The Bloom's syndrome helicase (BLM) interacts physically and functionally with p12, the smallest subunit of human DNA polymerase δ

Bloom's syndrome (BS) is a cancer predisposition disorder caused by mutation of the BLM gene, encoding a member of the RecQ helicase family. Although the phenotype of BS cells is suggestive of a role for BLM in repair of stalled or damaged replication forks, thus far there has been no direct evidence that BLM associates with any of the three human replicative DNA polymerases. Here, we show that BLM interacts specifically in vitro and in vivo with p12, the smallest subunit of human POL δ (hPOL δ). The hPOL δ enzyme, as well as the isolated p12 subunit, stimulates the DNA helicase activity of BLM. Conversely, BLM stimulates hPOL δ strand displacement activity. Our results provide the first functional link between BLM and the replicative machinery in human cells, and suggest that BLM might be recruited to sites of disrupted replication through an interaction with hPOL δ. Finally, our data also define a novel role for the poorly characterized p12 subunit of hPOL δ

Inborn and acquired metabolic defects in cancer

The observation that altered metabolism is the fundamental cause of cancer was made by Otto Warburg nearly a century ago. However, the subsequent identification of oncogenes and tumor suppressor genes has displaced Warburg's theory pointing towards genetic aberrations as the underlining cause of cancer. Nevertheless, in the last decade, cancer-associated mutations have been identified in genes coding for tricarboxylic acid cycle (TCA cycle, also known as Krebs cycle) and closely related enzymes that have essential roles in cellular metabolism. These observations have revived interest in Warburg's hypothesis and prompted a flurry of functional studies in the hope of gaining mechanistic insight into the links between mitochondrial dysfunction, metabolic alterations, and cancer. In this review, we discuss the potential pro-oncogenic signaling role of some TCA cycle metabolites and their derivatives (oncometabolites). In particular, we focus on their effects on dioxygenases, a family of oxygen and α-ketoglutarate-dependent enzymes that control, among other things, the levels and activity of the hypoxia-inducible transcription factors and the activity of DNA and histone demethylases

Central carbon metabolism in the progression of mammary carcinoma

There is a growing belief that the metabolic program of breast tumor cells could be a therapeutic target. Yet, without detailed information on central carbon metabolism in breast tumors it is impossible to know which metabolic pathways to target, and how their inhibition might influence different stages of breast tumor progression. Here we perform the first comprehensive profiling of central metabolism in the MCF10 model of mammary carcinoma, where the steps of breast tumor progression (transformation, tumorigenicity and metastasis) can all be examined in the context of the same genetic background. The metabolism of [U-13C]-glucose by a series of progressively more aggressive MCF10 cell lines was tracked by 2D NMR and mass spectrometry. From this analysis the flux of carbon through distinct metabolic reactions was quantified by isotopomer modeling. The results indicate widespread changes to central metabolism upon cellular transformation including increased carbon flux through the pentose phosphate pathway (PPP), the TCA cycle, as well as increased synthesis of glutamate, glutathione and fatty acids (including elongation and desaturation). The de novo synthesis of glycine increased upon transformation as well as at each subsequent step of breast tumor cell progression. Interestingly, the major metabolic shift in metastatic cells is a large increase in the de novo synthesis of proline. This work provides the first comprehensive view of changes to central metabolism as a result of breast tumor progression

A Gestational High Protein Diet Affects the Abundance of Muscle Transcripts Related to Cell Cycle Regulation throughout Development in Porcine Progeny

BACKGROUND: In various animal models pregnancy diets have been shown to affect offspring phenotype. Indeed, the underlying programming of development is associated with modulations in birth weight, body composition, and continual diet-dependent modifications of offspring metabolism until adulthood, producing the hypothesis that the offspring's transcriptome is permanently altered depending on maternal diet. METHODOLOGY/PRINCIPAL FINDINGS: To assess alterations of the offspring's transcriptome due to gestational protein supply, German Landrace sows were fed isoenergetic diets containing protein levels of either 30% (high protein--HP) or 12% (adequate protein--AP) throughout their pregnancy. Offspring muscle tissue (M. longissimus dorsi) was collected at 94 days post conception (dpc), and 1, 28, and 188 days post natum (dpn) for use with Affymetrix GeneChip Porcine Genome Arrays and subsequent statistical and Ingenuity pathway analyses. Numerous transcripts were found to have altered abundance at 94 dpc and 1 dpn; at 28 dpn no transcripts were altered, and at 188 dpn only a few transcripts showed a different abundance between diet groups. However, when assessing transcriptional changes across developmental time points, marked differences were obvious among the dietary groups. Depending on the gestational dietary exposure, short- and long-term effects were observed for mRNA expression of genes related to cell cycle regulation, energy metabolism, growth factor signaling pathways, and nucleic acid metabolism. In particular, the abundance of transcripts related to cell cycle remained divergent among the groups during development. CONCLUSION: Expression analysis indicates that maternal protein supply induced programming of the offspring's genome; early postnatal compensation of the slight growth retardation obvious at birth in HP piglets resulted, as did a permanently different developmental alteration and responsiveness to the common environment of the transcriptome. The transcriptome modulations are interpreted as the molecular equivalent of developmental plasticity of the offspring that necessitates adaptation and maintenance of the organismal phenotype

Metabolomic Profiling of Drug Responses in Acute Myeloid Leukaemia Cell Lines

Combined bezafibrate (BEZ) and medroxyprogesterone acetate (MPA) exert unexpected antileukaemic activities against acute myeloid leukaemia (AML) and these activities are associated with the generation of reactive oxygen species (ROS) within the tumor cells. Although the generation of ROS by these drugs is supported by preceding studies including our own, the interrelationship between the cellular effects of the drugs and ROS generation is not well understood. Here we report the use of NMR metabolomic profiling to further study the effect of BEZ and MPA on three AML cell lines and to shed light on the underlying mechanism of action. For this we focused on drug effects induced during the initial 24 hours of treatment prior to the onset of overt cellular responses and examined these in the context of basal differences in metabolic profiles between the cell lines. Despite their ultimately profound cellular effects, the early changes in metabolic profiles engendered by these drugs were less pronounced than the constitutive metabolic differences between cell types. Nonetheless, drug treatments engendered common metabolic changes, most markedly in the response to the combination of BEZ and MPA. These responses included changes to TCA cycle intermediates consistent with recently identified chemical actions of ROS. Notable amongst these was the conversion of α-ketoglutarate to succinate which was recapitulated by the treatment of cell extracts with exogenous hydrogen peroxide. These findings indicate that the actions of combined BEZ and MPA against AML cells are indeed mediated downstream of the generation of ROS rather than some hitherto unsuspected mechanism. Moreover, our findings demonstrate that metabolite profiles represent highly sensitive markers for genomic differences between cells and their responses to external stimuli. This opens new perspectives to use metabolic profiling as a tool to study the rational redeployment of drugs in new disease settings

SirT3 suppresses hypoxia inducible factor 1α and tumor growth by inhibiting mitochondrial ROS production

It has become increasing clear that alterations in cellular metabolism have a key role in the generation and maintenance of cancer. Some of the metabolic changes can be attributed to the activation of oncogenes or loss of tumor suppressors. Here, we show that the mitochondrial sirtuin, SirT3, acts as a tumor suppressor via its ability to suppress reactive oxygen species (ROS) and regulate hypoxia inducible factor 1α (HIF-1α). Primary mouse embryo fibroblasts (MEFs) or tumor cell lines expressing SirT3 short-hairpin RNA exhibit a greater potential to proliferate, and augmented HIF-1α protein stabilization and transcriptional activity in hypoxic conditions. SirT3 knockdown increases tumorigenesis in xenograft models, and this is abolished by giving mice the anti-oxidant N-acetyl cysteine. Moreover, overexpression of SirT3 inhibits stabilization of HIF-1α protein in hypoxia and attenuates increases in HIF-1α transcriptional activity. Critically, overexpression of SirT3 decreases tumorigenesis in xenografts, even when induction of the sirtuin occurs after tumor initiation. These data suggest that SirT3 acts to suppress the growth of tumors, at least in part through its ability to suppress ROS and HIF-1α

Gβγ and the C Terminus of SNAP-25 Are Necessary for Long-Term Depression of Transmitter Release

Short-term presynaptic inhibition mediated by G protein-coupled receptors involves a direct interaction between G proteins and the vesicle release machinery. Recent studies implicate the C terminus of the vesicle-associated protein SNAP-25 as a molecular binding target of Gβγ that transiently reduces vesicular release. However, it is not known whether SNAP-25 is a target for molecular modifications expressing long-term changes in transmitter release probability.This study utilized two-photon laser scanning microscopy for real-time imaging of action potential-evoked [Ca(2+)] increases, in single Schaffer collateral presynaptic release sites in in vitro hippocampal slices, plus simultaneous recording of Schaffer collateral-evoked synaptic potentials. We used electroporation to infuse small peptides through CA3 cell bodies into presynaptic Schaffer collateral terminals to selectively study the presynaptic effect of scavenging the G-protein Gβγ. We demonstrate here that the C terminus of SNAP-25 is necessary for expression of LTD, but not long-term potentiation (LTP), of synaptic strength. Using type A botulinum toxin (BoNT/A) to enzymatically cleave the 9 amino acid C-terminus of SNAP-25 eliminated the ability of low frequency synaptic stimulation to induce LTD, but not LTP, even if release probability was restored to pre-BoNT/A levels by elevating extracellular [Ca(2+)]. Presynaptic electroporation infusion of the 14-amino acid C-terminus of SNAP-25 (Ct-SNAP-25), to scavenge Gβγ, reduced both the transient presynaptic inhibition produced by the group II metabotropic glutamate receptor stimulation, and LTD. Furthermore, presynaptic infusion of mSIRK, a second, structurally distinct Gβγ scavenging peptide, also blocked the induction of LTD. While Gβγ binds directly to and inhibit voltage-dependent Ca(2+) channels, imaging of presynaptic [Ca(2+)] with Mg-Green revealed that low-frequency stimulation only transiently reduced presynaptic Ca(2+) influx, an effect not altered by infusion of Ct-SNAP-25.The C-terminus of SNAP-25, which links synaptotagmin I to the SNARE complex, is a binding target for Gβγ necessary for both transient transmitter-mediated presynaptic inhibition, and the induction of presynaptic LTD

Recent advances in the genetics of SDH-related paraganglioma and pheochromocytoma

The last 10 years have seen enormous progress in the field of paraganglioma and pheochromocytoma genetics. The identification of the first gene related to paraganglioma, SDHD, encoding a subunit of mitochondrial succinate dehydrogenase (SDH), was quickly followed by the identification of mutations in SDHC and SDHB. Very recently several new SDH-related genes have been discovered. The SDHAF2 gene encodes an SDH co-factor related to the function of the SDHA subunit, and is currently exclusively associated with head and neck paragangliomas. SDHA itself has now also been identified as a paraganglioma gene, with the recent identification of the first mutation in a patient with extra-adrenal paraganglioma. Another SDH-related co-factor, SDHAF1, is not currently known to be a tumor suppressor, but may shed some light on the mechanisms of tumorigenesis. An entirely novel gene associated with adrenal pheochromocytoma, TMEM127, suggests that other new paraganglioma susceptibility genes may await discovery. In addition to these recent discoveries, new techniques related to mutation analysis, including genetic analysis algorithms, SDHB immunohistochemistry, and deletion analysis by MLPA have improved the efficiency and accuracy of genetic analysis. However, many intriguing questions remain, such as the striking differences in the clinical phenotype of genes that encode proteins with an apparently very close functional relationship, and the lack of expression of SDHD and SDHAF2 mutations when inherited via the maternal line. Little is still known of the origins and causes of truly sporadic tumors, and the role of oxygen in the relationships between high-altitude, familial and truly sporadic paragangliomas remains to be elucidated