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M E Sunday

Publications and source records attributed to M E Sunday.

At least 37 records · Page 2Linked to original sources
PubMed1997

Generation and characterization of mice lacking gastrin-releasing peptide receptor.

Gastrin-releasing peptide (GRP) is a mammalian bombesin-like peptide which is widely distributed in the central nervous system as well as in the gastrointestinal tract. GRP binds to its high affinity receptor (GRPR) to elicit a wide spectrum of biological effects on behavior, digestion, and metabolism. To define the in vivo function of GRPR, we generated GRPR null mutant mice by gene targeting. The intracerebroventricular administration of GRP caused hypothermia in wild-type mice, but not in mutant mice. The GRPR deficient mice showed significantly increased locomotor activity during the dark period, and social responses scored by sniffing, mounting, and approaching behaviors against an intruder. Aggressive scores such as fighting and biting were not altered in the mutant mice. These phenotypes were observed in mice generated from two independent ES cell clones and backcrossed to a C57BL/6J background. The GRPR deficient mice should be useful for studying the bombesin system in vivo.

Animals↗
PubMed1997

Differential screening of a human chromosome 3 library identifies hepatocyte growth factor-like/macrophage-stimulating protein and its receptor in injured lung. Possible implications for neuroendocrine cell survival.

Transient pulmonary neuroendocrine cell hyperplasia and non-neuroendocrine lung tumors develop in nitrosaminetreated hamsters, which we hypothesized might modulate epithelial cell phenotype by expressing gene(s) homologous to human chromosome 3p gene(s) deleted in small cell carcinoma of the lung (SCLC). We differentially screened a chromosome 3 library using nitrosamine-treated versus normal hamster lung cDNAs and identified hepatocyte growth factor-like/macrophage-stimulating protein (HGFL/MSP) in injured lung. HGFL/MSP mRNA is low to undetectable in human SCLC and carcinoid tumors, but the HGFL/MSP tyrosine kinase receptor, RON, is present and functional on many of these neuroendocrine tumors. In H835, a pulmonary carcinoid cell line, and H187, a SCLC cell line, HGFL/ MSP induced adhesion/flattening and apoptosis. Using viable cell counts to assess proliferation after 14 d of treatment with HGFL/MSP, there is growth inhibition of H835 but not H187. Nitrosamine-treated hamsters also demonstrate pulmonary neuroendocrine cell apoptosis in situ during the same time period as expression of the endogenous HGFL/ MSP gene, immediately preceding the spontaneous regression of neuroendocrine cell hyperplasia. These observations suggest that HGFL/MSP might regulate neuroendocrine cell survival during preneoplastic lung injury, which could influence the ultimate tumor cell phenotype.

Animals↗
PubMed1997

Comparison of the ontogeny of protein gene product 9.5, chromogranin A and proliferating cell nuclear antigen in developing human lung.

Pulmonary neuroendocrine cell products, especially bombesin-like peptides, are important modulators of fetal lung growth, morphogenesis and maturation. In the present study, we describe the ontogeny of protein gene product 9.5 (PGP 9.5) in 28 midtrimester human fetal lungs, in comparison to chromogranin A (CGA), a marker of differentiated neuroendocrine cells, and proliferating cell nuclear antigen (PCNA), which is expressed by actively dividing cells. PGP 9.5 immunostaining colocalized with CGA in many cells, although the peak abundance of PGP 9.5 preceded that of CGA by 4 to 6 weeks. In addition, a novel staining pattern was noted for PGP 9.5: diffuse cytoplasmic staining of undifferentiated epithelial cells, which was demonstrated by all of the airways before 15 weeks gestation. After gestational week 15, only the smallest airways demonstrated this pattern. PCNA immunostaining demonstrated age-dependent regional variation. All samples had approximately 25% epithelial cells immunopositive for PCNA. Between 11 and 14 weeks gestation over 50% of the mesenchymal cells were PCNA positive. This mesenchymal staining decreased after 14 weeks, and was rare by week 19. There was no definite correlation between the immunostaining for PGP 9.5 and that for PCNA, although PGP 9.5 positive cells were usually PCNA negative. These observations suggest that other growth factors produced by non-neuroendocrine epithelial cells also participate in lung development.

Chromogranin A↗
PubMed1996

An intrinsic adrenergic system in mammalian heart.

We have identified a previously undescribed intrinsic cardiac adrenergic (ICA) cell type in rodent and human heart. Northern and Western blot analyses demonstrated that ICA cell isolates contain mRNA and protein of enzymes involved in catecholamine biosynthesis. Radioenzymatic catecholamine assays also revealed that the catecholamine profile of adult rat ICA cell isolates differed from that of sympathetic neurons. Unlike sympathetic neuronal cells, isolated ICA cells have abundant clear vesicles on electron microscopy. Endogenous norepinephrine and epinephrine constitutively released by ICA cells in vitro affect the spontaneous beating rate of neonatal rat cardiac myocytes in culture. Finally, ICA cells could be identified in human fetal hearts at a developmental stage before sympathetic innervation of the heart has been documented to occur. These findings support the concept that these cells constitute an ICA signaling system capable of participating in cardiac regulation that appears to be independent of sympathetic innervation.

Adult↗
PubMed1996

Embryonic mouse lung epithelial progenitor cells co-express immunohistochemical markers of diverse mature cell lineages.

Developmental expression of marker genes representative of different mature cell types can be used to study differentiation of cell lineages. We used immunohistochemistry to study expression in developing mouse lung of calcitonin gene-related peptide (CGRP), Clara cell 10-KD protein (CC10), and surfactant protein-A (SP-A), markers that are differentially expressed in neuroendocrine cells, Clara cells, and Type II alveolar cells. Two distinct developmental phases were revealed. The earlier phase (embryonic days 13-15; E13-E15) was characterized by CGRP, CC10, and SP-A immunostaining in all epithelial cells of the distal airways, with the three patterns being virtually identical in adjacent sections. The later phase (E16-E18) was characterized by emergence of staining of the differentiated cell types. These expression patterns were recapitulated in serumless organ culture, demonstrating that information necessary to generate both phases of gene expression is present within the lung analage by E11. We conclude that CGRP, CC10, and SP-A are co-expressed in most or all cells of the distal lung epithelium at E13-E15 and later become restricted to different cell lineages. This transient expression in progenitor cells of gene products characteristic of diverse differentiated cell types may reflect an underlying mechanism of gene regulation.

Animals↗
PubMed1995

Glucose-dependent insulinotropic peptide (GIP) gene expression in the rat salivary gland.

Previous studies have indicated that following nutrient ingestion, GIP is released principally from the upper small intestine. In addition to its presence in the rat small intestine, GIP transcripts have also been localized to the submandibular salivary gland (SSG). The present studies were directed to further characterize expression of the GIP gene in the SSG. Pregnant rats were sacrificed at gestational days 18 and 20, followed by the removal of rat fetuses. The duodenum pancreas, and SSG were then excised from the fetuses, as well as from neonatal pups at ages 1, 3, 7, 10, 14, and 21 days. RNA was extracted and measured by Northern blot analysis using specific rat GIP probes. GIP transcripts were first detected in the duodenum in the 18-day fetus and reached maximum levels at birth. In contrast, GIP mRNA was not observed in the SSG until 10 days postnatally and was not detected at all in either the fetal or neonatal pancreas. In situ hybridization of the SSG using an 35S-labelled antisense GIP RNA probe demonstrated expression of the GIP gene to be limited to ductal cells, with no transcripts present in acini. In separate experiments, rats fasted overnight were given water or 10% glucose. While no changes were detected in water-fed rats following oral glucose ingestion, small, but significant increases in SSG GIP gene expression were detected at 60 and 240 min. The results of these initial studies suggest the possibility of a functional role for GIP in the rat salivary gland by the demonstration of GIP mRNA in the SSG by Northern analysis and in situ hybridization, as well as by an increase in SSG GIP gene expression following a glucose meal.

Animals↗
PubMed1995

Bombesin and [Leu8]phyllolitorin promote fetal mouse lung branching morphogenesis via a receptor-mediated mechanism.

Pulmonary neuroendocrine cells are localized predominantly at airway branchpoints. Previous work showed that gastrin-releasing peptide (GRP), a major pulmonary bombesin-like peptide, occurred in neuroendocrine cells exclusively in branching human fetal airways. We now demonstrate that GRP and GRP receptor genes are expressed in fetal mouse lung as early as embryonic day 12 (E12), when lung buds are beginning to branch. By in situ hybridization, GRP receptor transcripts were at highest levels in mesenchymal cells at cleft regions of branching airways and blood vessels. To explore the possibility that bombesin-like peptides might play a role in branching morphogenesis, E12 lung buds were cultured for 48 hr in serum-free medium. In the presence of 0.10-10 microM bombesin, branching was significantly augmented as compared with control cultures, with a peak of 94% above control values at 1 microM (P < 0.005). The bombesin receptor antagonist [Leu13- psi(CH2NH)Leu14]bombesin alone (100 nM) had no effect on baseline branching but completely abolished bombesin-induced branching. A bombesin-related peptide, [Leu8]phyllolitorin also increased branching (65% above control values at 10 nM, P < 0.005). [Leu8]Phyllolitorin also significantly augmented thymidine incorporation in cultured lung buds. Fibronectin, which is abundant at branchpoints, induces GRP gene expression in undifferentiated cell lines. These observations suggest that BLPs secreted by pulmonary neuroendocrine cells may contribute to lung branching morphogenesis. Furthermore, components of branchpoints may induce pulmonary neuroendocrine cell differentiation as part of a positive feedback loop, which could account in part for the high prevalence of these cells at branchpoints.

Animals↗
PubMed1995

Differential display RT-PCR for identifying novel gene expression in the lung.

The unique identity of each cell is the result of differential gene expression. A new strategy for differential cDNA screening introduced by Liang and Pardee utilizes anchored oligo-dT primers and random 5' oligonucleotide 10-mers to carry out polymerase chain reaction (PCR) on reverse-transcribed RNA from different cell populations. The amplified cDNAs are displayed on a standard sequencing gel as 100-500 bands per lane on the resulting autoradiograms and comparisons are drawn between the different cell populations. The major advantages of this method over previous differential screening approaches are its high sensitivity, the small amounts of tissue required, and the ability to carry out rapid mRNA analyses using total RNA and to test multiple tissues in parallel. Its limitations are the need for many primer combinations for adequate representation of mRNAs and the large number of bands displayed. A screening strategy should include multiple positive and negative control samples and Northern blots to identify cDNAs differentially expressed. This approach should facilitate the identification of many novel genes expressed in a variety of physiological and pathological conditions.

Animals↗
PubMed1995

Histochemical characterization of non-neuroendocrine tumors and neuroendocrine cell hyperplasia induced in hamster lung by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone with or without hyperoxia.

Lung tumors induced by 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK) with or without hyperoxia have frequent K-ras mutations but only rare p53 mutations, suggesting that this may be a model for non-small cell lung cancers. The goals of the present study were (1) to characterize the histopathology of lung tumors induced in hamsters by NNK with or without O2 and (2) as a corollary, to quantitate the pulmonary neuroendocrine cell hyperplasia in the different treatment groups early and late in the treatment period. Lung tumors induced by NNK with or without O2 were 71% adenomas, 22% adenocarcinomas, approximately 4% bronchoalveolar carcinomas, and approximately 4% squamous/adenosquamous carcinomas. One-half of all tumors were positive for the Clara cell antigen CC10 and 21% of NNK-induced tumors were mucin positive, compared with 2% of NNK/O2-induced tumors (P = 0.003). Immunostaining for PGP9.5 was positive in 5% of tumors induced by NNK alone, but in none of NNK/O2-induced tumors (P = 0.024). Abundant proliferating cell nuclear antigen occurred in 55% of NNK-induced tumors, compared with 19% of NNK/O2-induced tumors (P = 0.009). These data indicate that NNK with or without O2 induces non-neuroendocrine lung tumors. Hyperoxia appears to inhibit cell proliferation and suppress mucinous and partial neuroendocrine differentiation in some of these tumors.

Animals↗
PubMed1994

Modulation of oncogene and tumor suppressor gene expression in a hamster model of chronic lung injury with varying degrees of pulmonary neuroendocrine cell hyperplasia.

BACKGROUND: Intense pulmonary neuroendocrine cell (PNEC) hyperplasia occurs during preneoplastic lung injury in hamsters treated with diethylnitrosamine (DEN) plus hyperoxia. Alterations in oncogene and tumor suppressor gene expression during this process have not been explored. EXPERIMENTAL DESIGN: Our goals were: (a) to analyze expression of genes potentially involved in growth and differentiation of PNECs and/or nonneuroendocrine pulmonary epithelial cells (non-PNECs) in hamsters treated for up to 20 weeks with hyperoxia and DEN or the major tobacco-derived nitrosamine, 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK); and (b) as a corollary, to determine which cells were most mitotically active by immunostaining for c-myc and proliferating cell nuclear antigen. RESULTS: Immunohistochemical analyses demonstrated intense PNEC hyperplasia after treatment with either DEN/O2 or NNK/O2. Whereas DEN/O2-induced PNEC hyperplasia spontaneously regressed, NNK/O2-induced PNEC hyperplasia continued to increase up to 20 weeks. Rb transcripts were decreased similarly in lungs from all treatment groups (NNK/O2 = DEN/O2 = DEN alone) in spite of large differences in PNEC hyperplasia between these groups. c-myc was overexpressed in lungs from animals treated with NNK/O2, DEN/O2 and DEN alone, in which c-myc protein immunostaining occurred predominantly in non-PNECs. Proliferating cell nuclear antigen immunostaining confirmed that non-PNECs were most mitotically active. CONCLUSIONS: These data indicate that PNEC hyperplasia is primarily due to PNEC differentiation, suggesting that this model is ideal for studying mechanisms of neuroendocrine differentiation. Paracrine effects of PNEC-derived growth factors may then contribute to dysregulation of non-PNEC growth preceding the ultimate development of non-neuroendocrine lung tumors in nitrosamine-treated hamsters.

Animals↗
PubMed1993

Anti-bombesin monoclonal antibodies modulate fetal mouse lung growth and maturation in utero and in organ cultures.

Fetal pulmonary neuroendocrine cells (PNECs) contain abundant gastrin-releasing peptide (GRP, mammalian bombesin-like peptide [BLP]). Previously, addition of bombesin resulted in increased fetal lung growth and maturation in utero and in organ cultures. A monoclonal antibody (mAb) to bombesin (2A11) blocked baseline automaturation of lung organ cultures in serum-free medium. In the present study, we analyze lung development following daily in utero administration of 2A11 from gestational days 15-18. Fetal lung treated with 2A11 and then harvested on day 18 demonstrated a dose-dependent decrease in surfactant phospholipid synthesis compared to controls treated with MOPC, an unreactive mAb. However, 2A11-treated fetal lung harvested on day 17 showed paradoxical increases in 3H-choline incorporation into saturated phosphatidylcholine, 3H-thymidine incorporation into DNA, and relative numbers of differentiated type II pneumocytes. In serum-containing day 17 lung organ cultures, 2A11 stimulated choline and thymidine incorporation. Since epidermal growth factor (EGF) is the only agent besides bombesin known to stimulate both fetal lung growth and maturation, we added EGF to serum-free cultures and reconstituted the stimulatory effects. A murine EGF receptor mAb (ERA) blocked 2A11-induced lung growth and maturation in serum-containing cultures, and this effect was overcome by adding EGF. In vivo, ERA also blocked stimulatory effects of 2A11 in fetal lung on day 17. These observations suggest that EGF receptor up-regulation may maintain lung growth and maturation if BLP levels are diminished on day 17. Nonetheless, BLPs appear to be involved in lung maturation on day 18, supporting a role for PNECs in normal lung development.

Animals↗
PubMed1993

CD10/neutral endopeptidase 24.11 regulates fetal lung growth and maturation in utero by potentiating endogenous bombesin-like peptides.

Bombesin-like peptides (BLPs) are mitogens for bronchial epithelial cells and small cell lung carcinomas, and increase fetal lung growth and maturation in utero and in organ cultures. BLPs are hydrolyzed by the enzyme CD10/neutral endopeptidase 24.11 (CD10/NEP) which is expressed in bronchial epithelium and functions to inhibit BLP-mediated growth of small cell lung carcinomas. To determine whether CD10/NEP regulates peptide-mediated lung development, we administered a specific CD10/NEP inhibitor, SCH32615, to fetal mice in utero from gestational days e15-17. Fetal lung tissues were evaluated on e18 for: (a) growth using [3H]thymidine incorporation into nuclear DNA; and (b) maturation using: [3H]-choline incorporation into surfactant phospholipids, electron microscopy for type II pneumocytes, and Northern blot analyses for surfactant apoproteins A, B, and C. Inhibition of CD10/NEP stimulated [3H]thymidine incorporation into DNA (70% above baseline, P < 0.005), [3H]choline incorporation into surfactant phospholipids (38% above baseline, P < 0.005), increased numbers of type II pneumocytes (36% above baseline, P = 0.07), and fivefold higher surfactant protein A transcripts (P < 0.05). CD10/NEP-mediated effects were completely blocked by the specific bombesin receptor antagonist, [D-Phe12, Leu14]bombesin. These observations suggest that CD10/NEP regulates fetal lung growth and maturation mediated by endogenous BLPs.

Animals↗
PubMed1992

Induction and spontaneous regression of intense pulmonary neuroendocrine cell differentiation in a model of preneoplastic lung injury.

Pulmonary neuroendocrine cell (PNEC) hyperplasia is associated with chronic lung diseases in humans, where it is thought to play a role in reparative responses to lung injury. To investigate the kinetics of strongly induced PNEC hyperplasia in an animal model, we exposed hamsters to a combination of hyperoxia (60% O2) and diethylnitrosamine (DEN) for up to 20 weeks. We thus demonstrate not only the induction but also spontaneous regression of intense PNEC differentiation and growth, which are much more intense than those observed with DEN alone. Lung tissues were immunostained for serotonin, calcitonin gene-related peptide (CGRP), calcitonin (CT), and gastrin-releasing peptide (GRP) (mammalian bombesin). Between 9 and 12 weeks of treatment, the number of CGRP- and serotonin-positive neuroepithelial bodies per cm airway epithelium increased over 10-fold, and CT became detectable. The number of neuroepithelial bodies immunostained for CGRP, serotonin, and CT peaked at 12-14 weeks of treatment, thereafter regressing to near-control levels by 20 weeks, in spite of continued DEN/O2 treatment. Simultaneously, by 6-7 weeks of treatment, there was a significant increase in the mean number of CGRP-positive cells per neuroepithelial body, which continued to rise up to double control levels, with a plateau at 13-20 weeks. GRP and pro-GRP immunostaining were not detectable at any time point. Polymerase chain reaction analyses of neuroendocrine-specific mRNAs demonstrated that CGRP, CT, and GRP mRNAs (normalized for beta-actin) peaked in lung tissues from most animals at 9-14 weeks after the beginning of DEN/O2 treatment, with decreased expression at 16-20 weeks. These data suggest that regulation of levels of these neuropeptides may be primarily transcriptional. This model may be a valuable system for analyzing mechanisms of induction and regression of normal PNEC differentiation and growth.

Animals↗
PubMed1992

CD10/neutral endopeptidase 24.11 in developing human fetal lung. Patterns of expression and modulation of peptide-mediated proliferation.

The cell membrane-associated enzyme CD10/neutral endopeptidase 24.11 (CD10/NEP) functions in multiple organ systems to downregulate responses to peptide hormones. Recently, CD10/NEP was found to hydrolyze bombesin-like peptides (BLP), which are mitogens for normal bronchial epithelial cells and small cell lung carcinomas. Growth of BLP-responsive small cell lung carcinomas was potentiated by CD10/NEP inhibition, implicating CD10/NEP in regulation of BLP-mediated tumor growth. BLP are also likely to participate in normal lung development because high BLP levels are found in fetal lung, and bombesin induces proliferation and maturation of human fetal lung in organ cultures and murine fetal lung in utero. To explore potential roles for CD10/NEP in regulating peptide-mediated human fetal lung development, we have characterized temporal and cellular patterns of CD10/NEP expression and effects of CD10/NEP inhibition in organ cultures. Peak CD10/NEP transcript levels are identified at 11-13 wk gestation by Northern blots and localized to epithelial cells and mesenchyme of developing airways by in situ hybridization. CD10/NEP immunostaining is most intense in undifferentiated airway epithelium. In human fetal lung organ cultures, inhibition of CD10/NEP with either phosphoramidon or SCH32615 increases thymidine incorporation by 166-182% (P < 0.025). The specific BLP receptor antagonist, [Leu13-psi(CH2NH)Leu14]bombesin abolishes these effects on fetal lung growth, suggesting that CD10/NEP modulates BLP-mediated proliferation. CD10/NEP expression in the growing front of airway epithelium and the effects of CD10/NEP inhibitors in lung explants implicate the enzyme in the regulation of peptide-mediated fetal lung growth.

Cell Differentiation↗
PubMed1992

Isoform-specific thyroid hormone receptor antibodies detect multiple thyroid hormone receptors in rat and human pituitaries.

There are three known isoforms of the thyroid hormone receptor (TR) in the rat: TR alpha-1, TR beta-1, and TR beta-2. The TR alpha-1 and TR beta-1 mRNAs are found in many tissues, whereas TR beta-2 mRNA is detected only in the pituitary gland. Thus far, TR alpha-1 and TR beta-1 mRNAs have been found in humans and are highly homologous to their counterparts in rats; however, TR beta-2 mRNA has not yet been demonstrated in humans. To examine the expression of these TRs at the protein level, we have raised isoform-specific polyclonal antibodies in female New Zealand White rabbits against the rat TRs and c-erbA alpha-2, a carboxy-terminal variant of TR alpha-1 that does not bind thyroid hormone. The rabbits were immunized with synthetic peptides that contained the following amino acid sequences: TR alpha-common-(10-31), c-erbA alpha-2-(428-442), TR beta-1-(73-93), and TR beta-2-(86-101, 113-133). All immune sera could bind specifically to their respective immunizing peptides on enzyme-linked immunosorbent assay as well as immunoprecipitate specifically in vitro translated rat and human TRs. Anti-TR beta-1 and anti-TR alpha-common antibodies could immunoprecipitate TR beta-1 or TR alpha-1, respectively, in transfected COS-7 cells. We also immunostained normal adult rat and human pituitary glands. Each isoform-specific antibody could immunostain almost all of the anterior pituitary cells, suggesting that TR alpha-1, TR beta-1, TR beta-2, and c-erbA alpha-2 are most likely expressed in all anterior pituitary cell types in rats and humans. The staining of rat pituitary glands by the anti-TR beta-2 antibodies demonstrates for the first time that TR beta-2 is expressed as a protein in pituitary cells. Furthermore, the staining of human pituitary glands by the anti-TR beta-2 antibodies suggests that there is a human homolog of the rat pituitary-specific TR beta-2 that shares similar epitopes with the rat TR beta-2. In summary, we have prepared isoform-specific antibodies against TRs that can recognize in vitro translated, transiently transfected, and in situ rat and human pituitary TRs. These antibodies will be useful in examining tissue- and cell type-specific expression of rat and human TRs at the protein level.

Animals↗