Jeane Silva

Selective apoptosis of pluripotent mouse and human stem cells by novel ceramide analogues prevents teratoma formation and enriches for neural precursors in ES cell–derived neural transplants

The formation of stem cell–derived tumors (teratomas) is observed when engrafting undifferentiated embryonic stem (ES) cells, embryoid body–derived cells (EBCs), or mammalian embryos and is a significant obstacle to stem cell therapy. We show that in tumors formed after engraftment of EBCs into mouse brain, expression of the pluripotency marker Oct-4 colocalized with that of prostate apoptosis response-4 (PAR-4), a protein mediating ceramide-induced apoptosis during neural differentiation of ES cells. We tested the ability of the novel ceramide analogue N-oleoyl serinol (S18) to eliminate mouse and human Oct-4(+)/PAR-4(+) cells and to increase the proportion of nestin(+) neuroprogenitors in EBC-derived cell cultures and grafts. S18-treated EBCs persisted in the hippocampal area and showed neuronal lineage differentiation as indicated by the expression of β-tubulin III. However, untreated cells formed numerous teratomas that contained derivatives of endoderm, mesoderm, and ectoderm. Our results show for the first time that ceramide-induced apoptosis eliminates residual, pluripotent EBCs, prevents teratoma formation, and enriches the EBCs for cells that undergo neural differentiation after transplantation.

Regulation of cell death in mitotic neural progenitor cells by asymmetric distribution of prostate apoptosis response 4 (PAR-4) and simultaneous elevation of endogenous ceramide

Cell death and survival of neural progenitor (NP) cells are determined by signals that are largely unknown. We have analyzed pro-apoptotic signaling in individual NP cells that have been derived from mouse embryonic stem cells. NP formation was concomitant with elevated apoptosis and increased expression of ceramide and prostate apoptosis response 4 (PAR-4). Morpholino oligonucleotide-mediated antisense knockdown of PAR-4 or inhibition of ceramide biosynthesis reduced stem cell apoptosis, whereas PAR-4 overexpression and treatment with ceramide analogs elevated apoptosis. Apoptotic cells also stained for proliferating cell nuclear antigen (a nuclear mitosis marker protein), but not for nestin (a marker for NP cells). In mitotic cells, asymmetric distribution of PAR-4 and nestin resulted in one nestin(−)/PAR-4(+) daughter cell, in which ceramide elevation induced apoptosis. The other cell was nestin(+), but PAR-4(−), and was not apoptotic. Asymmetric distribution of PAR-4 and simultaneous elevation of endogenous ceramide provides a possible mechanism underlying asymmetric differentiation and apoptosis of neuronal stem cells in the developing brain.

Lgi1 null mutant mice exhibit myoclonic seizures and CA1 neuronal hyperexcitability

LGI1 in humans is responsible for a predisposition to autosomal dominant partial epilepsy with auditory features (ADPEAF). However, mechanisms of how LGI1 mutations cause epilepsy remain unclear. We have used a mouse chromosome engineering strategy to create a null mutation for the gene ortholog encoding LGI1. The Lgi1 null mutant mice show no gross overall developmental abnormalities from routine histopathological analysis. After 12-18 days of age, the homozygous mutant mice all exhibit myoclonic seizures accompanied by rapid jumping and running and die shortly thereafter. The heterozygous mutant mice do not develop seizures. Electrophysiological analysis demonstrates an enhanced excitatory synaptic transmission by increasing the release of the excitatory neurotransmitter glutamate, suggesting a basis for the seizure phenotype. This mouse model, therefore, provides novel insights into the mechanism behind ADPEAF and offers a new opportunity to study the mechanism behind the role of LGI1 in susceptibility to myoclonic seizures.

Synthesis and characterization of novel ceramide analogs for induction of apoptosis in human cancer cells

A variety of anti-cancer drugs elevate endogenous ceramide, thereby inducing apoptosis in tumor cells. Recently, we have introduced novel ceramide analogs of the beta-hydroxy alkyl amide type, which trigger pro-apoptotic signaling pathways without prior elevation of endogenous ceramide. They induce apoptosis specifically in rapidly dividing neuroblastoma cells, but not in resting or differentiated cells. We characterize new ceramide mimics that have been derived from N-acylation of serinol (S), diethanolamine (B), propanolamine (P), and tris(hydroxy-methyl)methylamine (T) with myristic (14), palmitic (16), or oleic (18) acid. The water solubility of these compounds exceeds that of ceramide by more than 100-fold (up to 5 mM). Apoptosis of human neuroblastoma, glioma, medulloblastoma, and adenocarcinoma cells is induced by N-(2-hydroxy-1-(hydroxymethyl)ethyl)-palmitoylamide, C16-serinol (S16), N-(2-hydroxy-1-(hydroxymethyl)ethyl)-oleoylamide, C18-serinol (S18), N-bis(2-hydroxyethyl)-myristoyl-amide (B16), and N-tris(hydroxymethyl)methyl-oleoylamide (T18) within 60 min of incubation, and is completed even after removal of the compound from the medium. This is most likely due to a rapid uptake of the analogs followed by their slow release from the cells. Alteration of the acyl chain length to less than 14 methylene units, removal of the amino group, or reducing the number of hydroxyalkyl residues to less than two significantly lowers or eliminates the pro-apoptotic potential of these compounds. The target specificity of novel ceramide analogs for tumor cells, their water solubility, and fast pro-apoptotic mechanism indicates a high therapeutic potential for cancer treatment.

Long-chain ceramide is elevated in presenilin 1 (PS1M146V) mouse brain and induces apoptosis in PS1 astrocytes

The pro‐apoptotic sphingolipid ceramide plays an emergent role in the etiology of Alzheimers disease (AD), although its function for neurodegeneration is not known. We determined the concentration and composition of ceramide in hippocampal tissue from newborn presenilin 1 (PS1) knock‐in (PS1M146V) mice, a mouse model for early‐onset familial AD. We found that PS1 tissue contains 3.1 (±0.5)‐fold more total ceramide than wild‐type tissue. In particular, the proportion of C20 and C24 ceramide is increased by 4.0‐ or 8.5‐fold, respectively. The ceramide elevation in PS1 brain is consistent with a 3.7 (±0.5)‐fold increase of the protein level of the neurotrophin receptor p75NTR, which has been suggested to stimulate the hydrolysis of sphingomyelin to generate ceramide. The predominance of C20 and C24 ceramide is concurrent with the elevated gene expression of lass 2 and lass 4, two isoforms of ceramide synthase that generate dihydroceramide with long‐chain fatty acid. Our study indicates that primary cultured astrocytes but not neurons from PS1 mice undergo apoptosis when incubated with C20 ceramide. In contrast, wild‐type astrocytes remain unaffected. The sensitivity of PS1 astrocytes is most likely due to the 9.5 (±0.4)‐fold elevated expression of PAR‐4 (prostate apoptosis response‐4), a protein that inhibits atypical PKCζ/λ in the presence of ceramide. Our results suggest that astroglial death due to ceramide/PAR‐4‐induced apoptosis may critically contribute to the etiology of AD.

Lipids isolated from bone induce the migration of human breast cancer cells

Bone is the most common site to which breast cancer cells metastasize. We found that osteoblast-like MG63 cells and human bone tissue contain the bile acid salt sodium deoxycholate (DC). MG63 cells take up and accumulate DC from the medium, suggesting that the bone-derived DC originates from serum. DC released from MG63 cells or bone tissue promotes cell survival and induces the migration of metastatic human breast cancer MDA-MB-231 cells. The bile acid receptor farnesoid X receptor (FXR) antagonist Z-guggulsterone prevents the migration of these cells and induces apoptosis. DC increases the gene expression of FXR and induces its translocation to the nucleus of MDA-MB-231 cells. Nuclear translocation of FXR is concurrent with the increase of urokinase-type plasminogen activator (uPA) and the formation of F-actin, two factors critical for the migration of breast cancer cells. Our results suggest a novel mechanism by which DC-induced increase of uPA and binding to the uPA receptor of the same breast cancer cell self-propel its migration and metastasis to the bone.

Ceramide Regulates Atypical PKCζ/λ-mediated Cell Polarity in Primitive Ectoderm Cells A NOVEL FUNCTION OF SPHINGOLIPIDS IN MORPHOGENESIS

In mammals, the primitive ectoderm is an epithelium of polarized cells that differentiates into all embryonic tissues. Our study shows that in primitive ectoderm cells, the sphingolipid ceramide was elevated and co-distributed with the small GTPase Cdc42 and cortical F-actin at the apicolateral cell membrane. Pharmacological or RNA interference-mediated inhibition of ceramide biosynthesis enhanced apoptosis and impaired primitive ectoderm formation in embryoid bodies differentiated from mouse embryonic stem cells. Primitive ectoderm formation was restored by incubation with ceramide or a ceramide analog. Ceramide depletion prevented plasma membrane translocation of PKCζ/λ, its interaction with Cdc42, and phosphorylation of GSK-3β, a substrate of PKCζ/λ. Recombinant PKCζ formed a complex with the polarity protein Par6 and Cdc42 when bound to ceramide containing lipid vesicles. Our data suggest a novel mechanism by which a ceramide-induced, apicolateral polarity complex with PKCζ/λ regulates primitive ectoderm cell polarity and morphogenesis.

Novel FGFR inhibitor ponatinib suppresses the growth of non-small cell lung cancer cells overexpressing FGFR1.

Lung cancer is still the leading cause of cancer-related deaths worldwide. Identifying new oncogenic drivers and developing efficient inhibitors through molecular targeting approaches are crucial for improving therapies. The aim of this study was to investigate whether targeting fibroblast growth factor receptor 1 (FGFR1) with ponatinib inhibits the cell growth in both established and primary lung cancer cells overexpressing FGFR1. Eighty-eight non-small cell lung cancer (NSCLC) and paired normal tissue specimens were analyzed by real-time RT-PCR for FGFR1 gene expression. We identified four cell lines and two newly established primary lung cancer cultures that showed high FGFR1 expression levels, and evaluated the effect of the novel FGFR1 inhibitor ponatinib on cell growth. Approximately 50% (30 out of 59) NSCLC specimens expressed FGFR1>2-fold compared with their adjacent normal counterparts using quantitative RT-PCR. Ponatinib treatment of established NSCLC cell lines expressing higher levels of FGFR1 resulted in marked cell growth inhibition and suppression of clonogenicity. This growth inhibition was associated with inactivation of FGFR1 and its downstream targets. FGFR1 knockdown by shRNA achieved similar results when compared to treatment with ponatinib. Furthermore, ponatinib was able to significantly inhibit the growth of primary lung cancer cultures in vitro. Our data indicate that pharmacological inhibition of FGFR1 kinase activity with ponatinib may be effective for the treatment of lung cancer patients whose tumors overexpress FGFR1.

Nitric oxide enhances Ca2+-dependent K+ channel activity in rat carotid body cells

Abstract. The nitric oxide (NO) donor S-nitroso-acetylpenicillamine (SNAP) enhanced Ca2+-dependent K+ channel activity in rat carotid body chemoreceptor cells. Ca2+-dependent K+ channel activity was enhanced by SNAP in 38% (whole-cell configuration) and 67% (cell-attached mode) of the cells tested and was not affected by intracellular Ca2+ chelation with BAPTA-AM. Enhancement of Ca2+-dependent K+ channel activity by SNAP was blocked by the cGMP-dependent protein kinase G inhibitor 8-[(4-chlorophenyl)thio]-guanosine 3′,5′-cyclic monophosphothioate Rp diastereomer (Rp-8-pCPT-cGMPS). NO thus enhances Ca2+-dependent K+ channel activity through cGMP-dependent protein kinase G. The NO-mediated increase in Ca2+-dependent K+ channel activity is likely to alter the function of carotid body chemoreceptor cells and could explain the decreased chemosensitivity of the carotid body in response to NO released from efferent nerves or vascular endothelial cells.

Homozygous inactivation of the Lgi1 gene results in hypomyelination in the peripheral and central nervous systems

Mutations in the LGI1 gene in humans predispose to the development of autosomal dominant partial epilepsy with auditory features (ADPEAF). Homozygous inactivation of the Lgi1 gene in mice results in an epilepsy phenotype characterized by clonic seizures within 2–3 weeks after birth. Before onset of seizures, the 2–3‐week‐old null mutant mice show poor locomotor activity and neuromuscular strength. EM analysis of the sciatic nerve demonstrates impaired myelination of axons in the peripheral nervous system. Although heterozygous mutant mice do not show any locomotor phenotypes, they also demonstrate an intermediate level of hypomyelination compared with the wild‐type mice. Hypomyelination was also observed in the central nervous system, which, although relatively mild, was still significantly different from that of the wild‐type mice. These data suggest a role for LGI1 in the myelination functions of Schwann cells and oligodendrocytes.