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Germline mutations of TP53, which cause the cancer predisposition disorder Li-Fraumeni syndrome (LFS), can increase mitochondrial activity as well as fatty acid β-oxidation (FAO) in mice. Increased fatty acid metabolism can promote cancer malignancy, but its specific contribution to tumorigenesis in LFS remains unclear. To investigate this, we crossed LFS mice carrying the p53 R172H knock-in mutation (p53172H/H, homolog of the human TP53 R175H LFS mutation) with myoglobin-knockout (MB−/−) mice known to have decreased FAO. MB−/− p53172H/H double-mutant mice also showed mildly reduced FAO in thymus, a common site of T lymphoma development in LFS mice, in association with an approximately 40% improvement in cancer-free survival time. RNA sequencing profiling revealed that the p53 R172H mutation promotes mitochondrial metabolism and ribosome biogenesis, both of which are suppressed by the disruption of MB. The activation of ribosomal protein S6, involved in protein translation and implicated in cancer promotion, was also inhibited in the absence of MB. To further confirm the role of FAO in lymphomagenesis, mitochondrial FAO enzyme, carnitine palmitoyltransferase 2 (CPT2), was specifically disrupted in T cells of p53172H/H mice using a Cre-loxP–mediated strategy. The heterozygous knockout of CPT2 resulted in thymus FAO haploinsufficiency and an approximately 30% improvement in survival time, paralleling the antiproliferative signaling observed with MB disruption. Thus, this study demonstrates that moderating FAO in LFS can suppress tumorigenesis and improve cancer-free survival with potential implications for cancer prevention.Prevention Relevance:Mildly inhibiting the increased fatty acid oxidation observed in a mouse model of Li-Fraumeni syndrome, a cancer predisposition disorder caused by inherited mutations of TP53, dampens aberrant pro-tumorigenic cell signaling and improves the survival time of these mice, thereby revealing a potential strategy for cancer prevention in patients.

Inheritance of germline mutations in the tumor suppressor gene TP53 causes Li-Fraumeni syndrome (LFS), a cancer predisposition disorder. The arginine to histidine substitution at amino acid position 337 of p53 (R337H) is a founder mutation highly prevalent in southern and southeastern Brazil and is considered an LFS mutation. Although this mutation is of significant clinical interest, its role in tumorigenesis using animal models has not been described. Here, we generate a knockin mouse model containing the homologous R337H mutation (mouse R334H). De novo tumorigenesis was not significantly increased in either heterozygous (p53334R/H) or homozygous (p53334H/H) p53 R334H knockin mice compared with wild-type mice. However, susceptibility to diethylnitrosamine (DEN)-induced liver carcinogenesis was increased in a mutant allele dose-dependent manner. In parallel, p53334H/H mice exposed to DEN exhibited increased DNA damage but decreased cell-cycle regulation in the liver. Oligomerization of p53, which is required for transactivation of target genes, was reduced in R334H liver, consistent with its decreased nuclear activity compared with wild-type. By modeling a TP53 mutation in mice that has relatively weak cancer penetrance, this study provides in vivo evidence that the human R337H mutation can compromise p53 activity and promote tumorigenesis.Significance: A germline mutation in the oligomerization domain of p53 decreases its transactivation potential and renders mice susceptible to carcinogen-induced liver tumorigenesis. Cancer Res; 78(18); 5375–83. ©2018 AACR.

This Supplemental Information file contains the Materials and Methods for the supplementary tables and figures merged into this same file. These are Tables 1-3 which show primer sequences, observed genotypes and gender of p53 R334H mouse crossings, and incidence of adrenal abnormalities; and Figures S1-S9 which provide additional data on mouse phenotype, DEN metabolism, tumor LOH, DNA damage time course, oncogene-induced senescence in MEFs, p53 expression levels, and further oligomerization studies.

Alangium confertiflorum Y.H.Tan & H.B.Ding, a new species of Alangium section Alangium (Cornaceae) from China-Laos transboundary region, is described and illustrated here. The similarities and differences with closest allied species in the section Alangium, A. amplum W.J.de Wilde & Duyfjes and A. indochinense W.J.de Wilde & Duyfjes are discussed. The new species is assessed as Endangered (EN) following the IUCN Red List Categories and Criteria

SUMMARY The physiological effects of the many germline mutations of TP53, encoding the tumor suppressor protein p53, are poorly understood. Here we report generating a p53 R178C knockin mouse modeling the human TP53 R181C mutation, which is notable for its prevalence and prior molecular characterization. Consistent with its weak cancer penetrance in humans, homozygous p53178C/C mice show a modest increase in tumorigenesis but, surprisingly, are lean with decreased body fat content. They display evidence of increased lipolysis and upregulation of fatty acid metabolism in their inguinal white adipose tissue (iWAT). Gene expression and chromatin immunoprecipitation sequencing (ChIP-seq) analyses show that the mutant p53 bound and trans-activated Beta-3-Adrenergic Receptor (ADRB3), a gene that is known to promote lipolysis and is associated with obesity. This study reveals that a germline mutation of p53 can affect fat metabolism, which has been implicated in cancer development., Graphical Abstract, In Brief Knockin of the mouse homolog of a human TP53 germline mutation known to cause Li-Fraumeni syndrome, a cancer predisposition disorder, results in a mouse model characterized by lower body fat content. Kang et al. show that enhancing transactivation of the lipolytic gene ADRB3 by mutant p53 contributes to this phenotype.

Molecular oxygen possesses a dual nature due to its highly reactive free radical property: it is capable of oxidizing metabolic substrates to generate cellular energy, but can also serve as a substrate for genotoxic reactive oxygen species generation. As a labile substance upon which aerobic life depends, the mechanisms for handling cellular oxygen have been fine-tuned and orchestrated in evolution. Protection from atmospheric oxygen toxicity as originally posited by the Endosymbiotic Theory of the Mitochondrion is likely to be one basic principle underlying oxygen homeostasis. We briefly review the literature on oxygen homeostasis both in vitro and in vivo with a focus on the role of the mitochondrion where the majority of cellular oxygen is consumed. The insights gleaned from these basic mechanisms are likely to be important for understanding disease pathogenesis and developing strategies for maintaining health.

Although organ hypofunction and immunosuppression are life-threatening features of severe sepsis, the hypofunctioning organs and immune cells usually regain normal functionality if patients survive. Because tissue interstitial fluid can become acidic during the septic response, we tested the hypothesis that low extracellular pH (pHe) can induce reversible metabolic and functional changes in peritoneal macrophages from C57BL/6J mice. When compared with macrophages cultured at normal pHe, macrophages living in an acidic medium used less glucose and exogenous fatty acid to produce ATP. Lactate, glutamine, and de novo–synthesized fatty acids supported ATP production by mitochondria that gained greater mass, maximal oxygen consumption rate, and spare respiratory capacity. The cells transitioned to an M2-like state, with altered immune responses to LPS and slightly decreased phagocytic ability, yet they regained basal energy production, normal mitochondrial function, and proinflammatory responsiveness when neutral pHe was restored. Low pHe induces changes that support macrophage survival while rendering the cells less proinflammatory (more “tolerant”) and less able to phagocytose bacteria. Macrophage responses to low interstitial pH may contribute to the reversible organ hypofunction and immunoparalysis noted in many patients with sepsis.

Doxorubicin is a widely used chemotherapeutic agent that causes dose-dependent cardiotoxicity in a subset of treated patients, but the genetic determinants of this susceptibility are poorly understood. Here, we report that a noncanonical tumor suppressor activity of p53 prevents cardiac dysfunction in a mouse model induced by doxorubicin administered in divided low doses as in the clinics. While relatively preserved in wild-type (p53(+/+)) state, mice deficient in p53 (p53(−/−)) developed left ventricular (LV) systolic dysfunction after doxorubicin treatment. This functional decline in p53(−/−) mice was associated with decreases in cardiac oxidative metabolism, mitochondrial mass, and mitochondrial genomic DNA (mtDNA) homeostasis. Notably, mice with homozygous knockin of the p53 R172H (p53(172H/H)) mutation, which like p53(−/−) state lacks the prototypical tumor suppressor activities of p53 such as apoptosis but retains its mitochondrial biogenesis capacity, showed preservation of LV function and mitochondria after doxorubicin treatment. In contrast to p53-null state, wild-type and mutant p53 displayed distinct mechanisms of transactivating mitochondrial transcription factor A (TFAM) and p53-inducible ribonucleotide reductase 2 (p53R2), which are involved in mtDNA transcription and maintenance. Importantly, supplementing mice with a precursor of NAD(+) prevented the mtDNA depletion and cardiac dysfunction. These findings suggest that loss of mtDNA contributes to cardiomyopathy pathogenesis induced by doxorubicin administered on a schedule simulating that in the clinics. Given a similar mtDNA protection role of p53 in doxorubicin-treated human induced pluripotent stem cell (iPSC)-derived cardiomyocytes, the mitochondrial markers associated with cardiomyopathy development observed in blood and skeletal muscle cells may have prognostic utility.

Both the stress-response protein, SIRT1, and the cell cycle checkpoint kinase, CHK2, play critical roles in aging and cancer via the modulation of cellular homeostasis and the maintenance of genomic integrity. However, the underlying mechanism linking the two pathways remains elusive. Here, we show that SIRT1 functions as a modifier of CHK2 in cell cycle control. Specifically, SIRT1 interacts with CHK2 and deacetylates it at lysine 520 residue, which suppresses CHK2 phosphorylation, dimerization, and thus activation. SIRT1 depletion induces CHK2 hyperactivation-mediated cell cycle arrest and subsequent cell death. In vivo, genetic deletion of Chk2 rescues the neonatal lethality of Sirt1−/− mice, consistent with the role of SIRT1 in preventing CHK2 hyperactivation. Together, these results suggest that CHK2 mediates the function of SIRT1 in cell cycle progression, and may provide new insights into modulating cellular homeostasis and maintaining genomic integrity in the prevention of aging and cancer.

Germline mutations of TP53, which cause the cancer predisposition disorder Li-Fraumeni syndrome (LFS), can increase mitochondrial activity as well as fatty acid β-oxidation (FAO) in mice. Increased fatty acid metabolism can promote cancer malignancy, but its specific contribution to tumorigenesis in LFS remains unclear. To investigate this, we crossed LFS mice carrying the p53 R172H knock-in mutation (p53172H/H, homolog of the human TP53 R175H LFS mutation) with myoglobin-knockout (MB−/−) mice known to have decreased FAO. MB−/− p53172H/H double-mutant mice also showed mildly reduced FAO in thymus, a common site of T lymphoma development in LFS mice, in association with an approximately 40% improvement in cancer-free survival time. RNA sequencing profiling revealed that the p53 R172H mutation promotes mitochondrial metabolism and ribosome biogenesis, both of which are suppressed by the disruption of MB. The activation of ribosomal protein S6, involved in protein translation and implicated in cancer promotion, was also inhibited in the absence of MB. To further confirm the role of FAO in lymphomagenesis, mitochondrial FAO enzyme, carnitine palmitoyltransferase 2 (CPT2), was specifically disrupted in T cells of p53172H/H mice using a Cre-loxP–mediated strategy. The heterozygous knockout of CPT2 resulted in thymus FAO haploinsufficiency and an approximately 30% improvement in survival time, paralleling the antiproliferative signaling observed with MB disruption. Thus, this study demonstrates that moderating FAO in LFS can suppress tumorigenesis and improve cancer-free survival with potential implications for cancer prevention. Prevention Relevance: Mildly inhibiting the increased fatty acid oxidation observed in a mouse model of Li-Fraumeni syndrome, a cancer predisposition disorder caused by inherited mutations of TP53, dampens aberrant pro-tumorigenic cell signaling and improves the survival time of these mice, thereby revealing a potential strategy for cancer prevention in patients.

The homeostatic link between oxidative stress and autophagy plays an important role in cellular responses to a wide variety of physiological and pathological conditions. However, the regulatory pathway and outcomes remain incompletely understood. Here, we show that reactive oxygen species (ROS) function as signaling molecules that regulate autophagy through ataxia‐telangiectasia mutated (ATM) and cell cycle checkpoint kinase 2 (CHK2), a DNA damage response (DDR) pathway activated during metabolic and hypoxic stress. We report that CHK2 binds to and phosphorylates Beclin 1 at Ser90/Ser93, thereby impairing Beclin 1‐Bcl‐2 autophagy‐regulatory complex formation in a ROS‐dependent fashion. We further demonstrate that CHK2‐mediated autophagy has an unexpected role in reducing ROS levels via the removal of damaged mitochondria, which is required for cell survival under stress conditions. Finally, CHK2(−/−) mice display aggravated infarct phenotypes and reduced Beclin 1 p‐Ser90/Ser93 in a cerebral stroke model, suggesting an in vivo role of CHK2‐induced autophagy in cell survival. Taken together, these results indicate that the ROS‐ATM‐CHK2‐Beclin 1‐autophagy axis serves as a physiological adaptation pathway that protects cells exposed to pathological conditions from stress‐induced tissue damage.

Background Li-Fraumeni syndrome (LFS) is a highly penetrant autosomal dominant cancer predisposition disorder caused by germline TP53 pathogenic variants. Patients with LFS have increased oxidative phosphorylation capacity in skeletal muscle and oxidative stress in blood. Metformin inhibits oxidative phosphorylation, reducing available energy for cancer cell proliferation and decreasing production of reactive oxygen species that cause DNA damage. Thus, metformin may provide pharmacologic risk reduction for cancer in patients with LFS, but its safety in nondiabetic patients with germline TP53 pathogenic variants has not been documented. Methods This study assessed safety and tolerability of metformin in nondiabetic LFS patients and measured changes in metabolic profiles. Adult patients with LFS and germline TP53 variant received 14 weeks of metformin. Blood samples were obtained for measurement of serum insulin-like growth factor–1, insulin, and insulin-like growth factor binding protein 3. Hepatic mitochondrial function was assessed with fasting exhaled CO2 after ingestion of 13C-labeled methionine. Changes in serum metabolome were measured. All statistical tests were 2-sided. Results We enrolled 26 participants: 20 females and 6 males. The most common adverse events were diarrhea (50.0%) and nausea (46.2%). Lactic acidosis did not occur, and there were no changes in fasting glucose. Cumulative mean 13C exhalation was statistically significantly suppressed by metformin (P = .001). Mean levels of insulin-like growth factor binding protein 3 and insulin-like growth factor-1 were statistically significantly lowered (P = .02). Lipid metabolites and branched-chain amino acids accumulated. Conclusions Metformin was safe and tolerable in patients with LFS. It suppressed hepatic mitochondrial function as expected in these individuals. This study adds to the rationale for development of a pharmacologic risk-reduction clinical trial of metformin in LFS.

Li-Fraumeni syndrome (LFS) is a cancer predisposition disorder caused by germline mutations in TP53 that can lead to increased mitochondrial metabolism in patients. However, the implications of altered mitochondrial function for tumorigenesis in LFS are unclear. Here, we have reported that genetic or pharmacologic disruption of mitochondrial respiration improves cancer-free survival in a mouse model of LFS that expresses mutant p53. Mechanistically, inhibition of mitochondrial function increased autophagy and decreased the aberrant proliferation signaling caused by mutant p53. In a pilot study, LFS patients treated with metformin exhibited decreases in mitochondrial activity concomitant with activation of antiproliferation signaling, thus reproducing the effects of disrupting mitochondrial function observed in LFS mice. These observations indicate that a commonly prescribed diabetic medicine can restrain mitochondrial metabolism and tumorigenesis in an LFS model, supporting its further consideration for cancer prevention in LFS patients.

Under normal conditions, basal levels of wild-type p53 promote mitochondrial function through multiple mechanisms. Remarkably, some missense mutations of p53, in contrast to the null state, can result in the retention of its metabolic activities. These effects are particularly prominent in the mitochondria and demonstrate a functional role for mutant p53 in cancer metabolism. This review summarizes accumulating data on the mechanisms by which p53 missense mutations can regulate mitochondrial metabolism and promote the viability and survival of both normal and cancer cells, thus acting as a double edged sword for the host. Greater understanding of these mechanisms may provide insights for developing new treatment or preventive strategies against cancer.

Inheritance of germline mutations in the tumor suppressor gene TP53 causes Li-Fraumeni syndrome (LFS), a cancer predisposition disorder. The arginine to histidine substitution at amino acid position 337 of p53 (R337H) is a founder mutation highly prevalent in southern and southeastern Brazil and is considered an LFS mutation. Although this mutation is of significant clinical interest, its role in tumorigenesis using animal models has not been described. Here, we generate a knockin mouse model containing the homologous R337H mutation (mouse R334H). De novo tumorigenesis was not significantly increased in either heterozygous (p53334R/H) or homozygous (p53334H/H) p53 R334H knockin mice compared with wild-type mice. However, susceptibility to diethylnitrosamine (DEN)-induced liver carcinogenesis was increased in a mutant allele dose-dependent manner. In parallel, p53334H/H mice exposed to DEN exhibited increased DNA damage but decreased cell-cycle regulation in the liver. Oligomerization of p53, which is required for transactivation of target genes, was reduced in R334H liver, consistent with its decreased nuclear activity compared with wild-type. By modeling a TP53 mutation in mice that has relatively weak cancer penetrance, this study provides in vivo evidence that the human R337H mutation can compromise p53 activity and promote tumorigenesis. Significance: A germline mutation in the oligomerization domain of p53 decreases its transactivation potential and renders mice susceptible to carcinogen-induced liver tumorigenesis. Cancer Res; 78(18); 5375–83. ©2018 AACR.

The modification mechanism of hypereutectic Al-17.5%Si alloy with Nd was studied by OM and XRD. The results of OM analysis show that pure Nd could effectively refine primary in hypereutectic Al-17.5%Si alloy. When Nd addition is 0.3%, the average grain size of primary silicon reaches its minimum. The average grain size of primary silicon is reduced from 40~60μm to 10~30μm after modification. XRD patterns show that no new phase forms after Nd modification. The results of mechanical properties tests show that whole properties of modified samples are significantly improved. When Nd addition is 0.3%, tensile strength of the alloy reaches its maximum. Tensile strength is increased about 35.8% from 120 MPa to 163 MPa. Elongation was increased about 175% from 0.8% to 2.2%. The improvement of mechanical properties should attribute to fine primary after modification.

1 Center for Molecular Medicine, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA 2 College of Health Science, Eulji University, Seongnam, South Korea 3 Cardiovascular-Pulmonary Branch, NHLBI, NIH, Bethesda, MD, USA 4 Murine Phenotyping Core, NHLBI, NIH, Bethesda, MD, USA 5 School of Medicine, ChungnamNational University, Daejeon, South Korea 6 Hematology Branch, NHLBI, NIH, Bethesda, MD, South Korea J Mol Med (2016) 94:287–289 DOI 10.1007/s00109-016-1399-y

p53, a critical tumor suppressor, regulates mitochondrial respiration, but how a nuclear protein can orchestrate the function of an organelle encoded by two separate genomes, both of which require p53 for their integrity, remains unclear. Here we report that the mammalian homolog of the yeast mitochondrial disulfide relay protein Mia40 (CHCHD4) is necessary for the respiratory-dependent translocation of p53 into the mitochondria. In the setting of oxidative stress, increased CHCHD4 expression partitions p53 into the mitochondria and protects its genomic integrity while decreasing p53 nuclear localization and transcriptional activity. Conversely, decreased CHCHD4 expression prevents the mitochondrial translocation of p53 while augmenting its nuclear localization and activity. Thus, the mitochondrial disulfide relay system allows p53 to regulate two spatially segregated genomes depending on oxidative metabolic activity.

There is growing evidence that alterations in metabolism may contribute to tumorigenesis. Here, we report on members of families with the Li-Fraumeni syndrome who carry germline mutations in TP53, the gene encoding the tumor-suppressor protein p53. As compared with family members who are not carriers and with healthy volunteers, family members with these mutations have increased oxidative phosphorylation of skeletal muscle. Basic experimental studies of tissue samples from patients with the Li-Fraumeni syndrome and a mouse model of the syndrome support this in vivo finding of increased mitochondrial function. These results suggest that p53 regulates bioenergetic homeostasis in humans. (Funded by the National Heart, Lung, and Blood Institute and the National Institutes of Health; ClinicalTrials.gov number, NCT00406445.).

The new adiabatic multiple coating is composed of surface coating and ground coating. The main raw material of surface coating is diatomite and that of ground coating is zircon powder. This paper separately study on the formula of the surface and ground coating. The results show that the adiabatic and other properties of composite coating are good and the coating can be used on the metal mold to cast the ferritic nodular cast iron (ferrite% ＞95%). And the using life of metal mold is improved.

Although exercise is linked with improved health, the specific molecular mechanisms underlying its various benefits require further clarification. Here we report that exercise increases the nuclear localization and activity of p53 by acutely down-regulating coiled-coil-helix-coiled-coil-helix domain 4 (CHCHD4), a carrier protein that mediates p53 import into the mitochondria. This response to exercise is lost in transgenic mice with constitutive expression of CHCHD4. Mechanistically, exercise-induced nuclear transcription factor FOXO3 binds to the CHCHD4 promoter and represses its expression, preventing the translocation of p53 to the mitochondria and thereby increasing p53 nuclear localization. The synergistic increase in nuclear p53 and FOXO3 by exercise can facilitate their known interaction in transactivating Sirtuin 1 (SIRT1), a NAD+-dependent histone deacetylase that mediates adaptation to various stresses. Thus, our results reveal one mechanism by which exercise could be involved in preventing cancer and potentially other diseases associated with aging.

Oxysterol binding protein related protein 1S (ORP1S) is a member of a family of sterol transport proteins. Here we present evidence that ORP1S translocates from the cytoplasm to the nucleus in response to sterol binding. The sterols that best promote nuclear import of ORP1S also activate the liver X receptor (LXR) transcription factors and we show that ORP1S binds to LXRs, promotes binding of LXRs to LXR response elements (LXREs) and specifically enhances LXR-dependent transcription via the ME.1 and ME.2 enhancer elements of the apoE gene. We propose that ORP1S is a cytoplasmic sterol sensor, which transports sterols to the nucleus and promotes LXR-dependent gene transcription through select enhancer elements.

p53 regulates the cell cycle and deoxyribonucleic acid (DNA) repair pathways as part of its unequivocally important function to maintain genomic stability. Intriguingly, recent studies show that p53 can also transactivate genes involved in coordinating the two major pathways of energy generation to promote aerobic metabolism, but how this serves to maintain genomic stability is less clear. In an attempt to understand the biology, this review presents human epidemiologic data on the inverse relationship between aerobic capacity and cancer incidence that appears to be mirrored by the impact of p53 on aerobic capacity in mouse models. The review summarizes mechanisms by which p53 regulates mitochondrial respiration and proposes how this might contribute to maintaining genomic stability. Although disparate in nature, the data taken together suggest that the promotion of aerobic metabolism by p53 serves as an important tumor suppressor activity and may provide insights for cancer prevention strategies in the future. Antioxid. Redox Signal. 15, 1739–1748.

Rationale: Exercise capacity is a physiological characteristic associated with protection from both cardiovascular and all-cause mortality. p53 regulates mitochondrial function and its deletion markedly diminishes exercise capacity, but the underlying genetic mechanism orchestrating this is unclear. Understanding the biology of how p53 improves exercise capacity may provide useful insights for improving both cardiovascular as well as general health. Objective: The purpose of this study was to understand the genetic mechanism by which p53 regulates aerobic exercise capacity. Methods and Results: Using a variety of physiological, metabolic, and molecular techniques, we further characterized maximum exercise capacity and the effects of training, measured various nonmitochondrial and mitochondrial determinants of exercise capacity, and examined putative regulators of mitochondrial biogenesis. As p53 did not affect baseline cardiac function or inotropic reserve, we focused on the involvement of skeletal muscle and now report a wider role for p53 in modulating skeletal muscle mitochondrial function. p53 interacts with Mitochondrial Transcription Factor A ( TFAM ), a nuclear-encoded gene important for mitochondrial DNA (mtDNA) transcription and maintenance, and regulates mtDNA content. The increased mtDNA in p53 +/+ compared to p53 −/− mice was more marked in aerobic versus glycolytic skeletal muscle groups with no significant changes in cardiac tissue. These in vivo observations were further supported by in vitro studies showing overexpression of p53 in mouse myoblasts increases both TFAM and mtDNA levels whereas depletion of TFAM by shRNA decreases mtDNA content. Conclusions: Our current findings indicate that p53 promotes aerobic metabolism and exercise capacity by using different mitochondrial genes and mechanisms in a tissue-specific manner.

Previously we reported that when cell cholesterol is acutely lowered with beta-methyl-cyclodextrin the amount of activated ERK1/2 in caveolae dramatically increases. We traced the origin of this novel method of pERK1/2 accumulation to a macromolecular complex with dual specific phosphatase activity that contains the serine/threonine phosphatase PP2A, the tyrosine phosphatase HePTP, the oxysterol-binding protein OSBP and cholesterol. When cell cholesterol is lowered, or oxysterols is introduced, the complex disassembles and pERK1/2 increases. In an effort to better understand how OSBP functions as a cholesterol-regulated scaffolding protein, we have mapped the functional parts of the molecule. The command center of the molecule is a centrally located, 51 amino acids (408-459) long sterol-binding domain that can bind both cholesterol and 25-hydroxycholesterol. This domain is functional whether attached to the N- or the C-terminal half of OSBP. Introduction of a Y458S mutation impairs binding. Even though 25-hydroxycholesterol will compete for cholesterol binding to OSBP(408-809), it will not compete for cholesterol binding in full-length OSBP. Upon further analysis we found that a glycine-alaninerich region at the N-terminal end of OSBP works with the PH domain to control cholesterol binding without affecting 25-hydroxycholesterol binding. Finally, we found that HePTP and PP2A bind the C-terminal half of OSBP, HePTP binds a coiled-coil domain (amino acids 732-761), and PP2A binds neither the coiled-coil nor HePTP. On the basis of this information we propose a new model for how OSBP is able to sense both membrane cholesterol and oxidized sterols and link this information to the ERK1/2 signaling pathway.

Large population studies have shown that living at higher altitudes, which lowers ambient oxygen exposure, is associated with reduced cardiovascular disease mortality. However, hypoxia has also been reported to promote atherosclerosis by worsening lipid metabolism and inflammation. We sought to address these disparate reports by reducing the ambient oxygen exposure of ApoE-/- mice. We observed that long-term adaptation to 10% O2 (equivalent to oxygen content at ∼5000 m), compared to 21% O2 (room air at sea level), resulted in a marked decrease in aortic atherosclerosis in ApoE-/- mice. This effect was associated with increased expression of the anti-inflammatory cytokine interleukin-10 (IL-10), known to be anti-atherogenic and regulated by hypoxia-inducible transcription factor-1α (HIF-1α). Supporting these observations, ApoE-/- mice that were deficient in IL-10 (IL10-/- ApoE-/- double knockout) failed to show reduced atherosclerosis in 10% oxygen. Our study reveals a specific mechanism that can help explain the decreased prevalence of ischemic heart disease in populations living at high altitudes and identifies ambient oxygen exposure as a potential factor that could be modulated to alter pathogenesis. Key messages: Chronic low ambient oxygen exposure decreases atherosclerosis in mice. Anti-inflammatory cytokine IL-10 levels are increased by low ambient O2. This is consistent with the established role of HIF-1α in IL10 transactivation. Absence of IL-10 results in the loss of the anti-atherosclerosis effect of low O2. This mechanism may contribute to decreased atherosclerosis at high altitudes.

Regular exercise and a physically active lifestyle have favorable effects on health. Several issues related to this theme are addressed in this report. A comment on the requirements of personalized exercise medicine and in-depth biological profiling along with the opportunities that they offer is presented. This is followed by a brief overview of the evidence for the contributions of genetic differences to the ability to benefit from regular exercise. Subsequently, studies showing that mutations in TP53 influence exercise capacity in mice and humans are succinctly described. The evidence for effects of exercise on endothelial function in health and disease also is covered. Finally, changes in cardiac and skeletal muscle in response to exercise and their implications for patients with cardiac disease are summarized. Innovative research strategies are needed to define the molecular mechanisms involved in adaptation to exercise and to translate them into useful clinical and public health applications.

Host recognition and disposal of LPS, an important Gram-negative bacterial signal molecule, may involve intracellular processes. We have therefore analyzed the initial pathways by which LPS, a natural ligand of glycosylphosphatidylinositol (GPI)-anchored CD14 (CD14-GPI), enters CD14-expressing THP-1 cells and normal human monocytes. Exposure of the cells to hypertonic medium obliterated coated pits and blocked 125I-labeled transferrin internalization, but failed to inhibit CD14-mediated internalization of [3H]LPS monomers or aggregates. Immunogold electron microscope analysis found that CD14-bound LPS moved principally into noncoated structures (mostly tubular invaginations, intracellular tubules, and vacuoles), whereas relatively little moved into coated pits and vesicles. When studied using two-color laser confocal microscopy, internalized Texas Red-LPS and BODIPY-transferrin were found in different locations and failed to overlap completely even after extended incubation. In contrast, in THP-1 cells that expressed CD14 fused to the transmembrane and cytosolic domains of the low-density lipoprotein receptor, a much larger fraction of the cell-associated LPS moved into coated pits and colocalized with intracellular transferrin. These results suggest that CD14 (GPI)-dependent internalization of LPS occurs predominantly via noncoated plasma membrane invaginations that direct LPS into vesicles that are distinct from transferrin-containing early endosomes. A smaller fraction of the LPS enters via coated pits. Aggregation, which greatly increases LPS internalization, accelerates its entry into the nonclathrin-mediated pathway.

Although bacterial lipopolysaccharides (LPS) and several other microbial agonists can bind to mCD14 (membrane CD14), a cell-surface receptor found principally on monocytes and neutrophils, host-derived mCD14 ligands are poorly defined. We report here that phosphatidylinositol (PtdIns), phosphatidylinositol-4-phosphate, and other phosphatidylinositides can bind to mCD14. Phosphatidylserine (PS), another anionic glycerophospholipid, binds to mCD14 with lower apparent affinity than does PtdIns. LPS-binding protein, a lipid transfer protein found in serum, facilitates both PS- and PtdIns-mCD14 binding. PtdIns binding to mCD14 can be blocked by anti-CD14 monoclonal antibodies that inhibit LPS-mCD14 binding, and PtdIns can inhibit both LPS-mCD14 binding and LPS-induced responses in monocytes. Serum-equilibrated PtdIns also binds to mCD14-expressing cells, raising the possibility that endogenous PtdIns may modulate cellular responses to LPS and other mCD14 ligands in vivo.

Background: Metformin is FDA approved for the treatment of type II diabetes. Frequently reported side effects of metformin use in diabetics are abdominal cramping, nausea, and diarrhea. Lactic acidosis, a life-threatening accumulation of lactic acid in the blood, is also a potential adverse effect of metformin. The primary goal of this phase I study was to assess the safety and tolerability of metformin in non-diabetic patients with germline mutations in TP53 and a clinical diagnosis of LFS. LFS is a highly-penetrant, autosomal dominant, cancer predisposition disorder characterized by early onset of breast, sarcoma, adrenocortical, brain, and other cancers. Further, LFS patients have been shown to have increased oxidative phosphorylation capacity in skeletal muscle and oxidative stress markers. Evidence suggests metformin inhibits oxidative phosphorylation in mitochondria, reducing available energy for cancer cell proliferation, and decreasing the production of reactive oxygen species that can cause DNA damage. Although metformin is used safely in non-diabetic individuals for off -label indications such as polycystic ovarian syndrome and infertility, its use in non-diabetic patients with germline TP53 mutations has never been documented. Objectives: To assess safety and tolerability of metformin dosages from 500 mg to 2000 mg per day in patients with germline mutations in TP53 and a clinical diagnosis of LFS. Methodology: Adult LFS patients (?18 years old) with documented germline mutation in TP53 were enrolled for 20 weeks. Patients with a history of cancer had to be in remission (surgery and chemotherapy completed 6 and 12 months prior respectively). Patients could not have a history of diabetes or prior metformin use. Metformin was initiated at 500 mg per day and increased in 500 mg dose increments every two weeks to a maximum dose of 2000 mg by week 8. Patients were maintained on 2000 mg of metformin for six additional weeks (week 14). For the last six weeks of the study, patients were taken off metformin (week 20). The targeted accrual goal was ?22 subjects. Results: Of 24 non-diabetic, LFS patients enrolled, 18 (75%) were female, and 6 (25%) were male. Thirteen (54%) females and 1 male (16%) had a prior history of cancer. The age range for females and males was 25-52 and 20-54 years old respectively. One patient developed cancer, and 2 withdrew from the study before starting treatment; 1 dropped out of the study due to personal issues after reaching a dose of 1500 mg. Of the remaining 20 patients, 1 dropped out of the study due to intolerable dyspepsia/reflux at a dose of 500 mg and 1 patient could only tolerate a maximum dose of 1500 mg due to headaches and nausea; the remaining 18 patients tolerated 2000 mg of metformin. Of 22 patients who took metformin, 20 (90%) experienced abdominal pain/nausea/diarrhea. Five patients (22%) experienced headaches. Three patients (13%) experienced reflux/dyspepsia. Two patients experienced one episode of vomiting (9%). None of the patients experienced lactic acidosis. Fasting glucose at week 14 on 2000 mg of metformin daily for females ranged from 76-96 mg/dL and for males 86-98 mg/dL. Conclusions: Metformin was safe and tolerable in non-diabetic LFS patients with germline mutations in TP53. LFS patients report similar side effects as diabetic patients on metformin. No cases of lactic acidosis occurred. Citation Format: Farzana Lukmanji Walcott, Paul M. Hwang, Ping-yuan Wang, Sharon A. Savage, Phuong Mai, Seth M. Steinberg, Michael N. Pollak, Christina Annunziata, Antonio T. Fojo. Safety and tolerability of metformin for chemoprevention in Li-Fraumeni syndrome (LFS). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT156.

Human erythrocyte band 3 reconstituted into phospholipid vesicles has been used for studying the interaction of interdigitated lipid bilayer with an integral membrane protein. The interdigitated gel phase in DPPG/band 3 or DPPG/DMPC/band 3 systems was induced with polymyxin B (PMB) or Tris+. The phase transitions of the vesicles were detected with high-sensitivity differential scanning calorimetry (DSC). The results indicated that band 3 does not cause significant alterations in the interdigitated phase of phospholipids, with only a little decrease of the phase transition enthalpies. Fluorescence measurements showed that the transition of phospholipid/band 3 systems from the non-interdigitated to interdigitated phase is accompanied by marked intrinsic fluorescence changes of band 3. The interdigitated phase of DPPG or DPPG/DMPC vesicles increases the intrinsic fluorescence intensity of band 3 and significantly decreases the accessibility of certain tryptophan residues on the protein to Cs+.

Oxygen is not only required for oxidative phosphorylation but also serves as the essential substrate for the formation of reactive oxygen species (ROS), which is implicated in ageing and tumorigenesis. Although the mitochondrion is known for its bioenergetic function, the symbiotic theory originally proposed that it provided protection against the toxicity of increasing oxygen in the primordial atmosphere. Using human cells lacking Synthesis of Cytochrome c Oxidase 2 (SCO2 –/–), we have tested the oxygen toxicity hypothesis. These cells are oxidative phosphorylation defective and glycolysis dependent; they exhibit increased viability under hypoxia and feature an inverted growth response to oxygen compared with wild-type cells. SCO2 –/– cells have increased intracellular oxygen and nicotinamide adenine dinucleotide (NADH) levels, which result in increased ROS and oxidative DNA damage. Using this isogenic cell line, we have revealed the genotoxicity of ambient oxygen. Our study highlights the importance of mitochondrial respiration both for bioenergetic benefits and for maintaining genomic stability in an oxygen-rich environment.

Purpose: This phase I chemoprevention pilot study in LFS patients will: 1) assess tolerability of 2000 mg/day of oral metformin in patients with LFS, 2) assess the effect of metformin on circulating serum insulin biomarkers, and 3) use non-invasive technology to assess the effect of metformin on hepatic and skeletal muscle mitochondria in these patients. Background: LFS is a highly-penetrant, autosomal dominant, cancer predisposition disorder characterized by early onset of a spectrum of cancers. Approximately 70% of “classic” LFS families have germline TP53 mutations. Metformin is FDA approved for the treatment of type II diabetes. Epidemiologic studies in diabetic patients have associated metformin use with reduced cancer incidence and mortality, and preclinical research has shown metformin is selectively toxic to cancer cells with loss of p53 function. In non-diabetic patients metformin is well tolerated, does not cause hypoglycemia, and lowers levels of circulating insulin and IGF-1 (insulin-like growth factor 1). In p53+/- mouse models with decreased p53 activity, calorie restriction has resulted in reduction of plasma IGF-1 levels and delayed tumor development, suggesting that lowering IGF-1 may reduce or delay cancer development in individuals with decreased p53 function. Further, p53 also regulates nuclear transcription of mitochondrial proteins and maintains mitochondrial DNA and biogenesis. A recent clinical study indicates that LFS patients with TP53 germline mutations may have increased oxidative phosphorylation capacity in skeletal muscle. Metformin inhibits oxidative phosphorylation in the mitochondria, reducing energy available for rapidly-proliferating cells with mutant TP53. Thus, patients with germline TP53 mutations may benefit from metformin as a chemopreventive agent. Hypotheses/Methods: 1) Metformin will be safe and tolerable in LFS patients with germline mutations in TP53 in oral doses up to 2000 mg/day; 2) Metformin will modulate circulating IGF-1 (insulin-like growth factor 1), IGFBP3 (insulin-like growth factor binding protein 3) and insulin; 3) Metformin will decrease oxidative phosphorylation in both skeletal muscle and hepatic mitochondria. Serum biomarker levels will be assessed by ELISA assays. Hepatic mitochondrial function will be assessed by C-13 methionine breath test. Skeletal muscle mitochondrial function will be assessed by P-31 magnetic resonance spectroscopy. Non-diabetic adults (age >18) with deleterious germline TP53 mutations are eligible. The metformin starting dose is 500 mg/day, with 500 mg/day dose increments every two weeks to a maximum dose of 2000 mg/day. Total study time is 20 weeks. Patients will be on metformin for a total of 14 weeks, after which metformin will be discontinued for 6 weeks. Serum biomarkers and mitochondrial function will be assessed at baseline and weeks 2, 8, 14, and 20. The targeted accrual goal for patients undergoing full dose escalation is ≥22, which will provide an 80% power to detect a change from baseline in each of the three biomarkers equal to 0.75 standard deviations of the change (0.75 effect size) using a two-tailed 0.0167 level paired t-test. This study has been approved by the Institutional Review Board and is open to accrual. Citation Format: Farzana L. Walcott, Paul M. Hwang, Ping-yuan Wang, Sharon A. Savage, Phuong Mai, Seth M. Steinberg, Michael N. Pollak, Philip A. Dennis, Antonio Tito Fojo. Design of a phase I chemoprevention study of metformin and Li-Fraumeni syndrome (LFS). [abstract]. In: Proceedings of the AACR Special Conference: Cancer Susceptibility and Cancer Susceptibility Syndromes; Jan 29-Feb 1, 2014; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(23 Suppl):Abstract nr 29. doi:10.1158/1538-7445.CANSUSC14-29