Methods to study neuroepithelial bodies as airway oxygen sensors.
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Animals↗
Biomedical subjects
H Yeger
Publications and source records attributed to H Yeger.
In vitro cytoprotective activity of squalene on a bone marrow versus neuroblastoma model of cisplatin-induced toxicity. implications in cancer chemotherapy.
The development of a non-toxic selective cytoprotective agent that preferentially protects normal tissues from chemotherapy toxicity, without protecting malignant tissues, is a major challenge in cancer chemotherapy research. The available cytoprotective agents are either toxic or lack selective cytoprotective activity. Here, we report the in vitro selective cytoprotective activity of squalene, an isoprenoid molecule with antioxidant properties. Normal human bone marrow (BM) derived colony-forming unit (CFU) growth was increased by squalene in a dose-dependent manner. Squalene (12.5-25 microM) treatment significantly protected the CFUs from cisplatin-induced toxicity; the protective effect was equivalent to reduced glutathione (GSH), a known cytoprotective agent. Squalene also increased the long-term survival of cisplatin-treated 4-week-old CFUs. Cisplatin-induced apoptosis of CFUs as measured by the TUNEL assay was reduced by squalene. To examine the squalene-induced protection of tumours, several neuroblastoma cell lines, including five MYCN-amplified cell lines, were grown in monolayers, as well as in anchorage-independent cultures, in the presence of squalene and cisplatin. Squalene did not protect the neuroblastoma (NBL) cell lines from cisplatin-induced toxicity. In addition, squalene did not protect the NBL cells from carboplatin, cyclophosphamide, etoposide and doxorubicin-induced toxicity. In conclusion, our results suggest that squalene has a selective in vitro cytoprotective effect on BM-derived haematopoietic stem cells that is equipotent to GSH.
Antineoplastic Agents↗
Proposal for a unified CCN nomenclature.
A proposal is put forth to unify the nomenclature of the CCN family of secreted, cysteine rich regulatory proteins. In the order of their description in the literature, CCN1 (CYR61), CCN2 (CTGF), CCN3 (NOV), CCN4 (WISP-1), CCN5 (WISP-2), and CCN6 (WISP-3) constitute a family of matricellular proteins that regulate cell adhesion, migration, proliferation, survival, and differentiation, at least in part through integrin mediated mechanisms. This proposal is endorsed by the International CCN Society and will serve to eliminate confusion from the multiple names that have been given to these molecules.
Connective Tissue Growth Factor↗
Differential expression of E-cadherin and beta catenin in primary and metastatic Wilms's tumours.
BACKGROUND: The E-cadherin-catenin adhesion complex is crucial for intercellular adhesiveness and maintenance of tissue architecture. Its impairment is associated with poorly differentiated phenotype and increased invasiveness of carcinomas. AIMS: To evaluate E-cadherin, beta catenin, gamma catenin, and ezrin expression and its relation to histopathological features of primary and metastatic Wilms's tumours. METHODS: Immunohistochemistry was used to determine the expression and cellular distribution of E-cadherin, beta catenin, gamma catenin, and ezrin in primary and metastatic Wilms's tumours. Western blotting was used to determine polypeptide size and expression of E-cadherin and beta catenin in Wilms's tumours compared with normal kidney. RESULTS: Moderate expression of E-cadherin was found mainly in cytoplasm and occasionally cell membranes of dysplastic tubules, whereas low expression was seen in cytoplasm of blastemal cells. Primary and metastatic tumours showed moderate to high beta catenin expression in blastemal and epithelial cells, with predominantly membranous and cytoplasmic staining. Occasional nuclear staining was noted in metastatic tumours. Low to high gamma catenin and ezrin expression was seen in cytoplasm of blastemal and epithelial cells of primary and metastatic tumours. Higher amounts of 92 kDa beta catenin were detected in tumours than in normal kidney. Low expression of 120 kDa E-cadherin was seen in moderately differentiated tumours, whereas expression was lacking in poorly differentiated tumours. CONCLUSIONS: Compared with primary tumours, metastatic tumours showed lower expression of E-cadherin and gamma catenin, with nuclear staining for beta catenin. Low E-cadherin was associated with poorly differentiated tumours. These results suggest that abnormal expression of adhesion proteins correlates with the invasive and metastatic phenotype in Wilms's tumours.
Analysis of Variance↗
Expression of the human NOV gene in first trimester fetal tissues.
NOV, located on human chromosome 8q24.1, was originally cloned following discovery of its avian homolog as a consequence of over-expression in virally induced nephroblastoma. The gene product is a secreted, modular, protein and a member of the CCN gene family. Evidence to date indicates that the expression of the wild type protein is associated with cellular quiescence in normal embryonic fibroblasts yet produces growth stimulatory effects on established murine NIH 3T3 cells. Here we report the expression of NOV in the first trimester of human embryogenesis, between 5 and 10 weeks. In situ hybridisation and immunohistochemistry reveal widespread expression in derivatives of all three germ layers. The most abundant sites of expression are in the motor neurons and floor plate of the spinal cord, adrenal cortex, fusing skeletal, and smooth muscle, the urogenital system and the developing heart. Additionally, expression is seen in the cranial ganglia, differentiating chondrocytes, gonads, and lung. The sites of expression suggest strongly that autocrine or paracrine expression of NOV is associated with the process of cell differentiation.
Adrenal Cortex↗
Respiratory control in neonatal mice with NADPH oxidase deficiency.
A membrane bound cytochrome b(558) (NADPH oxidase) is a candidate for the oxygen sensor in pulmonary neuroepithelial bodies (NEBs) - putative airway chemoreceptors. Recent electrophysiological studies on NEB from mice with NADPH oxidase deficiency (OD; gp(91phox) knock-out) have shown lack of response of O(2) sensitive K(+) current to hypoxia challenge compared with wild-type (WT) control mice. To assess the effects of oxidase deficiency on the control of ventilation at the whole animal level, respiratory measurements were conducted under normoxic and hypoxic conditions in neonatal OD mice and compared that with the WT control group. Five-day-old OD mice were faster and shallower breathers during normoxia as well as hypoxia. In addition, the maximum hypoxic ventilatory response of the OD mice was lower than that of the WT control group and the peak increase in minute ventilation (.V(max)-.V(normoxia)) was greater in WT control than the OD mice (P=0.02). Since the lung development and NEB morphology in OD mice were comparable to the WT control mice, the observed differences implicate NADPH oxidase as an O(2) sensor involved in neonatal ventilatory control, possibly modulated via pulmonary NEBs.
Animals↗
Report and abstracts on the first international workshop on the CCN family of genes.
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Animals↗
Expression of CFTR and Cl(-) conductances in cells of pulmonary neuroepithelial bodies.
The pulmonary neuroendocrine cell system comprises solitary neuroendocrine cells and clusters of innervated cells or neuroepithelial bodies (NEBs). NEBs figure prominently during the perinatal period when they are postulated to be involved in physiological adaptation to air breathing. Previous studies have documented hyperplasia of NEBs in cystic fibrosis (CF) lungs and increased neuropeptide (bombesin) content produced by these cells, possibly secondary to chronic hypoxia related to CF lung disease. However, little is known about the role of NEBs in the pathogenesis of CF lung disease. In the present study, using a panel of cystic fibrosis transmembrane conductance regulator (CFTR)-specific antibodies and confocal microscopy in combination with RT-PCR, we demonstrate expression of CFTR message and protein in NEB cells of rabbit neonatal lungs. NEB cells expressed CFTR along with neuroendocrine markers. Confocal microscopy established apical membrane localization of the CFTR protein in NEB cells. Cl(-) conductances corresponding to functional CFTR were demonstrated in NEB cells in a fresh lung slice preparation. Our findings suggest that NEBs, and related neuroendocrine mechanisms, likely play a role in the pathogenesis of CF lung disease, including the early stages before establishment of chronic infection and chronic lung disease.
Animals↗
Differential sensitivity of various pediatric cancers and squamous cell carcinomas to lovastatin-induced apoptosis: therapeutic implications.
3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase is the rate-limiting enzyme of the mevalonate pathway, the diverse array of end products of which are vital for a variety of cellular functions, including cholesterol synthesis and cell cycle progression. We showed previously that this enzyme holds a critical role in regulating tumor cell fate, including cell death, as its expression is down-regulated in response to retinoic acid, a potent anticancer therapeutic. Indeed, direct inhibition of HMG-CoA reductase with lovastatin, a competitive inhibitor of this enzyme, induced a pronounced apoptotic response in neuroblastoma and acute myeloid leukemic cells. We have now extended this work and evaluated a wide variety and large number of tumor-derived cell lines for their sensitivity to lovastatin-induced apoptosis. These cell lines were exposed to a wide range (0-100 microM) of lovastatin for 2 days and assayed for cell viability using the 3,4,5-dimethyl thiazlyl-2,2,5-diphenyltetrazolium bromide assay and the induction of apoptosis by flow cytometric and ultrastructural analyses. Lovastatin induced a pronounced apoptotic response in cells derived from juvenile monomyelocytic leukemia, pediatric solid malignancies (rhabdomyosarcoma and medulloblastoma), and squamous cell carcinoma of the cervix and of the head and neck. Interestingly, the subset of malignancies that are particularly sensitive to lovastatin-induced apoptosis correspond to those tumor subtypes that are sensitive to the biological and antiproliferative effects of retinoids in vitro. The nature of the biologically active form of lovastatin has been challenged recently as the growth-inhibitory effects of this drug were attributed to its prodrug lactone form that does not inhibit HMG-CoA reductase function. In this report, we demonstrate that the apoptotic properties of lovastatin are triggered by the open ring acid form that is a potent inhibitor of HMG-CoA reductase activity. Thus, we have identified a subset of tumors that are sensitive to lovastatin-induced apoptosis and show HMG-CoA reductase as a potential therapeutic target of these cancers.
Adult↗
Selection of viable cardiomyocytes for cell transplantation using three-dimensional tissue culture.
BACKGROUND: We developed a three-dimensional cardiac tissue culture system to select viable and contractile cells for the purpose of cardiomyocyte transplantation. In this study we will show that reaggregation of cardiomyocytes in culture is an active process indicative of cardiomyocyte viability and functionality. METHODS: Myocardial tissue from newborn mice has been enzymatically digested, incubated in culture inserts, and studied by phase contrast microscopy, conventional histology, immunohistochemistry, electron microscopy, and TUNEL assay. RESULTS: Cells that are plated on nonadhesive surfaces, reaggregate to spontaneously contracting cell aggregates. The capacity to reaggregate was significantly dependent from the age of the tissue donor (P<0.0001) and on the method of enzymatic dissociation (P<0.0001). The majority of cells within the aggregates consisted of cardiomyocytes. After 24 hr incubation, significant amounts of laminin and fibronectin had been deposited between the cells. Ultrastructural analysis revealed viable cardiomyocytes attached to each other by tight junctions. The apoptotic rate within the aggregates was 11.4+/-4.6 vs. 44.5+/-10.5% immediately after dissociation (P<0.05). CONCLUSIONS: The capacity to form spontaneously contracting aggregates is an inherent characteristic of viable cardiomyocytes in 3-dimensional cultures, which could be successfully exploited for cellular cardiomyocyte transplantation.
Animals↗
The risk of fragile X premutation expansion is lower in carriers detected by general prenatal screening than in carriers from known fragile X families.
The Fragile X syndrome is the most common cause of inherited mental retardation. For a female premutation carrier, the risk of having a child with a full mutation is positively correlated with the size of the premutation. The current study was performed to evaluate the risk of premutation expansion in the offspring of average-risk carriers detected by general prenatal screening. Over a 4-year period, 9,660 women underwent DNA screening for FMR1 mutation/premutation at the Tel Aviv Sourasky Medical Center. A premutation was defined as a CGG repeat number >50 in the 5' untranslated region (UTR) of exon 1 in the FMR1 gene. The study included only individuals with no family history of X-linked mental retardation or known FMR1 mutations. A premutation was found in 85 women (1 in 114), 68 of whom consented to have prenatal diagnoses in 74 pregnancies. The abnormal allele was transmitted to the offspring in 44 pregnancies. Of these, no change in allele size was noted in 35 pregnancies (79.6%), and expansion within premutation range was evident in 4 pregnancies (9%). In 5 pregnancies (11.4%), expansion to the full mutation was noted. This occurred only in carriers having more than 90 repeats. We conclude that the likelihood of Fragile X premutation expansion to full mutation is significantly lower in individuals ascertained by general prenatal carrier testing than in those from known Fragile X families.
Female↗
Expression of the chloride channel ClC-2 in the murine small intestine epithelium.
The chloride channel ClC-2 has been implicated in neonatal airway chloride secretion. To assess its role in secretion by the small intestine, we assessed its subcellular expression in ileal segments obtained from mice and studied the chloride transport properties of this tissue. Chloride secretion across the mucosa of murine ileal segments was assessed in Ussing chambers as negative short-circuit current (I(sc)). If ClC-2 contributed to chloride secretion, we predicted on the basis of previous studies that negative I(sc) would be stimulated by dilution of the mucosal bath and that this response would depend on chloride ion and would be blocked by the chloride channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid but not by DIDS. In fact, mucosal hypotonicity did stimulate a chloride-dependent change in I(sc) that exhibited pharmacological properties consistent with those of ClC-2. This secretory response is unlikely to be mediated by the cystic fibrosis transmembrane conductance regulator (CFTR) channel because it was also observed in CFTR knockout animals. Assessment of the native expression pattern of ClC-2 protein in the murine intestinal epithelium by confocal and electron microscopy showed that ClC-2 exhibits a novel distribution, a distribution pattern somewhat unexpected for a channel involved in chloride secretion. Immunolabeled ClC-2 was detected predominantly at the tight junction complex between adjacent intestinal epithelial cells.
4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
Epithelial Na(+) channel (ENaC) expression in the developing normal and abnormal human perinatal lung.
Impaired lung epithelial Na(+) channel (ENaC) activity at the time of birth results in respiratory distress. To investigate potential mechanisms, the ontogeny and cellular distribution of the alphaENaC subunit mRNA expression was studied in normal, immature, and abnormal (hypoplastic) human fetal lungs using nonradioisotopic in situ hybridization. Surprisingly, alphaENaC expression was detected at the embryonic stage of normal lung development (4 to 5 wk gestation) when expression was localized to the fetal lung bud epithelium. By late gestation, ENaC was expressed in the conductive and respiratory airway epithelium, serous cells, and the distal lung unit in an alveolar type II (ATII) epitheliumlike distribution. Significant alphaENaC expression was found in newborn lung diseases associated with respiratory distress. One explanation is that alphaENaC mRNA is constitutively expressed, and that activity is regulated, at least in part, at the post-transcriptional level. Alternative explanations are that the expression of the beta or gammaENaC subunits may be impaired in certain newborn lung diseases or that alternate Na(+) permeant channels or transporters are important to lung liquid absorption in humans at birth.
Biological Transport, Active↗
Glypican-3-deficient mice exhibit developmental overgrowth and some of the abnormalities typical of Simpson-Golabi-Behmel syndrome.
Glypicans are a family of heparan sulfate proteoglycans that are linked to the cell surface through a glycosyl-phosphatidylinositol anchor. One member of this family, glypican-3 (Gpc3), is mutated in patients with the Simpson-Golabi-Behmel syndrome (SGBS). These patients display pre- and postnatal overgrowth, and a varying range of dysmorphisms. The clinical features of SGBS are very similar to the more extensively studied Beckwith-Wiedemann syndrome (BWS). Since BWS has been associated with biallelic expression of insulin-like growth factor II (IGF-II), it has been proposed that GPC3 is a negative regulator of IGF-II. However, there is still no biochemical evidence indicating that GPC3 plays such a role.Here, we report that GPC3-deficient mice exhibit several of the clinical features observed in SGBS patients, including developmental overgrowth, perinatal death, cystic and dyplastic kidneys, and abnormal lung development. A proportion of the mutant mice also display mandibular hypoplasia and an imperforate vagina. In the particular case of the kidney, we demonstrate that there is an early and persistent developmental abnormality of the ureteric bud/collecting system due to increased proliferation of cells in this tissue element. The degree of developmental overgrowth of the GPC3-deficient mice is similar to that of mice deficient in IGF receptor type 2 (IGF2R), a well characterized negative regulator of IGF-II. Unlike the IGF2R-deficient mice, however, the levels of IGF-II in GPC3 knockouts are similar to those of the normal littermates.
Abnormalities, Multiple↗
Identification of a novel zinc finger gene, zf5-3, as a potential mediator of neuroblastoma differentiation.
We established a unique parental neuroblastoma cell line, NUB-7, which mimics the bipotentiality of neuroblastoma in vivo along neuronal and Schwann cell lineages following dibutyryl cAMP and retinoic acid treatments, respectively. Differential display identified a putative novel zinc finger gene as a potential differentiation-responsive gene coincident with retinoic acid treatment of NUB-7. This cDNA clone, now designated zf5-3, was mapped to chromosome 19 using somatic cell hybrids, and a larger cDNA clone further localized this gene to band 13.1-13.2 by fluorescent in situ hybridization. zf5-3 possesses 4 characteristic zinc finger DNA-binding motifs as determined by its nucleic acid and proposed amino acid sequence. Expression of zf5-3 is restricted to fetal neuronal, hepatic and renal tissues and their tumor-derived cell lines, including 8/9 neuroblastomas and 2/2 malignant rhabdoid tumors of kidney. The restricted expression in the kidney of zf5-3 to collecting tubules and ureter epithelium is suggestive of an ectodermal histogenesis of malignant rhabdoid tumors of kidney. During development of the fetal human brain, high levels of zf5-3 mRNA are restricted to the mitotically active, undifferentiated neuroblasts. Morphological evidence of overt differentiation was generally accompanied by a marked loss in zf5-3 expression. Therefore, the neuronal tissue expression profile and the down-regulation coincident with retinoic acid-induced neuroblastoma maturation implicate zf5-3 as a potential mediator of their differentiation.
Amino Acid Sequence↗
Increased sensitivity of acute myeloid leukemias to lovastatin-induced apoptosis: A potential therapeutic approach.
We recently demonstrated that 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme of de novo cholesterol synthesis, was a potential mediator of the biological effects of retinoic acid on human neuroblastoma cells. The HMG-CoA reductase inhibitor, lovastatin, which is used extensively in the treatment of hypercholesterolemia, induced a potent apoptotic response in human neuroblastoma cells. This apoptotic response was triggered at lower concentrations and occurred more rapidly than had been previously reported in other tumor-derived cell lines, including breast and prostate carcinomas. Because of the increased sensitivity of neuroblastoma cells to lovastatin-induced apoptosis, we examined the effect of this agent on a variety of tumor cells, including leukemic cell lines and primary patient samples. Based on a variety of cytotoxicity and apoptosis assays, the 6 acute lymphocytic leukemia cell lines tested displayed a weak apoptotic response to lovastatin. In contrast, the majority of the acute myeloid leukemic cell lines (6/7) and primary cell cultures (13/22) showed significant sensitivity to lovastatin-induced apoptosis, similar to the neuroblastoma cell response. Of significance, in the acute myeloid leukemia, but not the acute lymphocytic leukemia cell lines, lovastatin-induced cytotoxicity was pronounced even at the physiological relevant concentrations of this agent. Therefore, our study suggests the evaluation of HMG-CoA reductase inhibitors as a therapeutic approach in the treatment of acute myeloid leukemia.
Acute Disease↗
Lovastatin-induced apoptosis of human medulloblastoma cell lines in vitro.
Medulloblastoma is a malignant paediatric central nervous system tumor with a poor prognosis, stimulating the evaluation of improved treatment strategies. Lovastatin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, is currently used to treat patients with hypercholesterolemia. This compound also inhibits the production of non-steroidal mevalonate derivatives that are implicated in the control of cellular proliferation, and can induce cell-cycle arrest in vitro. We recently showed that lovastatin inhibited growth and promoted apoptosis of neuroblastoma, the peripheral nervous system 'cousin' of medulloblastoma. Therefore the potential of lovastatin as a possible anticancer drug against medulloblastoma was evaluated in vitro. Four medulloblastoma cell lines, Daoy, UW228, D341 Med and D283 Med, were treated with 1-40 microM of lovastatin in vitro. Analysis of cell morphologic changes, cell viability, DNA fragmentation and flow cytometry in all four cell lines showed growth inhibition and induction of apoptosis with lovastatin treatment. As little as 10 microM of lovastatin was sufficient to cause a marked reduction in cell numbers, and more than 20 microM of lovastatin induced >90% cells to undergo apoptosis, after intervals ranging between 36 and 96 h, depending on the cell line. Lovastatin induced apoptosis in these cell lines was concomitant with cell cycle arrest in G1. The attached cell lines UW228 and Daoy were more sensitive to lovastatin than D283 Med and D341 Med. Daoy cells which survived several cycles of lovastatin treatment could still be induced to undergo apoptosis after longer treatment times. The efficient induction of apoptosis by lovastatin favours this drug as a potential new avenue of therapeutic intervention for medulloablastoma.
Apoptosis↗
Characterization and distribution of an oncofetal antigen (M2A antigen) expressed on testicular germ cell tumours.
M2A antigen is an oncofetal antigen associated with germ cell neoplasia, present in testis on fetal gonocytes and re-expressed on carcinoma in situ (CIS) and germ cell tumours. We developed a panel of monoclonal antibodies (mAb), M2A (IgG2a), D1-26 (IgG2b) and D2-40 (IgG1), to this antigen in order to characterize its structure and study its distribution among germ cell tumours. M2A antigen was purified by sequential lectin and antibody affinity chromatography and characterized as a monomeric M, 40 000 surface sialoglycoprotein, extensively glycosylated with O-linked carbohydrate structures, but devoid of N-linked sugars. Terminal sialic acid residues were required for reactivity with mAb M2A and D1-26, but not D2-40. Sections of 69 testicular germ cell tumours, fixed in formalin and embedded in paraffin, were stained with mAb D2-40 to examine the distribution of M2A antigen. Uniform membrane staining was observed in seminomas, and focal staining in 69% of embryonal carcinomas, 29% of teratomas and 25% of yolk sac tumours. CIS in the vicinity of all germ cell tumours also displayed uniform membrane staining. The characterization of M2A antigen, and the development of mAb which react with it in conventionally preserved archival specimens, provide important initiatives to study the origin and progression of germ cell neoplasia.
Animals↗