromosome 11q23-q25 and exists in two alternatively spliced forms, one of which, similarly to APP, contains a KPI domain APLP1 is located on chromosome 19q13.1 and, as yet, no spliced transcripts have been identified APP is a member of a highly conserved family of glycoproteins, which includes the APP-like proteins 1 (APLP1) and 2 (APLP2) in vertebrates, APPL in Despite the structural homology and conserved domain structure of APP family members, it has been shown that their subcellular localization differs strikingly. A recent study by Kaden et al. showed that APP and APLP2 mainly localize within intracellular compartments, such as the ER and endosomes, with only low levels at the plasma membrane. In contrast, APLP1 was found to mainly localize to the plasma membrane, corresponding to an increased tendency to form in-trans interactions at cell-cell contacts, highlighting an important role for APLP1 in cell adhesion Studies using knockout mice have revealed important insights into the relationship between different APP family members. Single knockout for APP, APLP1, or APLP2 cause minor phenotypes due to a clear redundancy within the APP gene family Both APLP1 and APLP2, like APP, can be cleaved by α-, β- and γ-secretase, and intracellular domains are released after γ-secretase cleavage at the ε-site Proteolytic processing of APP, APLP1 and APLP2 by γ-secretase-mediated ε-cleavage generates intracellular domains (ICDs), which are stabilized by binding to the adaptor protein Fe65 and, when coexpressed with Fe65, can transactivate Gal4-Tip60 constructs The members of the APP family–APP, APLP1, and APLP2–show a high degree of sequence homology. In particular, the intracellular domain of APP family members is highly conserved and contains common motifs such as a caspase cleavage site and a YENPTY domain for binding of adaptor proteins. We have previously shown that AICD bound to Fe65 translocates to the nucleus and forms, together with Tip60, spherical nuclear AFT complexes Coexpression of Fe65, Tip60, and APP in HEK cells resulted in the retention of Fe65-AICD complexes in the nucleus and the formation of AFT complexes as reported previously To exclude the possibility that the observed different nuclear signaling capabilities of APP family members were due to properties of the chosen cell line, experiments were repeated in N2a mouse neuroblastoma cells. Coexpression of Fe65, Tip60, and APP led to the formation of spherical nuclear AFT complexes , top rowTo further study the subcellular localization of the APP family members we expressed APP/APLP with N-terminal 3 myc tags, preceded by the APP signal peptide to ensure membrane insertion, and C-terminal 3HA tags . A GAPDHThe N-terminal 3 myc tag-constructs allowed cell-surface labeling of APP/APLPs on living cells . AntibodTo measure the half-lives of the APP family members we inhibited protein synthesis for different durations. HEK cells were transfected with C-terminally 3HA-tagged APP/APLP constructs, all driven by a GAPDH promoter to ensure comparable expression levels. 24 hours after transfection, protein synthesis was inhibited with cycloheximide (CHX). Cells were harvested after the indicated time of cycloheximide incubation and levels of full-length protein determined by Western blot . APP/APLTo investigate whether differences in nuclear localization capability of AICD and AL1ICD are mediated by properties of their extracellular or intracellular domains, we constructed chimeric APP/APLP1 expression plasmids. ICDs of APP and APLP1 were joined at the ε-cleavage site of APLP1 and APP, resulting in the chimeric constructs APLP1-AICD and APP-AL1ICD, respectively . ChimeriBecause the chimeric proteins carry a chimeric ε-cleavage site it is possible that absent nuclear signaling of ICDs is due to impaired γ-secretase cleavage. To investigate this possibility, HEK cells were transfected with APP, APLP1 or the chimeric APP/APLP1 proteins and treated with the γ-secretase inhibitor DAPT . DAPT trOur results show that nuclear signaling by the APP family members is determined by properties of the ICDs, which are highly conserved and share common motifs, such as a caspase cleavage site and the YENPTY motif . We reasCo-immunoprecipitation of streptavidin-binding protein (SBP)-tagged APP and APP(VML646LLR) showed that Fe65 binding is not impaired by exchange of N-terminal ICD residues . Since DSeveral reports have demonstrated that AICD is degraded by the proteasome Next, we asked whether the localization of APP family ICDs that do not form AFT complexes is changed after epoxomicin treatment. Both APP(VML646LLR)-Cit and APP-AL1ICD-Cit, coexpressed with Fe65 and Tip60, formed AFT complexes when treated with epoxomicin . These rAPLP1 does not signal to the nucleus but it nevertheless binds to Fe65. We therefore investigated the influence of APLP1 expression on the nuclear signaling of AICD. We used a myc-tagged Tip60 to be able to coexpress Cerulean and Citrine-tagged APLP1 and APP. AICD cleaved from APP again translocated to nuclear AFT complexes in cells coexpressing Tip60 and Fe65 . APLP1 bAlthough the APP family members share a high sequence homology and undergo similar processing, different properties and functions of the three proteins have been described We show that the ICDs released from APP and APLP2 localize together with Fe65 and Tip60 to spherical nuclear complexes. In contrast, the ICD released from APLP1 does not localize to the nucleus, although it is able to bind Fe65. We demonstrate that nuclear localization of AL1ICD is prevented at two steps of APLP1 processing. Firstly, slower turnover of full-length APLP1 compared to APP results in lower levels of CTFs, which are the direct precursors of ICDs. Secondly, AL1ICD undergoes faster proteasomal degradation compared to AICD. Both steps ultimately result in lower levels of AL1ICD. Our results also indicate that, although APLP1 does not signal directly to the nucleus, it has a regulatory function in AICD nuclear localization.The expression of full-length APP or APLP2, together with Fe65 and Tip60, results in the translocation of their ICDs to nuclear AFT complexes, which is dependent on cleavage by γ-secretase in vivoUsing cell surface labeling of living cells and determination of protein half-life time after cycloheximide-induced inhibition of translation, we clearly show that full-length APP and APLP2 have a much faster turnover than APLP1. The stability of APLP1 is evident in different experimental settings and in stark contrast to the rapid turnover of APP and APLP2, whose rapid turnover kinetics have also been reported It has been reported that AICD is degraded by insulin-degrading enzyme (IDE), the lysosomal pathway, and the proteasome, whereas degradation of ALICDs has only been poorly investigated Since the levels and proteasomal degradation of ICDs of APP(VML646LLR) and APLP1 behave in a similar way, our results suggest that the proteasomal degradation rate of ICDs is dependent on their N-terminal residues. Interestingly, work by Alexander Varshavsky and colleagues has revealed a proteasomal pathway in which the degradation of a protein is related to the identity of the protein’s N-terminal residues Walsh et al. reported higher stability for AL1ICD than AICD and AL2ICD when ICDs were expressed as soluble forms Our results help to explain the profound effects seen in knockout mouse models of the APP family. Animals with a double deficiency in both APP and APLP2 die shortly after birth We propose that AL1ICD does not have a direct physiological role in transcriptional regulation, but through sequestration of Fe65 by APLP1 this family member might act as a repressor of AICD-mediated nuclear signaling. We conclude that nuclear signaling is a prime function of the APP and APLP2 ICDs. Our results also caution the use of drugs inhibiting APP processing as treatment for AD because of possible interference with APP/APLP transcriptional function.All animal experiments were performed according to the Swiss animal protection law (TschG) and approved by the local animal committee .APP-Citrine, HA-Fe65, and CFP-Tip60 expressing constructs have been described previously 2 in Dulbecco’s modified eagle medium , supplemented with 10% fetal calf serum (FCS) and 1% Penicillin/Streptomycin (Invitrogen). For transfection Lipofectamine 2000 (Invitrogen) was used according to the manufacturer’s protocol. Medium was replaced after three hours and cells fixed or homogenized 20–24 h after transfection if not indicated differently. To inhibit protein synthesis, cells were treated with 100 µg/ml cycloheximide . Epoxomicin  or MG-132  were used to inhibit the proteasome, and DAPT  was used to inhibit γ-secretase. Primary cultures were prepared from cortices and hippocampi of 1-day-old C57Bl/6 mice as described Human embryonic kidney cells  and mouse neuroblastoma N2a (N2a) were purchased from DSMZ  and cultured at 37°C and 5% COCells were fixed for 20 minutes with 4% paraformaldehyde (PFA), washed with TBS containing 0.05% Triton X-100 and blocked for one hour with TBS containing 0.2% Triton X-100, 5% horse serum, and 5% goat serum. Fixed cells were incubated overnight with mouse anti-myc  or rat anti-HA (Roche) antibodies diluted 1∶100 in blocking solution. After washing, cells were incubated with Cy2, Cy3 or Cy5-conjugated secondary antibodies  and embedded in Mowiol (Sigma-Aldrich).HEK cells were, 24 hours after transfection, incubated with mouse anti-myc antibodies  at 4°C for 10 or 30 minutes. After washing with ice-cold PBS, cells were fixed and stained for HA and myc tags.Cell were lysed in a buffer, containing 150 mM NaCl, 10 mM Tris pH 7.6, 1% Triton X100, 0.25% NP40, and protease inhibitor (Roche) and centrifuged for 5 minutes at 14000 rpm Pull-down of proteins fused to streptavidin-binding protein (SBP) was done as described previously Images were acquired on a Leica TCS/SP2 confocal microscope  and FRET measurements performed as described previously Figure S1ICDs derived from APP and APLP2, but not APLP1, form nuclear AFT complexes in HEK cells. Confocal fluorescence images of HEK cells transfected with HA-Fe65, CFP-Tip60, and APP-Cit (row 1–2), HA-Fe65, CFP-Tip60, and APLP1-Cit (row 3–4), HA-Fe65, CFP-Tip60, and APLP2-Cit (row 5–6). AFT complex formation was observed in cells transfected with APP-Cit or APLP2-Cit. In contrast cells transfected with APLP1-Cit did not show AFT complex formation. Scale bar represents 13 µm.(TIF)Click here for additional data file.Figure S2ICDs derived from APP and APLP2, but not APLP1, form nuclear AFT complexes in N2a cells. Confocal fluorescence images of N2a cells transfected with HA-Fe65, CFP-Tip60, and APP-Cit (row 1–2), HA-Fe65, CFP-Tip60, and APLP1-Cit (row 3–4), HA-Fe65, CFP-Tip60, and APLP2-Cit (row 5–6). AFT complex formation was observed in cells transfected with APP-Cit or APLP2-Cit. In contrast cells transfected with APLP1-Cit did not show AFT complex formation. Scale bar represents 13 µm.(TIF)Click here for additional data file.Figure S3APP family members show different subcellular localization and heterodimerization. (A) Confocal fluorescence images of HEK cells transfected with APLP1-Cer and APP-Cit. Top row shows maximum projection and middle row single sections at different z positions. Note the intracellular localization of APP and the prominent localization of APLP1 at the plasma membrane. In contrast, the coexpression of APP and APLP2 shows a clear overlap and localization to the same intracellular compartments (bottom row). (B) Confocal fluorescence and FRET analysis of primary astrocytes expressing APP family members. APP-Cit was coexpressed with APP-Cer (top row), APLP1-Cer (second row), APLP2-Cer (bottom row). (C) Confocal fluorescence pictures and FRET analysis of HEK cells expressing APLP1-Cer and APP-Cit (top row), APLP2-Cer and APP-Cit (bottom row). (D) Confocal fluorescence pictures and FRET analysis of primary neurons expressing APP-Cer and APP-Cit (top row) and APLP1-Cer and APP-Cit (bottom row). In different cell types (B–D) coexpression of APP-Cit and APP-Cer revealed a strong FRET signal due to the presence of APP homodimers. Similarly, coexpression of APP-Cit and APLP2-Cer generated a FRET signal. In contrast, expression of APLP1-Cer and APP-Cit resulted in minimal FRET signal, indicating the near absence of APP/APLP1 heterodimerization. Scale bars represent 13 µm.(TIF)Click here for additional data file.Figure S4Schematic presentation of APP family ICD sequences and APP mutations used in this study.(TIF)Click here for additional data file.Figure S5Replacement of most AICD residues by the corresponding AL1ICD residues does not ablate nuclear signaling. Confocal fluorescence images of HEK cells cotransfected with HA-Fe65, CFP-Tip60 and the indicated APP-Cit mutants. Scale bar represents 13 µm.(TIF)Click here for additional data file.


In this study, sixty diverse tumor cell lines were exposed to GDC-0941 and classified by GI50 value as sensitive or resistant. The most sensitive and resistant cell lines were analyzed for their baseline levels of gene expression and phosphorylation of key signaling nodes. Phosphorylation or activation status of both the PI3K-Akt signaling axis and PARP were correlated with in vitro response to GDC-0941. A gene expression signature associated with in vitro sensitivity to GDC-0941 was also identified. Furthermore, in vitro siRNA-mediated silencing of two genes in this signature, OGT and DDN, validated their role in modulating sensitivity to GDC-0941 in numerous cell lines and begins to provide biological insights into their role as chemosensitizers. These candidate biomarkers will offer useful tools to begin a more thorough understanding of determinants of patient response to PI3K inhibitors and merit exploration in human cancer patients treated with PI3K inhibitors.The phosphoinositide 3-kinase (PI3K) signaling pathway is significantly altered in a wide variety of human cancers, driving cancer cell growth and survival. Consequently, a large number of PI3K inhibitors are now in clinical development. To begin to improve the selection of patients for treatment with PI3K inhibitors and to identify  PIP3 subsequently recruits 3-phosphoinositide dependent protein kinase 1 (Pdpk1) and the serine-threonine protein kinase Akt to the plasma membrane, culminating in the phosphorylation and activation of Akt. It is Akt which then mediates a cascade of phosphorylation events leading to the activation of downstream pathways promoting tumor survival and growth PI3KCB, PDPK1 and AKT1 and loss of heterozygosity and mutational inactivation of PTEN, a negative regulator of PI3K-signaling The PI3K (phosphoinositide 3-kinase) signaling cascade is one of the most frequently de-regulated pathways in human cancer The frequent activation of PI3K, and its downstream effectors, has prompted multiple independent efforts to develop selective inhibitors against the PI3K pathway as a chemotherapeutic strategy in cancer, and a number of these compounds are now in clinical development The goal of this study was to identify additional novel pathways and/or genes that confer GDC-0941 sensitivity or resistance amongst a panel of cell lines representing a highly diverse range of tumor types, to aid in the development of more generalizable predictors of drug response. To this end, we carried out a drug sensitivity screen using the NCI-60 panel of tumor cells to profile their cellular response to GDC-0941. The NCI-60 cancer cell line collection is a highly characterized panel of 60 diverse human cancer cell lines, representing multiple tumor types  which has been screened for drug sensitivity to >100,000 compounds de novo drug sensitivity. Prominent amongst genes significantly up-regulated in the GDC-0941 resistant cell lines were the genes O-linked N-acetylglucosamine transferase (OGT) and dendrin (DDN). Validation experiments using siRNA-mediated loss of expression of either OGT or DDN resulted in increased in vitro sensitivity to GDC-0941 in multiple cell lines, and concomitant alterations in effectors of both the PI3K and MAPK signaling cascades. These results demonstrate that OGT and DDN are novel in vitro regulators of cellular response to inhibition of PI3K signaling and highlight their potential roles as additional predictive biomarkers for de novo resistance to GDC-0941 and other PI3K inhibitors. We also found that GDC-0941 has broad anti-proliferative activity amongst the nine tumor types represented in the NCI-60 cell panel, suggesting its clinical utility against multiple tumor types.We stratified the cell lines into groups either sensitive or resistant to GDC-0941, and then correlated the phenotype of these cell lines with basal levels (in the absence of GDC-0941) of mRNA expression and the phosphorylation state of multiple signaling nodes to identify predictive biomarkers of ATCC). Upon receipt, the identities of all cell lines in the NCI-60 collection were independently authenticated via STR profiling performed by the ATCC Cell Authentication Testing Service using the PowerPlex® 18D System (Promega). All experiments conducted in this study were performed using the authenticated NCI-60 tumor cell line collection obtained directly from the NCI DTP The NCI-60 tumor cell line collection In vitro drug sensitivity testing of the NCI-60 tumor cell line collection against GDC-0941 was performed, with two biological replicates, by the National Cancer Institute's Developmental Therapeutics Program as previously described 50 was defined as the drug concentration resulting in a 50% reduction in net protein increase (as measured by SRB staining) as compared to control cells. Briefly, following treatment with a 5-point, 10-fold serial dilution with GDC-0941 for 48 hours, cells were fixed in TCA and a 0.4% (w/v) sulforhodamine B (SRB) solution in 1% acetic acid is added. Cells are washed with 1% acetic acid, air dried, bound stain solubilized with 10 mM trizma base, and absorbance measured at 515 nm 50 (z-score) ≥0.8 as being resistant and cell lines with a z-score ≤−0.8 as being sensitive to GDC-0941.2 mRNA expression data for the non-drug treated NCI-60 tumor cell line collection http://discover.nci.nih.gov/cellminer) and imported into GenePattern 3.3 for all downstream analyses (http://www.broad.mit.edu/cancer/software/genepattern). Missing values were estimated with a weighted K-nearest neighbors algorithm (KNNimpute), and two-way average linkage hierarchical cluster analysis was performed using a UPGMA algorithm, with the data being displayed relative to the median expression for each gene. To identify genes differentially expressed between GDC-0941 resistant- and sensitive-tumor cell lines, we utilized the Comparative Marker Selection Module P value cutoff of 0.01. All P values were corrected for multiple testing with the Bonferroni correction. The Gene Set Enrichment Analysis (GSEA) a priori defined sets of genes from biologically coherent pathways. To correct for multiple hypotheses testing, the false discovery rate (FDR) threshold was set at <0.25.Normalized logP values <0.05 were considered significant.Correlations between drug sensitivity and gene expression, levels of phosphorylation, mutational status, tumor type, or tissue of origin were estimated by Spearman's rank correlation method, and differences between groups were calculated with Student's t test using Prism 5.0 (GraphPad). All tests of significance were two-sided, and plus siRNAs (Thermo Scientific), containing pools of 4 siRNAs per gene, were utilized for siRNA knockdown experiments as described previously 5 cells/ml) were plated in black, clear-bottomed 96-well plates in antibiotic-free RPMI 1640 medium and allowed to adhere overnight. Cells were then transfected with siRNA using DharmaFECT transfection reagent (Thermo Scientific) at a final concentration of 25 nM. Following a 4 hour incubation, 10 µl per well of GDC-0941 or NVP-BEZ235 were then added for a total assay volume of 100 µl. Assays were performed in triplicate, with ON-TARGETplus Non-Targeting siRNA (Thermo Scientific) as a negative control, with biological replicates. For experiments with downstream protein analyses, cells were transfected with siRNAs as above. 60 hours post-transfection, cells were treated with 300 nM, 1 µM, or 4 µM GDC-0941 for 24 hours prior to harvesting. For experiments to determine the combined effects of siRNA knockdown and PI3K inhibition on cell proliferation, cell viability was measured at 24 hour intervals using the CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega) with the differences between cell numbers at 72 hours calculated using Student's t test using Prism 5.0 (GraphPad).ON-TARGET50 values for GDC-0941, and the effect of siRNA knockdown of OGT or DDN on sensitivity to GDC-0941, cells were transfected as above. Four hours post transfection, 10 µl per well of an 11-point, 2-fold serial dilution of GDC-0941 (10 µM maximum concentration) was then added, for a total assay volume of 100 µl. Assays were performed in triplicate, with ON-TARGETplus Non-Targeting siRNA (Thermo Scientific) as a negative control, with biological replicates. Cell viability was measured 72 h later using the CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega), and IC50 values calculated by fitting a four-parameter logistic curve of dose-response data using Prism 5.0 (GraphPad). Comparison of the fitted log IC50 values for GDC-0941 in drug treated cell lines versus drug treated cell lines in which OGT or DDN were knocked down by siRNA treatment was performed using the F-test to determine if the fitted midpoint (log IC50) of the curves were statistically significant, with a significance cutoff of P<0.05 sufficient to reject the null hypothesis. siRNA knockdown after 72 hours was validated by Western Blotting using primary antibodies to OGT (Sigma) or qRT-PCR using the Roche LightCycler480 as described previously 5′-TGAACAGTGGTAGCGACAGC-3′ (forward), 5′-GGAGCTATCTCGGTGCCTG-3′ (reverse), 5′-FAM-ATCCCCAAGCCAAAGCTACAGGGA-3′-BHQ (probe)) and to the endogenous control, ACTB (5′-CAGCAGATGTGGATCAGCAAG-3′ (forward), 5′-GCATTTGCGGTGGACGAT-3′ (reverse), 5′-FAM-AGGAGTATGACGAGTCCGGCCCC-3′-BHQ (probe)).For determination of IC450) was normalized to total Akt protein levels. For visualization as a heatmap, data were background-subtracted, median normalized, and converted into a heatmap using Java TreeView. Assays were performed in triplicate.Phosphorylation and/or cleavage status of Erk1/2 (T202/Y204), Mek1/2 (S217/S221), p38 MAPK (T180/Y182), Stat3 (Tyr705), Erbb2 (Y1221/Y1222), Erbb3 (panY), Akt1 (S473), Akt1 (T308), mTOR (S2448), 4E-BP1 (T37/T46), p70 S6 Kinase (T389), S6 (S235/S236), IRS1 (S307), IRS2 (panY), p53 (S15), Bad (S112), cleaved caspase-3, cleaved PARP, and Chk2 (T68) were determined using the PathScan Sandwich ELISA kits  as per manufacturer's instructions. Raw signal intensity /FoxO3a (T32), and total Cyclin D1. Anti-OGT and anti-O-GlcNAc antibodies were obtained from Sigma. All other antibodies were obtained from Cell Signaling. Quantitative analysis of pixel density for O-GlcNAc staining was determined using AlphaVIEW SA software (Cell Biosciences).For Western analysis, cells were washed with PBS, and lysate was collected using the M-PER extraction reagent (Thermo Scientific) containing Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific). 20 µg of protein per sample were separated by electrophoresis on 4–12% Bis-Tris NuPAGE Novex acrylamide gels (Invitrogen) and were then transferred to nitrocellulose membranes using the iBlot system (Invitrogen). Antibodies used were OGT, O-GlcNAc, β-actin, GSK-3β (S9), p2750 values, ranging from 0.011 µM to 3.80 µM  were generated using publicly available data http://discover.nci.nih.gov/cellminer). Following preliminary efforts to identify genes differentially expressed between all GDC-0941-sensitive and –resistant tumor cell lines , Akt1 (T308), mTOR (S2448), 4E-BP1 (T37/T46), p70 S6 Kinase (T389), and S6 (S235/S236). In addition, we assayed the activation status of MAP kinases, Erk1/2 (T202/Y204), Mek1/2 (S217/S221), and p38 MAPK (T180/Y182); transcriptional activators, Stat3 (Tyr705); growth factor and nutrient signaling, Erbb2 (Y1221/Y1222), Erbb3 (panY), IRS1 (S307), IRS2 (panY); and apoptosis signaling molecules, p53 (S15), Bad (S112), cleaved caspase-3, and cleaved PARP .s = −0.50, P = 0.02) or T308 , significantly correlated with sensitivity to GDC-0941. We also observed significant correlation between the level of phosphorylated 4E-BP1, at T37/T46, and sensitivity to GDC-0941 . These results suggest that increased baseline activation of several downstream effectors in the PI3K pathway (phosphorylated Akt1 (at S473 and/or T308), and phosphorylated 4E-BP1 at T37/T46) may serve as predictive markers for de novo sensitivity to GDC-0941. Finally, we observed a significant correlation between baseline levels of cleaved PARP and sensitivity to GDC-0941 .It is notable that levels of phosphorylated Akt1, either at S473 (N-acetylglucosamine transferase (OGT) (N-acetylglucosamine (O-GlcNAc), with the O-GlcNAc modification functioning as a regulatory switch in a manner analogous to phosphorylation Amongst the most differentially expressed genes we identified between GDC-0941-sensitive and GDC-0941–resistant tumor cell lines is O-linked β-se (OGT) ; Fig. 2.Kip1, relative to GDC-0941- or OGT siRNA-alone treated cells , increased phosphorylation of the cell cycle checkpoint regulator Chk2 was observed in both the MDA-MB-231 and OVCAR-4 cell lines upon treatment with GDC-0941 and OGT siRNA . To furtAs a corollary, we next tested the overall role of the O-GlcNAc modification in mediating tumor cell line response to inhibition of PI3K by GDC-0941, by using PUGNAc, a pharmacological inhibitor of OGA, the glycosidase that removes the O-GlcNAc moiety, thus increasing the overall cellular levels of O-GlcNAc de novo sensitivity to GDC-0941, we used siRNA pools targeting DDN to knockdown the expression of DDN in the GDC-0941-resistant MDA-MB-231 breast tumor cell line, and in a second breast tumor cell line, the GDC-0941-sensitive MCF-7 breast tumor cell line. Following knockdown of DDN and treatment with GDC-0941, we then assayed for cell proliferation and phosphorylation status of multiple signaling pathways, including downstream effectors of PI3K  . While i of PI3K . Knockdo of PI3K  resulted of PI3K . We then of PI3K .The PI3K (phosphoinositide 3-kinase) signaling cascade is one of the most frequently de-regulated pathways in human cancer de novo sensitivity to GDC-0941. As such, one caveat is that there may be heretofore uncharacterized mutations in additional genes that also drive de novo sensitivity to GDC-0941. Only three of the eight GDC-0941-sensitive tumor cell lines studied in-depth in this work had activating mutations in PI3KCA. Nevertheless, our results demonstrate that increased levels of phosphorylation of key downstream effectors of the PI3K pathway significantly correlate with increased sensitivity to GDC-0941 (de novo sensitive to GDC-0941 or other inhibitors of the PI3K and/or mTOR pathways.We observed a wide range of sensitivities (0.011 µM to 3.80 µM) to GDC-0941 across the NCI-60 human tumor cell line collection. In accordance with previous results in a large panel of breast tumor cell lines GDC-0941  and thatGDC-0941  may reflKip1 and FoxM1 OGT regulates nutrient sensing and multiple signaling pathways though its reversible posttranslational modification of proteins by the addition of the O-GlcNAc moiety and is now being appreciated as a possible regulatory switch, influencing protein-protein interactions, glucose metabolism, proteasome function, the transcriptional machinery, and mechanisms of cellular invasion and proliferation in breast cancer in cancer biology Common to the cell lines in which loss of OGT expression results in sensitization to PI3K inhibitors was increased phosphorylation of Chk2. This cell cycle regulatory protein has overlapping substrate specificity with the cell cycle checkpoint Chk1 but its role in regulating cell cycle progression is less well understood, and is likely to be subjective based on tissue and/or tumor type de novo sensitive MCF-7 and de novo resistant MDA-MB-231 cell lines . One potentially important cause of the low overlap between the genes identified here and in the study by O'Brien et al. is that in the latter case the investigators confined their search to breast tumor cell lines, whereas we did not introduce this restriction. The biomarkers identified in our work and other recent studies using panels of diverse tumor cell lines may have increased likelihood to overcome heterogeneity in clinical specimens It is of note that only one gene, VAV3, a guanine nucleotide exchange factor which has been shown to be an effector for multiple receptor tyrosine kinases in vitro and in vivo characterization of these candidate biomarkers as predictors of de novo sensitivity or resistance to PI3K inhibitors, as pharmacodynamics markers, as well providing additional insights into the PI3K pathway and its role in cancer biology. One significant caveat to this work is that the characterization of these candidate biomarkers is limited to tumor cell lines. The use of appropriate patient-derived tumor samples, either in vitro or ex vivo, would provide greater context in understanding the biomarkers described in this work as predictors of de novo response to PI3K inhibitors. Furthermore, prior to their use in the identification of patients likely to derive benefit from PI3K inhibitors, the candidate biomarkers of chemotherapeutic response identified here and in other studies need to be subjected to clinical validation using prospective retrospective analyses using patient samples from relevant clinical trials to determine their sensitivity, specificity and reproducibility This work serves as a starting point for further de novo sensitivity to GDC-0941 and validate two of these markers as in vitro regulators of cellular response to inhibition of PI3K signaling. In addition, that in vitro loss of DDN expression sensitizes both de novo GDC-0941-sensitive and -resistant tumor cell lines to PI3K inhibition suggests that the relationship between expression and response may be continuous, with a large dynamic range. However, further work will be required to more completely understand this relationship and to determine the utility of DDN (or OGT) as robust biomarkers. These data also highlight several biomarkers that may serve as pharamacodynamic markers of drug efficacy, or as additional drug targets to increase patient response to PI3K inhibitors.In conclusion, these data show that GDC-0941 has broad anti-proliferative activity amongst many tumor types represented in the NCI-60 cell panel, suggesting its clinical utility against multiple tumor types, and that a number of tumor cell lines with activated AKT1 are highly sensitive to inhibition of PI3K with GDC-0941. We have also identified several novel genomic and phosphoproteomic biomarkers of Figure S1In vitro sensitivity of the NCI-60 tumor cell line collection to GDC-0941 and correlation between in vitro and mutational status of PI3KCA, PTEN, or tumor type. A, GI50 (in µM) of GDC-0941 for 60 cell lines are organized by tumor of origin, as indicated below cell line names. Mean GI50 (0.66 µM) for all cell lines is indicated by dashed line. B, Correlation between GI50 (µM) to GDC-0941 and mutational status of PI3KCA or loss of PTEN protein, amongst all 60 tumor cell lines in the NCI-60 collection. C, Correlation between GI50 (µM) to GDC-0941 and mutational status of PI3KCA or loss of PTEN protein, amongst 16 GDC-0941-resistant and -sensitive tumor cell lines  analyzed in detail in this study. D, Correlation between GI50 (µM) to GDC-0941 and mutational status of AKT1, PDK1, or MTOR, amongst all 60 tumor cell lines in the NCI-60 collection. E, Correlation between GI50 (µM) to GDC-0941 and mutational status of AKT1, PDK1, or MTOR, amongst 16 GDC-0941-resistant and -sensitive tumor cell lines  analyzed in detail in this study. F, correlation between GI50 (µM) to GDC-0941 and tumor type of all 60 tumor cell lines in the NCI-60 collection. Tumor types are color coded as follows: Breast (peach), CNS (grey), Colon (green), Leukemia (red), Melanoma (orange), NSCLC (blue), Ovarian (purple), Prostate (indigo), and Renal (taupe).(TIF)Click here for additional data file.Figure S2Gene Set Enrichment Analysis (GSEA) identifies biologically-coherent pathways differentially expressed between GDC-0941-resistant and-sensitive tumor cell lines. A, Table of KEGG pathways upregulated in GDC-0941-resistant tumor cell lines, with number of genes (“SIZE”), enrichment score (“ES”), normalized enrichment score (“NES”), nominal p-value , and q-value  listed. B, Enrichment plot indicating the enrichment score for the KEGG proteasome gene set. C, Heatmap showing relative expression of genes in KEGG proteasome gene set amongst sixteen GDC-0941-resistant and –sensitive tumor cell lines.(TIF)Click here for additional data file.Figure S3Quantitative analysis of O-GlcNAc levels in OGT siRNA-treated cells, as determined by Western blotting. A, Pixel intensity  of total O-GlcNAc levels for MDA-MB-231 tumor cells transfected with OGT-targeting siRNA or non-silencing siRNA, in the presence or absence of 1 µM GDC-0941 for 24 hours. Each treatment condition is indicated below x-axis. Inset is the inverted image of Western blot in (TIF)Click here for additional data file.Figure S450 curves showing dose-response of the MDA-MB-231, OVCAR-4, and MCF7 tumor cell lines treated with GDC-0941.IC A, IC50 curves for MDA-MB-231 cells treated with GDC-0941 alone (green curve), NS siRNA+GDC-0941 (red curve), or siRNA targeting OGT+GDC-0941 (blue curve). B, IC50 curves for OVCAR-4 cells treated with GDC-0941 alone (green curve), NS siRNA+GDC-0941 (red curve), or siRNA targeting OGT+GDC-0941 (blue curve). C, IC50 curves for MCF7 cells treated with GDC-0941 alone (green curve), NS siRNA+GDC-0941 (red curve), or siRNA targeting DDN+GDC-0941 (blue curve). D, IC50 curves for MDA-MB-231 cells treated with GDC-0941 alone (green curve), NS siRNA+GDC-0941 (red curve), or siRNA targeting DDN+GDC-0941 (blue curve). Below each curve is the calculated IC50 value under for each condition, as well as the results of statistical comparison of the fitted IC50 values between conditions.(TIF)Click here for additional data file.Figure S5Loss of OGT expression increases sensitivity of the MDA-MB-231 tumor cell line but not the OVCAR-4 tumor cell line to the dual PI3K/mTOR inhibitor NVP-BEZ235. A, MDA-MB-231 cells transfected with OGT-targeting siRNA or non-silencing siRNA, in the presence or absence of 10 nM NVP-BEZ235. Cell viability was assayed at 0, 24, 48, and 72 hours post-transfection and treatment with NVP-BEZ235. B, OVCAR-4 cells transfected with OGT-targeting siRNA or non-silencing siRNA, in the presence or absence of 10 nM NVP-BEZ235. Cell viability was assayed at 0, 24, 48, and 72 hours post-transfection and treatment with NVP-BEZ235. All above data represent mean ± SEM (n = 3);*, P<0.05;**, P<0.01;***, P<0.001.(TIF)Click here for additional data file.Figure S6Increased cellular levels of O-GlcNAc increases resistance of the T-47D tumor cell line to GDC-0941 and alters the phosphorylation state of ERBB3. A, T-47D cells treated with 100 µM PUGNAc, in the presence or absence of 100 nM GDC-0941. Cell viability was assayed at 0, 24, 48, and 72 hours post-transfection and treatment with GDC-0941. B, Immunoblot analysis of whole cell lysates from T-47D cells using anti-OGT or anti-O-GlcNAc antibodies. Cells were treated with 100 µM PUGNAc or DMSO control for 24 hours, as indicated. C, Immunoblot analysis of whole cell lysates from T-47D cells with indicated antibodies. Cell treatments are as indicated. D, Quantitative phosphorylation analysis of ERBB3 (PanY) in T-47D cells treated with 100 µM PUGNAc, in the presence or absence of 100 nM GDC-0941 for 24 hours. Cells were treated with 100 nM GDC-0941 or DMSO control for 24 hours, as indicated. E, Quantitative phosphorylation analysis of Mek1/2 (S217/S221) in T-47D cells treated with 100 µM PUGNAc, in the presence or absence of 100 nM GDC-0941 for 24 hours. Cells were treated with 100 nM GDC-0941 or DMSO control for 24 hours, as indicated. All above data represent mean ± SEM (n = 3);*, P<0.05;**, P<0.01;***, P<0.001.(TIF)Click here for additional data file.Table S1In vitro sensitivity of the NCI-60 tumor cell line collection to GDC-0941. GI50 values (in µM) of GDC-0941 for 60 cell lines are rank ordered from lowest (most sensitive) to highest (resistant).(XLSX)Click here for additional data file.Table S2221 genes most differentially expressed between GDC-0941-sensitive and GDC-0941-resistant tumor cell lines .(XLSX)Click here for additional data file.


We also examined the relationship between altered expression of hedgehog pathway mediators and survival in a well-characterized cohort of metastatic melanoma patients with prospectively collected follow up information. Studies revealed that over 40% of the melanoma cell lines examined harbored significantly elevated levels of the hedgehog pathway mediators SMO, GLI2, and PTCH1 compared to melanocytes (p < 0.05). SMO inhibition using siRNA and the small molecule inhibitor, NVP-LDE-225, suppressed melanoma growth in vitro, particularly in those cell lines with moderate SMO and GLI2 expression. NVP-LDE-225 also induced apoptosis in vitro and inhibited melanoma growth in a xenograft model. Gene expression data also revealed evidence of compensatory up-regulation of two other developmental pathways, Notch and WNT, in response to hedgehog pathway inhibition. Pharmacological and genetic SMO inhibition also downregulated genes involved in human embryonic stem cell pluripotency. Finally, increased SMO expression and decreased expression of the hedgehog pathway repressor GLI3 correlated with shorter post recurrence survival in metastatic melanoma patients. Our data demonstrate that hedgehog pathway inhibition might be a promising targeted therapy in appropriately selected metastatic melanoma patients.Previous reports have demonstrated a role for hedgehog signaling in melanoma progression, prompting us to explore the therapeutic benefit of targeting this pathway in melanoma. We profiled a panel of human melanoma cell lines and control melanocytes for altered expression of hedgehog pathway members and determined the consequences of both genetic and pharmacological inhibition of the hedgehog pathway activator Smoothened (SMO) in melanoma, both  Melanoma incidence is quickly rising, with an estimated lifetime risk of 1 in 50 ,3. The ain vitro [in vitro and in vivo [in vitro and in vivo, and we report, for the first time, the association of aberrant hedgehog pathway activity with decreased survival in melanoma patients.Over the past five years, three independent groups have described the importance of yet another embryonic signaling cascade, the hedgehog pathway, in melanoma progression ,15,16. Tin vitro . Most re in vivo . These sPrimary melanoma lines WM35, WM1552c, WM1575, WM98.1, WM115, WM278, WM-793, WM-853, WM-902, WM-983, WM-1366, WM1361A, and WM3248 were purchased from the Wistar Institute  and cultured in Mel 2% medium [The U133A 2.0 Affymetrix array was performed on four melanocyte controls , four primary and 14 metastatic cell lines as described in reference . A curatSHH, GLI1, GLI2, GLI3, SMO, or PTCH1 were associated with significantly decreased post-recurrence survival. RNA was isolated from the 30 additional metastatic melanoma specimens using the RNAeasy Kit .The 30 additional metastatic melanoma patients were identified through the Interdisciplinary Melanoma Cooperative Group database at New York University School of Medicine . Of the 30 specimens from 30 patients, 11 were lymph node metastases, 15 were skin metastases, and four were visceral metastases. The median follow-up time for the cohort from the time of primary diagnosis to last follow-up date was 70.08 months. The study was approved by the New York University Institutional Review Board, and all patients signed informed consent before enrollment. Relevant clinicopathologic, demographic, and survival data were recorded for all patients.Using the previously published Human Genome U133A 2.0 Affymetrix expression profile of 44 metastatic melanoma samples  from patDescriptive statistics were calculated for baseline demographic and clinicopathologic characteristics. Cox proportional hazards model dichotomized at the median expression value was used to examine the association between hedgehog pathway mediatory transcript levels and post-recurrence survival (time from first recurrence to death). Kaplan-Meier curves were generated using Graph Pad Prism 5.0 Software .4 cells per well in a 12-well dish in triplicate in DMEM medium. The day after (day 0), the medium was replaced, and DMSO, cyclopamine , the oral Smoothened inhibitor in Phase II clinical trials, NVP-LDE-225, (Novartis Pharma AG), or Vemurafenib  at indicated concentrations were added. At the indicated time points (3–12 days), cells were fixed in 10% formalin solution and stored in PBS at 4 °C. After the final time point, all the plates were stained with crystal violet. After color elution with 10% acetic acid, optical density was read at 590 nm. A representative curve of three independent experiments is reported.The indicated cell lines were seeded at a density of 1 × 105 A375 cells were plated in 10 cm plates, and 24 h later were treated with DMSO or 5 µM NVP-LDE-225. After 72 h of treatment, cells were trypsinized, washed with PBS, and fixed in 70% ethanol. Prior to FACS analysis, fixed cells were stained with propidium iodide in PBS (25 µg/mL) containing 250µg/ml RNase A. Pan-caspase activation and changes in mitochondrial potential were determined in A375 and WM3248 cells after 72 h of treatment with 10 µM NVP-LDE-225 using the dual sensor MitoCasp™ assay  according to the manufacturer’s protocol. Stained cells were evaluated in an LSRII flow cytometer and analyzed using Flowing Software version 2.4 .5 × 104 melanoma cells were plated in antibiotic free medium into each well of a 12 well plate. 18 h later, 40 pmol of ON-Target plus SMPARTpool Human SMO (L-005726-00-0005), Human GLI1 (L-003896-00-0005), or Human GLI2 (L-006468-00-0005) siRNA  diluted in Opti-Mem I reduced serum media were transfected into the cells using Lipofectamine 2000 . The medium was changed after 6 h and cells were trypsinized for quantitative real time PCR or stained with crystal violet at indicated time points.2–4 × 106 A375 cells were injected into the flank of NOD/Scid/IL2 gamma receptor -/- (NOG) mice (n = 20). Once tumors were palpable (6 days after injection), mice were randomized into two groups and vehicle (n = 10) or NVP-LDE-225 was administered orally at 60 mg/kg/day. After one week of treatment, tumors were excised, weighed, and RNA was isolated from tumors for RT-PCR. Effective pathway inhibition was confirmed with qPCR of PTCH1 transcript levels in the tumors of NVP-LDE-225 treated mice as compared to vehicle treated mice.1.5 × 10The transcriptional profile of NVP-LDE-225 and SMO siRNA treated WM3248 and A375 melanoma cells was generated using a Human Genome U133 Plus 2.0 genechip. The WM3248 and A375 cell lines were treated for 24 h with either 5 µM NVP-LDE-225 or SMO siRNA. The significantly downregulated canonical signaling pathways in SMO inhibitor treated WM3248 and A375 cell lines, as compared to DMSO or non-targeting siRNA treated cells, respectively, were generated with Ingenuity Pathway Analysis software . Canonical pathways analysis identified the pathways from the IPA library of canonical pathways that were most significant to the data set. Genes from the data set that met the fold change cutoff of 1.2 and were associated with a canonical pathway in the Ingenuity Knowledge Base were considered for the analysis. The significance of the association between the data set and the canonical pathway was measured in 2 ways: (1) A ratio of the number of molecules from the data set that map to the pathway divided by the total number of molecules that map to the canonical pathway is displayed. (2) Fisher’s exact test was used to calculate a p‐value determining the probability that the association between the genes in the dataset and the canonical pathway is explained by chance alone.WNT5A and GLI2 were found to be significantly higher than melanocytes in over 50% of the melanoma cell lines,  (p < 0.05).We analyzed the expression of 77 genes from a curated gene signature of hedgehog pathway activation using our microarray expression profile of 18 melanoma cell lines and four melanocyte controls . Array ectively)   (p < 0.05). There was also loss of expression of negative regulators of the hedgehog pathway such as protein kinase A (PRKA) and dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 (DYRK2) in a subset of melanoma cell lines  (p < 0.05). 11 cell lines expressed high levels of both WNT5a and GLI2 as compared to the melanocytes controls and 10/11 of these cell lines (91%) displayed loss of both DYRK2 and PRKA  melanoma cell lines and SMO expression was elevated in 13/26 (50%) cell lines. Six of the cell lines overexpressing GLI2 as compared to melanocytes concomitantly overexpressed SMO. The GLI2 transcriptional target WNT5A was found to upregulated in 22/26 (85%) melanoma cell lines as compared to melanocyte controls. Finally, the hedgehog pathway transcriptional target PTCH1 was elevated in 10/26 (39%) melanoma cell lines as compared to immortalized melanocytes. Of note, in our microarray analyses, GLI1 was not found upregulated in our panel of melanoma cells as compared to melanocytes. GLI1 was found to be expressed at much lower levels than GLI2, SMO, or PTCH1 mRNA as measured by quantitative qRT-PCR  , and in the WM278 cell line, which harbors a one copy GLI2 and two copy SMO allele gain as detected by a DNA SNP array [50 values of 47.5 and 91.9 μM, respectively)  b. CyclopNP array  and overctively) b. IntereTo account for potential off-target effects of small molecule SMO inhibition, we employed SMO siRNA to determine whether genetic interference with the hedgehog pathway would also inhibit growth. Transient transfection of siRNA against SMO demonstrated a similar antiproliferative effect at 72 h as three days of 5μM cyclopamine treatment in the WM3248 cell line a. siRNA 50 of less than 10 μM and a lack of antiproliferative effect in cells that displayed an IC50 greater than 45 μM . However, cell death analysis using a pan-caspase detection assay and a mitochondrial potential assay revealed an increased percentage of apoptotic cells after 72 h of NVP-LDE-225 treatment in both the A375 and WM3248 cells (p = 0.0025)  c.50 < 10 µM)  to hedgehog pathway inhibition. Our data revealed that 36% (6/16) of the V600E B-Raf mutant melanoma cell lines had modest expression of GLI2 and SMO as compared to melanocytes a,b. In o< 10 µM) . 50%  and tumor weight (p = 0.0979) as compared to the vehicle treated mice . We isolated RNA from 30 additional metastatic melanoma specimens and performed U133 2.0 Plus Affymetrix array on these samples. Upon examining the pooled expression values from all 74 samples, there remained a statistically significant association between elevated SMO and decreased survival in the expanded cohort (p = 0.0374). SHH, GLI1, GLI2, PTCH1, and WNT5 were not associated with decreased survival in this expanded cohort. While the hedgehog pathway transcriptional repressor GLI3 was not detected by the U133A platform in any of our first 44 melanoma specimens, the U133 2.0 Plus array analysis did detect GLI3 transcript expression and revealed differential expression among the 30 metastatic melanoma specimens analyzed. Elevated expression of the hedgehog pathway transcriptional repressor GLI3 (as defined as above the median expression level) in the 30 metastatic tumor specimens associated with better survival in metastatic melanoma patients (p = 0.015). Microarray expression levels of GLI3 and SMO in patient tumors were confirmed by quantitative real time PCR . In the A375 cell line, both siRNA to SMO and NVP-LDE-225 downregulated the growth factor PDGF-alpha and the protein translation mediator eIF4a2, while upregulating the apoptosis mediator annexin A9 and the melanoma antigen MAGE-E1 (p < 0.05). Interestingly, in both the A375 and WM3248 cell lines, NVP-LDE-225 downregulated the Ras signaling adaptor GRAP  and upregulated XAF1 , a mediator of programmed cell death. In both cell lines tested, the notch ligand DLL3  and the WNT5a receptor RORA were upregulated by NVP-LDE-225 treatment .To determine the mechanism of SMO inhibitor-induced apoptosis, we performed gene expression profiling of melanoma cells treated with NVP-LDE-225 and SMO siRNA treated melanoma cells. Microarray expression profiles of WM3248 and A375 cell lines treated with siRNA to SMO or the small molecule NVP-LDE-225 were compared to non-targeting siRNA or DMSO treated controls, respectively. In the WM3248 cell line, both genetic interference and NVP-LDE-225 treatment for 24 h led to downregulation of cell adhesion molecules claudin20 and ICAM2, as well as the metalloproteases BMP1 and MMP28, while mediators of programmed cell death such as caspase I, forkhead BoxO3 pseudogene, FOS-like antigen 2, and BCL2 interacting protein 3 were upregulated (p < 0.05). The role of NANOG in Mammalian Embryonic Stem Cell Pluripotency pathway and the role of Oct4 in Mammalian Embryonic Stem Cell Pluripotency pathway were also significantly downregulated in both SMO siRNA and inhibitor treated A375 and WM3248 cells (p < 0.05).Ingenuity Pathway Analysis (IPA) was also performed on the transcriptional profile of NVP-LDE-225 and SMO siRNA treated melanoma cells. The pathways from the IPA canonical pathway library that were significantly downregulated by both SMO siRNA and NVP-LDE-225 in both the A375 and WM3248 cell lines are pictured in p < 0.05). NVP-LDE-225 upregulated the Death Receptor Signaling Pathway, MYC Mediated Apoptosis Signaling, and Apoptosis Signaling in melanoma cell lines (p < 0.05).A heatmap of differentially expressed genes from the IPA human stem cell pluripotency pathway in SMO siRNA and NVP-LDE-225- treated cells as compared to their respective controls is pictured in in vitro and in vivo. Our findings that NVP-LDE-225 inhibits proliferation and induces apoptosis in melanoma cell lines agree with those of Jalili et al. However, in contrast to their study, we observed a more dramatic antiproliferative effect in B-RAF mutant cell lines as compared to B-RAF wild type cells and we did not observe a cell cycle arrest upon NVP-LDE-225 treatment. The relative resistance of B-RAF wild type melanoma cell lines and the lack of cell cycle arrest observed in our study may be secondary to the different melanoma cell lines used. While Jalili et al. observed cell cycle arrest in NVP-LDE-225 treated UACC 257 and LOX IMVI cell lines, we observed no cell cycle alterations in NVP-LDE-225 treated WM3248 and A375 melanoma cell lines. Similarly, while Jalili et al. demonstrated sensitivity of MEWO and MEL FH wild type B-RAF cell lines to NVP-LDE-225, the three wild type B-RAF cell lines  used in our study were resistant to NVP-LDE-225, with IC50s > 50 µM. Finally, the more robust in vivo antitumor response observed in Jalili et al.’s xenograft model may be secondary to the direct intratumoral delivery of NVP-LDE-225 in their animal model as compared to the oral gavage delivery method used in our study.Here we show, for the first time, that elevated levels of hedgehog pathway components correlate with worse survival in melanoma patients. We also demonstrate that NVP-LDE-225, a small molecule SMO inhibitor already in clinical trials in other solid tumors, inhibits the hedgehog pathway effectively and has antitumor activity against B-RAF mutant melanoma both in vivo are necessary to better define a clinically useful regimen. Whether SMO inhibition might be effective in vemurafenib resistant B-RAF mutant tumors is currently under investigation in our laboratory.Our observations suggest that hedgehog pathway inhibitors may be an additional therapeutic strategy against B-RAF mutant melanoma. As activated RAS/MAPK signaling has been shown to induce hedgehog pathway activity via translocation of GLI1 and GLI2 into the nucleus, it is possible that B-RAF mutation is augmenting hedgehog pathway activity in our melanoma cell lines and that inhibiting both B-RAF and the hedgehog pathway may result in an enhanced therapeutic effect. Our data demonstrate a modest but significant effect of combining B-RAF and hedgehog inhibitors, but additional studies of the dosage and sequence of administration in vitro and in vivo. In ongoing clinical trials in advanced basal cell carcinoma, this daily dosed oral inhibitor has been well tolerated with little toxicity. Thus, our findings could be readily translated into clinical trials of a SMO inhibitor in combination with vemurafenib in patients whose B-RAF mutant tumors express GLI2 and SMO at moderate levels.Although other groups have reported upregulation of hedgehog pathway members in metastatic tissue as compared to primary specimens, this is the first report of an association of SMO levels with significantly decreased survival in metastatic melanoma patients. The protective effect of elevated GLI3 levels underscores the role of hedgehog pathway activation in more aggressive disease. We also report that a clinically relevant inhibitor already in phase II clinical trials inhibits melanoma growth i.e., B-RAF), but also the embryonic signaling pathways promoting self renewal in a subset of melanoma cells. Inhibition of melanoma initiating cell self-renewal might extend the durability of the robust, albeit short-lived, antitumor responses achieved with vemurafenib in metastatic melanoma patients.Ingenuity pathway analysis revealed that silencing of the hedgehog pathway by either chemical or genetic means significantly inhibited the expression of human embryonic stem cell pluripotency related genes. This result indicates that SMO inhibitors may effectively target the melanoma stem cell compartment and supports a recent study showing that melanoma-initiating cells require hedgehog signaling in order to self-renew . In ordeDLL3 and the WNT5 receptor ROR in both the A375 and WM3248 cell lines. As both of the cell lines tested demonstrated upregulation of the programmed cell death mediator XAF1 in response to both small molecule and genetic SMO inhibition, this protein is likely a key mediator in hedgehog inhibitor-induced apoptosis. The upregulation of the notch ligand DLL3 and WNT5 that we observed in our microarray analysis indicate a compensatory upregulation of alternative stem cell signaling pathways in response to SMO inhibition. Compensatory upregulation of alternate stem cell pathways is a phenomenon previously reported with Notch inhibitors in neural tumors [NVP-LDE-225 induced a programmed cell death gene expression profile in both cell lines tested, and also paradoxically upregulated the notch ligand l tumors . Schreckl tumors . Hedgehol tumors . In lighFurthermore, we identified overexpression of GLI2 and SMO as a signature of resistance to smoothened inhibition in melanoma, as has been reported recently in human medulloblastoma using both NVP-LDE-225 and Vismodegib, an oral SMO inhibitor that is FDA-approved in advanced basal cell carcinoma. Two studies have reported amplifications of chromosomal regions containing GLI2 in Vismodegib and NVP-LDE-225-resistant medulloblastoma cell lines ,32,33. OAs NVP-LDE-225 enhanced the cytotoxic effects of single agent B-RAF inhibition, SMO inhibition may be effective in combination with vemurafenib in patients whose tumors express moderate levels of GLI2 and SMO. Based on the responses to NVP-LDE-225 in our melanoma cell line panel, it appears that a subset of patients whose tumors harbor either V600E B-RAF mutations or N-RAS mutation may benefit most from inhibition of SMO.In summary, our pre-clinical studies support SMO as a therapeutic target in melanoma and offer insights into the clinical potential of this new treatment strategy both as a single agent and in combination with other targeted agents in metastatic melanoma patients.


Ovarian cancer is the leading cause of death among gynaecological malignancies. Extracellular matrix (ECM) can affect drug resistance by preventing the penetration of the drug into cancer cells and increased resistance to apoptosis. This study demonstrates alterations in the expression levels of ECM components and related genes in cisplatin-, doxorubicin-, topotecan-, and paclitaxel-resistant variants of the A2780 ovarian cancer cell line. Affymetrix Gene Chip Human Genome Array Strips were used for hybridisations. The genes that had altered expression levels in drug-resistant sublines were selected and filtered by scatter plots. The genes that were up- or downregulated more than fivefold were selected and listed. Among the investigated genes, 28 genes were upregulated, 10 genes were downregulated, and two genes were down- or upregulated depending on the cell line. Between upregulated genes 12 were upregulated very significantly—over 20-fold. These genes included COL1A2, COL12A1, COL21A1, LOX, TGFBI, LAMB1, EFEMP1, GPC3, SDC2, MGP, MMP3, and TIMP3. Four genes were very significantly downregulated: COL11A1, LAMA2, GPC6, and LUM. The expression profiles of investigated genes provide a preliminary insight into the relationship between drug resistance and the expression of ECM components. Identifying correlations between investigated genes and drug resistance will require further analysis. Among gynaecologic malignancies ovarian cancer is the leading cause of deaths. The average 5-year survival is approximately 40%, but patients with advanced disease (stages III and IV according to FIGO classification) have a significantly lower survival rate of only 10–20% , 4.The main reason of low chemotherapy effectiveness is drug resistance of cancer cells. Cellular mechanisms of drug resistance are various. They include lower accumulation of the drug in the cells, change in localization of the drug in the cell, slower inactivation of the drug, faster repair of damage by the drug DNA and cellular membranes as well as increased ability for tolerance of these damages, and changes in molecular targets, which make them insensitive or less sensitive to the drugs' actions, changes in gene expression, and changes in regulation of apoptosis. However, the most significant and frequently occurring mechanism of drug resistance is multiple drug resistance (MDR). It means the ability of cancer cells to actively remove drugs from the cell via transport proteins. The most important proteins taking part in this process are proteins belonging to ABC family, and among them the best known is glycoprotein P .Although we know a lot about chemotherapy resistance, sometimes it is difficult to explain response of cancer cells to cytostatic drugs on the basis of expression profile of genes involved in drug resistance. This indicates that other unknown yet genes can also participate in cancer drug resistance. Genome wide expression analysis by oligonucleotide microarray is a powerful molecular tool for the discovery of new genes involved in molecular processes including drug resistance. in vivo [Extracellular matrix (ECM) is made up of ground substance and fibers. The ground substance consists of proteoglycans like syndecan and lumican, multiadhesive glycoproteins like fibronectin and laminin, and glycosaminoglycans. Between fibers we can distinguish between collagen and elastin fibers. These molecules control many aspects of cell life such as gene expression, cell proliferation, differentiation, migration, adhesion, and cancer metastasis . Express in vivo  and in d in vivo . in vivo but not in vitro. This can be related to tumour microenvironment and expression of ECM [It has been reported that ECM can affect drug resistance by preventing the penetration of the drug in the cancer cell –11. Usinn of ECM . Some ren of ECM . Changesn of ECM , 18.This study shows alterations in the gene expression levels of ECM proteins in the Cis-resistant , paclitaxel (Pac)-resistant , Dox-resistant , and Top-resistant  variants of the A2780 ovarian cancer cell line.v/v) foetal bovine serum, 2 pM L-glutamine, penicillin (100 units/mL), streptomycin (100 units/mL), and amphotericin B (25 μg/mL)) at 37°C in a 5% CO2 atmosphere.A2780 sublines that were resistant to Cis , Pac , Dox , and Top  were generated by the exposure of the A2780 cell line to incremental increases in the concentrations of the relevant drugs. The final concentrations of each drug were 1000 ng/mL Cis, 1100 ng/mL Pac, 100 ng/mL Dox, and 24 ng/mL Top. These concentrations were based on the work of Dietel et al. in 1993 [w/v denaturing agarose gel. Additionally, all samples were checked on a Bioanalyser 2100 . The evaluated RIN was between 8.5 and 10, with average of 9.2. Each RNA sample was diluted to 100 ng/μL with an OD260/OD280 ratio of 1.8–2.0. All RNA samples were prepared in triplicate. cDNA was synthesised in two steps (first strand synthesis and second strand synthesis) using the Affymetrix GeneChip 3′IVT Express Kit , according to the manufacturer's instructions. Biotin-labelled cRNA synthesis (IVT Labelling) and cRNA fragmentation were performed using the Affymetrix GeneChip Kit reagents, according to the procedure described in the Affymetrix GeneAtlas 3′IVT Express Kit technical manual.RNA was isolated from A2780 and all the resistant sublines using the TRI Reagent , according to the manufacturer's instructions. The absorbance values  were measured for RNA quantification by spectrophotometry. The intactness of the extracted RNA was checked by electrophoresis using a 1% Biotin-labelled and fragmented target cRNA samples were loaded into Affymetrix GeneChip (Human Genome U219) Array Strips together with controls cRNAs and oligo B2. The hybridisation procedure was conducted at 45°C for 16 h in an AccuBlock Digital Dry Bath  hybridisation oven. The washing and staining procedure was performed using an Affymetrix GeneAtlas Fuidics Station according to the instructions in the technical manual. An Affymetrix GeneAtlas Imaging Station was used for scanning the arrays.P value indicating the significance of the enrichment score, a nonparametric statistical Mann-Whitney U-test was used (α = 0.05). Genes whose expression was significantly different between the resistant sublines and the parental A2780 cell line were listed. The upregulated and downregulated genes were selected. The genes were filtrated by scatter plot  was used for further data analysis and evaluation. Using quality control checkpoints and statistical analysis of gene fold-change significances, a table of the most important changes in gene expression was constructed. Next, the generated table was imported to Pathway Studio Explore  where proper statistical analyses were carried out. To evaluate the ter plot  and the The quality of all GeneChip expression data were in “good sample” limits according to preliminary data analysis parameters such as background and noise averages, percentage of present calls, presence of internal hybridization controls in increasing signals, presence of poly-A controls as decreasing signals, and GAPDH to beta actin 3′/5′ signal ratios.The genes that are related to ECM structure and metastatic property of cancers were selected. Analysis of these gene expressions in eight drug-resistant ovarian cancer cell lines can give some information about response of cancer cells to different cytostatic drugs treatment. Tables Collectively, expression of 40 genes encoding ECM proteins, integrin receptors, matrix metallopeptidases, and related genes was changed in drug-resistant cell lines. 28 genes were upregulated in at least one drug-resistant cell lines. 10 genes were downregulated in at least one drug-resistant cell line and two genes were down- or upregulated dependent on the cell line. The most variable cell lines were one of the Pac-resistant cell line A2780PR1 and one of the Top-resistant cell line A2780TR2. In these cell lines we observed changes in 19 and 14 gene expression, respectively. The most stable cell line was Dox-resistant cell line A2780DR2; only three genes were upregulated.Six genes were upregulated in both Top-resistant cell lines : ITGB8, COL1A2, TGFBI, LAMA4, HAPLN1, and MGP. One gene COL11A1 was downregulated in both Top-resistant cell lines. One gene LAMB1 was upregulated in both Pac-resistant cell lines . Three genes COL11A1, FN1, and KERA were downregulated in both Pac-resistant cell lines. One gene TIMP3 was upregulated and three genes COL11A1, FBN1, and KERA were downregulated in both Cis-resistant cell lines . Only one gene EPYC was upregulated in both Dox-resistant cell lines .From 40 analysed genes, expression of 12 was upregulated very significantly—over 20-fold increase. These genes included COL1A2, COL12A1, COL21A1, LOX, TGFBI, LAMB1, EFEMP1, GPC3, SDC2, MGP, MMP3, and TIMP3. Four genes were very significantly downregulated: COL11A1, LAMA2, GPC6, and LUM. Changes in expression levels of all genes are summarised in Tables This paper presents expression of genes encoding ECM and related proteins in eight drug-resistant ovarian cancer cell lines. The genes with fold-change values between 5 and 0.2 were considered to not be altered significantly, and the relationships between these genes and drug resistance will not be discussed.The MDR phenotype of cancer cells is mainly related to expression of drug transporters from ABC family and among them the main players are glycoprotein P (P-gp) and breast cancer resistant protein (BCRP) . Express in vivo but also in vitro in drug resistance cell lines [ in vitro, these molecules are likely to be important in drug resistance of cancer cells.However, increasing body of evidences indicates that tumour microenvironment  and exprll lines . If canc in vitro. This effect can result from interaction of collagen with cellular receptors leading to increased resistance to apoptosis [We observed upregulation of five collagen genes: COL1A2, COL16A1, COL17A1, COL18A1, and COL21A1 in A2780PR1 cell lines. In contrast only one collagen gene COL4A1 was upregulated in the A2780PR2 cell line. Thus expression of these genes seems not to be induced specifically by Pac but rather is one of the possibilities of cell response to this drug treatment. Similarly expression of COL12A1 was observed only in one of Cis- and one of Dox-resistant cell lines but not in the others. Very high expression level of COL1A2 in both Top-resistant cell lines suggests that expression of this collagen can be a specific cellular response to Top treatment. Taken together overexpression of at least one collagen gene was observed in six from eight drug-resistant cell lines. In four from these six cell lines expression was very high—over 20-fold increase. It has been observed by Netti et al. that more penetration-resistant tumours have extended collagen network . Dense apoptosis . One morHigh expression level of LOX in A2780PR1 cell lines seems to be related to expression of many COL genes in this cell line. LOX is lysine oxidase responsible for crosslinking of collagens and elastin . Overexpβig-H3 and keratoepithelin) plays a role in a wide range of physiological and pathological conditions including tumorigenesis [Transforming growth of factor-beta-induced protein , decortin, epiphycan, keratocan (KERA), lumican (LUM), and syndecan (SDC) are ECM proteoglycans. Some glypicans play a role in cell proliferation and survival. Expression of GPC6 has been observed in drug-resistant breast cancer cell lines . In contKERA and LUM are members of the small leucine-rich proteoglycan (SLRP) family. KERA downregulation in Pac- and Cis-resistant cell lines is difficult to explain. To our knowledge relation of this gene to drug resistance has not been described so far. It can result from unspecific effect on these genes expression. Very high LUM downregulation in Pac-resistant cell line in the context of drug resistance is difficult to explain. LUM overexpression has been reported in Cis-resistant head and neck squamous cell carcinoma cell lines and in patients not responding to treatment with Cis-based combination chemotherapy . Thus thMatrix gla protein (MGP) is a protein found in numerous body tissues. The encoded protein is found in the organic matrix of bone and cartilage and likely acts as an inhibitor of bone formation. The role of this protein in drug resistance has not been described so far. We observe its expression in four drug resistance cell lines with very high expression in Pac and Top resistance cell lines. These cell lines also express high level of laminin and collagen genes. It is possible that MGP can play a role in collagen and laminin metabolism.Matrix metalloproteinase (MMP) is a family of zinc proteases involved in degradation of extracellular matrix in physiological and in disease condition, such as arthritis and metastasis. They are also responsible for cleaving number of bioactive molecules like cell surface receptors and play an important role in cell proliferation, differentiation, migration, angiogenesis, and apoptosis. In our study we observe overexpression of three MMPs: MMP1, MMP3, and MMP12. MMP1 is responsible for degradation of collagen types I, II, and III. In our study we observe overexpression of MMP1 in A2780PR1 and A2780TR2 cell lines. Both cell lines overexpress COL1A2. Thus overexpression of MMP1 in these cell lines can be related to COL1A2 overexpression and more invasive character of these cell lines. MMP1 overexpression has been reported in Dox-resistant breast cancer cell line . MMP1 ovTIMP3 belongs to tissue inhibitor of metalloproteinases gene family which includes inhibitors of the matrix metalloproteinases, involved in degradation of the extracellular matrix (ECM). In our cell lines we observe strong overexpression of TIMP3 in A2780CR1 cell line and some increase in expression in A2780CR2 cell line, both resistant to Cis. However we do not observe increase of metalloproteinases expression in these cell lines. Our result is contradictory to results from drug-resistant breast cancer cell lines. In this study strong downregulation of TIMP3 was observed in Doc- and Dox-resistant cell line . In thisIn our study we have shown changes in expression of many genes encoding ECM and related proteins in eight drug-resistant ovarian cancer cell lines. The expression profiles presented here provide preliminary insight into differential expression of ECM and related genes in drug-resistant ovarian cancer cell lines. We did not observe any general response to cytostatic treatment. Changes in genes expression seem to be rather cytostatic dependent than general response to chemotherapy. However, the similarity between two cell lines resistant to the same drug is also not prominent. Significance of these gene expressions in drug resistance requires further analysis and should be confirmed on other ovarian cancer cell lines.