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.

Background: Drug resistance is a common problem in cancer chemotherapy. Results: Transcriptomic and metabolomic data show that resistant leukemia cells exhibit reduced glutamine dependence, enhanced glucose dependence, and altered fatty acid metabolism. Conclusion:The metabolism of resistant leukemia cells is fundamentally rewired. Significance: Understanding the metabolic cost of resistance can lead to novel therapeutic strategies.

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.

BackgroundFree 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 FindingsWe 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).ConclusionsOur 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.

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 (spo11⌬ ndt80⌬), 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. M EIOTIC chromosome pairing and recombination heterochromatic pairing plays an important role (reviewed by Walker and Hawley 2000). are two processes that are generally necessary forIn organisms that require pairing, recombination, proper segregation of homologous chromosomes and and synapsis for normal chromosome segregation there mixing of parental genomes in the first meiotic division.are examples of mutants with obstructed, but not Both processes are closely linked to the appearance of blocked, meiotic progress. In a mutant strain of budding a proteinaceous structure, the synaptonemal complex yeast that lacks a major component of the SC, Zip1, (SC), which forms between the homologs along their some recombination still occurs, showing that the SC entire length (synapsis; reviewed in Roeder 1997). Alis not absolutely required for recombination in yeast though valid for a large majority of eukaryotes, the sim- (Sym et al. 1993;Storlazzi et al. 1996). The spore viabilple and straightforward view that all the meiotic events ity in the zip1 null mutants is 60%, showing that the (pairing, recombination, and synapsis) are needed to chromosome segregation is reasonably good (Tung and assure proper chromosome segregation is not true for Roeder 1998). While it seems as if the SC is not required all organisms. There are examples of accurate chromofor recombination, it has been generally believed that some division without either synapsis or recombination. the initial step of recombination, the formation of douIn fission yeast, Schizosaccaromyces pombe, both crossing over ble-strand breaks (DSB...

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 H 2 O 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.

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.

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 μM 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.