BE patients have an increased risk of developing esophageal adenocarcinoma (EAC). As many aspects of this condition are still unknown, there is a need for To review the literature on cell lines and incubation conditions for studying BE development.A literature search was performed using PubMed, EMBASE and the Cochrane library, combining the words esophagus, cell line, culture, Barrett’s, bile, acid, exposure, reflux and adenocarcinoma.A wide range of cell lines and incubation conditions to study BE development have been reported. The most commonly used cell lines are derived from epithelium from patients with BE or EAC. A 25-minute incubation with 200 μM bile salts induced cell proliferation and Akt phosphorylation. However, increased CDX2 and MUC2 expression was only observed with longer incubations or higher bile salt concentrations. Two-hundred μM bile at pH 6 showed a higher toxicity to EAC cells than the same concentration at pH 7. Multiple 5-minute exposures with 200 μM bile at pH 4 or pH 7 increased CK8/18 and COX2 in BE epithelial cells.in vitro setup for studying the development of BE.Two-hundred μM conjugated primary or secondary bile salts at pH 4 for multiple short exposures is able to induce BE specific factors in BE cell lines. In SQ and EAC cell lines; however, higher concentrations of secondary bile salts for 8 h are needed to induce BE specific molecules. Due to the high variability in reported methods, it is difficult to determine the most effective  Physiological reflux of gastric content from the stomach into the esophagus occurs in the majority of individuals . When thIt has been shown that the refluxate of BE patients also consists of higher concentrations of acid and bile salts compared to patients with GERD , 13. SinBarrett’s epithelium is characterized by the expression of several factors that distinguishes it from the normally present squamous epithelium in the distal esophagus, for example Caudal type Homeobox transcription factor 2 (CDX2), Cyclin D1, Cyclooxygenase-2 (COX2), Mucin 2 (MUC2) and Bone morphogenetic protein 4 (BMP4) –19. Othein vitro model, in which reflux of acid, bile or a combination, and its effects can be simulated and inhibited, would be useful. Over the last few years, several studies using various types of cell cultures to investigate the underlying mechanisms of bile and/or acid involved in BE development have been published. In this review, we will discuss these cell culture models with specific emphasis on cell lines, reflux components and incubation conditions.The metaplastic process causing esophageal squamous epithelial cells to transform into intestinal metaplastic columnar-lined epithelium (BE), followed by progression to EAC is complex . In ordeex vivo cultures, colon cancer cell lines and esophageal squamous carcinoma cell lines, papers reporting patient studies, animal studies and previous reviews were excluded. Our query resulted in 159 papers. Fourty-six publications finally met the inclusion criteria.A systematic search of the English-language literature indexed in PubMed, the Cochrane library and EMBASE was conducted that included a combination of the following search queries: esophagus, Barrett’s, cell line, culture, bile, acid, exposure, reflux and adenocarcinoma. The search was performed for the period 1990 until October 2011. In addition, a manual search of citations in relevant articles was performed. In order to limit the focus of this review, papers using ex vivo cultures of esophageal squamous epithelial or Barrett’s epithelial cells. In a study by Palanca-Wessels et al., the majority of primary (ex vivo) cultures of Barrett’s epithelium were maintained for 1 week, whereas only a small number of cultures extended for longer periods, up to 4 months   BE develops. It has been suggested that at least partly a genetic basis is involved , 86; howex vivo and in vitro models. The latter are easiest to use, as in vitro models have the advantage that they are widely available, can be cultured for longer periods and are not dependent on the presence of patients with BE. It is important that there is consensus on which in vitro model to use, in order to combine the results of different studies.Therefore, until there are animal models available that are validated for studying BE and EAC, we need to rely on in vitro models that have been reported to induce factors normally expressed in BE. This review shows that many different cell lines and incubation conditions are available, and, in addition, the measured outcome parameters are often different. It is therefore difficult to conclude that there is a single most perfect in vitro model to study BE development.In this review, we reported a large variety of in vitro model. For other research questions, particularly the development of esophageal adenocarcinoma, BAR-T or OE33 cells can be used. It is, however, important to keep in mind that cell lines are usually transformed in order to keep the cells continuously growing, and therefore may behave differently compared to esophageal cells growing in vivo. Moreover, the microenvironment is also not present in an in vitro system, i.e., cells do not interact with other cell types, particularly inflammatory cells, or with products secreted by the surrounding stroma, i.e., growth factors and inflammatory mediators, which also play a role in the grow pattern and functioning of esophageal lining cells.It seems likely that the choice for a specific cell line to investigate pathogenetic mechanisms in BE largely depends on the research question. If the aim is to study the process of metaplasia of normal esophageal squamous epithelial cell to a Barrett’s like cell type, esophageal squamous epithelium cell lines, such as Het-1A cells, are most appropriate to use in an in vitro bile salts at either a low or neutral pH are required to induce the changes that are seen in BE, such as NF-κB activation and increased cell proliferation. Based on our review, we suggest that a combination of bile salts and acid, i.e., 100 or 200 μM conjugated primary bile salts at pH 4 for 1 h, seems the most optimal culture condition to induce a Barrett’s like phenotype. This concentration of bile salts at low pH is similar to what is found in the refluxate of BE patients and has been shown to induce DNA damage [in vitro model includes multiple short exposures to bile salts and/or acid, which is comparable to the gastroesophageal reflux episodes that occur in GERD [From the reviewed studies it can be concluded that A damage , 34. In A damage , 56, 58. in GERD , 50, 79.in vitro cell line systems are able to induce the expression of markers that are specific for Barrett’s epithelium. When these models are used it may well be that our knowledge on the pathogenesis of the development of BE and its progression towards EAC can be improved.In conclusion, this data suggests that some selected


In the present study using human breast cancer cell lines, the biological activity of 1,25D-d6 administered alone and in combination with two commonly used antineoplastic agents, 5-fluorouracil and etoposide, was evaluated. Using an MTT assay, flow cytometry, and western blots, our data demonstrated that 1,25D-d6 has effects similar to the natural hormone on cell proliferation, cell cycle, and apoptosis. Furthermore, the combination of 1,25D-d6 and etoposide enhances the antitumoral effects of both compounds. Interestingly, the antitumoral effect is higher in the more aggressive MDA-MB-231 breast cancer cell line. Our data indicate that 1,25D-d6 administered alone or in combination with chemotherapy could be a good experimental method for accurately quantifying active 1,25D levels in cultures or in biological fluids, on both in vitro breast cancer cell lines and in vivo animal experimental models. It has been demonstrated that 1,25-dihydroxyvitamin D This has led to the study of its properties in several processes such as cancer [et al. in several mammary tumor cell lines [i.e., morphology, cytoarchitecture, size), proliferation, sensitivity to apoptotic stimuli, adhesiveness, migration, invasion, and the expression of marker genes associated with the inhibition of myoepithelial characteristics and with decreased malignancy. All of this may contribute to the vitamin’s protective action against neoplasia. In addition to its well known role in calcium homeostasis, numerous studies have demonstrated that 1,25-dihydroxyvitamin Ds cancer . In breas cancer . Furthers cancer ,8. Most ll lines . They shi.e., cisplatin and doxorubicin) [i.e., paclitaxel) [i.e., etoposide) [i.e., 5-fluorouracil) [1,25D and its analogues can also enhance, either synergistically or additively, the antitumor properties of several antineoplastic agents . These prubicin) ,12, micrlitaxel) , topoisooposide)  and withouracil) . 6 or a mixture of 1,25D-d6 and non-labeled 1,25D could allow accurately quantifying its concentration and bio-availability in an experimental model. One of these deuterated analogs of vitamin D is the 26,26,26,27,27,27-hexadeuterated 1,25-dihydroxyvitamin D3   A,B. 6) on human breast cancer cells, these compounds were administered alone and in combination with two antineoplastic agents, 5-fluorouracil and etoposide, and then citotoxicity, cell cycle, apoptosis, and three-dimensional cell growth was evaluated.To compare the effects of 1,25D and its deuterated form , inducing significantly decreased cell viability in all cell lines, in relation to control cells . To evaluate cell viability after 1,25D, 1,25D-d6 does not significantly reduce cell viability as compared with administration of each compound alone (6 could not be explained by vitamin D receptor (VDR) levels because these cells had lower VDR expression, compared with the other less aggressive MCF-7 and SKBR-3cell lines [5 analogue, as occurs in cells with high levels of VDR, suggesting that other factors are involved in 1,25D-mediated inhibition of cell proliferation [As shown in nd alone A–C. Decrll lines . Lower Vll lines . VDR levferation . 6 plus etoposide, we focused our next experiments on this cell line and this antineoplastic agent. The lower cell viability in MDA-MB-231 cells after treatment with 1,25D and 1,25D-d6 plus etoposide could be explained by reduced cell proliferation or increased cell death. Therefore, we next investigated the effect of both hormones and etoposide on cell cycle and apoptosis. It is well known that etoposide is cell-cycle dependent and phase specific, inducing G2/M arrest and activation of p53 [p < 0.001). In addition, Western blot indicated that etoposide also increased cyclin A, cyclin B, and p53 expression . Administration of 1,25D plus etoposide  significantly enhances the effect on apoptosis with respect to administration of 1,25D , but not with respect to administration of etoposide alone. Administration of 1,25D-d6 plus etoposide enhances, but not significantly, early apoptosis with respect the administration of each compound . We also used Western blot to analyse the expression levels of several proteins involved in the apoptosis pathway. Our data indicates that no significant changes were found in the anti-apoptotic Bcl-2 and the pro-apoptotic Bid proteins after administration either alone or in combination of etoposide, 1,25D, and 1,25D-d6, in relation to control cells  in sphere diameter compared with control cells, and this effect increased when etoposide was combined with 1,25D-d6 (p = 0.002). We also explored three-dimensional growth of MDA-MB-231 cells. 6 when combined with etoposide. It has been demonstrated that etoposide inhibits the ability of 1,25D to cause accumulation of 25-hydroxyvitamin D3 24-hydroxylase mRNA(CYP24), an enzyme that catabolizes this hormone, thus increasing the bioavailability of 1,25D [6 had similar effects to 1,25D plus etoposide on cell viability, cell proliferation, and apoptosis. Thus, it seems reasonable to assume that similar mechanisms are involved with 1,25D-d6, and to speculate that etoposide could also increase its bioavailability and providing it with similar biological activity, at least in the mammary tumor cell lines used in our experimental approach.Previous studies have reported that some deuterated vitamin D analogs have similar or lower effects than their non-deuterated forms on clonal growth and cell differentiation in several leukemia cell lines, in addition to stimulating intestinal calcium absorption and bone calcium mobilization . Howeverof 1,25D . Althoug6, instead of the non-deuterated form, could be a better method for accurately quantifying its concentration in breast cancer cell lines or in animal experimental models in vivo. In summary, our results seems to indicate that administration of etoposide (and perhaps other antineoplastic agents) plus 1,25D-d2 (95:5) atmosphere at 37 °C. Confluent cells were washed twice with PBS and harvested by a brief incubation with trypsin-EDTA solution  in PBS. 1,25D and 1,25 D-d6 were provided by Profs. Antonio Mouriño and Miguel Maestro . 5-Fluorouracil and etoposide were obtained from Ferrer Farma  and Teva Genéricos Española, S.L. . 5-Fluorouracil and etoposide were dissolved in PBS and used at 200 nM and 500 nM, respectively. The human breast carcinoma cell lines MCF-7, SK-BR-3, and MDA-MB-231, obtained from the European Collection of Cell Cultures , were grown in 90 mm petri dishes in DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 mg/mL streptomycin, and 2 mM L-glutamine in an air-CO6 at 100 nM. The two antineoplastic agents, etoposide and 5-fluorouracil were used at 500 nM and 200 nM, respectively. The same doses were used when combined with 1,25D or 1,25D-d6.Cell lines were treated either with 1,25D or 1,25D-d4 cells/mL) were seeded in a volume of 0.5 mL into 24-well tissue culture plates. MCF-7, SKBR-3, and MDA-MB231 cell lines were treated after 24 h as described above. The absorbance of the samples was recorded 48 h after treatment at 570 nm in a multiwell plate reader . Results were plotted for each cell line as the mean ± SD values of quadruplicates from at least two independent experiments.Cell viability experiments were carried out using MTT assay. Cells , and propidium iodide  for 60 min in darkness, for cell cycle evaluation. Apoptosis analyses were performed using Annexin V (Ann)-FITC. Cells (2 × 105) were harvested, washed twice with PBS, and resuspended in 1 × binding buffer (10 mM Hepes (pH 7.4), 140 mM NaCl, and 2.5 mM CaCl2). 5 µL of FITC-Annexin V was added and incubated for 15 min at room temperature in darkness. Finally, 400 µL of 1 × binding buffer was added to each tube, and analyzed. Cell cycle and apoptosis assays were carried out by using a Guava flow cytometer . Briefly, 2 × 10Western blotting of MDA-MB231 cells was performed as previously described . Briefly3 cells per 100 µL of culture medium, supplemented with 2% (vol/vol) of Matrigel, was carefully placed on the coverslips on top of the solidified Matrigel and incubated at 37 °C for 30 min. Coverslips were then placed in six-well plates with 500 µL of culture medium per well. After 10 days, cells were treated with 1,25D-d6 and etoposide alone or in combination for one week. Photographs of the 3D cultures were taken with a Nikon Eclipse Ti-S inverted microscope  equipped with a ProgRes C3 camera and the ProgRess Capture Pro 2.7 software [For 3D cell culture, 12 mm coverslips were coated with 60 µL of ice-cold Matrigel (BD Biosciences) and incubated at 37 °C for 20 min to allow the Matrigel to solidify. Cells were treated for 5 min with 0.25% trypsin-EDTA solution . A single cell suspension containing 5 × 10p values of less than 0.05 were considered statistically significant. The MATLAB R2011a Version7.1 software [Each experiment was performed at least three times. Values are expressed as mean ± standard deviation. Means were compared using one-way ANOVA, with the Tukey’s test for post hoc comparisons. software  was used6 had similar bioactivity to the natural hormone 1,25D when combined with the antineoplastic drug, etoposide. Combined with 1,25D-d6, etoposide enhanced the antitumoral activity of each drug on cell viability in the breast cancer cell lines MCF-7, SKBR-3 and MDA-MB-231. Interestingly, the antitumor effect was higher in the more aggressive cell line MDA-MB-231. Our results support that 1,25D-d6 administered alone or in combination with chemotherapy could be a good experimental method for accurately quantifying its concentration in culture medium or biological fluids. Our data indicates that 1,25D-d


Given MG-132's strong inhibitory effect on LMS cell viability, we hypothesized that LMS cells may be sensitive to treatment with other proteasome inhibitors and demonstrated that bortezomib, a clinically-approved proteasome inhibitor not included in the original cmap screen, potently inhibited the viability of the LMS cell lines. These findings suggest that systematically linking LMS subtype-specific expression signatures with drug-associated expression profiles represents a promising approach for the identification of new drugs for LMS.Leiomyosarcoma (LMS) is a malignant, soft-tissue tumor for which few effective therapies exist. Previously, we showed that there are three molecular subtypes of LMS. Here, we analyzed genes differentially expressed in each of the three LMS subtypes as compared to benign leiomyomas and then used the Connectivity Map (cmap) to calculate enrichment scores for the 1309 cmap drugs in order to identify candidate molecules with the potential to induce a benign, leiomyoma-like phenotype in LMS cells. 11 drugs were selected and tested for their ability to inhibit the growth of three human LMS cell lines. We identified two drugs with  Leiomyosarcoma (LMS) is a malignant neoplasm of smooth muscle that accounts for approximately one quarter of all soft-tissue sarcomas. Most frequently, LMS occurs in the uterus or the retroperitoneum, but these tumors can also present in a number of soft tissues throughout the body. Current treatment protocols for LMS consist of surgery with adjuvant doxorubicin-based chemotherapy . There aLeiomyoma (LM) is a benign smooth muscle neoplasm that, like its malignant counterpart LMS, frequently occurs in the uterus. While LM is a significant cause of hospitalizations for gynecological disorders and is the most frequent reason for hysterectomies among US women, these growths virtually never metastasize . We hypoin vitro , a reference collection of gene expression profiles from cultured human cell lines  treated with a large and diverse library of small molecules , 5. Previn vitro . To generate gene signatures of each LMS subtype, we performed Significance Analysis of Microarrays  and idenhttp://www.broad.mit.edu/cmap/), which is a publically available resource designed to find connections between disease-associated gene expression signatures and drug response signatures [To identify drugs to target the different LMS subtypes, we utilized the Connectivity Map ). Next, we treated the LMS cell lines with each drug for 72 hours, using a concentration range of 0 μM to 10 μM, before assessing cell viability , we found no statistically significant association between in vitro drug response and cmap enrichment scores . To ascertain whether the magnitude of cmap enrichment, and not the positivity or negativity of the score, was predictive of in vitro drug response, we similarly calculated the correlation between cell viability and cmap scores, this time using only the absolute value of the cmap enrichment scores. Similarly, we found no statistically significant overall association between the magnitude of a drug's cmap enrichment score and its ability to inhibit in vitro LMS cell viability .To determine whether cmap enrichment scores were associated with in vitro drug response as a binary variable, we performed a Fisher's exact test where a significant cmap enrichment score was defined as having an absolute value greater than 0.75 and a significant drug response was defined as having greater than 50% cell viability inhibition at a 10 μM concentration. While we found no statistically significant association between a significant cmap enrichment score and inhibition of cell viability, our data did show a trend suggesting that a highly nonzero cmap enrichment score to LMS subtype II is more likely to be predictive of in vitro drug response ; Supplemental Table  S3).To specifically evaluate whether highly nonzero enrichment  were associated with in vitro drug response in LMS. However, we note that we selected a set of 11 drugs highly enriched for enrichment scores near 1 or −1, and we did identify 2 of 11 drugs with strong in vitro activity. These drugs represent novel candidate drugs for treatment of LMS.These findings suggest that further work that would include a larger number of observations is needed to systematically evaluate the relationship between cmap enrichment score and There are currently no targeted therapies available for the treatment of LMS. Treatment of LMS typically consists of surgery with doxorubicin-based chemotherapy and consideration for adjuvant ifosfamide and radiotherapy in selected cases. Doxorubicin-based therapy has only shown a marginal association with improved overall survival, thereby making it important to evaluate additional therapeutic molecules for the treatment of these tumors .Previously, we have shown that there exist three distinct subtypes of LMS, characterized by unique genomic, transcriptional, and protein expression characteristics  and we hHomo sapiens genome [in vitro efficacy demonstrated in the current work may provide a starting point for a more rigorous in vitro and in vivo exploration of Cantharidin or other phosphatase inhibitors for the treatment of LMS.Of the 11 small molecules evaluated experimentally in our current study, two drugs, Cantharidin and MG-132, were able to strongly inhibit cell viability in all three LMS cell lines tested. Cantharidin is an ancient Chinese medicine that has been demonstrated to have anticancer activity through its inhibition of protein phosphatases –12. InteMG-132 is a potent inhibitor of the proteasome with an ability to specifically reduce the degradation of ubiquitin-conjugated proteins in mammalian cells . Given tSeveral reports in the literature have utilized comparative gene expression profiling studies between normal and diseased tissues to identify and validate drugs with therapeutic potential. Two such studies utilized cmap to identify potential therapeutic molecules for neuroblastoma and colorectal cancer , 7. A siin vitro in the 11 drugs that we tested. It is important to note that the primary goal of our analysis was to identify new therapeutic drugs for LMS and not to systematically evaluate cmap, and consequently the set of 11 drugs we selected was significantly enriched for drugs with highly nonzero enrichment scores. This design may have increased our ability to identify effective drugs, but gave us little statistical power to rigorously evaluate associations between drug response and cmap score, as we evaluated few drugs with enrichment scores near zero. The two drugs that showed in vitro efficacy showed highly divergent enrichment scores, with Cantharidin showing a highly negative score while MG132 showed a strongly positive score. Although our study was not well-powered to identify significant overall associations between enrichment scores and in vitro efficacy in LMS, the rate at which we were able to identify drugs that could inhibit cell viability using cmap  was higher than similarly designed studies that screened entire chemical libraries without a priori predictions of efficacy. For example, Rickardson and colleagues observed a 4.4% hit rate  for small molecules that could inhibit myeloma cell line growth, and Zhang and colleagues observed a 0.6% hit rate  in a screen of new therapeutic compounds that could inhibit thyroid cancer growth [In the present work, we show that while using cmap we were able to identify two drugs that potently inhibited LMS cell growth, there was no overall statistically significant association between cmap enrichment scores and actual cell viability inhibition r growth , 18. WhiFurther, while the gene expression profiles of the three LMS cell lines evaluated showed the strongest similarity to LMS Subtype II, it is likely still necessary to evaluate a broader spectrum of cell lines in order to ascertain whether, and to what degree, our LMS patient tumor subtyping is applicable to immortalized cultured cells. Unfortunately, there exist very few human LMS cell lines available for study, thereby limiting our ability to more rigorously investigate the relationship between subtype specificity and drug response. As more LMS clinical specimens are immortalized and made available to the research community for cell culture studies, it would be informative to ascertain their subtype specificity and to characterize their drug response profiles.Our findings do suggest that further work is needed to systematically assess the relationship between cmap enrichment scores and drug effects in a variety of cancer cell model systems. It will be valuable for future studies of drug candidates identified using computational approaches to document both the identified candidates that are successfully validated as well as the candidates that do not show efficacy in follow-up experiments.in vitro assays. Our study identified two drugs, Cantharidin and MG-132, that showed strong antigrowth effects in LMS cell lines and that may form the starting point for a more focused evaluation of these and similar drugs for the treatment of LMS.In conclusion, we utilized gene expression profiles to predict novel drug candidates for LMS, and we functionally tested 11 of these drugs using http://smd.stanford.edu/) [The clinico-pathologic features of the 51 LMS cases have been described previously . The 19 P values to estimate the probability of observing a perturbagen's enrichment score due to chance; however, only a minority of perturbagens in the database contained nonnull P values, due to insufficient replicates, less than 50% nonnull connectivity scores for a perturbagen's instances, or a mean connectivity score of zero. To allow us to use the full database of perturbagens to generate hypotheses, we focused our analysis on perturbagen enrichment scores and not the perturbagen P values.The “upsignature” and “downsignature” generated for each of the 3 LMS subtypes were uploaded into cmap , 5. In c2 and the medium was replaced every 2-3 days.LMS03, LMS04, and LMS05 cell lines were derived from human LMS clinical specimens and have been described previously , 21. LMSDoxorubicin, Cantharadin, tyrophostin, 6-thioguanine, oxamic acid, 2-Deoxy-d-glucose, and DMOG were purchased from Sigma-Aldrich. MG132 was purchased from AG Scientific. Gefitinib and Metformin HCl were purchased from Tocris Bioscience. LY294002 was purchased from Cell Signaling Technology. Sirolimus was purchased from Cayman Chemical. Bortezomib was purchased from Selleck Chemicals. All drugs were reconstituted in DMSO (Sigma-Aldrich) to create stock concentrations of 10 mM.μM was used. Cell viability was determined using the WST-1 Cell Proliferation Assay (Roche Diagnostics) according to manufacturer's protocols; average signals were plotted.Cells were seeded at a density of 4,000 cells per well in clear 96-well plates , incubated for 24 h to allow adherence to the surface, and then treated in quadriplicate for 72 h with nine-point dilution series of the compounds. For all drugs, a concentration range of 0 to 10 For each of the 3 LMS cell lines, we computed the Pearson correlation between the drug's percent inhibition and the drug's enrichment score. To assess whether the magnitude (and not direction) of the score was associated with inhibition, we used the absolute value of the enrichment score in the correlation analysis. To assess whether a discretized enrichment score showed a significant association with discretized cell viability, we discretized the values at a threshold  and computed a Fisher's exact test.∼0 to ∼1, they are enriched for drugs with higher absolute scores.Supplemental Figure 1: Boxplot of distribution of absolute value of cmap enrichment scores in the 1309 perturbagen's in cmap (left side) vs. the 11 drugs evaluated in LMS cell line (right side). Although the evaluated drug's scores range from Supplemental Table S1: Correlation between cmap scores and percent cell viability.Supplemental Table S2: Correlation between magnitude  of cmap scores and percent cell viability.Supplemental Table S3: Fisher's exact test 2x2 contingency table.Supplemental Table S4: Full results of the Connectivity Map analysis.Click here for additional data file.


Steroid hormone receptors represent a major target in drug discovery. As ligand inducible transcription factors, their activity can be modulated by small lipophilic molecules. Here we describe two panels of potent and selective luciferase reporter cell lines based on cells with low endogenous steroid receptor activity (U2OS). The panels contain reporter cell lines for estrogen receptors α and β, androgen, glucocorticoid, mineralocorticoid, and progesterone receptors. In the first panel, the activation of either synthetic, steroid response elements containing promoter or viral promoter is mediated by full-length steroid receptors. The second panel is based on the expression of the chimeric receptor, which was created by the replacement of the N-terminal part of the molecule by Gal4 DBD and that binds to multiple UAS sites in the reporter promoter. Both panels were extensively characterized by profiling 28 ligands in dose response manner in agonist and antagonist mode. We have analyzed and compared the responses to tested ligands from both panels and concluded that in general both systems generated similar qualitative response in terms of potency, efficacy, partial agonism/antagonism, mixed agonistic/antagonistic profiles and the rank of potencies was well conserved between both panels. However, we have also identified some artifacts introduced by the Gal4/LBD reporter assays in contrast to their full-length receptor reporter counterparts. Keeping in mind the advantages and drawbacks of each reporter format, these cell lines represent powerful and selective tools for profiling large compound libraries (HTS) and for detailed study of mechanisms by which compounds exert their biological effects. Steroid hormone receptors are ligand-inducible transcription factors belonging to nuclear hormone receptor superfamilly and include two receptors for estrogens (ERα and ERβ), androgen receptor (AR), glucocorticoid receptor (GR), mineralocorticoid receptor (MR) and progesterone receptor (PR) . They coSteroid hormone receptor activity can be regulated in many different ways including posttranslational modifications resulting from cell signaling activity, for example phosphorylation of target proteins. Expression of different set of coactivator or corepressor molecules and the activity of other transcription factors can be responsible for different transcriptional regulation by steroid receptors in different cellular contexts . Howeverluc2 gene. The second format relies on the chimeric steroid receptor where the N-terminal part of the receptor containing AF-1 and DNA binding domain (DBD) was replaced by the DBD from the yeast transcription factor Gal4. This construct was cotransfected to the cells together with reporter vector containing 9 copies of Gal4 Upstream Activator Sequences (UAS) coupled to minimal promoter upstream of the luc2P gene coding for the luciferase with PEST destabilizing sequence (pGL4.35 [luc2P/9XGAL4UAS/Hygro]). This system was described recently, and its utility was illustrated by preparation of ERα and GR reporter assays in HEK293 cells  was created by inserting an oligonucleotide comprising three copies of estrogen responsive elements (ERE) CCAGGTC ACAGTGACCTGAGT in the pGL4.26 [luc2/minP/Hygro] reporter vector . In this synthetic estrogen responsive promoter the centers of the perfect palindromic inverted repeats are separated by 21 bp from each other. The 3xERE containing oligonucleotide was inserted in the XhoI/BglII restriction sites of pGL4.26 [luc2/minP/Hygro] reporter vector. pGL4.26-3xGRE [luc2/Hygro] was constructed in a similar way by insertion of an oligonucleotide containing three copies of glucocorti-coid responsive elements (GRE) GGTACATTTTGTT CTAGCCAG in the XhoI/BglII restriction sites of pGL4.26 [luc2/minP/Hygro] reporter vector.pGL4.26-3xERE [pGL4.35 [luc2P/9XGAL4UAS/Hygro] and pGL4.36 [luc2P/MMTV/Hygro] reporter vectors were described previously .Final constructs were verified by restriction digests and sequencing.luc2/Hygro], pGL4.26-3xGRE [luc2/ Hygro], pGL4.36 [luc2P/MMTV/Hygro] or pGL4.35 [luc2P/ 9XGAL4UAS/Hygro]) using PEI 25kDa  transfection. Two days after transfection, selection of stable transfectants was initiated by adding Hygromycin B  to the culture medium at final concentration of 100 μg/ml. Cells were grown in the Hygromycin B-containing medium for three weeks, and after that time cells were frozen and were used later for transfection with expression vectors for steroid receptors.The production of stable reporter cell lines for both full-length steroid hormone receptors and chimeric steroid LBD receptors in U2OS cells was done in two steps. In the first step, U2OS cells were transfected with reporter vector alone  to the culture medium at final concentration of 250 μg/ml and 100 μg/ml respectively. Cells were propagated in the medium containing antibiotics for at least two months before being frozen and used for compound profiling.U2OS cell line stably transfected with pGL4.35 [luc2/Hygro] was transfected with expression vectors encoding full-length steroid receptors: pcDNA3-hERα and pcDNA3-ERβ, creating U2OS-ERα/3xERE and U2OS-hERβ/3xERE cell lines, respectively. U2OS cell line stably transfected with pGL4.26-3xGRE [luc2/Hygro] was transfected with expression vectors encoding full-length steroid receptors: pcDNA3-hAR and pcDNA3-hGR, creating U2OS-AR/3xGRE and U2OS-GR/3xGRE cell lines, respectively. U2OS cell line stably transfected with pGL4.36 [luc2P/MMTV/Hygro] was transfected with expression vectors encoding full-length steroid receptors: pcDNA3-hAR, pcDNA3-hGR and pcDNA3-hMR to generate U2OS-AR/MMTV, U2OS-GR/MMTV, U2OS-MR/MMTV reporter cell lines, respectively. Two days after transfection, selection of stable transfectants was initiated by adding G418 and Hygromycin B  to the culture medium at final concentration of 250 μg/ml and 100 μg/ml respectively. Cells were propagated in the antibiotics containing medium for at least two months before being frozen in liquid nitrogen and profiled with different compounds.U2OS cell line stably transfected with pGL4.26-3xERE , while pGL4.26-3xGRE [luc2/Hygro] can be used to monitor activity of the remaining steroid receptors: GR, MR, AR and PR. Although all of these receptors can activate the luciferase gene in the presence of the agonist by binding to GRE elements in the reporter vector, the response was not equally efficient in all of these cases. For instance, MR showed only weak transactivation on the 3xGRE-containing synthetic promoter. To overcome these limitations, we combined these receptors with pGL4.36 [luc2P/MMTV/Hygro] reporter vector, which contains the enhancer region from the mouse mammary tumor virus long terminal repeat (MMTV LTR) promoter. This region was originally isolated as progesterone and glucocorticoid responsive enhancer [To build the full-length steroid receptor reporter system we created two reporter vectors carrying synthetic promoters with three copies of either estrogen responsive element (ERE) or glucocorticoid responsive element (GRE) coupled to a minimal promoter upstream of the coding sequence for the luciferase. ERE and GRE are constituted by the perfect palindromic repeats with a three bp spacing of variable bases. The centers of palindromes are separated by 21 bp from each other, and the distance of the third repeat from the minimal promoter is 50 bp. ERα and ERβ bind to EREs in the promoter of the reporter vector pGL4.26-3xERE [enhancer , but it 1). Not surprisingly it was difficult to find a cell line with null background activity for all steroid receptors. For instance, COS7 cells derived from kidney of African green monkey and CHO cells from Chinese hamster ovary are often used in the study of ERs, which is in agreement with our observation that they have no ER activity. On the other hand COS7 cells are responsive to progesterone and CHO cells to cortisol and aldosterone when transfected with GRE containing reporter vector suggesting that they express nonnegligible levels of PR and GR/MR, respectively. In HEK 293 cells we found GR activity. No detectable activity of any steroid receptor was found in U2OS cells that was in agreement with previous observations [Cross-talk with different signaling pathways is not the only technical problem that needs to be carefully considered during data analysis. Cross-talk with endogenously expressed steroid receptors can be even more important issue because ERα and ERβ bind to the same response elements, and GR, MR, AR and PR activate more or less efficiently promoters in both reporter vectors. When creating selective reporter cell lines, it is essential to express exogenous steroid receptors in cell lines with low background activity of other members of this receptor class. Unfortunately many of the cell lines traditionally used in the individual steroid receptor research do not fulfill this condition, and they could be only used in the study of small subset of receptors in the same time due to the endogenous expression of other steroid receptors. In order to create robust and selective reporter assays that would ideally be based on single parental cell line, we screened several commonly used cell lines for endogenous steroid receptor activity . To make sure that all cell lines are stable and do not change properties such as dynamic range or potency when exposed to ligands, they were cultured in the antibiotics containing medium for at least two months before freezing them and using them for compound profiling.As a starting point we have created four master cell lines stably transfected by following reporter vectors: pGL4.26-3xERE [50/LogIC50 values calculated from dose response curves  are summarized in the Table 1.To validate these cell lines with respect to their utility to detect agonistic and antagonistic properties of compounds, we used them for profiling a set of 28 well-established ligands, comprising both natural ligands and synthetic compounds, commonly used in clinics or as potent tools in the study of steroid receptors. The profiling was performed in the agonist and antagonist mode, and LogEC50 values varied between 1 pM and 100 pM for the following combinations ERα/E2, ERβ/E2, AR/DHT and MR/aldosterone and the potency of GR-expressing reporter cell lines was close to 1 nM for dexamethasone: EC50 (U2OS-GR/3xGRE) = 1.66 nM and EC50 (U2OS-GR/MMTV) = 1.03 nM (Table 1e). The lower potency of dexamethasone and glucocorticoids more generally on GR  in comparison to potencies observed in the 17β-estradiol induced transactivation by ERα and ERβ  or dihydrotestosterone induced transactivation by AR  was expected and is consistent with data in the literature [50(U2OS-ERα/3xERE) = 4 pM and EC50(U2OS-ERβ/3xERE) = 5 pM (Table 1a). Similarly both AR reporter cell lines respond to dihydrotestosterone in the picomolar range: EC50(U2OS-AR/3xGRE) = 4 pM and EC50(U2OS-AR/MMTV) = 3 pM (Table 1c) and MR to aldosterone in the subnanomolar range: EC50(U2OS-MR/MMTV) = 88 pM (Table 1g). We also compared data generated with our cell lines to values in the literature and found our data comparable to literature reports for all receptor/promoter combinations [Generally all U2OS reporter cell lines expressing full-length steroid receptors were sensitive to their commonly used agonists and antagonists. ECterature . GR geneinations , 19-21.The same cellular background represented by U2OS cell type devoid of any endogenous steroid receptor activity enabled the construction of a consistent reporter system allowing monitoring activity of unique receptors with exceptional selectivity. Our data confirm this premise and the selectivity is illustrated by the fact that we did not find any sign of luciferase activity when treating ERα and ERβ reporter cell lines with glucocorticoid activity-exhibiting compounds  even at 10 μM concentration.1e, g). Since these receptors are generally inhibited by estrogens, androgens and progestagens at higher concentrations, we detected their antagonistic activity especially in MR reporter cell lines. In the tested concentration range they can be ranked according their decreasing antagonistic potency to MR: progesterone > testosterone > dihydrotestosterone > 17β-estradiol > danazol, PPT.Similarly no agonistic activity was observed with GR and MR reporter cell lines treated with estrogens or androgens . In addition it was stronger than in the master reporter cell lines U2OS-/3xGRE and U2OS-/MMTV, and we did not detect the same activity with U2OS-AR-LBD / 9xGal4UAS cell line, which is however less sensitive as discussed below. Therefore we hypothesize that the weak androgenic activation by glucocorticoids results from mixed activity of very low level of endogenously expressed GR and cross reactivity of glucocorticoids with AR.2=0.89, Fig. 4a). Generally it can be noticed that U2OS-AR/MMTV is slightly more sensitive than U2OS-AR/3xGRE with some dose response curves shifted slightly to lower concentrations. Both cell lines show the same mixed agonistic/antagonistic profiles for cyproterone acetate, progesterone and mifepristone with comparable partial activities .To estimate to what extent promoter context contributes to different responses provoked by the same ligand and receptor we compared compound potencies from reporter cell lines which contain either synthetic promoter containing 3xGRE or more complex viral promoter from MMTV. Two AR reporter cell lines show almost identical response to tested compounds considering the potency and efficacy of the dose response . We could identify few different responses in the antagonist mode where U2OS-GR/MMTV was less sensitive to detect the antagonistic properties of some compounds like spironolactone or mifepristone. However, U2OS-GR/MMTV cell line provides about three times higher dynamic range and better data reproducibility (data not shown).Very similar profiles were also obtained with GR reporter cell lines . There is very little difference between the response from U2OS-ERα/3xERE and U2OS-ERαwt-LBD in both agonist and antagonist mode as illustrated by few examples in the Fig. (f) and high correlation of LogEC50, LogIC50 values obtained with these two different reporter formats . An exception is probably somewhat consistently higher potency of androgenic compounds in ERα-LBD reporters about 1 log but these compounds activate ERα in the micromolar and higher concentration range.For ERα we have prepared two Gal4/LBD reporter cell lines: U2OS-ERαrecently  and carr4d, Table 1a). This shift is noticeable mainly in the agonist mode and is consistent in the whole compound collection; hence the rank of potencies is conserved between LBD and full-length ERβ reporters. It is worth noting that ERβ is considerably weaker transactivator than ERα due to lower affinity for ERE half-sites [The response to compounds mediated by ERβ-LBD is shifted of about 1.5 logs to higher concentrations compared to full-length ERβ reporter . When looking closely at data from both formats of GR reporters, we concluded that U2OS-GR-LBD/9xGal4UAS cell line generally tends to be more sensitive to antagonist effect of compounds. For example mifepristone is partial agonist/full antagonist on both full-length GR reporter cell lines but very potent pure full antagonist on LBD format. Also we didn´t detect very weak partial agonistic activity of progesterone using U2OS-GR-LBD/9xGal4UAS. Similarly, MR-LBD expressing reporter cell line is in contrast to its full-length MR counterpart only weakly and partially activated by glucocorticoids such as dexamethasone, betamethasone and beclomethasone, and interestingly these compounds have pronounced full antagonist effect in the LBD format, while there is no sign of antagonistic activity in the full-length receptor format. The correlation between  obtained from different formats of GR reporters is lower than in case of ERα or ERβ mainly due to the artifacts in the antagonist mode . These data suggest that MR-LBD and GR-LBD based reporter cell lines generally reproduce well the activity profiles of tested compounds as obtained using reporter cell lines expressing full-length receptors but in addition can produce false positives when assayed in the antagonist mode.With GR and MR both formats gave similar profiles with tested compounds in the agonist mode as shown in Table 3a, b, c) and Table 1c. The dose response curve is shifted in average about 1 log to higher concentrations for high affinity ligands. We also noticed that the dose response profile of low affinity ligands diverged more substantially from profiles obtained by full-length AR reporter cell lines as illustrated by partial activation of AR-LBD by E2  and other compounds like spironolactone, cyproterone acetate and danazol.It was previously shown that the main transactivation activity inducible by ligand resides in AF-1 of the N-terminus of the AR molecule instead of AF-2 -27 and tWe have created and subsequently characterized two panels of U2OS-based luciferase steroid receptor reporter cell lines using two different reporter formats: Gal4/LBD reporter cell lines and full-length steroid receptor reporter cell lines. We have characterized these cell lines by profiling them using a set of 28 well established ligands and analyzed their properties in terms of compound potency, efficacy, selectivity and reproducibility of results among different formats.The main advantage of the whole system is that it is based on single cell type: osteosarcoma U2OS cell line, and therefore it has unified and constant cellular background that is defined by the expression of the same type and amounts of enzymes involved in the metabolism of compounds and the same coactivators and corepressors regulating the steroid receptor transcription response. Thus results obtained from different reporter cell lines can be directly compared. This is also closely related to the selectivity of cell lines and to the question to what extent is the compound activity mediated by one given receptor. The use of uniform cellular background makes the estimation of contribution of different receptors to the measured compound activity easier since it is possible to test the compound on all receptors and compare with values from master reporter cell lines carrying the reporter vector alone. In addition we have shown that U2OS cells have no detectable activity of any of the steroid receptors and therefore there is no cross-talk with endogenously expressed receptors. This was demonstrated by showing selective activation of specific receptors when exposed to selective ligands. Taken together and considering the fact that U2OS cells are robust and easy to maintain in culture, these properties makes the whole reporter system highly consistent, selective and robust.via binding sites for different cellular transcription factors.As a next step we have carefully compared and analyzed data obtained using both assay formats. The advantage of full-length steroid receptor reporter system is that it is based on the native fully functional receptors that can bind to high affinity response elements in the promoter of the reporter vectors. Because some receptors interact with the DNA and change their conformation according to the response element, we have also prepared reporter cell lines for AR, GR and MR where the defined response element (3xGRE) was replaced by the complex viral promoter derived from the MMTV LTR. Although we have noticed subtle differences between the cell lines using MMTV instead of 3xGRE, generally the use of MMTV provided the same information about the compound activity, improved dynamic range of the assay and reproducibility of data but also increased the risk of integrating signals from other signaling pathways The problem of using overexpression of the fully biologically active full-length steroid receptors is potential lower tolerance of higher expression levels by the cells. As a result cells expressing higher levels of the specific receptor are usually unstable in the culture and the dynamic range of the assay might be limited from this reason.Another problem with full-length receptors is that they respond to multiple stimuli from within the cell, and their activity can be modulated indirectly by posttranslational modifications mainly on the N-terminal part of the molecule as a result of activation of the other signaling pathways such as MAPK and PKA, for example.These problems are not associated with the Gal4/LBD reporter format since the activity is mediated through Gal4/UAS response elements foreign to most of the mammalian transcription factors, and consequently this format has generally low background activity. Moreover the N-terminal AF-1 with regulatory sequences was removed from the chimeric receptors, and therefore the connection between the biological activity of the compound and the measured luciferase activity is more tightly associated with the specific activity of the steroid receptor as a result of direct compound binding.G420C-LBD/9xGal4UAS, U2OS-ERβ-LBD/9xGal4UAS and U2OS-AR-LBD/9xGal4UAS the rank of potencies of tested compounds is surprisingly well conserved. We have however identified few weak points of this format in U2OS-GR-LBD/9xGal4UAS and U2OS-MR-LBD/9xGal4UAS. Inactive or purely agonistic compounds in the full-length format tended to show antagonistic properties when assayed with U2OS-GR-LBD/9xGal4UAS. The effect was even more pronounced with U2OS-MR-LBD/9xGal4UAS where some compounds exhibited both decreased potency and efficacy in the agonist mode and became full antagonists in the opposite mode. In other words MR-LBD and GR-LBD-based reporters generally reproduce the activity profiles of some compounds compared to full-length receptor reporters, but can produce false positives when tested in the antagonist mode.The comparison of compound profiles from Gal4/LBD reporter assays with those from full-length receptor reporter assays showed that there is a high consensus between these two formats especially for ERα, ERβ and GR. Although some Gal4/LBD reporter cell lines suffer from slightly decreased potency (0.5-1.5 logs) like U2OS-ERαG420C-LBD/9xGal4UAS and U2OS-ERβ-LBD/9xGal4UAS assay and partial agonist in U2OS-ERβ-LBD/9xGal4UAS assay reflecting clearly and perfectly the response from full-length receptors (data not shown). Similarly weak partial agonism of selective estrogen receptor modulators (SERMs) such as 4-hydroxy-tamoxifen, tamoxifen citrate or raloxifene hydrochloride was also detected using Gal4/LBD assays although it was less pronounced than in full-length receptor reporter format (Table 1a).On the other hand Gal4/LBD reporters reflected surprisingly reliably some subtle activity characteristics of compounds such as partial agonism. For example genistein, ERβ selective isoflavone was almost full agonist in U2OS-ERα/3xERE assay, partial agonist in U2OS-ERβ/3xERE assay, full agonist in both U2OS-ERαThe expression of chimeric receptors is generally well tolerated by the host cell and considerably improves the dynamic range of the reporter assays compared to its full-length receptor counterpart with one exception being AR, where the removal of N-terminus causes almost complete silencing of the ligand induced transactivation as discussed above.1b, supplementarymaterial). Z´-factor exceeding 0.5 is commonly accepted criterion for the satisfactory assay quality for the HTS [wt-LBD/9xGal4UAS and U2OS-AR-LBD/9xGal4UAS. The relatively low fold induction of the ERαwt-LBD reporter assay causing lower Z´-factor (0.65) was mentioned above and can be overcome by the introduction of the G420C mutation to the ERα molecule (full-length receptor amino acid numbering). The resulting reporter assay  has 10 times higher fold induction (data not shown) and higher Z´-factor (0.76). In addition the Gal4/LBD reporter assays can benefit from the constitutive expression of a Renilla luciferase-neoR fusion gene which can be used as an internal control in the HTS and can be helpful in compensating for variability between samples caused by difference in cell number for example by random pipetting errors or by nonspecific interference with the luciferase reporter readout and cytotoxic effects of compounds [From these reasons Gal4/LBD reporter format is especially well suited for HTS campaigns. We have successfully carried out the compound profiling using all reporter cell lines in the 384-well format using instruments for HTS (Pin tool robotic compound transfer). In order to assess the reproducibility of the recorded data we have calculated Z´-factor for all the reporter cell lines in the Gal4/LBD reporter format (Table ,  the HTS . Four ouompounds .Because each of these formats represents specific set of advantages and drawbacks, altogether they embody a powerful and complete tool for profiling of large compound libraries and identifying modulators showing interesting and useful activity profiles over the whole family of steroid hormone receptors. They are also sophisticated tools for the detailed study of mechanisms by which these compounds modulate the activity of the specific steroid receptors.Supplementary material is available on the publishersWeb site along with the published article.


The molecular heterogeneity of human cancer cells at the level of signaling protein activities remains poorly understood. Using a panel of 64 colorectal (CRC) cancer cell lines the activity status of the MAP kinases Erk1 and Erk2 was investigated. Erk1/2 activity varied greatly within the CRC cell line panel and was not detectably associated with the speed of cell growth in 10 CRC lines analyzed. As expected, mutations in K-Ras or B-Raf were often, albeit not always, linked to high Erk1/2 activity. The phosphorylation of several known Erk1/2 targets investigated did not generally reflect Erk1/2 activity in the 10 CRC lines analyzed. However, the reduction of Erk1/2 activity with MEK inhibitors generally abolished cell growth but only led to an increase of cellular p27Kip1 levels in CRC cells with high Erk1/2 activity levels. The results indicate that high Erk1/2 activation is utilized by some CRC lines to override the cell cycle brake p27Kip1, while others presumably rely on different mechanisms in order to inactivate this important cell cycle brake. Such detailed knowledge of the molecular diversity of cancer cell signaling mechanisms may eventually help to develop molecularly targeted, patient-specific therapeutic strategies and treatments. The limited knowledge about the heterogeneity of cancers on the signaling protein activity level is a major obstacle for better, individualized cancer therapies with signal transduction-modulating drugs. It is now well feasible to comprehensively analyze mutations and mRNA expression changes in tumor biopsies and isolated tumor cells with high-throughput techniques. By contrast, in-depth biochemical analyses of signaling protein activities are currently all but impossible with patient biopsy material. However, important insight into the individual diversity of cancers can be gained by analyzing large panels of cancer cells from a specific tumor type -3.Erk1 and 2 are multifunctional kinases which are employed in a very wide range of normal and pathological cell types, in many cases in order to regulate cell proliferation or differentiation -6. HowevIn this study we have analyzed 64 different CRC cell lines for the activity status of Erk1 and 2 , further supporting a key role for additional modifiers in determining the activity of Erk1/2 within a subset of CRC cell lines.Ras GTP-loading assays and data base searches The total Erk1/2 levels are similar in all 10 cell lines. Unexpectedly, the apparent activity of MEK1/2, analyzed by western blotting with a pMEK1/2 (pS217/pS221) antibody, did not correlate well with Erk1/2 activity (data not shown). We are at present unable to provide a molecular explanation for this, but a potential reason could again be the impact of Erk phosphatases, such as those of the MKP family.Comparing the growth rates in the 10 cell lines failed to show any correlation between Erk1/2 activity and proliferation speed Figure , initialIn order to address the latter possibility, selected Erk substrates and targets, including Elk1, Msk1, Myc and p90Rsk, were analyzed with phosphoepitope specific antibodies in the 10 CRC lines. It is not possible to perform a comprehensive analysis at this point, since more than 160 substrates and targets of Erk have been reported so far  and phosp27Kip1 is an important cell cycle regulatory protein previously discussed as a direct or indirect target of Erk1/2, with phosphorylation leading to its proteasomal destruction -16. TherIndeed, inhibiting Erk activation through the potent MEK1/2 inhibitor U0126, led to an increased expression of p27Kip1 in all 5 CRC lines with high Erk activity and none of the lines with low Erk activity Figure . HRA-19 The simplest explanation for these findings is that a subset of CRC cells utilizes the strong activation of Erk1/2 to down-regulate p27Kip1 expression. This may be mediated by targeting the Thr187 residue on p27Kip1, which is linked to its ubiquitinylation and degradation -16.Despite these interesting results, we can, of course, by no means exclude the possibility that high Erk1/2 activity is also required to phosphorylate other key substrates in this subset of 'high Erk' CRC cells.Erk1 and Erk2 boast a plethora of known substrates  and veryNevertheless, from our study it would appear that down-regulation of p27Kip1 expression is at least a common, if not ubiquitous occurrence in CRC cells with high Erk1/2 activity. Similar findings have been reported in other tumor types . In someInterestingly, the absolute expression levels of p27Kip1 do not correlate with Erk activity levels  or Erk kinase; U0126: a MEK inhibitor compound.The authors declare that they have no competing interests.TK designed and conducted most experiments, analyzed data and wrote the manuscript. TR co-supervised TK, analyzed data and wrote the manuscript. SF conceived the project, designed and conducted experiments, analyzed data and wrote the manuscript. All authors read and approved the final version of the manuscript.Origin of CRC cell lines.Click here for fileMethods description.Click here for fileExpression levels of p27Kip1 in 10 CRC cell lines with high or low Erk activity.Click here for fileEffect of the MEK1/2 U0126 on the morphology of HCT 116 cells.Click here for file


To determine the expression of breast metastasis suppressor 1 (BRMS1) in human uveal melanoma (UM) tissues and cell lines. In addition, we intend to establish a possible association between BRMS1 expression and the presence of metastatic disease.Thirty-one formalin-fixed paraffin-embedded tissues from enucleated eyes of patients with UM were immunostained. Clinical–pathological data were obtained, including age, tumour location, largest dimension, cell type, and occurrence of metastasis. The expression of BRMS1 mRNA in four human UM cell lines was determined by real-time reverse transcriptase polymerase chain reaction, and protein expression was assessed by immunocytochemistry and western blot. The association between BRMS1 immunostaining and location, largest tumour dimension, and tumour cell type was determined using the correlation coefficient test. The association between BRMS1 immunostaining and the incidence of metastasis was assessed using Kaplan–Meier analysis.P = 0.69), tumour cell type (P = 0.68), largest tumour dimension (P = 0.75), and tumour location (P = 0.11).Of the 31 cases of UM, 24 (77.42%) stained positive and seven (22.58%) negative for BRMS1. From the positively stained tumours, 21 (87.50%) showed cytoplasmatic staining. Macrophages were usually positive when present in the tumour and staining intensity was generally higher than in UM cells. BRMS1 mRNA was present in all four human UM cell lines, as well as cytoplasmatic immunoexpression of BRMS1. Immunoblotting showed variable BRMS1 protein levels between the different cell lines. No statistically significant correlation was found between BRMS1 protein expression and survival (BRMS1 is expressed in UM both at the mRNA and protein level; however, neither was associated with any of the prognosticor outcome parameters that we tested. Uveal melanoma (UM) represents approximately 5% of all reported melanomas , with anet al [The traditional concept that metastasis occurs during late stages of tumourigenesis  has beenet al  recordedet al [KISS1, revealed a strong correlation between KISS1 immunoreactivity and lower risk of metastatic disease in UM and a better survival rate [The metastatic cascade involves complex, interrelated, and essential steps . Genes tet al  have shoet al . Anotherval rate . To dateet al [Breast metastasis suppressor 1 (BRMS1) is a gene that maps to chromosome 11q13.1–q13.2 , 24 and et al  have coret al , 27. SinThe metastasis suppressor BRMS1 has not been studied in UM. Therefore, our purpose is to investigate the immunohistochemical expression of BRMS1 in human UM specimens, and to establish if there is an association between its expression and metastatic disease. In addition, we aim to determine BRMS1 mRNA and protein expression in human UM cell lines.Thirty-one formalin-fixed paraffin-embedded blocks of enucleated primary tumours from patients with UM were collected from the archives of the Henry C. Witelson Ocular Pathology Laboratory and Registry, McGill University, Montreal, Canada.Tumour cell type, as recorded in the original pathology report, was reclassified according to the modified Callender’s classification of UM . TumoursFormalin-fixed paraffin-embedded sections of the collected blocks were stained with haematoxylin and eosin for histopathological assessment.Immunohistochemistry was performed using the Ventana BenchMark LT fully automated machine . The fully automated processing of bar code labelled slides included baking of the slides, solvent-free deparaffinisation, and CC2 (Citrate buffer pH 6.0) antigen retrieval. Slides were incubated with the mouse antihuman monoclonal antibody against BRMS1 (M01), clone 2D4-2G11 , at a dilution of 1:500 for 40 min at room temperature, followed by application of biotinylated secondary antibody , then an avidin/streptavidin enzyme conjugate complex . Finally, the antibody was detected by Fast Red chromogenic substrate and counterstained with haematoxylin.As a positive control, sections of breast cancer were used, as this cancer has been shown to express BRMS1 protein . For negA single experienced pathologist blinded to the patients’ follow-up data analysed the stained sections. The sections were classified as group 1 when more than 30% of the neoplastic cells stained for BRMS1 and as group 2 when less than 30% stained. Both groups were subclassified into cytoplasmatic, nuclear, or nuclear–cytoplasmatic staining and into diffuse or focal staining.g for 10 min to form a pellet. Cells were then suspended in 1 mL of medium and counted in trypan blue using a haemocytometer.Four human UM cell lines  were incubated at 37 ºC in a humidified 5% CO2-enriched atmosphere. The cells were cultured in RPMI-1640 medium , supplemented with 5% heat inactivated foetal bovine serum (Invitrogen), 1% fungizone (Invitrogen), and 1% penicillin–streptomycin (Invitrogen). Cells were cultured as a monolayer in 25-cm2 flasks  and observed twice weekly, at every media change, for normal growth by phase contrast microscopy. The cultures were grown to confluence and passaged by treatment with 0.05% trypsin in ethylenediaminetetraacetic acid  at 37 ºC and washed in 7 mL RPMI-1640 media before being centrifuged at 120in vitro studies [The UM cell lines 92.1, SP6.5, MKT-BR, and OCM-1 were established by Dr. Jager , Dr. Pelletier , Dr. Belkhou , and Dr. Albert , respectively. The characteristics of these cell lines are described elsewhere, and their metastatic potential (92.1 > SP6.5 > OCM-1 > MKT-BR) was determined using an immunosuppressed rabbit model  and late studies .Total cellular RNA was extracted from the four human UM cell lines using the Qiagen RNeasy kit following the manufacturer’s instructions. Briefly, the cells were disrupted and homogenised using the included lysate buffer and extracted with a 20.5 gauge syringe. The lysate was then centrifuged to remove any insoluble material. One volume of 70% ethanol was added to the lysate and mixed, before the solution was added to the included RNeasy mini column. Following centrifuging, the columns were washed twice using the included buffer solutions. Total cellular RNA was then eluted using RNase free water.Expression level of BRMS1 mRNA was subsequently determined by quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR) performed in triplicate using Quantitect one-step SYBR Green PCR method  following the manufacturer’s instructions. A Chromo4 thermocycler (MJ Research) was used for all experiments and the results were analysed using the GeneEx software. QuantiTect primer assay pairs (Qiagen) for BRMS1. The β-actin housekeeping gene was also used in all experiments for the purpose of normalisation.6 cells/mL, and 300 μL was placed in each spin to be evenly plated on each slide. The spins were fixed with 2% paraformaldehyde. All slides were then immunostained with a mouse anti-human monoclonal antibody against BRMS1 (M01), clone 2D4-2G11 (Abnova Corporation), diluted 1:100, using the Ventana BenchMark LT fully automated machine  programmed to use a standard Avidin–Biotin Complex method.Cytospins of four human UM cell lines with different metastatic potentials  were prepared using Cytospin3 centrifuge . Cells from culture were diluted to a concentration of 1 × 10g. Protein concentration was determined by Bio-Rad protein assay . Protein aliquots of 10 μg were loaded onto 12% SDS-Page gel and then transferred to polyvinylidene fluoride-Millipore (PVDF) membrane.Cells from the four human UM cell lines were lysed on ice in TNESV  containing a protease inhibitor cocktail (Sigma) and the lysate was cleared by centrifugation for 10 min at 12000The membrane was blotted for a mouse anti-human monoclonal antibody against BRMS1 (M01), clone 2D4-2G11 , at a dilution of 1:1000. Horseradish peroxidase conjugated goat anti-mouse secondary antibody  was applied. Detection was performed using enhanced chemiluminescence with Pierce SuperSignal West Femto Maximum Sensitivity Substrate  and captured with autoradiographic film . β-actin was used for loading controls.P-value of less than 0.05 was considered statistically significant. All data accumulation was in accordance with country and provincial laws, and the tenets of the Declaration of Helsinki.Statistical analysis was performed using a computer-based statistical package . The association between BRMS1 immunostaining and tumour location, largest tumour dimension, and tumour cell type was determined using the correlation coefficient test. The association between BRMS1 immunostaining and the incidence of metastasis was assessed using the Kaplan–Meier survival analysis. A Thirty-one patients were included in our study. The mean age at the time of primary tumour diagnosis of UM patients was 61.13 years ± 12.61 (mean ± standard deviation) in both groups. In The RT-PCR showed that the BRMS1 mRNA was present in the four human UM cell lines. The relative expression across the cell lines was similar .Immunoexpression of BRMS1 was evident in all four UM cell lines to a similar degree, independent of their metastatic potentials with a cytoplasmic staining pattern.Immunoblotting showed a positive band at ~26.8 kDa for all cell lines, with variable BRMS1 protein levels between the different cell lines. MKT-BR, the least metastatic of the four assayed cell lines, had the highest protein expression, and SP6.5, the second-most metastatic cell line, had the lowest protein expression .P = 0.69), tumour cell type (P = 0.68), largest tumour dimension (P = 0.75), or tumour location (P = 0.11).Of the UM cases studied, 24/31 were positive and 7/31 were negative for anti-BRMS1, forming, respectively, groups 1 and 2. From group 1, 21 of the tumours demonstrated cytoplasmatic staining, two had nuclear–cytoplasmatic staining, and one had nuclear staining ; two of For the first time, we have shown that BRMS1 is expressed in human UM tissue as well as in UM cell lines.It is known that the metastatic cascade involves the coordinated expression of multiple genes to result in an established metastatic nodule, while it can only take one gene to block this process . Since tKISS1 [The only MSGs previously described in UM are NM23 and KISS1 –23. BRMSKISS1 , 25–27.et al [BRMS1 was first analysed at the mRNA level in four human UM cell lines with different metastatic potentials. BRMS1 mRNA was present in the four cell lines and their relative expression was similar. This differs from what has been published regarding BRMS1 mRNA levels in other cancers. In human breast carcinoma cell lines, for example, it was shown that the amount of BRMS1 mRNA present in the cell lines is inversely correlated with their metastatic potential . Seraj eet al , in a puet al .BRMS1 was present in the four UM cell lines as demonstrated by immunocytochemistry. Western blot analysis was used to evaluate levels of BRMS1 protein expression. The immunoblotting demonstrated a positive band at ~26.8 kDa in all cell lines, and it distinguished the amount of protein between them; MKT-BR, the least metastatic of the four, had the highest protein expression. These findings agree with the attribution made to BRMS1 of suppressing metastasis: the least metastatic cell line expresses more BRMS1 than the more metastatic ones. In human breast carcinoma cell lines, it has been shown that the degree of metastasis suppression generally correlates with the level of BRMS1 protein expression . Howeveret al [The mean age at the time of primary tumour diagnosis of our UM patients was 61 years, which is in agreement with the findings of other studies , 14, 16.et al  demonstret al [et al [Seraj et al  investiget al . In suppl [et al  correlatet al [et al [et al [et al showed a differential expression of BRMS1 in the nucleus and cytoplasm of cutaneous melanoma cells. The authors hypothesised that cytoplasmic BRMS1 restricts melanoma progression while possibly nuclear BRMS1 promotes melanoma cell invasion [in vivo effect, which could possibly explain our cytoplasmatic staining in all of the cell lines and in 87.50% of the UM specimens. It is important to note that, like many other proteins, BRMS1 may have different roles in cutaneous and UM [Seraj et al  further l [et al  found BRl [et al , 37. NF-l [et al –40, actil [et al . Using tl [et al  demonstrl [et al . It seeml [et al . These finvasion . BRMS1 hinvasion . Althougs and UM . Furtheret al [KAI-1, another MSG, is so highly expressed in macrophages [In UM, tumour-infiltrating macrophages are common and are associated with increased melanoma-specific mortality . It was et al , studyinrophages  that at rophages . We cannrophages , 48.et al [et al [The occurrence of metastasis was similar between groups 1 and 2. This differs from what would be expected from a gene that suppresses metastasis. The expression of NM23 in UM, for example, has shown to be inversely correlated with the development of liver metastasis in nude mice . No statet al  also foul [et al  demonstrIn vitro studies showed that the least metastatic cell line had higher levels of BRMS1. However, when patient samples were analysed, no statistically significant correlation could be established between BRMS1 protein expression and survival, tumour cell type, largest tumour dimension, and tumour location. More studies are needed to further dissect the roles BRMS1 has in UM.This is the first study to assess the MSG BRMS1 in UM cell lines and patient specimens; BRMS1 is expressed in UM at both the mRNA and protein levels.


The combination of systemic cisplatin with local and regional radiotherapy as primary treatment of head and neck squamous cell carcinoma (HNSCC) leads to cure in approximately half of the patients. The addition of cisplatin has significant effects on outcome, but despite extensive research the mechanism underlying cisplatin response is still not well understood.TP53 mutational status of each cell line and investigated the expression levels of 11 potentially relevant genes by quantitative real-time PCR. In addition, we measured cisplatin accumulation and retention, as well as the level of platinum-DNA adducts.We examined 19 HNSCC cell lines with variable cisplatin sensitivity. We determined the 50 value was significantly correlated with the platinum-DNA adduct levels that accumulated during four hours of cisplatin incubation (p = 0.002). We could not find a significant correlation between cisplatin sensitivity and any of the other parameters tested, including the expression levels of established cisplatin influx and efflux transporters. Furthermore, adduct accumulation did not correlate with mRNA expression of the investigated influx pumps (CTR1 and OCT3) nor with that of the examined DNA repair genes .We found that the ICOur findings suggest that the cisplatin-DNA adduct level is the most important determinant of cisplatin sensitivity in HNSCC cells. Imaging with radio-labeled cisplatin might have major associations with outcome. Head and neck squamous cell carcinomas (HNSCCs) arise in the mucosal linings of the upper aerodigestive tract. Early stage tumors are treated with surgery or radiotherapy and these treatment modalities are combined to treat more advanced tumors. Since recent years chemoradiation protocols are increasingly applied to treat locally advanced carcinomas, consisting of systemic application of cisplatin (cis-diammine-dichloroplatinum II) combined with locoregional radiotherapy. The addition of cisplatin causes a significant increase in survival rates CTR1 was identified as an important cisplatin influx transporter ATP7AATP7BSeveral mechanisms of cisplatin resistance have been described previously  is one of the pathways known to remove cisplatin lesions from the DNA TP53 gene, an important player in cellular homeostasis, has also been associated with cisplatin response. It was demonstrated that mutated and inactivated TP53 influences cisplatin sensitivity TP53 mutations, loss of CDKN2A (p16INK4a) or gain of CCND1 (Cyclin D1). This might result in the entrance of tumor cells into S-phase despite the presence of cisplatin adducts, leading to cell death.Furthermore, the Despite the importance of the aforementioned genes in the pharmacodynamics and anti-tumor effect of cisplatin, the actual mechanism of cisplatin response in HNSCC is still not understood. Many genes seem to be important in this response, but their relevance may vary from one tumor model to another and affect cisplatin activity at different levels . In the present study we characterized cisplatin sensitivity and the cellular characteristics of 19 HNSCC cell lines, the largest panel tested as far as we know. We investigated the expression levels of several genes that are implicated in cisplatin toxicity and measured cisplatin accumulation and retention rates as well as cisplatin-DNA adduct levels in these cell lines. Finally, we investigated potential correlations between the different parameters studied and the cellular sensitivity to cisplatin.2 at 37°C. Cell lines of sporadic tumors were established as described previously FANCC), VU-SCC-1365  and OHSU-0974 (with mutations in FANCA). Cell line VU-SCC-9917 was newly established from a sporadic oral cavity tumor, after written informed consent was obtained. The studies involving clinical specimens were according to the Dutch legislations on research with human material. This study was approved by the Institutional Review Board (Medisch Ethische Toetsingscommissie VUmc).HNSCC cell lines used were all cultured in Dulbecco's modified Eagle's medium (DMEM), 5% fetal calf serum (FCS) and 2 mM L-glutamine, in a humidified atmosphere of 5% COCell lines UM-SCC-6, UM-SCC-11B, UM-SCC-14A, UM-SCC-14B, UM-SCC-14C, UM-SCC-22A, UM-SCC-22B and UM-SCC-38 were obtained from Dr. T. Carey (University of Michigan) Cisplatin was obtained from Teva Pharmachemie  in a concentration of 1 mg/ml.2. Cell viability was determined by 2 hours incubation with 20 µl of a 1∶1 dilution of CellTiter-Blue  with culture medium. Fluorescence was measured using an Infinite 200 microplate reader .Cells were plated in 96 well plates in concentrations that allow the cells to grow till 70% confluence after 5 days . After 24 hours, cells were treated with 18 different concentrations of cisplatin (0–666 µM). Plates were incubated for 72 hours at 37°C/5% COTP53 (exons 5–9) was performed on all 19 HNSCC cell lines as described previously Direct dideoxynucleotide sequencing of the evolutionary conserved regions of GUSB) was used as a housekeeping gene GUSB using the Delta/Delta Ct method Cell pellets were made from cultures grown to 70% confluence. RNA was extracted using TRIzol , and quality control tests were performed using the Nanodrop . Complementary DNA was synthesized from 250 ng of RNA template using AMV reverse transcriptase  and a custom designed reverse primer specific for the gene of interest. Amplification of the cDNA was performed on the ABI/Prism 7500 Sequence Detector System  with 1× Power SYBR Green PCR Master Mix  and custom designed primers for each gene of interest. The primer sequences are displayed in Cells were seeded in triplicate in flasks. When 70% confluence was reached, 2 flasks were incubated with 75 µM cisplatin, and the medium of the third flask was refreshed. After four hours, the cells of one cisplatin-incubated flask (the accumulation flask) and the control flask were harvested. The other cisplatin-incubated flask was washed twice with PBS and incubated with fresh medium for another 3 hours, in order to analyze retention of the platinum compound. After counting, cells were resuspended in 0.5 ml 2 M NaOH for overnight incubation at 55°C. Subsequently, 1 ml 1 M HCl was added. Platinum was measured using flameless atomic absorption spectrometry (FAAS) using a Perkin Elmer AS-800 with a THGA graphite furnace . The instrument response was calibrated across the range 0.2 to 3.0 µM for the whole cell lysate determinations and from 50 to 250 pmol per extract for the DNA adduct determinations (the DNA isolation procedure is specified below). With the graphite furnace 20 µl of individual samples was dried at 110–130°C, ashing was performed at 1300°C and atomization at 2200°C with a read time of 5 seconds.Cells were treated as described above for the accumulation and retention experiments. Cells were harvested in culture medium, centrifuged and cell pellets were stored at −20°C until further use. The QiaAmp DNA mini kit  was used to extract genomic DNA from the cell pellets, and the quality was controlled by OD260/280 nm analysis on a Nanodrop . Resulting DNA samples were analyzed for cisplatin content using FAAS as described above.50) and the tumor characteristics of the cell lines are displayed in 50 values of 0.15 µM and 0.80 µM, respectively. This was not unexpected as both these cell lines were established from patients carrying a mutation in one of the Fanconi anemia (FA) genes. On the other hand, cell line OHSU-0974, also established from a FA patient, did not show an extremely sensitive phenotype . Cell line UM-SCC-38 showed the highest IC50 value with 7.57 µM.Nineteen head and neck squamous cell carcinoma (HNSCC) cell lines were subjected to treatment with 18 different concentrations of cisplatin, ranging from 0.01 to 666 µM. Examples of resulting curves are shown in 50 value . Cell lines UM-SCC-14B, UM-SCC-14C and UM-SCC-22B were left out of this analysis since these were not established from independent primary tumors. When we grouped the cell lines according to their tumor stage (early stage tumors (T1–T2) or late stage (T3–T4)), a significant correlation with cisplatin sensitivity was not found either .There was no significant correlation between the stage of the tumor from which the cell lines were derived and the ICTP53 mutation status in all 19 cell lines  showed mutations in the TP53 gene. Nine cell lines  had truncating TP53 mutations according to the classification described by Lindenbergh-van der Plas et al. TP53 mutations; one missense and one truncating mutation. For further analyses, we only counted the truncating mutation since this mutation has most impact on TP53 functioning. The other eight (47%) cell lines showed one or more missense TP53 mutations. The TP53 mutation status  did not significantly correlate to cisplatin sensitivity , even when UM-SCC-14B, UM-SCC-14C and UM-SCC-22B were eliminated from the analysis .We investigated the ll lines . Two celCTR1, OCT1, OCT2 and OCT3), efflux  or are involved in DNA crosslink repair . We used quantitative real-time PCR to investigate the expression of these genes in our cell line panel , no correlations were found. Furthermore, the type of TP53 mutation  also showed no correlation with the expression of the nine genes tested. Remarkably, there was a highly significant correlation  between BRCA1 and BRCA2 expression in the 19 cell lines  nor to any of the mRNA expression levels, among which were the assigned influx pumps CTR1 and OCT3. Elimination of cell lines with a known FA defect from the analysis did not change this observation. The percentage retention also did not show a significant correlation to any of the characteristics of the cell lines, including the mRNA expression of efflux pumps ATP7A and ATP7B. Remarkably, we found a significantly inverse correlation between the cisplatin accumulation and retention , indicating that cells with high cisplatin accumulation also show a strong efflux.Intriguingly, the large range in accumulation did not correlate to the ICDNA isolated from cell cultures incubated with 75 µM cisplatin for four hours was subjected to flameless atomic absorption spectrometry (FAAS) in order to determine the level of platinum adducts in the DNA. The resulting data are displayed in 50 value , even when we excluded the three cell lines with a known mutation in one of the FA genes . This indicates that cells with a low IC50 value for cisplatin contain significantly more DNA adducts compared to cells that are more resistant to cisplatin  showed a significant correlation, but with the current literature at hand no likely explanation for these correlations could be suggested.When comparing the expression levels of the genes tested in our HNSCC panel, we found several significant correlations. Some correlations were explainable when looking at the gene functions. We found a very strong correlation between the expression of 50 values harbor less platinum-DNA adducts. This suggests that the DNA adduct level resulting from cisplatin incubation is the most important determinant of cisplatin sensitivity. Therefore, imaging of radio-labeled cisplatin in patients might be an excellent indicator for treatment outcome. A genome-wide functional siRNA screen might be a useful tool to determine all the genes that specifically determine the cisplatin response and consequently the adduct accumulation level.From these results we can conclude that the mechanism of cisplatin response seems a relatively simple process in HNSCC. Cells with high ICTable S1Primer sequences for quantitative real-time PCR.(DOC)Click here for additional data file.Table S250 values) and TP53 mutation status of 19 HNSCC cell lines.Inhibitory cisplatin concentrations (IC(DOC)Click here for additional data file.Table S3Relative mRNA expression of the indicated genes in 19 HNSCC cell lines.(XLS)Click here for additional data file.


The advent of tyrosine kinase inhibitor (TKI) therapy has considerably improved the survival of patients suffering chronic myelogenous leukemia (CML). Indeed, inhibition of BCR-ABL by imatinib, dasatinib or nilotinib triggers durable responses in most patients suffering from this disease. Moreover, resistance to imatinib due to kinase domain mutations can be generally circumvented using dasatinib or nilotinib, but the multi-resistant T315I mutation that is insensitive to these TKIs, remains to date a major clinical problem. In this line, ponatinib (AP24534) has emerged as a promising therapeutic option in patients with all kinds of BCR-ABL mutations, especially the T315I one. However and surprisingly, the effect of ponatinib has not been extensively studied on imatinib-resistant CML cell lines. Therefore, in the present study, we used several CML cell lines with different mechanisms of resistance to TKI to evaluate the effect of ponatinib on cell viability, apoptosis and signaling. Our results show that ponatinib is highly effective on both sensitive and resistant CML cell lines, whatever the mode of resistance and also on BaF3 murine B cells carrying native BCR-ABL or T315I mutation. We conclude that ponatinib could be effectively used for all types of TKI-resistant patients. Chronic myelogenous leukemia (CML) is a disease of the hematopoietic stem cell, characterized by the t9 ; 22) q34 ; q11) translocation encoding the oncoprotein BCR-ABL [1 translo2 q34 ; qin vitro [http://www.clinicaltrials.gov).Regarding BCR-ABL kinase mutations, the multi-resistant T315I one remains a crucial clinical challenge, since until recently, there is no effective treatment for the patients carrying this particular type of mutation. Ponatinib is a multi-targeted kinase inhibitor that exhibits high activity against the T315I mutation but also other BCR-ABL kinase mutants in vitro , 13. In in vitro , 14, 15 Surprisingly, although ponatinib represents a promising molecule for patients with BCR-ABL mutations, its mechanism of action has not been extensively studied, more particularly in imatinib-resistant CML cells with no BCR-ABL mutation. In this line, we have previously generated several imatinib-resistant cell lines from the parental K562, JURLMK1 and Lama CML cell lines , 16, 17.In the present study, we took advantage of the availability of these cell lines to evaluate the effect of ponatinib in comparison to other TKIs on cell metabolism, proliferation and apoptosis. In addition, we used the murine BaF3 cell line carrying either a wild-type BCR-ABL protein or its T315I and G250E mutated counterparts to decipher the mechanisms of action of this TKI. We show that ponatinib is highly efficient to induce cell growth inhibition and induction of apoptosis on different imatinib CML cell lines, whatever their mode of resistance.The effect of ponatinib on the viability of several imatinib-resistant cell lines was evaluated in comparison with that of imatinib and dasatinib. Cell lines were incubated for 48h with increasing concentrations of imatinib, dasatinib or ponatinib and cell viability was assessed using the XTT assay. We first investigated the effect of these ITKs on the viability of murine Ba/F3 cells, carrying wild type (WT) BCR-ABL, T315I or G250E-BCR-ABL mutation. As shown in Figure The IC50 values for the ponatinib effect's in parental CML cells and for the different imatinib-resistant cells lines were very close (1 to 6nM versus 8 to 30nM) Figure . The IC5Moreover, the IC50 values for parental K562 CML cells (6nM) and Lama CML cells (1nM) were also in good agreement with the one previously reported in the literature  .It is well established that TKIs induced different types of cell death, including apoptosis in CML cell lines , 19, 20.To investigate the effect of ponatinib on CML cell growth, we next performed clonogenic assays using the different Imatinib-sensitive and resistant cell lines described above. BaF3 cells carrying WT BCR-ABL protein failed to form colonies in soft agar in the presence of imatinib  was highly effective on CD34+ cells from the same patients with only a minor effect on PBMC at 30nM Figure . The effThe findings described in the present study confirm and extend the work previously reported by O’ Hare et al. in sensitive CML cell lines and BaF3 cells carrying native and mutated BCR-ABL . HoweverCRKL is a direct substrate of BCR-ABL and therefore the status of CRKL phosphorylation is sought to be a reflect of the one of BCR-ABL. Although dephosphorylation of BCR-ABL by ponatinib was nearly complete at 10-30nM, only a higher concentration of ponatinib (100nM) was capable to efficiently dephosphorylate CRKL. These results are in good agreement with those of O Hare et al. . One posIn conclusion, we show here for the first time that ponatinib is highly efficient to induce cell death in different imatinib-resistant CML cell lines. Phase II clinical trial evaluation of oral ponatinib in patients with refractory CML and other hematologic malignancies are ongoing. Our results suggest that in addition to T315I patients, ponatinib could be widely used in resistant CML patients, whatever the supposed mode of resistance to first line ITKs.Imatinib mesylate  was purchased from Enzo Life Sciences , and Dasatinib and Ponatinib were purchased from Selleckchem . RPMI 1640 medium, IMDM medium and fetal calf serum (FCS) were from life technologies . Sodium fluoride, sodium orthovanadate, phenyl-methyl-sulfonyl fluoride (PMSF), aprotinin, leupeptin were purchased Sigma-Aldrich (France). Anti-HSP60 and anti-ERK antibodies were from Santa Cruz Biotechnology . HRP conjugated anti-mouse and anti-goat antibodies were from Dakopatts . Anti-PARP, anti-phospho-ERK, anti-CRKL, anti-phospho-CRKL, anti-ABL, anti-phospho-ABL and peroxydase-conjugated anti-rabbit antibodies were obtained from Cell Signaling Technology .Human CML cell lines K562, Lama, and JURLMK-1 were grown at 37°C under 5% CO2 in RPMI supplemented with 10% FCS, 50 U/ml penicillin, 50 μg/ml streptomycin, and 1mM sodium pyruvate. K562, Lama and JURLMK-1 cells imatinib-resistant have been described earlier , 11. TheCells (15'103 cells/100ml) were incubated in a 96 well plate with different effectors for the times indicated in the figure legends. Fifty microliters of sodium 3-[After stimulation, cells were washed with ice-cold PBS and were stained with the annexin-V-fluos staining kit  according to the manufacturer's procedure. Fluorescence was measured by using the FL2 channels of a fluorescence-activated cell sorter apparatus (Miltenyi cytometer).After stimulation, cells were harvested and lysed in buffer containing 1% Triton X-100 and supplemented with protease and phosphatase inhibitors (Roche Diagnostics). Lysates were pelleted, and 50μg of protein were analyzed by SDS-PAGE as described previously .TKI was added to cell lines growing in semisolid methylcellulose medium . Colonies were detected after 10 days of culture by adding 1 mg/ml of 3--2,5-diphenyltetrazolium bromide (MTT) reagent and were scored by Image J quantification software .Blood samples were collected from patients newly diagnosed with CML. All patients were part of an institutional protocol. Peripheral blood mononuclear cells were isolated by density centrifugation . CD34+ cells were prepared by magnetic bead separation as described previously . CD34+ c