Proton magnetic resonance spectroscopy ( 1 H MRS) consistently detects significant differences in choline phospholipid metabolites of malignant versus benign breast lesions. It is critically important to understand the molecular causes underlying these metabolic differences, because this may identify novel targets for attack in cancer cells. In this study, differences in choline membrane metabolism were characterized in breast cancer cells and normal human mammary epithelial cells (HMECs) labeled with [1,2-13 C]choline, using 1 H and 13 C magnetic resonance spectroscopy. Metabolic fluxes between membrane and water-soluble pool of choline-containing metabolites were assessed by exposing cells to [1,2-13 C]choline for long and short periods of time to distinguish between catabolic and anabolic pathways in choline metabolism. Gene expression analysis using microarrays was performed to understand the molecular mechanisms underlying these changes. Breast cancer cells exhibited increased phosphocholine (PC; P < 0.001), total choline-containing metabolites (P < 0.01), and significantly decreased glycerophosphocholine (P < 0.05) compared with normal HMECs. Decreased 13 C-enrichment was detected in choline (P < 0.001) and phosphocholine (P < 0.05, P < 0.001) of breast cancer cells compared with HMECs, indicating a higher metabolic flux from membrane phosphatidylcholine to choline and phosphocholine in breast cancer cells. Choline kinase and phospholipase C were significantly overexpressed, and lysophospholipase 1, phospholipase A2, and phospholipase D were significantly underexpressed, in breast cancer cells compared with HMECs. The magnetic resonance spectroscopy data indicated that elevated phosphocholine in breast cancer cells was primarily attributable to increased choline kinase activity and increased catabolism mediated by increased phospholipase C activity. These observations were consistent with the overexpression of choline kinase and phospholipase C detected in the microarray analyses.

Mitral valve prolapse (MVP) is a common human phenotype, yet little is known about the pathogenesis of this condition. MVP can occur in the context of genetic syndromes, including Marfan syndrome (MFS), an autosomal-dominant connective tissue disorder caused by mutations in fibrillin-1. Fibrillin-1 contributes to the regulated activation of the cytokine TGF-β, and enhanced signaling is a consequence of fibrillin-1 deficiency. We thus hypothesized that increased TGF-β signaling may contribute to the multisystem pathogenesis of MFS, including the development of myxomatous changes of the atrioventricular valves. Mitral valves from fibrillin-1-deficient mice exhibited postnatally acquired alterations in architecture that correlated both temporally and spatially with increased cell proliferation, decreased apoptosis, and excess TGF-β activation and signaling. In addition, TGF-β antagonism in vivo rescued the valve phenotype, suggesting a cause and effect relationship. Expression analyses identified increased expression of numerous TGF-β-related genes that regulate cell proliferation and survival and plausibly contribute to myxomatous valve disease. These studies validate a novel, genetically engineered murine model of myxomatous changes of the mitral valve and provide critical insight into the pathogenetic mechanism of such changes in MFS and perhaps more common nonsyndromic variants of mitral valve disease. IntroductionMarfan syndrome (MFS) is a common, autosomal-dominant, systemic disorder of connective tissue, with an estimated prevalence of 1 in 5,000-10,000 individuals (1). It is caused by mutations in FBN1, the gene encoding fibrillin-1 (2), the principal component of extracellular matrix microfibrils. Clinical manifestations of MFS include skeletal deformities, ocular lens dislocation, lung pathology, and cardiac complications, such as aortic dissection and mitral valve prolapse (MVP) and dysfunction. Mitral valve disease is the leading indication for surgery and cause of death in young children with MFS, and there are currently no known or proposed medical therapies for prophylactic treatment of valve disease. While infants presenting with the most severe and rapidly progressive form of MFS can show mitral valve dysfunction at birth, manifestations are highly variable in the classic form of MFS, and myxomatous changes are not easily quantified using noninvasive imaging modalities, precluding a precise understanding of the natural history of disease. To our knowledge, neither recapitulation nor mechanistic exploration of mitral valve pathology in mouse models of MFS has previously been documented.

Astrocyte heterogeneity remains largely unknown in the CNS due to lack of specific astroglial markers. In this study, molecular identity of in vivo astrocytes was characterized in BAC ALDH1L1 and BAC GLT1 eGFP promoter reporter transgenic mice. ALDH1L1 promoter is selectively activated in adult cortical and spinal cord astrocytes, indicated by the overlap of eGFP expression with ALDH1L1 and GFAP, but not with NeuN, APC, Olig2, IbaI, PDGFRα immunoreactivity in BAC ALDH1L1 eGFP reporter mice. Interestingly, ALDH1L1 expression levels (protein, mRNA, and promoter activity) in spinal cord were selectively decreased during postnatal maturation. In contrast, its expression was up-regulated in reactive astrocytes in both acute neural injury and chronic neurodegenerative (G93A mutant SOD1) conditions, similar to GFAP, but opposite of GLT1. ALDH1L1 + and GLT1 + cells isolated through fluorescence activated cell sorting (FACS) from BAC ALDH1L1 and BAC GLT1 eGFP mice share a highly similar gene expression profile, suggesting ALDH1L1 and GLT1 are co-expressed in the same population of astrocytes. This observation was further supported by overlap of the eGFP driven by the ALDH1L1 genomic promoter and the tdTomato driven by a 8.3kb EAAT2 promoter fragment in astrocytes of BAC ALDH1L1 eGFP X EAAT2-tdTomato mice. These studies support ALDH1L1 as a general CNS astroglial marker and investigated astrocyte heterogeneity in the CNS by comparing the molecular identity of the ALDH1L1 + and GLT1 + astrocytes from astroglial reporter mice. These astroglial reporter mice provide useful in vivo tools for the molecular analysis of astrocytes in physiological and pathological conditions.

Mitral valve prolapse (MVP) is a common human phenotype, yet little is known about the pathogenesis of this condition. MVP can occur in the context of genetic syndromes, including Marfan syndrome (MFS), an autosomal-dominant connective tissue disorder caused by mutations in fibrillin-1. Fibrillin-1 contributes to the regulated activation of the cytokine TGF-β, and enhanced signaling is a consequence of fibrillin-1 deficiency. We thus hypothesized that increased TGF-β signaling may contribute to the multisystem pathogenesis of MFS, including the development of myxomatous changes of the atrioventricular valves. Mitral valves from fibrillin-1-deficient mice exhibited postnatally acquired alterations in architecture that correlated both temporally and spatially with increased cell proliferation, decreased apoptosis, and excess TGF-β activation and signaling. In addition, TGF-β antagonism in vivo rescued the valve phenotype, suggesting a cause and effect relationship. Expression analyses identified increased expression of numerous TGF-β-related genes that regulate cell proliferation and survival and plausibly contribute to myxomatous valve disease. These studies validate a novel, genetically engineered murine model of myxomatous changes of the mitral valve and provide critical insight into the pathogenetic mechanism of such changes in MFS and perhaps more common nonsyndromic variants of mitral valve disease. IntroductionMarfan syndrome (MFS) is a common, autosomal-dominant, systemic disorder of connective tissue, with an estimated prevalence of 1 in 5,000-10,000 individuals (1). It is caused by mutations in FBN1, the gene encoding fibrillin-1 (2), the principal component of extracellular matrix microfibrils. Clinical manifestations of MFS include skeletal deformities, ocular lens dislocation, lung pathology, and cardiac complications, such as aortic dissection and mitral valve prolapse (MVP) and dysfunction. Mitral valve disease is the leading indication for surgery and cause of death in young children with MFS, and there are currently no known or proposed medical therapies for prophylactic treatment of valve disease. While infants presenting with the most severe and rapidly progressive form of MFS can show mitral valve dysfunction at birth, manifestations are highly variable in the classic form of MFS, and myxomatous changes are not easily quantified using noninvasive imaging modalities, precluding a precise understanding of the natural history of disease. To our knowledge, neither recapitulation nor mechanistic exploration of mitral valve pathology in mouse models of MFS has previously been documented.

The mechanism of CD4 ؉ T-cell depletion during chronic human immunodeficiency virus type 1 (HIV-1) infection remains unknown. Many studies suggest a significant role for chronic CD4 ؉ T-cell activation. We assumed that the pathogenic process of excessive CD4

Arsenic has played a key medicinal role against a variety of ailments for several millennia, but during the past century its prominence has been displaced by modern therapeutics. Recently, attention has been drawn to arsenic by its dramatic clinical efficacy against acute promyelocytic leukemia. Although toxic reactive oxygen species (ROS) induced in cancer cells exposed to arsenic could mediate cancer cell death, how arsenic induces ROS remains undefined. Through the use of gene expression profiling, interference RNA, and genetically engineered cells, we report here that NADPH oxidase, an enzyme complex required for the normal antibacterial function of white blood cells, is the main target of arsenic-induced ROS production. Because NADPH oxidase enzyme activity can also be stimulated by phorbol myristate acetate, a synergism between arsenic and the clinically used phorbol myristate acetate analog, bryostatin 1, through enhanced ROS production can be expected. We show that this synergism exists, and that the use of very low doses of both arsenic and bryostatin 1 can effectively kill leukemic cells. Our findings pinpoint the arsenic target of ROS production and provide a conceptual basis for an anticancer regimen.A lthough arsenic has played a significant therapeutic role in various diseases for Ͼ2,000 years (1, 2), it was not used clinically for decades, until recently when clinical trials worldwide confirmed its dramatic therapeutic effects in acute promyelocytic leukemia (APL) (3, 4). APL is a subtype of acute myelocytic leukemia with most cases carrying the characteristic chromosomal translocation t(15, 17) that results in the PML-RAR␣ fusion protein (5). Although APL is highly responsive to arsenic, the presence of PML-RAR␣ fusion protein is neither absolutely necessary nor sufficient for sensitivity to arsenic (3, 6, 7). The mechanism by which arsenic is effective against APL remains elusive, despite studies suggesting that arsenic can promote degradation of the oncogenic PML-RAR␣ fusion protein (8, 9). Paradoxically, arsenic is also an established human carcinogen that can induce reactive oxygen species (ROS), leading to DNA damage or cell death (10-13).Some previous mechanistic studies (14, 15) were limited to exposure of cells other than myeloid cells, or to arsenite rather than arsenic trioxide for brief periods, and hence do not reflect the clinical setting for cytotoxic effects of arsenic on APL cells. To explore the molecular mechanisms of arsenic's therapeutic effects in the treatment of APL patients with daily continuous infusion of arsenic trioxide, we treated a human APL cell line, NB4, for Ͼ1 week with arsenic trioxide at a dose lower than the plasma trough level achieved in APL patients. We reported previously that arsenic at this dose was able to down-regulate human telomerase hTERT transcription (16). In this report, we determined changes in gene expression profiles by using oligonucleotide microarrays, and we found that NADPH oxidase components were dramatically up-regulated within days in m...

Cohesion establishment and maintenance are carried out by proteins that modify the activity of Cohesin, an essential complex that holds sister chromatids together. Constituents of the replication fork, such as the DNA polymerase ␣-binding protein Ctf4, contribute to cohesion in ways that are poorly understood. To identify additional cohesion components, we analyzed a ctf4⌬ synthetic lethal screen performed on microarrays. We focused on a subset of ctf4⌬-interacting genes with genetic instability of their own. Our analyses revealed that 17 previously studied genes are also necessary for the maintenance of robust association of sisters in metaphase. Among these were subunits of the MRX complex, which forms a molecular structure similar to Cohesin. Further investigation indicated that the MRX complex did not contribute to metaphase cohesion independent of Cohesin, although an additional role may be contributed by XRS2. In general, results from the screen indicated a sister chromatid cohesion role for a specific subset of genes that function in DNA replication and repair. This subset is particularly enriched for genes that support the S-phase checkpoint. We suggest that these genes promote and protect a chromatin environment conducive to robust cohesion. INTRODUCTIONIn budding yeast, Cohesin is a four subunit protein complex (Mcd1/Scc1, Scc3, Smc1, and Smc3) that depends on the activity of regulatory proteins for its chromosome association, activation, and destruction in each cell cycle (reviewed in Nasmyth, 2001). Sister chromatid association by Cohesin must be established during S phase (Skibbens et al., 1999;Toth et al., 1999) and is maintained until separation of sister chromatids at anaphase. Many studies indicate a role for the replication fork in establishment of robust sister chromatid cohesion, in addition to its traditional role of semiconservative DNA duplication. Several replication-associated proteins from budding yeast are known to support robust cohesion, including the products of nonessential genes CTF4, CTF8, CTF18, DCC1, TRF4, and TRF5 (Wang et al., 2000b;Hanna et al., 2001;Mayer et al., 2001). Null mutants for these genes lead to metaphase cohesion failure at frequencies of ϳ25-35% in cells that are held at metaphase in the absence of microtubules.These nonessential replication fork constituents represent several subcomplexes at the replication fork and contribute independent primary molecular functions that are not well understood. Ctf4 protein forms an association with DNA polymerase ␣ (Formosa and Nittis, 1999) that may compete with binding of a chromatin remodeling subunit Cdc68/ Pob3 (Wittmeyer and Formosa, 1997). Ctf18 protein is a component of an alternative RF-C complex in which it replaces Rfc1, and is joined by Ctf8 and Dcc1 subunits (Hanna et al., 2001;Mayer et al., 2001). The orthologous human RF-C CTF18 complex can load PCNA onto DNA and promote Pol␦ activity in vitro (Bermudez et al., 2003;Kanellis et al., 2003;Merkle et al., 2003). The nonessential proteins Trf4 and Trf5 together co...

Although the differentiation of ES cells to cardiomyocytes has been firmly established, the extent to which corresponding cardiac precursor cells can contribute to other cardiac populations remains unclear. To determine the molecular and cellular characteristics of cardiac-fated populations derived from mouse ES (mES) cells, we isolated cardiac progenitor cells (CPCs) from differentiating mES cell cultures by using a reporter cell line that expresses GFP under the control of a cardiac-specific enhancer element of Nkx2-5, a transcription factor expressed early in cardiac development. This ES cell-derived CPC population initially expressed genetic markers of both stem cells and mesoderm, while differentiated CPCs displayed markers of 3 distinct cell lineages (cardiomyocytes, vascular smooth muscle cells, and endothelial cells) -Flk1 (also known as Kdr), c-Kit, and Nkx2-5, but not Brachyury -and subsequently expressed Isl1. Clonally derived CPCs also demonstrated this multipotent phenotype. By transcription profiling of CPCs, we found that mES cell-derived CPCs displayed a transcriptional signature that paralleled in vivo cardiac development. Additionally, these studies suggested the involvement of genes that we believe were previously unknown to play a role in cardiac development. Taken together, our data demonstrate that ES cell-derived CPCs comprise a multipotent precursor population capable of populating multiple cardiac lineages and suggest that ES cell differentiation is a valid model for studying development of multiple cardiac-fated tissues.