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.

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.

Despite the known longevity of human hematopoietic stem and progenitor cells (HSC), numerous functional impairments of these cells can be observed in an age-dependent manner. However, the molecular alterations associated with aging of HSC are largely unknown. Therefore, we scrutinized gene expression patterns of HSC from newborn, young and old healthy donors. CD34+ HSC were isolated via immuno-magnetic separation and evaluated by FACS analysis. We performed cDNA macroarray analyses on a first set of CD34+ samples (n=13). We found the genes encoding KU-antigen 70 kD (KU70), microsomal glutathione S-transferase 1 (MGST1) and BCL2-interacting killer (BIK) to possess age-related mRNA expression levels. KU70 is a DNA repair gene and part of the DNA-dependent protein kinase (DNA-PK) complex. Its expression was negatively correlated with donor age showing highest expression levels in newborn, 2.6-fold lower levels in young and 6.3-fold lower levels in old donors. The transcription levels of MGST1, a gene protecting against oxidative stress, progressively increased with age. Expression was lowest in newborn, 2.6-fold higher in young and 4.3-fold higher in old donors. BIK is a proapoptotic gene and its expression was positively correlated with donor age: lowest in newborn, 1.8-fold higher in young and 4.1-fold higher in old donors. These findings were confirmed with an independent, second set of CD34+ samples (n=16) by means of quantitative real-time RT-PCR. Elucidation of age-dependent molecular alterations in healthy HSC facilitate a better understanding of functional impairments in hematopoiesis and may become valuable for anti-aging drug development and the emerging field of regenerative medicine.

Historically X-linked sideroblastic anemia, with rare exceptions, was thought to be manifested only in males. Since the discovery of the erythroid-specific isoform of 5-aminolevulinate synthase (ALAS2) and the cloning of its gene (ALAS2) 15 years ago, mutation analysis has revealed that clinical expression of this X-linked disorder is prevalent in females as well. However, presence of the disease in both genders within affected kindreds appears to be very uncommon. We report a unique family with the disorder in three women who have had widely disparate clinical courses. The anemia is associated with a previously unrecognized ALAS2 mutation (Arg436Trp) and is unresponsive to pyridoxine. To clarify the varied clinical courses of the patients, X-chromosome inactivation patterns were examined in hematopoietic and non-hematopoietic cells. We observed inactivation patterns supporting the conclusions that one daughter has a mild phenotype at age 31 because of moderate constitutive skewed X-chromosome inactivation, another daughter with clinical onset at age 16 is severely affected due to extreme constitutive X-skewing, whereas the mother developed progressive anemia in the fifth decade as she acquired an age-related non-random X-inactivation in hematopoietic cells. In addition, we observed random X-inactivation in reticulocytes of all three women that contrasted with a markedly skewed inactivation pattern in bone marrow erythroid cells. This discordance is attributable to apoptosis of erythroid precursors derived from progenitor cells with an active X-chromosome bearing the ALAS2 mutation. The features of the disorder in this family are also instructive in regard to the differential diagnosis of sideroblastic anemias in women.

The influence of Exisulind on the viability and apoptosis of CD34(+) stem cells from patients with advanced myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML)/MDS was investigated. In eight out of 10 patient samples Exisulind reduced the fraction of viable cells by inducing apoptosis. We found evidence that Exisulind-mediated apoptosis depends on c-Jun NH(2)-terminal kinase (JNK) activation. Addition of a specific JNK-inhibitor to Exisulind-treated advanced MDS and AML/MDS cells partly abrogated apoptosis. We propose that Exisulind is tested in clinical phase I/II trials for the treatment of advanced MDS and AML/MDS.

Treatment of patients suffering from myelodysplastic syndromes and secondary acutemyeloid leukemia after MDS is often unsuccessful. Pro-apoptosis with arsenic trioxide hasrecently been proposed as a novel therapeutic approach. Exisulind is another potentially proapoptoticagent, 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 healthyCD34+ bone marrow cells. Treatment of sAML/MDS cells with Exisulind clearly inhibitedcolony formation in the CFU-assays. Interestingly, Exisulind did not alter the percentages ofsAML/MDS cells in G1-, G2-, M- or S-phase, but reduced proliferation and inducedapoptosis 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 withExisulind. Adding a specific JNK-inhibitor to Exisulind-treated sAML/MDS cells partiallyabrogated apoptosis, thus proving that Exisulind-mediated apoptosis in sAML/MDS cells isdependent on JNK activation. We conclude that JNK is one mediator of apoptosis insAML/MDS cells treated with Exisulind. Moreover, our data strongly suggests to explore thepotential use of Exisulind as a novel, pro-apoptotic therapy for patients with MDS andsAML/MDS.

Abstract 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.

Treatment of patients suffering from myelodysplastic syndromes (MDS) is difficult and frustrating, particularly when in progression or already progressed to secondary acute myeloid leukemia (sAML). In these cases, the bone marrow contains an increasing number of immature cells, which are characterized by reduced apoptotic and increased proliferative activity. Therefore, pro-apoptosis has recently been proposed as a novel therapeutic approach. Exisulind is a potentially pro-apoptotic agent, and therefore, we investigated its influence on proliferation, differentiation, cell cycle and apoptosis in peripheral blood-derived CD34+ stem cells (PBSC) obtained from 10 patients suffering from refractory anaemia with excess of blasts in transformation (RAEB-T), two sAML/MDS cell lines, one de-novo AML cell line and healthy CD34+ bone marrow cells. Incubation 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-, S-, G2-, M-phase, but reduced proliferation and induced apoptosis in this cell type. Likewise, in 8 out of 10 RAEB-T samples Exisulind significantly inhibited proliferation and increased the rate of apoptotic cells. 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 and RAEB-T cells partly abrogated apoptosis, thus proving that Exisulind-mediated apoptosis in sAML/MDS and RAEB-T cells is dependent on JNK activation. We conclude that JNK is one mediator of apoptosis in 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 RAEB-T and sAML/MDS.

There is a higher incidence of hematological clonal stem cell disorders in elderly persons. Age-related alterations of hematopoietic stem and progenitor cells (HSC) may represent one factor underlying this observation. However, the molecular changes related to stem cell aging are largely unknown. Therefore, we scrutinized gene expression patterns of HSC from umbilical cord blood (CB) as well as bone marrow from young (BM-Y, mean age 32,8, SD 12,4) and old (BM-O, mean age 88,8, SD 4,4) healthy donors. CD34+ HSC were isolated via immuno-magnetic separation and CD34+ purity was evaluated by FACS analysis. Thereafter we performed cDNA array analyses on a first set of samples (n=18). We found that the BCL2-interacting killer gene (BIK) and the gene encoding KU Antigen 70kD (KU70) show age-related mRNA expression levels. BIK is a proapoptotic gene and its expression was positively correlated with donor’s age, i.e. lowest in CB, 1.8-fold higher in BM-Y and 4.2-fold higher in BM-O. KU70 is a DNA repair gene and part of the DNA dependent protein kinase (DNA-PK). Its expression was negatively correlated with donor’s age showing highest expression levels in CB, 2.2-fold lower levels in BM-Y and 5.3-fold lower levels in BM-O. These findings were confirmed with a second set of samples (n=16) by means of quantitative RT-PCR. Elucidation of age-dependent molecular alteration in healthy HSC might facilitate a better understanding of hematological malignancies in elderly persons.

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‐κB 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.

Surface enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF MS) has facilitated disease-specific serum protein profiles, which may become instrumental for diseases that are difficult to diagnose. The diagnosis of MDS can be very difficult since bone marrow dysplasia and peripheral cytopenia, the hallmarks of MDS, can be observed due to many reasons other than MDS. Moreover, cytomorphological evaluation of dysplasia requires extensive experience and is thus dependent on the examiner. Between 2–16% of the patients have a fibrotic bone marrow, which can impede appropriate bone marrow aspiration and smear. Finally, while cytogenetic examination can strongly support the diagnosis of MDS, only 40–50% of the patients have an abnormal karyotype. In the present study, we employed a SELDI-TOF MS-based procedure termed Pattern Track™ in order to analyze a total of 218 serum samples. We generated serum protein profiles from a first sample set comprising 74 patients with MDS, 39 control patients with cytopenia for reasons other than MDS, and 24 healthy persons. We fractionated their serum by means of anion exchange chromatography and applied the resulting serum fractions to weak cationic exchange as well as to reversed phase chromatography ProteinChip™ arrays. We randomly split this dataset into a learning (n=72) and a first independent validation set (n=41). Then, we used a k-nearest-neighbor algorithm to build a class predicting profile that consisted of 81 protein peaks. That profile was tested by leave-one-out cross validation and predicted the diagnosis MDS with an accuracy of 81.9% in the learning set (Fisher's test, P=0.0000003). Then, we tested the profile on our first independent validation set and obtained a similar accuracy of 80.5% (P=0.0002). Its diagnostic performance and long-term reproducibility were confirmed by successfully applying it to a prospectively collected second independent validation set consisting of 81 new samples (P=0.0000006). Eventually, following serial chromatography, 1D gel electrophoresis, and tryptic peptide fingerprinting, we discovered the identity of 2 members of the profile. We conclude that our predicting serum protein profile represents a novel, non-invasive aid in distinguishing patients with MDS from patients with cytopenia for reasons other than MDS.