Objective: The marked increase in the prevalence of obesity in the United States has recently been attributed to the increased fructose consumption. To determine if and how fructose might promote obesity in an animal model, we measured body composition, energy intake, energy expenditure, substrate oxidation, and several endocrine parameters related to energy homeostasis in mice consuming fructose. Research Methods and Procedures:We compared the effects of ad libitum access to fructose (15% solution in water), sucrose (10%, popular soft drink), and artificial sweetener (0% calories, popular diet soft drink) on adipogenesis and energy metabolism in mice. Results: Exposure to fructose water increased adiposity, whereas increased fat mass after consumption of soft drinks or diet soft drinks did not reach statistical significance (n ϭ 9 each group). Total intake of energy was unaltered, because mice proportionally reduced their caloric intake from chow. There was a trend toward reduced energy expenditure and increased respiratory quotient, albeit not significant, in the fructose group. Furthermore, fructose produced a hepatic lipid accumulation with a characteristic pericentral pattern. Discussion: These data are compatible with the conclusion that a high intake of fructose selectively enhances adipogenesis, possibly through a shift of substrate use to lipogenesis.

Separase, an endopeptidase required for the separation of sister-chromatides in mitotic anaphase, triggers centriole disengagement during centrosome duplication. In cancer, separase is frequently overexpressed, pointing to a functional role as an aneuploidy promoter associated with centrosomal amplification and genomic instability. Recently, we have shown that centrosomal amplification and subsequent chromosomal aberrations are a hallmark of chronic myeloid leukemia (CML), increasing from chronic phase (CP) toward blast crisis (BC). Moreover, a functional linkage of p210BCR-ABL tyrosine kinase activity with centrosomal amplification and clonal evolution has been established in long-term cell culture experiments. Unexpectedly, therapeutic doses of imatinib (IM) did not counteract; instead induced similar centrosomal alterations in vitro. We investigated the influence of IM and p210BCR-ABL on Separase as a potential driver of centrosomal amplification in CML. Short-term cell cultures of p210BCR-ABL-negative (NHDF, UROtsa, HL-60, U937), positive (K562, LAMA-84) and inducible (U937p210BCR-ABL/c6 (Tet-ON)) human cell lines were treated with therapeutic doses of IM and analyzed by qRT-PCR, Western blot analysis and quantitative Separase activity assays. Decreased Separase protein levels were observed in all cells treated with IM in a dose dependent manner. Accordingly, in all p210BCR-ABL-negative cell lines, decreased proteolytic activity of Separase was found. In contrast, p210BCR-ABL-positive cells showed increased Separase proteolytic activity. This activation of Separase was consistent with changes in the expression levels of Separase regulators (Separase phosphorylation at serine residue 1126, Securin, CyclinB1 and PP2A). Our data suggest that regulation of Separase in IM-treated BCR-ABL-positive cells occurs on both the protein expression and the proteolytic activity levels. Activation of Separase proteolytic activity exclusively in p210BCR-ABL-positive cells during IM treatment may act as a driving force for centrosomal amplification, contributing to genomic instability, clonal evolution and resistance in CML.

Background: Phenylacetic acid (PAA) is a recently described uremic toxin that inhibits inducible nitric oxide synthase expression and plasma membrane calcium ATPase and may therefore also be involved in remodeling of arteries. Such vascular effects have not been evaluated yet in patients with chronic kidney disease stage 5. Method: We prospectively measured the plasma concentrations of PAA using nuclear magnetic resonance spectroscopy in 50 patients with chronic kidney disease stage 5 (37 men, 13 women) on maintenance hemodialysis. Arterial vascular properties were quantified by the reflective index obtained from digital photoplethysmography. Results: During the hemodialysis session the plasma PAA concentration was reduced from 3.38 ± 0.24 mmol/l (mean ± SEM; median, 2.85 mmol/l; interquartile range, 2.02–4.52 mmol/l) to 2.25 ± 0.11 mmol/l (median, 2.06 mmol/l; interquartile range, 1.62–2.86 mmol/l; n = 50; p < 0.001). There was a significant correlation between the PAA concentration and the reflective index before the start of the hemodialysis session. Conclusion: The study demonstrates an association of PAA and arterial vascular properties in patients with chronic kidney disease stage 5.

ESPL1/Separase, an endopeptidase, is required for centrosome duplication and separation of sister-chromatides in anaphase of mitosis. Overexpression and deregulated proteolytic activity of Separase as frequently observed in human cancers is associated with the occurrence of supernumerary centrosomes, chromosomal missegregation and aneuploidy. Recently, we have hypothesized that increased Separase proteolytic activity in a small subpopulation of tumor cells may serve as driver of tumor heterogeneity and clonal evolution in chronic myeloid leukemia (CML). Currently, there is no quantitative assay to measure Separase activity levels in single cells. Therefore, we have designed a flow cytometry-based assay that utilizes a Cy5- and rhodamine 110 (Rh110)-biconjugated Rad21 cleavage site peptide ([Cy5-D-R-E-I-M-R]2-Rh110) as smart probe and intracellular substrate for detection of Separase enzyme activity in living cells. As measured by Cy5 fluorescence the cellular uptake of the fluorogenic peptide was fast and reached saturation after 210 min of incubation in human histiocytic lymphoma U937 cells. Separase activity was recorded as the intensity of Rh110 fluorescence released after intracellular peptide cleavage providing a linear signal gain within a 90–180 min time slot. Compared to conventional cell extract-based methods the flow cytometric assay delivers equivalent results but is more reliable, bypasses the problem of vague loading controls and unspecific proteolysis associated with whole cell extracts. Especially suited for the investigaton of blood- and bone marrow-derived hematopoietic cells the flow cytometric Separase assay allows generation of Separase activity profiles that tell about the number of Separase positive cells within a sample i.e. cells that currently progress through mitosis and about the range of intercellular variation in Separase activity levels within a cell population. The assay was used to quantify Separase proteolytic activity in leukemic cell lines and peripheral blood samples from leukemia patients.

Unbalanced (major route) additional cytogenetic aberrations (ACA) at diagnosis of chronic myeloid leukemia (CML) indicate an increased risk of progression and shorter survival. Moreover, newly arising ACA under imatinib treatment and clonal evolution are considered features of acceleration and define failure of therapy according to the European LeukemiaNet (ELN) recommendations. On the basis of 1151 Philadelphia chromosome positive chronic phase patients of the randomized CML-study IV, we examined the incidence of newly arising ACA under imatinib treatment with regard to the p210BCR-ABL breakpoint variants b2a2 and b3a2. We found a preferential acquisition of unbalanced ACA in patients with b3a2 vs. b2a2 fusion type (ratio: 6.3 vs. 1.6, p = 0.0246) concurring with a faster progress to blast crisis for b3a2 patients (p = 0.0124). ESPL1/Separase, a cysteine endopeptidase, is a key player in chromosomal segregation during mitosis. Separase overexpression and/or hyperactivity has been reported from a wide range of cancers and cause defective mitotic spindles, chromosome missegregation and aneuploidy. We investigated the influence of p210BCR-ABL breakpoint variants and imatinib treatment on expression and proteolytic activity of Separase as measured with a specific fluorogenic assay on CML cell lines (b2a2: KCL-22, BV-173; b3a2: K562, LAMA-84). Despite a drop in Separase protein levels an up to 5.4-fold increase of Separase activity under imatinib treatment was observed exclusively in b3a2 but not in b2a2 cell lines. Mimicking the influence of imatinib on BV-173 and LAMA-84 cells by ESPL1 silencing stimulated Separase proteolytic activity in both b3a2 and b2a2 cell lines. Our data suggest the existence of a fusion type-related feedback mechanism that posttranslationally stimulates Separase proteolytic activity after therapy-induced decreases in Separase protein levels. This could render b3a2 CML cells more prone to aneuploidy and clonal evolution than b2a2 progenitors and may therefore explain the cytogenetic results of CML patients.

Centrosomes play important roles in the maintenance of genetic stability and centrosomal aberrations are a hallmark of cancer. Deregulation of centriole duplication leads to supernumerary centrosomes, sister chromatid missegregation and could result in chromosomal instability (CIN) and aneuploidy. CIN is a common feature in at least 45% of patients with myelodysplastic syndromes (MDS). Therefore, we sought to investigate the centrosomal status and its role for development of CIN in bone marrow (BM) cells of MDS patients. BM cells of 34 MDS patients were examined cytogenetically. Furthermore, cells were immunostained with a centrosome-specific antibody to pericentrin to analyze the centrosomal status. Umbilical cord blood specimens and BM cells of healthy persons (n = 11 and n = 4) served as controls. In addition, the protein expression of the protease separase responsible for genetic stability was examined by western blot analysis. Centrosome abnormalities were detected in 10% (range, 4-17%) of cells of MDS samples, but in only 2% (range, 0-4%) of cells of healthy controls. Normal karyotypes were found in control cells and in BM cells of 16/34 MDS patients. The incidence of centrosomal alterations was higher in BM cells of patients with cytogenetic alterations (mean, 12%) compared to BM cells of patients without cytogenetic changes (mean, 7%). Our results indicate that centrosome alterations are a common and early detectable feature in MDS patients and may contribute to the acquisition of chromosomal aberrations. We assume that centrosome defects could be involved in disease progression and may serve as a future prognostic marker.

No abstract

3839 Introduction: Centrosomes play important roles in maintenance of genetic stability and centrosomal aberrations are a common hallmark of cancer. Deregulation of centriole duplication during the cell cycle leads to supernumerary centrosomes, sister chromatide missegregation and could result in chromosomal instability (CIN) and aneuploidy. CIN is a common feature in at least 50% of patients with myelodysplastic syndromes (MDS). Therefore, we sought to investigate the centrosomal status and its role for development of CIN in bone marrow (BM) cells of MDS patients. Furthermore, deregulation of the protease Separase is known as a driver of aneuploidy. It is considered as one of the master key players in centriole duplication and overexpression has been associated with the formation of supernumerary centrosomes in many cancers. Therefore, deregulated Separase could also serve as a marker for genetic instability and was investigated. Patients and methods: BM cells of 34 MDS patients were cytogenetically examined by G-banding technique. Furthermore, cells were immunostained with a centrosome-specific antibody to pericentrin followed by a Cy3-conjugated secondary antibody to analyze the centrosomal status. Umbilical cord blood specimens (CB; n=15) served as controls. In addition, Separase protein levels were analyzed in BM cells of four MDS patients and in CB cells of four healthy controls. Results: BM cells of all MDS patients displayed centrosome alterations as compared with corresponding controls. Centrosome abnormalities were detected in 10% (range, 4–17%) of analyzed cells of MDS patients but in only 2% (range, 0–4%) of cells of healthy donors (p≤0.0001). Normal karyotypes were found in all CB metaphases and in BM metaphases of 16/34 MDS patients. The incidence of centrosomal alterations was higher in bone marrow cells of patients with cytogenetic alterations (mean, 12%) compared to BM cells of patients without cytogenetic changes (mean, 7%). In BM cells of MDS patients Separase protein levels were lower (60% decrease) as compared to CB cells of the healthy control (p≤0.01). Conclusions: We could show that centrosome aberrations in BM cells of MDS patients occur before chromosomal changes are detectable. Therefore, centrosomal instability is an early step in MDS and may contribute to the acquisition of chromosomal alterations. Increase of aberrant centrosomes significantly correlates with karyotype instability and aneuploidy. It seems that centrosomal instability precedes karyotype instability via chromosomal missegregation and could contribute to the development of chromosomal changes and accelerate malignant transformation. Separase is one of the master key players in centriole duplication and chromatide segregation. Therefore, deregulated Separase could lead to genetic instability and malignant transformation. In future studies centrosomal alterations may serve as an additional prognostic biomarker for future diagnostics in MDS. Disclosures: No relevant conflicts of interest to declare.