Differential gene expression of bone marrow-derived CD34+ cells is associated with survival of patients suffering from myelodysplastic syndrome.

One feature of the molecular pathology of myelodysplastic syndromes (MDS) is aberrant gene expression. Such aberrations may be related to patient survival, and may indicate to novel diagnostic and therapeutic targets. Therefore, we aimed at identifying aberrant gene expression that is associated with MDS and patient survival. Bone marrow-derived CD34+ hematopoietic progenitor cells from six healthy persons and 16 patients with MDS were analyzed on cDNA macroarrays comprising 1,185 genes. Thereafter, our patients were followed-up for 54 months. We found differential expression of genes that were hitherto unrecognized in the context of MDS. Differential expression of 10 genes was confirmed by quantitative real-time RT-PCR. Hierarchical cluster analysis facilitated the separation of CD34+ cells of normal donors from patients with MDS. More importantly, it also distinguished MDS-patients with short and long survival. Scrutinizing our cDNA macroarray data for genes that are associated with short survival, we found, among others, increased expression of six different genes that encode the proteasome subunits. On the other hand, the most differentially down-regulated gene was IEX-1, which encodes an anti-apoptotic protein. We confirmed its decreased expression on RNA and protein level in an independent validation set of patient samples. The presented data broadens our notion about the molecular pathology of MDS and may lend itself to better identify patients with short survival. Furthermore, our findings may help to define new molecular targets for drug development and therapeutic approaches for patients with poor prognosis.

Reproducibility of proteomic profiles over 3 years in postmenopausal women not taking postmenopausal hormones.

Most proteomics studies examine one blood specimen per participant; however, it is unknown how well measures at one time point reflect an individual's long-term proteome pattern. Therefore, we examined the stability of the proteome over 3 years in postmenopausal women not taking hormones for at least 3 months using surface-enhanced laser desorption and ionization time-of-flight mass spectrometry. Using the Nurses' Health Study blood cohort, we randomly selected 60 women from a subset providing 2 to 3 blood samples over 3 years. Four different protein chip surfaces/plasma fractions were examined: unfractionated plasma on a CM10 and H50 chip, pH >/= 9, plasma fraction on a CM10 chip, and the organic fraction on the H50 chip, all with a low- and high-energy transfer protocol. Participant and quality control samples were aligned to a reference sample and then peak intensity was assessed for all peaks identified in the reference sample. The average coefficient of variation (CV) of the peak intensity within conditions ranged from 16% (H50, organic, low protocol) to 63% (CM10, pH > or = 9, high protocol). Generally, the CV and mean peak intensity of the quality control samples were inversely correlated (median -0.48). The mean intraclass correlation (ICC) within conditions ranged from 0.37 (H50, unfractionated, low protocol) to 0.68 (CM10, unfractionated, high protocol). For a signal-to-noise cutoff of 2.0, we observed 334 peaks, of which 241 (72%) had an ICC of > or =0.40. Although we observed a large range of CVs and ICCs, sufficient numbers of peaks had reasonable ICCs to suggest that protein peak reproducibility over 3 years was reasonable among postmenopausal women not taking hormones.

Serum proteomics and biomarkers in hepatocellular carcinoma and chronic liver disease.

PURPOSE: Proteomic profiling using surface enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF MS) enables the identification of biomarkers for cancer. We evaluated the sensitivity and specificity of SELDI-TOF MS for detection of established hepatocellular cancer (HCC) and compared it against alpha-fetoprotein (AFP), Lens culinaris agglutinin-reactive AFP (AFP-L3), and prothrombin induced by vitamin K absence-II (PIVKA-II). EXPERIMENTAL DESIGN: Forty-one patients with HCC and 51 patients with hepatitis C cirrhosis were enrolled. Serum was analyzed by SELDI-TOF MS using three Ciphergen protein array types. RESULTS: An 11-peak algorithm for HCC detection was identified. Using the AFP cutoff of 20 ng/mL, the sensitivity was 73% and the specificity was 71%. Using the AFP-L3 cutoff of 10% yielded a sensitivity of 63% and a specificity of 94%. Using the PIVKA-II cutoff of 125 milliabsorbance units (mAU), the sensitivity was 84% and the specificity was 69%. Overall, the sensitivity and specificity of SELDI-TOF MS for HCC were 79% and 86%, respectively. In multivariate analysis, the 11-peak SELDI profile was predictive of HCC independent of AFP, PIVKA, and AFP-L3. Among eight patients with the largest tumor size of <2 cm, SELDI-TOF MS correctly identified seven whereas AFP, AFP-L3, and PIVKA-II identified only three, one, and one, respectively. One of the 11 peaks in the SELDI-TOF MS 11-peak predictor from SELDI-TOF MS was identified as cystatin C. CONCLUSIONS: SELDI-TOF MS accurately distinguished patients with HCC from those with hepatitis C virus cirrhosis, was more accurate than traditional biomarkers in identifying small tumors, and should be further evaluated.

Proteomic identification of interleukin-2 therapy response in metastatic renal cell cancer.

PURPOSE: To detect a predictive protein profile that distinguishes interleukin-2 therapy responders and nonresponders among patients with metastatic renal cell carcinoma we used surface-enhanced laser desorption/ionization time-of-flight mass spectrometry. MATERIALS AND METHODS: Protein extracts from 56 patients with metastatic clear cell patients renal cell carcinoma obtained from radical nephrectomy specimens before interleukin-2 therapy were applied to protein chip arrays of different chromatographic properties and analyzed using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry. A class prediction algorithm was applied to identify a subset of protein peaks with expression values associated with interleukin-2 response status. Multivariate analysis was performed to assess the association between the proteomic profile and interleukin-2 response status, controlling for the effect of lymphadenopathy. RESULTS: From 513 protein peaks we discovered a predictor set of 11 that performed optimally for predicting interleukin-2 response status with 86% accuracy (Fisher's p <0.004, permutation p <0.01). Results were validated in an independent data set with 72% overall accuracy (p <0.05, permutation p <0.01). On multivariate analysis the proteomic profile was significantly associated with the interleukin-2 response when corrected for lymph node status (p <0.04). CONCLUSIONS: We identified and validated a proteomic pattern that is an independent predictor of the interleukin-2 response. The ability to predict the probability of the interleukin-2 response could permit targeted selection of the patients most likely to respond to interleukin-2, while avoiding unwanted toxicity in patients less likely to respond. This proteomic predictor has the potential to significantly aid clinicians in the decision making of appropriate therapy for patients with metastatic renal cell carcinoma.

ESE-1 is a potent repressor of type II collagen gene (COL2A1) transcription in human chondrocytes.

The epithelium-specific ETS (ESE)-1 transcription factor is induced in chondrocytes by interleukin-1beta (IL-1beta). We reported previously that early activation of EGR-1 by IL-1beta results in suppression of the proximal COL2A1 promoter activity by displacement of Sp1 from GC boxes. Here we report that ESE-1 is a potent transcriptional suppressor of COL2A1 promoter activity in chondrocytes and accounts for the sustained, NF-kappaB-dependent inhibition by IL-1beta. Of the ETS factors tested, this response was specific to ESE-1, since ESE-3, which was also induced by IL-1beta, suppressed COL2A1 promoter activity only weakly. In contrast, overexpression of ETS-1 increased COL2A1 promoter activity and blocked the inhibition by IL-1beta. These responses to ESE-1 and ETS-1 were confirmed using siRNA-ESE1 and siRNA-ETS1. In transient cotransfections, the inhibitory responses to ESE-1 and IL-1beta colocalized in the -577/-132 bp promoter region, ESE-1 bound specifically to tandem ETS sites at -403/-381 bp, and IL-1-induced binding of ESE-1 to the COL2A1 promoter was confirmed in vivo by ChIP. Our results indicate that ESE-1 serves a potent repressor function by interacting with at least two sites in the COL2A1 promoter. However, the endogenous response may depend upon the balance of other ETS factors such as ETS-1, and other IL-1-induced factors, including EGR-1 at any given time. Intracellular ESE-1 staining in chondrocytes in cartilage from patients with osteoarthritis (OA), but not in normal cartilage, further suggests a fundamental role for ESE-1 in cartilage degeneration and suppression of repair.

IGF axis gene expression patterns are prognostic of survival in epithelial ovarian cancer.

The IGF axis has documented growth-promoting effects in various malignancies, but its role in epithelial ovarian cancer (EOC) has not been adequately examined. We studied the expression of the IGF axis genes in relation to outcome in EOC. Microarray expression profiles from 64 patients with advanced-stage EOC were used. Two multi-gene subsets were chosen, one upstream of the IGF receptor ('IGF family') and the other downstream of the IGF receptor ('IGF signaling pathway'), and analyzed in relation to survival. In addition, expression patterns of the two gene subsets were analyzed in relation to favorable and unfavorable prognosis categories identified in a previous study by whole-genome expression profiling. In a gene-by-gene analysis, IGF binding protein 4 and IGF-II receptor gene expression was inversely associated with survival. Using hierarchical clustering, the two multi-gene subsets separated the patient cohort into two groups with different median survival (IGF family: 33 vs 63 months, P=0.02 and IGF signaling pathway: 41 vs 63 months, P=0.05). Furthermore, the two multi-gene subsets were differentially expressed between the previously defined favorable and unfavorable prognosis tumors (Kolmogorov-Smirnov permutation: P=0.0005 and 0.003 for the IGF family and signaling pathway respectively), and individual genes (including IGF-I, IGF-I receptor, and several genes downstream of the receptor) were overexpressed in unfavorable prognosis tumors (permutation P<0.05). The expression patterns of several genes in the IGF axis are associated with survival in EOC, and expression changes of these genes may be underlying previously proposed microarray-derived clinical prognostic models. Future studies are needed to more precisely determine the diagnostic and potential therapeutic significance of these findings.

Reduced PDEF expression increases invasion and expression of mesenchymal genes in prostate cancer cells.

The epithelium-specific Ets transcription factor, PDEF, plays a role in prostate and breast cancer, although its precise function has not been established. In prostate cancer, PDEF is involved in regulating prostate-specific antigen expression via interaction with the androgen receptor and NKX3.1, and down-regulation of PDEF by antiproliferative agents has been associated with reduced PDEF expression. We now report that reduced expression of PDEF leads to a morphologic change, increased migration and invasiveness in prostate cancer cells, reminiscent of transforming growth factor beta (TGFbeta) function and epithelial-to-mesenchymal transition. Indeed, inhibition of PDEF expression triggers a transcriptional program of genes involved in the TGFbeta pathway, migration, invasion, adhesion, and epithelial dedifferentiation. Our results establish PDEF as a critical regulator of genes involved in cell motility, invasion, and adhesion of prostate cancer cells.

Serum proteome profiling detects myelodysplastic syndromes and identifies CXC chemokine ligands 4 and 7 as markers for advanced disease.

Myelodysplastic syndromes (MDS) are among the most frequent hematologic malignancies. Patients have a short survival and often progress to acute myeloid leukemia. The diagnosis of MDS can be difficult; there is a paucity of molecular markers, and the pathophysiology is largely unknown. Therefore, we conducted a multicenter study investigating whether serum proteome profiling may serve as a noninvasive platform to discover novel molecular markers for MDS. We generated serum proteome profiles from 218 individuals by MS and identified a profile that distinguishes MDS from non-MDS cytopenias in a learning sample set. This profile was validated by testing its ability to predict MDS in a first independent validation set and a second, prospectively collected, independent validation set run 5 months apart. Accuracy was 80.5% in the first and 79.0% in the second validation set. Peptide mass fingerprinting and quadrupole TOF MS identified two differential proteins: CXC chemokine ligands 4 (CXCL4) and 7 (CXCL7), both of which had significantly decreased serum levels in MDS, as confirmed with independent antibody assays. Western blot analyses of platelet lysates for these two platelet-derived molecules revealed a lack of CXCL4 and CXCL7 in MDS. Subtype analyses revealed that these two proteins have decreased serum levels in advanced MDS, suggesting the possibility of a concerted disturbance of transcription or translation of these chemokines in advanced MDS.

Perspectives of proteomics in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a frequent hematological malignancy. Despite enormous therapeutic efforts that range from various cytotoxic agents to allogeneic stem cell transplantation, overall survival of patients with AML remains unsatisfying. The poor survival rates are mainly due to therapy-related mortality, failure of induction chemotherapy and early relapses. Therefore, novel therapeutic agents that are more efficient and better tolerated are eagerly sought after. For existing therapeutic strategies, there is a lack of markers that are capable of reliably predicting prognosis or the therapeutic response prior to treatment. There is hope that elucidation of the AML-specific proteome will prompt the discovery of novel therapeutic targets and biomarkers in AML. Modern mass-spectrometry instrumentation has achieved excellent performance in terms of sensitivity, resolution and mass accuracy; however, so far, the contribution of proteomics to the care of patients with AML is virtually zero. This might be partly because mass spectrometry instrumentation and protein fractionation still lack true high-throughput capabilities with highest levels of reproducibility, thus hampering large-scale translational studies with clinical samples. Since mass-spectrometry instruments are very intricate devices, their successful operation will hinge on the willingness and ability of mass-spectrometry experts and clinical researchers to adopt new views, learn from each other and cooperate in order to ultimately benefit the patient suffering from AML. This review highlights some clinical problems circumventing the treatment of patients with AML. Furthermore, it provides a brief overview of the technical background of standard proteomics approaches and describes opportunities, challenges and pitfalls of proteomic studies with regards to AML.

Urine proteomic profiling of pediatric nephrotic syndrome.

The prognosis of pediatric nephrotic syndrome (NS) correlates with the responsiveness to glucocorticoid therapy. Steroid-resistant NS (SRNS) patients progress to end-stage renal disease, while steroid-sensitive NS (SSNS) and steroid-dependent (SDNS) patients do not. We have performed proteomic profiling of urine samples from a cross section of pediatric and adolescent subjects with SSNS, SRNS, and orthostatic proteinuria (OP) to identify urinary biomarkers of steroid resistance. We performed surface-enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF MS) on urine from 19 subjects with SSNS/SDNS in remission, 14 with SSNS/SDNS in relapse, 5 with SRNS in relapse, and 6 with OP. Genetic algorithm search of principal component space revealed a group of five peaks distinguishing SRNS subjects, with mass/charge (m/z) values of 3,917.07, 4,155.53, 6,329.68, 7,036.96, and 11,117.4. Our analyses identified the peak at m/z 11,117.4 with an accuracy of 95% for classifying SRNS. Multidimensional protein fractionation and mass spectrometric analysis of SRNS urine samples combined with immunodepletion identified the 11,117.4 protein as beta2-microglobulin (B2M). Using an unbiased protein profiling approach, we have validated previously reported findings of B2M as a biomarker associated with SRNS. Prospective studies are warranted to establish additional biomarkers that would be predictive of SRNS.

The nonsteroidal anti-inflammatory drug Exisulind selectively induces apoptosis via JNK in secondary acute myeloid leukemia after myelodysplastic syndrome.

Treatment of patients suffering from myelodysplastic syndromes and secondary acute myeloid leukemia after MDS is often unsuccessful. Pro-apoptosis with arsenic trioxide has recently been proposed as a novel therapeutic approach. Exisulind is another potentially pro-apoptotic agent, and therefore, we investigated its influence on proliferation, differentiation, cell cycle and apoptosis in two sAML/MDS cell lines, one de-novo AML cell line and healthy CD34+ bone marrow cells. Treatment of sAML/MDS cells with Exisulind clearly inhibited colony formation in the CFU-assays. Interestingly, Exisulind did not alter the percentages of sAML/MDS cells in G1-, G2-, M- or S-phase, but reduced proliferation and induced apoptosis in this cell type. Exisulind had no effect on de-novo AML or normal CD34+ cells. We detected increased c-Jun NH2-terminal kinase activity in sAML/MDS cells treated with Exisulind. Adding a specific JNK-inhibitor to Exisulind-treated sAML/MDS cells partly abrogated apoptosis, thus proving that Exisulind-mediated apoptosis in sAML/MDS cells is dependent on JNK activation. We conclude that JNK is one mediator of apoptosis in sAML/MDS cells treated with Exisulind. Moreover, our data strongly suggests to explore the potential use of Exisulind as a novel, pro-apoptotic therapy for patients with MDS and sAML/MDS.

Optimization and evaluation of surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) with reversed-phase protein arrays for protein profiling.

Surface-enhanced laser desorption/ionization (SELDI) time-of-flight mass spectrometry with protein arrays has facilitated the discovery of disease-specific protein profiles in serum. Such results raise hopes that protein profiles may become a powerful diagnostic tool. To this end, reliable and reproducible protein profiles need to be generated from many samples, accurate mass peak heights are necessary, and the experimental variation of the profiles must be known. We adapted the entire processing of protein arrays to a robotics system, thus improving the intra-assay coefficients of variation (CVs) from 45.1% to 27.8% (p<0.001). In addition, we assessed up to 16 technical replicates, and demonstrated that analysis of 2-4 replicates significantly increases the reliability of the protein profiles. A recent report on limited long-term reproducibility seemed to concord with our initial inter-assay CVs, which varied widely and reached up to 56.7%. However, we discovered that the inter-assay CV is strongly dependent on the drying time before application of the matrix molecule. Therefore, we devised a standardized drying process and demonstrated that our optimized SELDI procedure generates reliable and long-term reproducible protein profiles with CVs ranging from 25.7% to 32.6%, depending on the signal-to-noise ratio threshold used.

The Ets transcription factor ESE-1 mediates induction of the COX-2 gene by LPS in monocytes.

Cyclooxygenase-2 (COX-2) is a key enzyme in the production of prostaglandins that are major inflammatory agents. COX-2 production is triggered by exposure to various cytokines and to bacterial endotoxins. We present here a novel role for the Ets transcription factor ESE-1 in regulating the COX-2 gene in response to endotoxin and other pro-inflammatory stimuli. We report that the induction of COX-2 expression by lipopolysaccharide (LPS) and pro-inflammatory cytokines correlates with ESE-1 induction in monocyte/macrophages. ESE-1, in turn, binds to several E26 transformation specific (Ets) sites on the COX-2 promoter. In vitro analysis demonstrates that ESE-1 binds to and activates the COX-2 promoter to levels comparable to LPS-mediated induction. Moreover, we provide results showing that the induction of COX-2 by LPS may require ESE-1, as the mutation of the Ets sites in the COX-2 promoter or overexpression of a dominant-negative form of ESE-1 inhibits LPS-mediated COX-2 induction. The effect of ESE-1 on the COX-2 promoter is further enhanced by cooperation with other transcription factors such as nuclear factor-kappa B and nuclear factor of activated T cells. Neutralization of COX-2 is the goal of many anti-inflammatory drugs. As an activator of COX-2 induction, ESE-1 may become a target for such therapeutics as well. Together with our previous reports of the role of ESE-1 as an inducer of nitric oxide synthase in endothelial cells and as a mediator of pro-inflammatory cytokines in vascular and connective tissue cells, these results establish ESE-1 as an important player in the regulation of inflammation.

NF-kappa B-mediated repression of growth arrest- and DNA-damage-inducible proteins 45alpha and gamma is essential for cancer cell survival.

The NF-kappaB/IkappaB signaling pathway is a critical regulator of cell survival in cancer. Here, we report that combined down-regulation of growth arrest- and DNA-damage-inducible proteins (GADD)45alpha and gamma expression by NF-kappaB is an essential step for various cancer types to escape programmed cell death. We demonstrate that inhibition of NF-kappaB in cancer cells results in GADD45alpha- and gamma-dependent induction of apoptosis and inhibition of tumor growth. Inhibition of GADD45alpha and gamma in cancer cells by small interfering RNA abrogates apoptosis induction by the inhibitor of NF-kappaB and blocks c-Jun N-terminal kinase activation, whereas overexpression of GADD45alpha and gamma activates c-Jun N-terminal kinase and induces apoptosis. These results establish an unambiguous role for the GADD45 family as an essential mediator of cell survival in cancer cells with implications for cancer chemotherapy and novel drug discovery.

Responses to the proinflammatory cytokines interleukin-1 and tumor necrosis factor alpha in cells derived from rheumatoid synovium and other joint tissues involve nuclear factor kappaB-mediated induction of the Ets transcription factor ESE-1.

OBJECTIVE: To investigate the expression of the novel Ets transcription factor ESE-1 in rheumatoid synovium and in cells derived from joint tissues, and to analyze the role of nuclear factor kappaB (NF-kappaB) as one of the central downstream targets in mediating the induction of ESE-1 by proinflammatory cytokines. METHODS: ESE-1 protein expression was analyzed by immunohistochemistry using antibodies in synovial tissues from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). ESE-1 messenger RNA (mRNA) levels were analyzed by reverse transcriptase-polymerase chain reaction or Northern blotting in human chondrocytes, synovial fibroblasts, osteoblasts, and macrophages, before and after exposure to interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNFalpha), or lipopolysaccharide (LPS) with or without prior infection with an adenovirus encoding the inhibitor of nuclear factor kappaB (IkappaB). The wild-type ESE-1 promoter and the ESE-1 promoter mutated in the NF-kappaB site were cloned into a luciferase reporter vector and analyzed in transient transfections. Electrophoretic mobility shift assays (EMSAs) and supershift assays with antibodies against members of the NF-kappaB family were conducted using the NF-kappaB site from the ESE-1 promoter as a probe. RESULTS: Immunohistochemical analysis showed specific expression of ESE-1 in cells of the synovial lining layer and in some mononuclear and endothelial cells in RA and OA synovial tissues. ESE-1 mRNA expression could be induced by IL-1beta and TNFalpha in cells such as synovial fibroblasts, chondrocytes, osteoblasts, and monocytes. Transient transfection experiments and EMSAs showed that induction of ESE-1 gene expression by IL-1beta requires activation of NF-kappaB and binding of p50 and p65 family members to the NF-kappaB site in the ESE-1 promoter. Overexpression of IkappaB using an adenoviral vector blocked IL-1beta-induced ESE-1 mRNA expression. Chromatin immunoprecipitation further confirmed that NF-kappaB binds to the ESE-1 promoter in vivo. CONCLUSION: ESE-1 is expressed in synovial tissues in RA and, to a variable extent, in OA, and is specifically induced in synovial fibroblasts, chondrocytes, osteoblasts, and monocyte/macrophages by IL-1beta, TNFalpha, or LPS. This induction relies on the translocation of the NF-kappaB family members p50 and p65 to the nucleus and transactivation of the ESE-1 promoter via a high-affinity NF-kappaB binding site. ESE-1 may play a role in mediating some effects of proinflammatory stimuli in cells at sites of inflammation.

ESE-1, an enterocyte-specific Ets transcription factor, regulates MIP-3alpha gene expression in Caco-2 human colonic epithelial cells.

We have previously shown that colonic epithelial cells are a major site of MIP-3alpha production in human colon and that enterocyte MIP-3alpha protein levels are elevated in inflammatory bowel disease. The aim of this study was to determine the molecular mechanisms regulating MIP-3alpha gene transcription in Caco-2 intestinal epithelial cells. We show that a kappaB element at nucleotides -82 to -93 of the MIP-3alpha promoter binds p50/p65 NF-kappaB heterodimers and is a major regulator of basal and interleukin-1beta (IL-1beta)-mediated gene activation. Scanning mutagenesis of the MIP-3alpha 5'-flanking region also identified two additional binding elements: Site X (nucleotides -63 to -69) and Site Y (nucleotides -143 to -154). Site X (CGCCTTC) bound Sp1 and regulated basal MIP-3alpha gene transcription. Overexpression of Sp1 increased basal luciferase activity, whereas, substitutions in the Sp1 element significantly reduced reporter activity. In contrast, Site Y (AAGCAGGAAGTT) regulated both basal and cytokine-induced gene activation and bound the Ets nuclear factor ESE-1. Substitutions in the Site Y element markedly reduced inducible MIP-3alpha reporter activity. Conversely, overexpression of ESE-1 significantly up-regulated MIP-3alpha luciferase levels. Taken together, our findings demonstrate that co-ordinate activation and binding of ESE-1, Sp1, and NF-kappaB to the MIP-3alpha promoter is required for maximal gene expression by cytokine-stimulated Caco-2 human intestinal epithelial cells.