Studies of IGF-I and Klotho Protein in Relation to Anabolic-Androgenic Steroid and Growth Hormone Administrations.

It has been suggested to longitudinally monitor Insulin-like growth factor I (IGF-I) as a biomarker for the detection of recombinant growth hormone (GH). Subsequently, it is of interest to understand any confounders of endogenous IGF-I. Herein we have studied if serum IGF-I concentration is affected by the intake of anabolic androgenic steroids (AAS) and the potential connection between IGF-I and klotho protein. Moreover, the usefulness of klotho as a biomarker for recombinant GH intake was assessed in healthy male volunteers. An ongoing administration of AAS did not affect the levels of IGF-I. Klotho protein was ~30% higher in men with an ongoing AAS use compared to those with previous (>2 months ago) AAS use, and the serum klotho protein correlated negatively with luteinizing hormone (LH) (r s = -0.38, p = 0.04) and follicle stimulating hormone (FSH) (r s = -0.35, p = 0.05) levels. Serum IGF-I and klotho concentrations showed no correlation in the AAS using population but showed a strong negative correlation in healthy volunteers (r s = -0.86, p = 0.006). The intake of recombinant GH did not affect the serum concentrations of the klotho levels. In conclusion, IGF-I was not affected by supra-physiological AAS doses in men. Interestingly, an association between AAS intake and serum klotho was seen. The usefulness of klotho as an androgen biomarker warrants further studies, whereas klotho can be discarded as a promising biomarker for GH doping.

The intra-individual stability of GH biomarkers IGF-I and P-III-NP in relation to GHRH administration, menstrual cycle, and hematological parameters.

The intra-individual stability of growth hormone (GH) biomarkers IGF-I, P-III-NP, calculated GH-2000 score in relation to growth hormone-releasing hormone (GHRH) (Somatorelin) administration, menstrual cycle, and hematological parameters were investigated in four men and eight women, respectively. Moreover, the hematological parameters hemoglobin (Hb) and percentage of reticulocyte (RET%) were statistically analyzed in relation to the GH biomarker parameters for the GHRH administration study and the menstrual cycle study. Longitudinal monitoring of IGF-I and/or GH-2000 score proved to be a viable approach to detect the GHRH intake in men, as all four participants show values above individually calculated thresholds (calculated as mean ± 3SD from three baseline samples). The intra-individual variation for IGF-I, P-III-NP, and calculated GH-2000 score in women, over two consecutive menstrual cycles, was investigated and established to be higher (coefficients variations [CVs] between 12% and 186%) than in men (CVs between 3% and 12%). The GHRH administration did not influence the hematological parameters. A strong positive correlation between Hb and IGF-I (Rs = 0.73, p < 0.0001) and a borderline weak correlation between RET% and IGF-I (Rs = 0.28, p = 0.054) were noticed in the women. No correlation for the P-III-NP and the hematological parameters was seen for the females in the menstrual cycle study. The results fortify previous studies that longitudinal monitoring of IGF-I and/or GH-2000 score may be a promising method to detect doping with GH and GH stimulating agents in men, whereas the large intra-individual variation noted in women indicates that longitudinal monitoring of these biomarker may be harder to evaluate in women.

Longitudinal studies of putative growth hormone (GH) biomarkers and hematological and steroidal parameters in relation to 2 weeks administration of human recombinant GH.

The detection of low doses of recombinant growth hormone is a challenge in antidoping testing. Future testing may lead toward the longitudinal monitoring of IGF-I and P-III-NP in an endocrine module. Additional biomarkers, for example vitamin D binding protein, alpha-HS-glycoprotein, fibronectin 1, and decorin have been identified in different omics studies. This was a longitudinal study of the usefulness of these putative biomarkers in relation to 2 weeks administration of low doses of recombinant growth hormone in healthy male volunteers. Moreover, the hematological parameters included in the athlete biological passport were studied as well as the serum concentration of testosterone and dihydrotestosterone. Fibronectin 1 increased by 20% during the treatment period (P ˂ 0.05), confirming the previous finding. Alpha-HS-glycoprotein decreased by 25% up to 3 weeks after treatment (P ˂ 0.05), contradicting previous results. The addition of fibronectin 1 increased the likelihood of detecting recombinant growth hormone intake based on individual calculated thresholds in some of the participants compared with the GH2000, IGF-I, and P-III-NP. The multiplication of fibronectin 1 concentration by IGF-I resulted in the most profound (up to 4-fold) changes. A minor 15% increase (P = 0.003) in the reticulocyte percentage was observed, but the changes did not lead to any atypical profile based on individual passport thresholds. Vitamin D binding protein, decorin, testosterone, and dihydrotestosterone were not affected by growth hormone. Dihydrotestosterone sulfate was negatively correlated with IGF-I at baseline (R = -0.50, P = 0.003) and post dose (R = -0.59, P = 0.01). In conclusion, fibronectin 1 was verified as a promising future biomarker for detecting low doses of recombinant growth hormone.

A simple method to immunopurify erthyropoiesis stimulating agents from urine, aiming to optimize erythropoietin screening by SAR-PAGE.

The efficiency of the immunopurification step of urinary erythropoietin (EPO) and recombinant forms is important for their optimal detection in antidoping screening. Previous investigations of immunopurification techniques have been done for immunomagnetic beads, EPO Purification Kit (EPK) columns (MAIIA Diagnostics), and enzyme-linked immunosorbent assay (ELISA) microplates (Stemcell Technologies) conjugated/coated with anti-EPO antibodies. In this study, a new immunopurification technique using anti-EPO sepharose gel beads, developed by MAIIA Diagnostics, to simplify and minimize sample handling was evaluated. This EPO Purification Gel Kit (EPGK) was compared with our current routine EPK for limit of detection (LOD). Linearity, recovery, repeatability, sample incubation time, and sample volume were also evaluated for EPGK. The LODs and linearity for EPK and EPGK were comparable to each other and the recovery for BRP, NESP, CERA, and EPO-Fc were within the range of other studies, and concentration of the sample eluate improved the recovery results. Little variation was seen within days, between days, and between operators. A 90 minute incubation of the sample with the sepharose gel beads is sufficient for most of the erythropoiesis stimulating agents (ESAs) tested, with 10 mL being an optimal sample volume for EPGK. The improved sample handling, higher sample throughput and the reduced working time demonstrate that the EPGK is a better alternative to the current MAIIA EPK immunopurification method for urine. The EPO Purification Gel Kit (from MAIIA Diagnostics) was evaluated and validated for immunopurification of endogenous erythropoietin and exogenous erythropoiesis stimulating agents from urine samples. The kit was a better alternative to that currently used (EPO Purification Kit) in many antidoping laboratories because it improves sample handling and increases sample throughput.

Longitudinally monitoring of P-III-NP, IGF-I, and GH-2000 score increases the probability of detecting two weeks' administration of low-dose recombinant growth hormone compared to GH-2000 decision limit and GH isoform test and micro RNA markers.

To detect doping with growth hormone (GH), GH isoform and biomarkers tests are available. Both methods use population-based decision limits. Future testing in anti-doping is progressing toward individual-based reference ranges, and it is possible that with such an approach the sensitivity to detect GH doping may increase. In addition to monitoring different proteins, the use of miRNAs as future GH biomarkers has been discussed. Here we have longitudinally studied the serum concentrations of IGF-I, P-III-NP and the different GH isoforms in nine healthy men prior to, during and after two weeks' administration with low doses (1 and 4 IU/day) of recGH. Moreover, three putative miRNAs were analyzed. The results show that 80% of the participants were identified as atypical findings using the GH isoform test. However, the participants were only positive 1.5-3 hours directly after an injection. Only one of the participants reached a GH-2000 score indicative of doping when a population-based decision limit was applied. When IGF-I and P-III-NP were longitudinally monitored, 88% of the participants were identified above an individual upper threshold arbitrarily calculated as three standard deviations above the mean values of four baseline samples. The miRNA levels displayed large intra-subject variations that did not change in relation to recGH administration. Our results show that the GH isoform test is very sensitive in detecting low doses of recGH but with a short detection window. Moreover, longitudinally monitoring of IGF-I and P-III-NP may be a promising future approach to detect GH doping.

Parallel Algorithms for Switching Edges in Heterogeneous Graphs.

An edge switch is an operation on a graph (or network) where two edges are selected randomly and one of their end vertices are swapped with each other. Edge switch operations have important applications in graph theory and network analysis, such as in generating random networks with a given degree sequence, modeling and analyzing dynamic networks, and in studying various dynamic phenomena over a network. The recent growth of real-world networks motivates the need for efficient parallel algorithms. The dependencies among successive edge switch operations and the requirement to keep the graph simple (i.e., no self-loops or parallel edges) as the edges are switched lead to significant challenges in designing a parallel algorithm. Addressing these challenges requires complex synchronization and communication among the processors leading to difficulties in achieving a good speedup by parallelization. In this paper, we present distributed memory parallel algorithms for switching edges in massive networks. These algorithms provide good speedup and scale well to a large number of processors. A harmonic mean speedup of 73.25 is achieved on eight different networks with 1024 processors. One of the steps in our edge switch algorithms requires the computation of multinomial random variables in parallel. This paper presents the first non-trivial parallel algorithm for the problem, achieving a speedup of 925 using 1024 processors.

Acute Myelogenous Leukemia Cells Secrete Factors that Stimulate Cellular LDL Uptake via Autocrine and Paracrine Mechanisms.

Leukemic cells isolated from most patients with acute myelogenous leukemia (AML) have higher low density lipoprotein (LDL) uptake than normal mononuclear blood cells. Little is known, however, about the mechanism behind the elevated LDL uptake. We investigated if AML cells secrete factors that stimulate cellular LDL uptake. Mononuclear blood cells were isolated from peripheral blood from 42 patients with AML at diagnosis. Cellular LDL uptake was determined from the degradation rate of 125I-labelled LDL. Conditioned media from AML cells stimulated the LDL degradation in the leukemic cell lines KG1 and HL60, and in isolated AML cells. The stimulatory effect correlated with the LDL degradation in the AML cells directly after isolation from blood. Conditioned media also autostimulated LDL degradation in the AML cells themselves. Concentrations of IL-6 and IL-8 in AML cell conditioned media correlated with the LDL degradation in AML cells directly after isolation from blood. Addition of R-TNF-α, but not IL-6 or IL-8, stimulated LDL degradation in HL60, KG1, and AML cells. The LDL degradation in AML cells could be inhibited by a LDL receptor blocking antibody. AML cells secrete factors that stimulate LDL uptake in a paracrine and autocrine pattern which open up therapeutic possibilities to inhibit the uptake of LDL by administration of antibodies to these factors.

Increased lanosterol turnover: a metabolic burden for daunorubicin-resistant leukemia cells.

The cholesterol metabolism is essential for cancer cell proliferation. We found the expression of genes involved in the cholesterol biosynthesis pathway up-regulated in the daunorubicin-resistant leukemia cell line CEM/R2, which is a daughter cell line to the leukemia cell line CCRF-CEM (CEM). Cellular (2)H2O labelling, mass spectrometry, and isotopomer analysis revealed an increase in lanosterol synthesis which was not accompanied by an increase in cholesterol flux or pool size in CEM/R2 cells. Exogenous addition of lanosterol had a negative effect on CEM/R2 and a positive effect on sensitive CEM cell viability. Treatment of CEM and CEM/R2 cells with cholesterol biosynthesis inhibitors acting on the enzymes squalene epoxidase and lanosterol synthase, both also involved in the 24,25-epoxycholesterol shunt pathway, revealed a connection of this pathway to lanosterol turnover. Our data highlight that an increased lanosterol flux poses a metabolic weakness of resistant cells that potentially could be therapeutically exploited.

Rewired metabolism in drug-resistant leukemia cells: a metabolic switch hallmarked by reduced dependence on exogenous glutamine.

Cancer cells that escape induction therapy are a major cause of relapse. Understanding metabolic alterations associated with drug resistance opens up unexplored opportunities for the development of new therapeutic strategies. Here, we applied a broad spectrum of technologies including RNA sequencing, global untargeted metabolomics, and stable isotope labeling mass spectrometry to identify metabolic changes in P-glycoprotein overexpressing T-cell acute lymphoblastic leukemia (ALL) cells, which escaped a therapeutically relevant daunorubicin treatment. We show that compared with sensitive ALL cells, resistant leukemia cells possess a fundamentally rewired central metabolism characterized by reduced dependence on glutamine despite a lack of expression of glutamate-ammonia ligase (GLUL), a higher demand for glucose and an altered rate of fatty acid ß-oxidation, accompanied by a decreased pantothenic acid uptake capacity. We experimentally validate our findings by selectively targeting components of this metabolic switch, using approved drugs and starvation approaches followed by cell viability analyses in both the ALL cells and in an acute myeloid leukemia (AML) sensitive/resistant cell line pair. We demonstrate how comparative metabolomics and RNA expression profiling of drug-sensitive and -resistant cells expose targetable metabolic changes and potential resistance markers. Our results show that drug resistance is associated with significant metabolic costs in cancer cells, which could be exploited using new therapeutic strategies.

Comparison of uptake mechanisms for anthracyclines in human leukemic cells.

AIMS: The mechanisms behind cellular anthracycline uptake are not completely understood. Knowledge about uptake mechanisms could be used to increase the selectivity of the drugs. We compared the uptake patterns of, daunorubicin (DNR), doxorubicin (DOX), epirubicin (EPI), idarubicin (IDA), and pirarubicin (PIRA) by cultured leukemic cells and investigated possible involvement of specific carriers. METHODS: HL-60 cells were incubated with anthracyclines for 1 hour in the absence or presence of transport inhibitors, suramin, or nucleosides and cellular drug uptake was determined. Cell survival was also determined. MCF-7 breast cancer cells were used as a negative control for concentrative nucleoside transporters (CNTs). Anthracycline concentration was determined with HPLC and fluorometric detection and apoptosis was determined with propidium iodide and flow cytometry. RESULTS: DNR, IDA, and PIRA had higher uptake than DOX and EPI with a prominent increase in uptake at concentrations > 1 µM. Uptake of all anthracyclines was greatly reduced at 0°C. Suramin, a purinergic-2-receptor inhibitor, strongly inhibited the uptake of all anthracyclines except PIRA and increased cell survival. Dipyridamole, an equilibrative NT (ENT) inhibitor, significantly inhibited the uptake of DNR only. The addition of nucleosides significantly inhibited the uptake of DNR, IDA, and PIRA but not in MCF-7 cells lacking functional CNTs. CONCLUSION: Our results suggest different uptake mechanisms for the anthracyclines studied. We found evidence for carrier mediated uptake mechanisms, supporting involvement of NTs in transmembrane transport of DNR, IDA, and PIRA. The results also showed a strong inhibition of suramin on anthracycline uptake by so far unknown mechanisms.

The FUN30 chromatin remodeler, Fft3, protects centromeric and subtelomeric domains from euchromatin formation.

The chromosomes of eukaryotes are organized into structurally and functionally discrete domains. This implies the presence of insulator elements that separate adjacent domains, allowing them to maintain different chromatin structures. We show that the Fun30 chromatin remodeler, Fft3, is essential for maintaining a proper chromatin structure at centromeres and subtelomeres. Fft3 is localized to insulator elements and inhibits euchromatin assembly in silent chromatin domains. In its absence, euchromatic histone modifications and histone variants invade centromeres and subtelomeres, causing a mis-regulation of gene expression and severe chromosome segregation defects. Our data strongly suggest that Fft3 controls the identity of chromatin domains by protecting these regions from euchromatin assembly.

Uptake of anthracyclines in vitro and in vivo in acute myeloid leukemia cells in relation to apoptosis and clinical response.

AIMS: To study anthracycline-induced apoptosis in leukemic cells isolated from patients with acute myelogenous leukemia (AML) in vitro and to compare intracellular anthracycline concentrations causing apoptosis in vitro with those obtained in vivo during anthracycline treatment. METHODS: Mononuclear blood cells from AML patients were isolated before (n = 20) and after anthracycline infusion (n = 24). The pre-treated cells were incubated in vitro with daunorubicin (DNR) and/or idarubicin (IDA). Anthracycline concentrations were determined by high-performance liquid chromatography, and apoptosis was detected by propidium iodine staining using a flow cytometer. RESULTS: There was a clear concentration-response relationship between intracellular anthracycline levels and apoptosis albeit with a large interindividual variation. Intracellular levels >1200 muM always led to high apoptosis development (>60%) in vitro. The intracellular concentrations of DNR in vivo (n = 24) were more than tenfold lower than the concentrations needed to induce effective apoptosis in vitro, although a significant relation between in vivo concentrations and clinical remission was found. We also found a significant relation between apoptosis induction in leukemic cells by IDA in vitro and clinical remission. CONCLUSIONS: Our results indicate that intracellular anthracycline levels in vivo are suboptimal and that protocols should be used that increase intracellular anthracycline levels.

ApoB100-LDL acts as a metabolic signal from liver to peripheral fat causing inhibition of lipolysis in adipocytes.

BACKGROUND: Free fatty acids released from adipose tissue affect the synthesis of apolipoprotein B-containing lipoproteins and glucose metabolism in the liver. Whether there also exists a reciprocal metabolic arm affecting energy metabolism in white adipose tissue is unknown. METHODS AND FINDINGS: We investigated the effects of apoB-containing lipoproteins on catecholamine-induced lipolysis in adipocytes from subcutaneous fat cells of obese but otherwise healthy men, fat pads from mice with plasma lipoproteins containing high or intermediate levels of apoB100 or no apoB100, primary cultured adipocytes, and 3T3-L1 cells. In subcutaneous fat cells, the rate of lipolysis was inversely related to plasma apoB levels. In human primary adipocytes, LDL inhibited lipolysis in a concentration-dependent fashion. In contrast, VLDL had no effect. Lipolysis was increased in fat pads from mice lacking plasma apoB100, reduced in apoB100-only mice, and intermediate in wild-type mice. Mice lacking apoB100 also had higher oxygen consumption and lipid oxidation. In 3T3-L1 cells, apoB100-containing lipoproteins inhibited lipolysis in a dose-dependent fashion, but lipoproteins containing apoB48 had no effect. ApoB100-LDL mediated inhibition of lipolysis was abolished in fat pads of mice deficient in the LDL receptor (Ldlr(-/-)Apob(100/100)). CONCLUSIONS: Our results show that the binding of apoB100-LDL to adipocytes via the LDL receptor inhibits intracellular noradrenaline-induced lipolysis in adipocytes. Thus, apoB100-LDL is a novel signaling molecule from the liver to peripheral fat deposits that may be an important link between atherogenic dyslipidemias and facets of the metabolic syndrome.

Meiotic chromosome synapsis in yeast can occur without spo11-induced DNA double-strand breaks.

Proper chromosome segregation and formation of viable gametes depend on synapsis and recombination between homologous chromosomes during meiosis. Previous reports have shown that the synaptic structures, the synaptonemal complexes (SCs), do not occur in yeast cells with the SPO11 gene removed. The Spo11 enzyme makes double-strand breaks (DSBs) in the DNA and thereby initiates recombination. The view has thus developed that synapsis in yeast strictly depends on the initiation of recombination. Synapsis in some other species (Drosophila melanogaster and Caenorhabditis elegans) is independent of recombination events, and SCs are found in spo11 mutants. This difference between species led us to reexamine spo11 deletion mutants of yeast. Using antibodies against Zip1, a SC component, we found that a small fraction (1%) of the spo11 null mutant cells can indeed form wild-type-like SCs. We further looked for synapsis in a spo11 mutant strain that accumulates pachytene cells (spo11Delta ndt80Delta), and found that the frequency of cells with apparently complete SC formation was 10%. Other phenotypic criteria, such as spore viability and homologous chromosome juxtaposition measured by FISH labeling of chromosomal markers, agree with several previous reports of the spo11 mutant. Our results demonstrate that although the Spo11-induced DSBs obviously promote synapsis in yeast, the presence of Spo11 is not an absolute requirement for synapsis.

Lateral elements inside synaptonemal complex-like polycomplexes in ndt80 mutants of yeast bind DNA.

The synaptonemal complex (SC) keeps the synapsed homologous chromosomes together during pachytene in meiotic prophase I. Structures that resemble stacks of SCs, polycomplexes, are sometimes found before or after pachytene. We have investigated ndt80 mutants of yeast, which arrest in pachytene. SCs appear normal in spread chromosome preparations, but are only occasionally found in intact nuclei examined in the electron microscope. Instead, large polycomplexes occur in almost every ndt80 mutant nucleus. Immunoelectron microscopy using DNA antibodies show strong preferential labeling to the lateral element parts of the polycomplexes. In situ hybridization using chromosome-specific probes confirms that the chromosomes in ndt80 mutants are paired and attached to the SCs. Our results suggest that polycomplexes can be involved in binding of chromosomes and possibly also in synapsis.