Mary K. Short

Magmas expression in neoplastic human prostate.

Magmas, is a 13-kDa mitochondrial protein which is ubiquitously expressed in eukaryotic cells. It was identified as a granulocyte-macrophage-colony stimulating factor (GM-CSF) inducible gene in hematopoietic cells and has a key role in the transport of mitochondrial proteins in yeast. Because GM-CSF receptor levels are elevated in prostate cancer, Magmas expression was examined in normal and neoplastic tissue. Magmas protein levels were barely detectible in non-neoplastic prostate glands. Increased amounts were observed in some samples of intraepithelial neoplasia. Approximately one half of the adenocarcinoma samples examined had weak Magmas expression, while the remainder had intermediate to high levels. The increased Magmas observed in malignant tissue was a result of higher protein expression and not from changes in mitochondrial content. Interestingly, in some patients, the normal prostate tissue had more Magmas message than the malignant portion. The results indicated that Magmas expression in prostate cancer is heterogeneous and independent of clinical stage and Gleason score. Further studies are needed to determine if Magmas expression has prognostic significance in prostate cancer.

Identification and characterization of Magmas, a novel mitochondria-associated protein involved in granulocyte-macrophage colony-stimulating factor signal transduction

OBJECTIVE The aim of this study was to identify granulocyte-macrophage colony-stimulating factor (GM-CSF) responsive genes. MATERIALS AND METHODS Potential GM-CSF responsive genes were identified by comparing the mRNA expression pattern of the murine myeloid cell line PGMD1 grown in either interleukin-3 (IL-3) or GM-CSF by differential display. Human and murine cDNA clones of one of the bands having increased expression in GM-CSF were isolated. mRNA expression of the gene was examined by Northern blot. Immunohistochemistry and studies with a green fluorescent fusion protein were used to determine its intracellular location. Growth factor-stimulated proliferation of PGMD1 cells transfected with constitutively expressed sense and anti-sense cDNA constructs of the gene was measured by 3H-thymidine incorporation. RESULTS A gene, named Magmas (mitochondria-associated granulocyte macrophage CSF signaling molecule), was shown to be rapidly induced when cells were switched from IL-3 to GM-CSF. Analysis of the amino acid sequence of Magmas showed it contained a mitochondrial signal peptide, but not any other known functional domains. The human and murine clones encode nearly identical 13-kDa proteins that localized to the mitochondria. Magmas mRNA expression was observed in all tissues examined. PGMD1 cells that overexpressed Magmas proliferated similarly to untransfected cells when cultured in IL-3 or GM-CSF. In contrast, cells with reduced protein levels grew normally in IL-3, but had impaired proliferation in GM-CSF. CONCLUSION Magmas is a mitochondrial protein involved in GM-CSF signal transduction.

Developmental Expression of Magmas in Murine Tissues and Its Co-expression with the GM-CSF Receptor

Magmas is a protein that is involved in GM-CSF signaling in a myeloid cell line. Its precise role in the signal transduction process is unclear. To accurately characterize Magmas expression in a variety of cells, mouse embryos and adult murine tissues were analyzed for both mRNA and protein content. Magmas expression was detected as early as the day 6.5 embryo. The level of expression was developmentally regulated. During embryo-genesis, elevated Magmas was observed in several structures, including heart, liver, notochord, choroid plexus, cervical ganglion, and nasal mucosa. Muscle, pancreas, intestinal mucosa, and testes were among the adult tissues with high Magmas expression. Most cell types, including hepatocytes and skeletal, smooth, and cardiac myocytes, also expressed the GM-CSF receptor (GMR) but the relative tissue levels of GMR were not always proportional to Magmas. The expression patterns suggest that Magmas has a role in both developing and mature tissues.

Essential role of Drosophila black-pearl is mediated by its effects on mitochondrial respiration

Black‐pearl (Blp) is a highly conserved, essential inner‐mitochondrial membrane protein. The yeast Blp homologue, Magmas/Pam16, is required for mitochondrial protein transport, growth, and survival. Our purpose was to determine the role of Drosophila Blp in mitochondrial function, cell survival, and proliferation. To this end, we performed mitotic recombination in Drosophila melanogaster, RNAi‐mediated knockdown, MitoTracker staining, measurement of reactive oxygen species (ROS), flow cytometry, electron transport chain complex assays, and hemocyte isolation from Drosophila larvae. Proliferation‐defective, Blp‐deficient Drosophila Schneider cells exhibited mitochondrial membrane depolarization, a 60% decrease in ATP levels, increased amounts of ROS (3.5‐fold), cell cycle arrest, and activation of autophagy that were associated with a selective 65% reduction of cytochrome c oxidase activity. N‐acetyl cysteine (NAC) rescued Blp‐RNAi‐treated cells from cell cycle arrest, indicating that increased production of ROS is the primary cause of the proliferation and survival defects in Blp‐depleted cells. blp hypomorph larvae had a 35% decreased number of plasmatocytes with a 45% reduced active mitochondrial staining and their viability was increased 2‐fold by administration of NAC, which blocked melanotic lesions. Loss of Blp decreases cytochrome c oxidase activity and uncouples oxidative phosphorylation, causing ROS production, which selectively affects mitochondria‐rich plasmatocyte survival and function, leading to melanotic lesions in Blp‐deficient flies.—Roy, S., Short, M. K., Stanley, E. R., Jubinsky, P. T. Essential role of Drosophila black‐pearl is mediated by its effects on mitochondrial respiration. FASEB J. 26, 3822–3833 (2012). www.fasebj.org

Design, synthesis, and biological activity of novel Magmas inhibitors.

Magmas (mitochondria associated, granulocyte-macrophage colony stimulating factor signaling molecule), is a highly conserved and essential gene, expressed in all cell types. We designed and synthesized several small molecule Magmas inhibitors (SMMI) and assayed their effects on proliferation in yeast. We found that the most active compound 9 inhibited growth at the 4 μM scale. This compound was shown by fluorometric titration to bind to Magmas with a K(d)=33 μM. Target specificity of the lead compound was established by demonstrating direct binding of the compound to Magmas and by genetic studies. Molecular modeling suggested that the inhibitor bound at the predicted site in Magmas.

New roles for mononuclear phagocytes in cancer biology.

The primary focus in the pathogenesis and treatment of human malignancies has been the tumor cell. However, the biologic properties of a malignancy are not all intrinsically determined. Interactions between heterogeneous cell populations influence the growth and survival of both normal and malignant cells. Studies defining the origin of endothelial cells involved in tumor angiogenesis first demonstrated the contributions of normal cellular environment. Recently, the mononuclear phagocyte lineage has been found to have biologically and clinically significant tumor enhancing and tumor suppressive effects. This article reviews the multiple roles of mononuclear phagocytes in cancer biology. A companion manuscript (J Pediatr Hematol Oncol. 2008, in press) describes the targeting of these cells for therapeutic benefit. Incorporating these strategies into future childhood cancer protocols could be an innovative approach for improving patient outcome.

The Yeast Magmas Ortholog Pam16 Has an Essential Function in Fermentative Growth That Involves Sphingolipid Metabolism

Magmas is a growth factor responsive gene encoding an essential mitochondrial protein in mammalian cells. Pam16, the Magmas ortholog in Saccharomyces cerevisiae, is a component of the presequence translocase-associated motor. A temperature-sensitive allele (pam16-I61N) was used to query an array of non-essential gene-deletion strains for synthetic genetic interactions. The pam16-I61N mutation at ambient temperature caused synthetic lethal or sick phenotypes with genes involved in lipid metabolism, perixosome synthesis, histone deacetylation and mitochondrial protein import. The gene deletion array was also screened for suppressors of the pam16-I61N growth defect to identify compensatory pathways. Five suppressor genes were identified (SUR4, ISC1, IPT1, SKN1, and FEN1) and all are involved in sphingolipid metabolism. pam16-I61N cells cultured in glucose at non-permissive temperatures resulted in rapid growth inhibition and G1 cell cycle arrest, but cell viability was maintained. Altered mitochondria morphology, reduced peroxisome induction in glycerol/ethanol and oleate, and changes in the levels of several sphingolipids including C18 alpha-hydroxy-phytoceramide, were also observed in the temperature sensitive strain. Deletion of SUR4, the strongest suppressor, reversed the temperature sensitive fermentative growth defect, the morphological changes and the elevated levels of C18 alpha-hydroxy phytoceramide in pam16-I61N. Deletion of the other four suppressor genes had similar effects on C18 alpha-hydroxy-phytoceramide levels and restored proliferation to the pam16-I61N strain. In addition, pam16-I61N inhibited respiratory growth, likely by reducing cardiolipin, which is essential for mitochondrial function. Our results suggest that the pleiotropic effects caused by impaired Pam16/Magmas function are mediated in part by changes in lipid metabolism.

A Syndrome of Holoprosencephaly, Recurrent Infections, and Monocytosis

We describe three siblings with holoprosencephaly, recurrent infections, and increased peripheral blood monocytes. These children were born to apparently healthy parents in a family with one unaffected child. Affected individuals had microcephaly, severe developmental delay, failure to thrive, and brachydactyly. The clinical courses were complicated by endocrine dysfunction, multiple respiratory, and skin infections. Laboratory studies showed normal karyotypes, normal lymphocyte function, and a peripheral blood monocytosis with markedly abnormal morphology. Mutation analysis of the seven genes (SHH, ZIC2, SIX3, TGI, FTDGF1, GLI2, and PTCH) known to be involved in holoprosencephaly was normal. This is the first report demonstrating an association between abnormal mononuclear phagocytes and holoprosencephaly.