Isobaric Tags For Relative Quantification Research Articles

Proteomic analysis of the similarities and differences of soil drought and polyethylene glycol stress responses in wheat (Triticum aestivum L.).

Our results reveal both soil drought and PEG can enhance malate, glutathione and ascorbate metabolism, and proline biosynthesis, whereas soil drought induced these metabolic pathways to a greater degree than PEG. Polyethylene glycol (PEG) is widely used to simulate osmotic stress, but little is known about the different responses of wheat to PEG stress and soil drought. In this study, isobaric tags for relative quantification (iTRAQ)-based proteomic techniques were used to determine both the proteomic and physiological responses of wheat seedlings to soil drought and PEG. The results showed that photosynthetic rate, stomatal conductance, intercellular CO2 concentration, transpiration rate, maximum potential efficiency of PS II, leaf water content, relative electrolyte leakage, MDA content, and free proline content exhibited similar responses to soil drought and PEG. Approximately 15.8% of differential proteins were induced both by soil drought and PEG. Moreover, both soil drought and PEG inhibited carbon metabolism and the biosynthesis of some amino acids by altering the accumulation of glyceraldehyde-3-phosphate dehydrogenase, ribulose-bisphosphate carboxylase, and phosphoglycerate kinase, but they both enhanced the metabolism of malate, proline, glutathione, and ascorbate by increasing the accumulation of key enzymes including malate dehydrogenase, monodehydroascorbate reductase, pyrroline-5-carboxylate dehydrogenase, pyrroline-5-carboxylate synthetase, ascorbate peroxidase, glutathione peroxidase, and glutathione S-transferase. Notably, the latter five of these enzymes were found to be more sensitive to soil drought. In addition, polyamine biosynthesis was specifically induced by increased gene expression and protein accumulation of polyamine oxidase and spermidine synthase under PEG stress, whereas fructose-bisphosphate aldolase and arginase were induced by soil drought. Therefore, present results suggest that PEG is an effective method to simulate drought stress, but the key proteins related to the metabolism of malate, glutathione, ascorbate, proline, and polyamine need to be confirmed under soil drought.

Thrombospondin-1, Platelet Factor 4, and Galectin-1 Are Associated with Engraftment in Patients with Sickle Cell Disease Who Underwent Haploidentical HSCT

BackgroundSickle cell disease (SCD) is an inherited red blood cell disorder which leads to significant morbidity and early mortality. The most widely available curative approach remains hematopoietic stem cell transplantation (HSCT). However, <15% SCD patients have human leukocyte antigen (HLA)- matched sibling donors. HLA-haploidentical (haplo) HSCT expands the donor pool considerably and is a practical alternative for these patients, but with an increased risk of allograft rejection. Biomarkers in patient plasma could potentially be helpful in predicting HSCT outcome and allow treatment at an early stage so that graft rejection could be reversed or prevented.ObjectiveThe purpose of the study was to detect variations in the plasma proteomes of engrafted patients as compared to those who rejected their grafts and elucidate mechanisms associated with engraftment.MethodsPatients were conditioned with alemtuzumab, 400cGy total body irradiation, and a dose escalation of post-transplant cyclophosphamide ranging from 0-100mg/kg (Fitzhugh CD et al. Blood Advances, 2017). Tandem Mass Tag (isobaric tags for relative quantification) was performed on plasma samples from engrafted (N=9) and rejected patients (N=10) who underwent haplo HSCT. Proteins with p-values <0.05, relative difference in protein concentration between groups >25%, and total number identified peptide sequences >2 were considered statistically significant. Bioinformatics tools (DAVID, GeneMANIA, STRING and LENS) were used to analyze the identified proteins. Biologically relevant proteins were further evaluated by ELISA to verify their association with the allograft status.ResultsThe proteomics analysis revealed 44 differentially expressed proteins between engrafted and rejected groups. Seven candidate proteins, namely thrombospondin-1 (TSP-1), platelet factor 4 (PF4), talin-1, moesin, cell division control protein 42 homolog, galectin-1 (GAL-1), and CD9 were selected for further analysis. Pathway enrichment analysis revealed that the differentially expressed proteins were mostly related to focal adhesion, rap1 signaling pathway, regulation of actin cytoskeleton platelet activation, and leukocyte transendothelial migration. The interaction network of the identified proteins indicated that the proteins were partially biologically connected.ELISA results of the 7 proteins revealed that TSP-1, PF4, and GAL-1 were significantly higher in the engrafted group as compared with the rejected group, African-American healthy volunteer group, and SCD group who had not been transplanted (see Figure 1). TSP-1 has been found to promote the survival of corneal transplants and is a major activator of transforming growth factor (TGF)-b signaling pathway (Saban DR et al. J Immunol, 2010), an important player in the initiation of immunological tolerance. PF4 has been observed to be associated with improved outcome in cardiac and kidney transplants (Shi G et al. J Clin Invest, 2014 and Zhang L et al. Inflammation, 2015), to enhance proliferation of regulatory T-cells, and to decrease Th17 differentiation (Shi G et al. J Clin Invest, 2014). Lastly, GAL-1 has been found to induce regulatory T- and regulatory B-cell function (Alhabbab R et al. Sci Rep, 2018).ConclusionsWe have shown for the first time that TSP-1, PF4,and GAL-1 are associated with engraftment in patients with SCD who undergo haplo HSCT. The study warrants further validation in a larger sample size as well as in vitro and in vivo analyses to evaluate whether our candidate proteins are associated with tolerance induction. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Proteomic profiling of rectal cancer reveals acid ceramidase is implicated in radiation response

BackgroundNeoadjuvant chemoradiotherapy (CRT) is used in locally advanced rectal cancer when tumours threaten the circumferential resection margin, with varying response to treatment. This experimental study aimed to identify significantly differentially expressed proteins between patients responding and not responding to CRT, and to validate any proteins of interest. MethodsMass spectrometry (with isobaric tagging for relative quantification) analysis of rectal cancers pre- and post-CRT, and at resection. Validation of proteins of interest was performed by assessing tissue microarray (TMA) immunohistochemistry expression in a further 111 patients with rectal cancer. ResultsProteomic data are available via ProteomeXchange with identifier PXD008436. Reduced abundance of contributing peptide ions for acid ceramidase (AC) (log fold change −1.526, p = 1.17E−02) was observed in CRT responders. Differential expression of AC was confirmed upon analysis of the TMAs. Cancer site expression of AC in stromal cells from post-CRT resection specimens was observed to be relatively low in pathological complete response (p = 0.003), and relatively high with no response to CRT (p = 0.017). ConclusionAC may be implicated in the response of rectal cancer to CRT. We propose its further assessment as a novel potential biomarker and therapeutic target. SignificanceThere is a need for biomarkers to guide the use of chemoradiotherapy in rectal cancer, as none are in routine clinical use. We have determined acid ceramidase may have a role in radiation response, based on novel proteomic profiling and validation in a wider dataset using tissue microarrays. The ability to predict or improve response would positively select those patients who will derive benefit, prevent delays in the local and systemic management of disease in non-responders, and reduce morbidity associated with chemoradiotherapy.

Evaluation of NQO1 as a predictive biomarker and therapeutic target in metastatic colorectal cancer

BackgroundA knowledge of the biology of colorectal liver metastases would be invaluable to inform clinical decision making; however, deriving this information from metastatic lesions is not feasible until after resection. We aimed to use proteomic analysis to establish the degree of biological similarity across disease sites and identify and validate biomarkers in the primary tumour that predict response to neoadjuvant chemotherapy in liver metastases. MethodsTissue from primary colorectal tumour and liver metastases (n=16) were subjected to proteomic analysis with isobaric tagging for relative quantification. Data were analysed with ProteinPilot software (Ab Sciex, Framingham, MA USA) with stratification of patients into low or high response to chemotherapy. These biomarkers were investigated by immunohistochemistry on a tissue microarray of 56 patients. Their therapeutic potential was investigated by dosing of SW480 cells with irinotecan or fluorouracil with or without inhibition by small interfering RNA (siRNA) or a competitive inhibitor (dicoumarol). FindingsWe identified 5766 discrete proteins, of which 2·54% were differentially expressed between primary and metastatic tumours. There were 170 potential response biomarkers in the primary tumour and 27 in the metastases. Lambda-crystallin homolog and NQO1 were common to both tissue types and showed consistent dysregulation. Immunostaining of NQO1 in metastatic tissue was lower in patients responding than in those not responding to chemotherapy (p=0·041), with a significant correlation between primary and metastatic disease sites (r=0·44, p=0·001). Knockdown of NQO1 with siRNA followed by treatment with irinotecan or fluorouracil reduced the IC50 from 100·1 to 49·8 μM and from 200·1 to 25·0 μM, respectively. Treatment of cells with dicoumarol before incubation in irinotecan or fluorouracil reduced the IC50 from 100·0 μM to 50·0 μM and from 183·7 μM to 49·9 μM, respectively. InterpretationWe show that proteomic sequencing of matched metastatic colorectal cancer samples is feasible with high coverage. The high degree of similarity between the primary and secondary tumours suggests that the primary tissue is predictive of the metastatic phenotype. NQO1 expression in the primary tumour predicts response to neoadjuvant chemotherapy in liver metastases, and inhibition of this protein at both genetic and functional levels improves chemosensitivity. FundingCancer Research UK.

PWE-305 Nqo1 is a predictive biomarker and therapaeutic target in metastatic colorectal cancer

<h3>Introduction</h3> A knowledge of the biological phenotype of colorectal liver metastases would be invaluable to inform clinical decision making; however, deriving this information from the metastatic lesions is not feasible until after resection. We aimed to use proteomic analysis to identify biomarkers in the primary tumour which predict response to neoadjuvant chemotherapy in liver metastases. <h3>Method</h3> Fresh tissue from both primary colorectal tumour and liver metastases from 17 patients was subjected to proteomic analysis using isobaric tagging for relative quantification, with stratification of patients into those showing low or high response to chemotherapy permitting the identification of potential predictive biomarkers. These markers were subsequently validated by immunohistochemistry on a tissue microarray of 57 patients and further investigated in an <i><i>in vitro</i></i>model. <h3>Results</h3> We identified 5768 discrete proteins, 5 of which predicted histopathological response to fluorouracil-based chemotherapy regimens. Immunostaining of NQO1 in the metastases was lower in those responding to chemotherapy (p = 0.041), with a significant correlation between primary and metastatic disease sites (r = 0.44, p = 0.001). Knockdown of NQO1 with small interfering RNA followed by treatment with irinotecan and 5FU reduced the IC50 from 100.1µM to 49.8µM and from 200.1µM to 25.0µM respectively. Pre-treating cells with dicoumarol (a known competitive inhibitor) prior to incubation in irinotecan and 5FU reduced the IC50 from 100.0µM to 50.0µM and from 183.7µM to 49.9µM respectively. <h3>Conclusion</h3> These results show that proteomic sequencing of matched metastatic colorectal cancer samples is feasible, with high protein coverage. The high degree of similarity between the primary and secondary proteomes suggests that primary tissue is predictive of the metastatic phenotype. NQO1 expression in the primary tumour predicts response to neoadjuvant chemotherapy in the liver metastases, and inhibition of this protein at both genetic and functional levels improves chemosensitivity. Further validation in an <i><i>in vivo</i></i>model is planned. <h3>Disclosure of interest</h3> P. Sutton Grant/ Research Support from: Cancer Research UK, J. Evans: None Declared, R. Jones: None Declared, C. Goldring: None Declared, D. Palmer: None Declared, N. Kitteringham: None Declared, H. Malik: None Declared, D. Vimalachandran: None Declared.

Proteomic analysis to identify biomarkers in the primary tumour that predict response to neoadjuvant chemotherapy in liver metastases

BackgroundColorectal cancer is the fourth commonest cancer in the UK, and the second commonest cause of cancer-related death. A knowledge of the biological phenotype of colorectal liver metastases would be invaluable to inform clinical decision making; however, deriving this information from the metastatic lesions is not feasible until after resection. We aimed to use proteomic analysis to identify biomarkers in the primary tumour that predict response to neoadjuvant chemotherapy in liver metastases. MethodsFresh tissue from both primary colorectal tumour and liver metastases from 17 patients was subjected to proteomic analysis using isobaric tagging for relative quantification. Data were analysed with Protein Pilot (Ab Sciex, Framingham, MA, USA), with stratification of patients into those showing low or high response to chemotherapy permitting the identification of potential predictive biomarkers. These markers were subsequently validated by immunohistochemistry on a tissue microarray of 63 patients. FindingsWe identified 5768 discrete proteins. Five of them predicted histopathological response to fluorouracil-based chemotherapy regimens, of which the FAD binding protein NQO1 was subsequently validated by immunohistochemistry. When compared with the chemotherapeutic agent alone, knockdown of the corresponding gene with small interfering RNA decreased cell viability when co-incubated with fluorouracil (77·1% vs 46·6%, p=0·037) and irinotecan (41·7% vs 24·4%, p=0·006). Similar results were also seen after inhibition of protein activity by pretreating cells with dicoumarol. InterpretationThese results show that proteomic sequencing of matched metastatic colorectal cancer samples is feasible, with high protein coverage. The high degree of similarity between the primary and secondary proteomes suggests that primary tissue is predictive of the metastatic phenotype. NQO1 expression in the primary tumour predicts response to neoadjuvant chemotherapy in the liver metastases, and inhibition of this protein at both genetic and functional levels improves chemosensitivity. FundingCancer Research UK.

Phenotypic analysis of primary colorectal cancer to inform management of metastatic disease

BackgroundColorectal cancer is the fourth commonest cancer in the UK, and the second commonest cause of cancer-related death. Liver surgery for colorectal liver metastases is increasing, because neoadjuvant chemotherapy has brought a greater number of patients to resection. A knowledge of the biological phenotype of colorectal liver metastases would be invaluable in informing clinical decision making; however, deriving this information from the metastatic lesions is not feasible until after resection. By contrast, material from the primary tumour is routinely available. We aimed to establish the feasibility of using biological information from a primary colorectal tumour to inform and predict response to neoadjuvant chemotherapy for liver metastases. MethodsFresh tissue from both primary colorectal tumour and liver metastases, and from normal colonic mucosa and liver parenchyma, were acquired at the time of resection in 17 patients and subjected to comparative proteomic analysis using isobaric tagging for relative quantification. Data were analysed with Protein Pilot (Ab Sciex, Framingham, MA, USA); proteins with expression significantly different across the different tissue types were subjected to pathway analysis with Metacore software. Stratification of patients to those showing low or high response to chemotherapy allowed the identification of proteins that were differentially expressed in the two groups and thus represented potential response biomarkers. Findings5768 discrete proteins were identified, with 1814 present in all samples and therefore used for analysis. Principal component analysis of relative protein abundance separated liver parenchyma from other tissue types, but did not separate individual patients. No proteins were significantly different between primary and metastatic tumours. 25 proteins were significantly (p<0·05) differentially expressed in the primary tumour compared with normal colon, whereas 53 were different between the liver metasases and normal colon. Five candidate proteins that predict favourable histopathological response to fluorouracil-based chemotherapy regimens were identified. InterpretationProteomic sequencing of matched metastatic colorectal cancer samples is feasible with high protein coverage. Analysis of dysregulated proteins revealed putative pathways that might be implicated in carcinoma progression and metastasis and identify potential novel drug targets. The high degree of similarity between the primary and secondary proteomes suggests that information from primary tissue is predictive of the metastatic phenotype. Further targeted analysis of proteins involved in drug activation and metabolism, as well as the validation of candidate biomarkers, is underway. FundingCancer Research UK.

P53 and c-Myc Are Critical Signaling Hubs That Maintain Chronic Myeloid Leukemia

Abstract Chronic myeloid leukemia (CML) is a clonal disorder of the hematopoietic system, leading to increased production of mature and progenitor myeloid cells. Although protein tyrosine kinase inhibitors (TKI) have been successful in managing the disease, there are exceptions where drug resistance and onset of blast crisis occur. Furthermore TKIs are ineffective against leukemic stem cells (LSC) that are responsible for disease initiation and maintenance. We have shown mRNA changes in primitive hematopoietic cells do not correlate directly to protein changes. Therefore to elucidate fundamental cellular differences between CML and normal cells we employed a proteomic approach (mass spectrometry with isobaric tagging for relative quantification). This approach permits unbiased analyses using direct comparative quantification of peptides and thus proteins from chronic phase CML and normal CD34+ human samples. Systematic data analysis identified that the majority of deregulated proteins are connected and regulated by two oncogenes with well defined roles in human disease, p53 and c-myc. The direction of regulation inferred suppression of p53 and up-regulation of c-myc. Altered expression of key proteins was validated using western blotting and immuno-fluorescence approaches. All (6/6) candidate/hub proteins identified using mass spectrometry were confirmed using these orthogonal approaches. Based on our systematic analysis, we targeted the candidate hubs using the drugs RITA (activates p53) and CPI-203 (inhibits c-myc expression; provided by Constellation Pharmaceuticals). In CML CD34+ cells, RITA reduced cell expansion in a concentration-dependent manner and induced significant levels of apoptosis as confirmed by positive staining of Annexin V and 4',6-diamidino-2-phenylindole (DAPI) using flow cytometry. CPI-203 also reduced cell expansion, but importantly induced differentiation in addition to apoptosis, as supported by flow cytometric monitoring of levels of carboxyfluorescein succinimidyl ester (CFSE) and CD34. Overlays of CFSE plots for untreated control vs. CPI-203 demonstrated that as cells divided in the presence of CPI-203, there was clear and rapid loss of CD34 expression which was not seen with RITA treatment. By measuring the dose-effect relationship of each drug alone and in combination, we demonstrated potent synergy with combination index (CI) values ranging from 0.034-0.286 based on loss of cell viability. Using flow cytometry we gated on CD34+38- CML cells to enable quantification of the differential effects of each drug alone and in combination against the most primitive and quiescent 1-5% of total CD34+ cells. Critically the apoptotic effect was inclusive of primitive CD34+38- cells and quiescent CFSEmax populations. In addition, experiments combining RITA and CPI-203 demonstrated undetectable colony forming cell units at the highest concentrations of drug used. Importantly, it appears that combining these two drugs has negligible effects on normal CD34+ cell counts, apoptosis and CFSE profiles. Currently NOD-SCID IL2R gamma null (NSG) repopulation assays are underway to determine if these drugs affect stem cells capable of engrafting immunocompromised mice. Our systems biology approach suggests that altered c-myc and p53 function underlie the most significant cellular differences within CML CD34+ cells, which has not been previously demonstrated. We confirm that in CML, p53 and c-myc hub proteins have the ability to modulate downstream defined target proteins thereby enhancing survival and proliferation and thus allowing maintenance of disease. Disclosures: No relevant conflicts of interest to declare.

Quantitative proteomic analysis of uveal melanoma reveals potential therapeutic targets

AbstractPurpose To identify potential therapeutic targets in uveal melanoma by global proteomic analysis of tumours with high‐ (HR) or low‐ (LR) risk of developing metastatic disease.Methods Proteins were extracted from fresh‐frozen tissues from 10 HR and 10 LR UM and from 22 normal choroid samples, subjected to isobaric tagging for relative quantification (iTRAQ) and analysed by nanoLC‐MS/MS. Peptide identification was performed using Protein Pilot with a False Discovery Rate set at 5%. Differential expression of proteins and their relative abundance between the groups was investigated by supervised and unsupervised hierarchical clustering and outlier analyses.Results We identified 2651 unique proteins. Compared to normal choroid, the tumours showed 672 proteins up‐regulated more than 1.5‐fold and 459 down‐regulated more than 1.5‐fold. Of these, 18 proteins with known functions in tumour development/progression were identified and shown to be differentially expressed between HR and LR samples. Four of these have been further validated by immunohistochemistry in a larger cohort of patient samples.Conclusion Using quantitative proteomic analysis and immunohistochemistry we have identified novel potential therapeutic targets in UM. One of these in particular is a tumour suppressor associated with chemosensitivity in other cancers, but not yet described in UM. This opens a novel therapeutic avenue that is being further investigated.

A Strategy for Large-Scale Phosphoproteomics and SRM-Based Validation of Human Breast Cancer Tissue Samples

Protein phosphorylation is a key mechanism of cellular signaling pathways and aberrant phosphorylation has been implicated in a number of human diseases. Thus, approaches in phosphoproteomics can contribute to the identification of key biomarkers to assess disease pathogenesis and drug targets. Moreover, careful validation of large-scale phosphoproteome analysis, which is lacking in the current protein-based biomarker discovery, significantly increases the value of identified biomarkers. Here, we performed large-scale differential phosphoproteome analysis using IMAC coupled with the isobaric tag for relative quantification (iTRAQ) technique and subsequent validation by selected/multiple reaction monitoring (SRM/MRM) of human breast cancer tissues in high- and low-risk recurrence groups. We identified 8309 phosphorylation sites on 3401 proteins, of which 3766 phosphopeptides (1927 phosphoproteins) were able to be quantified and 133 phosphopeptides (117 phosphoproteins) were differentially expressed between the two groups. Among them, 19 phosphopeptides were selected for further verification and 15 were successfully quantified by SRM using stable isotope peptides as a reference. The ratio of phosphopeptides between high- and low-risk groups quantified by SRM was well correlated with iTRAQ-based quantification with a few exceptions. These results suggest that large-scale phosphoproteome quantification coupled with SRM-based validation is a powerful tool for biomarker discovery using clinical samples.

A Novel Method for the Simultaneous Enrichment, Identification, and Quantification of Phosphopeptides and Sialylated Glycopeptides Applied to a Temporal Profile of Mouse Brain Development

We describe a method that combines an optimized titanium dioxide protocol and hydrophilic interaction liquid chromatography to simultaneously enrich, identify and quantify phosphopeptides and formerly N-linked sialylated glycopeptides to monitor changes associated with cell signaling during mouse brain development. We initially applied the method to enriched membrane fractions from HeLa cells, which allowed the identification of 4468 unique phosphopeptides and 1809 formerly N-linked sialylated glycopeptides. We subsequently combined the method with isobaric tagging for relative quantification to compare changes in phosphopeptide and formerly N-linked sialylated glycopeptide abundance in the developing mouse brain. A total of 7682 unique phosphopeptide sequences and 3246 unique formerly sialylated glycopeptides were identified. Moreover 669 phosphopeptides and 300 formerly N-sialylated glycopeptides differentially regulated during mouse brain development were detected. This strategy allowed us to reveal extensive changes in post-translational modifications from postnatal mice from day 0 until maturity at day 80. The results of this study confirm the role of sialylation in organ development and provide the first extensive global view of dynamic changes between N-linked sialylation and phosphorylation.

A Systems Biology Approach to Restrictive Cardiomyopathy in Drosophila

Drosophila melanogaster possess a simple linear heart tube and constitute an excellent genetic model system with which to investigate the effects of cardiomyopathic mutation. The Mhc5 myosin heavy chain mutation is located in the ‘transducer’domain and elicits hypercontractile function at the molecular level characterized by high ATPase activity and enhanced in vitro motility properties. Additionally, its expression impairs diastolic relaxation of the cardiac tube reminiscent of restrictive cardiomyopathy in humans. We have investigated the effect of the Mhc5 mutation on cardiac structure/function by quantitative proteomics using isobaric tags for relative quantification (iTRAQ). Excised fly hearts from yw (control) and Mhc5 strains were digested with trypsin, reduced, alkylated and labeled with ITRAQ reagent. Peptides from each pool were mixed together prior to fractionation by strong cation exchange chromatography and subsequent reversed-phase HPLC coupled to tandem mass spectrometry. This approach identified approximately 600 proteins, of which 94 were upregulated and 86 were downregulated in Mhc5 hearts relative to yw hearts (p<0.05). Ontological cluster analysis of the genes encoding the regulated proteins revealed that myofibrillar disarray in Mhc5 hearts likely stems from overexpression of actin with concomitant reduction of myofibrillar assembly proteins such as spectrin, and other actin-binding proteins. Structural remodeling was also characterized by increased expression of extracellular matrix proteins. Upregulation of proteins involved in mitochondrial oxidative phosphorylation and fatty acid catabolism suggests further bioenergetic remodeling. The proteomic, structural and ultrastructural data are consistent with a model whereby the elevated ATPase activity caused by Mhc5 mutation increases energetic demand, thereby stimulating a concerted compensatory metabolic response to maintain energetic homeostasis. Ongoing protein-network/interactome analysis will help to further refine the model.

Quantitative Proteomic Analysis Implicates An Altered B-Cell Differentiation State as a Mechanism Underlying GC-Resistance in An Acute Lymphoblastic Leukaemia (ALL) Cell Line Model.

Abstract 1736Poster Board I-762 IntroductionGlucocorticoids (GC) are pivotal agents used in the treatment of childhood acute lymphoblastic leukaemia (ALL). GC-resistance is a significant prognostic indicator of a poor treatment outcome in childhood ALL, but the underlying molecular basis remains unclear. Previous studies using cell lines have identified mutation/deletion of the glucocorticoid receptor (GR) as a mechanism of GC-resistance. However, genetic aberration of the GR is rare in clinical samples1. This disparity may be due to the mismatch repair deficient status of many ALL cell lines which consequently have a greater likelihood of acquiring mutations under GC-selection. We have used a discovery proteomics approach for hypothesis generation on potential mechanisms for resistance. To achieve this, we compared a well-characterized mismatch repair proficient GC-sensitive cell line, PreB 697, and a GC-resistant sub-clone (R3F9) both bearing wildtype GR, in a comparative proteomics experiment using 4-channel isobaric tagging for relative and absolute quantitation (the iTRAQ approach). MethodsCells were treated with either vehicle control or 0.1μM dexamethasone for 24 hours and subjected to subcellular fractionation to prepare a nuclear fraction. Each sample was labelled with a distinct isobaric tag for relative quantification and analysed by 2-dimensional liquid chromatography/ tandem mass spectrometry. The proteins were identified and relatively quantified using Protein Pilot software (Applied Biosystems). Ratios were calculated for dexamethasone-treated ‘versus' control vehicle for each cell line and an ITRAQ ratio of greater than or equal to ± 1.2 or less than 0.8 fold change were considered to be differentially expressed. ResultsThe comparative dataset highlighted two transcription factors which are involved in B-cell differentiation, PAX5 and IRF4, to be differentially expressed in the PreB 697 compared to the R3F9 cell line. The GC-resistant R3F9 cell line had reduced PAX5 and IRF4 protein expression compared to the parental cell line and this was further validated in other GC-resistant sub-clones derived from the PreB 697 cell line by western blot analysis. The reduced PAX5 level in the GC-resistant cell lines was not due to monoallelic loss, as measured by a QRT-PCR method or mutation as determined by DHPLC analysis of ‘hot-spot' exons. In addition, PAX5 mRNA levels were not significantly altered, thus suggestive of a post-transcriptional mechanism for PAX5 protein reduction. To test the direct role of PAX5 in GC-resistance, we reduced PAX5 mRNA and protein levels using RNA interference in the parental GC-sensitive, PreB 697 cell line. PAX5 protein levels were reduced by at least 80% and were maintained for 48 hours post-transfection. The PreB 697 cell line was transfected with siRNA directed to PAX5 using electroporation, the cells were allowed to recover for 24 hours and the levels of cell kill were assessed in response to a 48 hour incubation with 1 μM dexamethasone by Annexin V staining and the MTS assay. Paradoxically, PAX5 knockdown increased GC-sensitivity (mean 60.4% apoptosis, S.D. 16.8, N=3) in comparison to a non-specific siRNA (mean 31.0% apoptosis, S.D. 5.2, N=3) but did not influence sensitivity to either vincristine or daunorubicin. Thus, this response was specific to glucocorticoids. ConclusionUsing a proteomic approach we have shown alterations in PAX5 protein levels are associated with a GC-resistant phenotype which an mRNA-based technology would fail to detect. Modulation of PAX5 in ALL cells may influence the response to GC-therapy. It is known that GC-sensitivity alters during B-cell development, with early lymphoid precursors being highly sensitive and more mature B cells being highly resistant to GC-induced apoptosis. We propose that reduced PAX5 protein levels may reflect an altered differentiation state of the sub-clones of PreB 697 which are associated with a GC resistant phenotype. 1Irving et al, Cancer Res, 20052Schmidt et al, FASEB, 2006 DisclosuresNo relevant conflicts of interest to declare.