Biomedical subjects

F Cuttitta

Publications and source records attributed to F Cuttitta.

At least 55 records · Page 3Linked to original sources

PubMed1998

Coordinate expression of transforming growth factor-beta1 and adrenomedullin in rodent embryogenesis.

Transforming growth factor-beta (TGFbeta) and adrenomedullin (AM) are multifunctional regulatory peptides that are secreted by a variety of normal and malignant cells. The TGFbetas are expressed in developing organs and adults, and their tissue distribution pattern has possible significance for signaling roles in many epithelial-mesenchymal interactions. AM is also expressed in a variety of embryonic and adult tissues. The present study reports a comparison of the patterns of expression of the proteins and messenger RNAs (mRNAs) for TGFbeta1 and AM in the developing mouse embryo. Immunohistochemical and in situ hybridization analyses were performed on formalin-fixed paraffin-embedded sections of developing embryonic mouse tissues using specific antibodies and complementary RNA probes for TGFbeta1 and AM. The early placenta, including the giant trophoblastic cells, showed high levels of staining and hybridization for TGFbeta1 and AM proteins and mRNAs. The heart was the first organ that showed expression of TGFbeta1 and AM during embryogenesis. The spatio-temporal patterns of expression of TGFbeta1 and AM in cardiovascular, neural, and skeletal-forming tissues as well as in the main embryonic internal organs showed striking similarities. The lung, kidney, and intestine, in which epithelial-mesenchymal interactions occur, showed similar patterns of TGFbeta1 and AM expression. These data show colocalization of TGFbeta1 and AM in specific cell types associated with several tissues in the developing mouse embryo. Additionally, RT-PCR amplification and Northern blot hybridization showed expression of TGFbeta1 and AM mRNAs in all embryonic and adult mouse and rat tissues examined. Our data show that the expression of TGFbeta1 and AM is regulated in a spatial and temporal manner such that overlapping patterns of expression of TGFbeta1 and AM occur in several tissues at the same stage of development and in the same cellular location in rodent embryogenesis.

Adrenomedullin↗

PubMed1998

Local production and action of adrenomedullin in the rat adrenal zona glomerulosa.

This study was designed to investigate the synthesis and action of adrenomedullin in the rat adrenal gland. The results obtained from in situ hybridization and immunocytochemical studies suggest that adrenomedullin is synthesized not only in the medulla, but also within the zona glomerulosa of the rat adrenal cortex. Findings from in situ hybridization and binding studies also suggested that specific adrenomedullin receptors are expressed in the zona glomerulosa, and that low levels are present in the inner zones of the cortex. The Kd of the zona glomerulosa adrenomedullin receptor (5.5 nmol/l) suggests that it may respond to locally produced adrenomedullin rather than circulating concentrations of the peptide, which are in a lower range. It was found that adrenomedullin acted on zona glomerulosa cells in vitro to stimulate aldosterone release and cAMP formation, but in this tissue did not stimulate inositol phosphate turnover. The effect of adrenomedullin on aldosterone secretion was significantly attenuated by a protein kinase A inhibitor, suggesting that cAMP mediates the effects of adrenomedullin on aldosterone secretion. Adrenomedullin did not significantly affect the response of zona glomerulosa cells to stimulation by either ACTH or angiotensin II. Adrenomedullin did not affect the release of catecholamines, either adrenaline or noradrenaline, by intact adrenal capsular tissue. These data suggest that both adrenomedullin and its specific receptor are expressed in the rat adrenal zona glomerulosa, leading to the hypothesis that adrenomedullin may have an autocrine/paracrine role in the regulation of the rat adrenal zona glomerulosa.

Adrenomedullin↗

PubMed1998

Concurrent and distinct transcription and translation of transforming growth factor-beta type I and type II receptors in rodent embryogenesis.

The transforming growth factor-betas (TGF-betas) are multifunctional regulatory polypeptides that play a crucial role in many cell processes and function through a set of cell surface protein receptors that includes TGF-beta type I (RI) and type II (RII). The present study reports a comprehensive comparison of the patterns of expression of TGF-beta RI and RII proteins and mRNAs in the developing mouse embryo using immunohistochemical and in situ hybridization analyses. Although widespread expression of both TGF-beta receptors was detected throughout the embryonic development period so that many similarities occur in localization of the TGF-beta receptors, TGF-beta RI was expressed in a well-defined, non-uniform pattern that was different in many respects from that of TGF-beta RII. Whereas higher levels of TGF-beta RI compared to TGF-beta RII were detected in some tissues of the embryo at the beginning of organogenesis, the level of TGF-beta RII increased more dramatically than that of TGF-beta RI during late organogenesis; this was especially true in many neural structures where TGF-beta RI and RII were comparable by day 16. The lung, kidney and intestine, in which epithelial-mesenchymal interactions occur, showed a complex pattern of TGF-beta RI and Rll expression. Additionally, northern blot hybridization and reverse transcription-polymerase chain reaction (RT-PCR) amplification showed non-uniform expression of the transcripts for TGF-beta RI and RII in embryonic and adult mouse and rat tissues. These data show that regulation of TGF-beta1 RI and RII occurs concurrently, but distinctly, in a spatial and temporal manner in rodent embryogenesis which may allow control of signal transduction of TGF-beta during development.

Activin Receptors, Type I↗

PubMed1997

Adrenomedullin binds with high affinity, elevates cyclic AMP, and stimulates c-fos mRNA in C6 glioma cells.

The effects of adrenomedullin (ADM) on C6 glioma cells were investigated. [125I]ADM bound with high affinity (Kd = 24 nM) to a single class of sites (Bmax = 36,000/cell) in C6 cells. Specific [125I]ADM binding was inhibited with high affinity by ADM (IC50 value of 10 nM) but not ADM(22-52) or pro-adrenomedullin N-terminal 20 peptide (PAMP). By RT-PCR, ADM receptors were detected in C6 cells. ADM elevated cAMP (ED50 value of 10 nM) whereas PAMP and ADM(22-52) did not. ADM stimulated transiently c-fos mRNA in a concentration-dependent manner. Monoclonal antibody G6, which neutralizes ADM, significantly inhibited C6 proliferation and decreased the ability of ADM to elevate c-fos mRNA. These data suggest that ADM is a regulatory peptide of C6 cells.

Adrenomedullin↗

PubMed1997

New approaches to the integrated management of early lung cancer.

We have briefly surveyed some the developments in the field of molecular diagnostics that provide a basis for cautious optimism about progress in population-based early lung cancer screening. The sound lung cancer management strategies that were formulated several decades ago failed in clinical trials because the necessary tools to implement the strategies were not yet available. Technology is beginning to emerge that makes population-based screening achievable. This same technology may be used to define a comprehensive marker panel including the most informative markers from the long list of candidate markers. Validation studies will define more clearly the strengths and limitations of new molecular diagnostics and provide leads for further research attention. The clinical community can expedite this process if these validation efforts are aggressively pursued. Parallel developments are clearly needed in refining the range of therapeutic intervention for early cancer management. The success of both diagnostic and intervention tools is interwoven in the ultimate goal of reducing lung cancer mortality. This article is an invitation to think expansively about new approaches to cancer care that integrate the fruits of our hard-learned lessons.

Humans↗

PubMed1997

Adrenomedullin receptor expression in human lung and in pulmonary tumors.

Adrenomedullin (AM) is a multifunctional regulatory peptide that stimulates cyclic AMP production in many target tissues and is highly expressed in the lung. Analysis of the distribution of the recently cloned AM receptor (AM-R) by non-radioactive in situ hybridization revealed abundant expression in the basal cells of the airway epithelium and Type II pneumocytes. The expression of AM-R in the two cell types involved in epithelial regeneration of the lung suggests that AM may be relevant in such functions as organ development, wound repair, and epithelial turnover. AM-Rs are also synthesized in vivo and in vitro by a variety of tumor cells that also express the ligand, suggesting the existence of an autocrine loop that may be involved in tumor growth stimulation. The present findings suggest that the AM/AM-R regulatory system plays a major role in respiratory physiology and lung carcinogenesis and that new functions for AM remain to be identified.

Adenocarcinoma↗

PubMed1997

Expression of adrenomedullin and its receptor during embryogenesis suggests autocrine or paracrine modes of action.

The present study reports the developmental patterns of expression of adrenomedullin (AM) in rat and mouse embryos. AM is a novel multifunctional peptide recently isolated from a human pheochromocytoma, which has been shown to promote growth in a variety of mammalian cell lines. We have applied several techniques to investigate the localization of both the AM peptide and its receptor throughout development. Immunocytochemical detection has been performed using different specific antibodies against AM and its gene-related peptide pro-AM N-terminal 20 peptide. In situ hybridization showed the localization of the messenger RNAs for AM and its receptor. Western blot analysis together with reverse transcription-PCR gave further support to the localization of AM and its receptor in a variety of embryonic tissues. The localization of the receptor paralleled that of AM itself, suggesting an autocrine or paracrine mode of action. The spatio-temporal pattern of expression of AM in cardiovascular, neural, and skeletal-forming tissues as well as in the main embryonic internal organs is described. The primitive placenta, especially the giant trophoblastic cells, shows high levels of AM and AM receptor. The heart is the first organ that expresses AM during development. The kidney, lung, and developing tooth, in which epithelial-mesenchymal interactions are taking place, show specific patterns of AM expression. In several regions of the embryo, the patterns of AM expression correspond to the degree of differentiation. The possible involvement of AM in the control of embryonic invasion, proliferation, and differentiation is discussed.

Adrenomedullin↗

PubMed1997

Expression of adrenomedullin and its receptor in normal and malignant human skin: a potential pluripotent role in the integument.

Adrenomedullin (AM) is a multifunctional peptide involved in a variety of physiological functions, including growth regulation and antimicrobial activity. We have determined by immunohistochemistry and in situ hybridization that AM and its receptor are present in all the epithelial cells of the normal skin, including keratinocytes of the epidermis and hair follicles, as well as cells of the glands and secretory ducts. We also have detected AM in the sweat, by RIA. In addition, AM and its receptor were found in skin tumors of different histologies. The presence of AM and its receptor in normal and neoplastic skin was confirmed by RT-PCR and Western blot analysis performed on cell extracts from human skin cell lines. Radiolabeled AM bound to specific sites in cultured cells with a Kd of 9 nM. This binding was blocked by the addition of cold AM but not by related peptides such as AM 22-52, pro-AM 20 N-terminal peptide, calcitonin gene-related peptide, calcitonin gene-related peptide 8-37, or amylin. Finally, exposure to synthetic AM resulted in an increase of thymidine intake by skin cells. These results implicate AM as a potential player in skin defense against infectious microorganisms and as a possible autocrine growth factor in normal skin physiology and tumor development.

Adrenomedullin↗

PubMed1997

Central action of adrenomedullin to inhibit gastric emptying in rats.

The central action of human adrenomedullin (AM) to influence gastric emptying and the peripheral mechanisms involved were studied in conscious rats. The 20-min rate of gastric emptying of a methylcellulose solution was assessed after intracisternal (i.c.) injection of AM or rat alpha-calcitonin gene-related peptide (alphaCGRP). AM and alphaCGRP dose-dependently inhibited gastric emptying with i.c. ED50 values of 120 and 100 pmol, respectively. Human proadrenomedullin N-terminal 20 peptide (150-600 pmol, i.c.) and AM (150 pmol, i.v.) had no effect. The inhibitory actions of AM and alphaCGRP (150 pmol, i.c.) were completely blocked by the CGRP antagonist, human CGRP-(8-37) injected i.c. at 30 microg, but not at 15 microg. The CRF antagonist, [D-Phe12,Nle(21,38),C(alpha)MeLeu37]CRF-(12-41) (10 microg/rat) injected i.c. prevented i.c. rat/human CRF (150 pmol)-induced 53% inhibition of gastric emptying while not modifying the effect of AM. The action of AM (150 pmol, i.c.) was abolished by bilateral adrenalectomy or the beta-adrenergic blocker, propranolol (1 mg/kg, i.p.), but was not altered by indomethacin (5 mg/kg, i.p.) or subdiaphragmatic vagotomy. These results indicate that i.c. AM and alphaCGRP equipotently inhibit gastric emptying through mechanisms similarly antagonized by a high dose of CGRP-(8-37). The central AM action is mediated through adrenal-dependent, beta-adrenergic pathways independently from activation of central CRF receptors.

Adrenalectomy↗

PubMed1997

Antitumor activity of a monoclonal antibody directed against gastrin-releasing peptide in patients with small cell lung cancer.

BACKGROUND: Small cell lung cancer (SCLC) cells express and secrete gastrin-releasing peptide (GRP) which binds to receptors and stimulates growth of these cells. A murine monoclonal antibody, 2A11, which binds GRP with high affinity, decreased growth of SCLC cells in vitro and in athymic nude mice. A phase 1 trial and pharmacokinetic modeling in patients with lung cancer has defined the phase 2 dose of 2A11 but the antitumor activity in patients is unknown. METHODS: Thirteen patients with previously treated SCLC received 2A11 at 250 mg/m2 over 1 h three times per week for 4 weeks. Serum GRP, urine GRP, serum levels of 2A11, and human antimouse antibodies (HAMA) were determined. RESULTS: One of 12 (8%; 95% confidence interval, 0 to 38%) evaluable patients had complete resolution of radiographically detectable tumor lasting 4 months. Four patients (33%) had stable disease. No toxic reactions were observed. The pretreatment serum GRP level of the responding patient was 3.1 fmol/mL and the median of nine nonresponding patients was 7.3 fmol/mL (range, <1.0 to 29.0). The mean trough serum 2A11 level was 49+/-18 microg/mL in the responding patient and 32 to 487 mg/mL (median, 117) in 10 nonresponding patients. HAMA did not increase during 2A11 administration in any patient. CONCLUSIONS: Interruption of the GRP autocrine growth factor loop with 2A11 results in clinical antitumor activity in a minority of patients with previously treated SCLC. Further evaluation of the antitumor effects of 2A11 is warranted to define characteristics associated with response to 2A11.

Animals↗

PubMed1997

Adrenomedullin expression in the mouse mammary gland: evidence for the mature form in milk.

Adrenomedullin (AM) is a recently identified amidated peptide produced by a variety of tissue types. We have investigated the involvement of AM and its receptor (AM-R) in developing mouse mammary glands and have examined what influence ovarian hormones have on AM and AM-R expression in this system. Tissues from ductal morphogenesis, virgin adult, pregnancy, and lactation stages were assessed for AM and AM-R by molecular, biochemical and immunohistochemical techniques. Results from these studies indicated that messenger RNA for AM and AM-R and immunoreactivity for AM were expressed in the luminal epithelium of small and large ducts and in terminal end buds. Immunoreactive AM was identified as a cytoplasm component of ductal cells, with some cells also having nuclear staining. Western blot analysis of mammary gland tissues yielded two molecular mass species (M(r) 14,000 and 18,500) of AM immunoreactivity in the mammary gland for the above developmental stages, consistent with processed intermediate and prohormone forms respectively. Ovariectomy alone or followed by hormonal treatments did not alter the expression pattern for these two proteins. By Western blot, the fully processed AM form (M(r) 6000) was identified in milk extracts from lactating glands. These data suggest a potential role for AM and its receptor in the maintenance of mammary gland homeostasis and suggests a potential role for AM in development of the newborn.

Adrenomedullin↗

PubMed1996

Adrenomedullin expression in human tumor cell lines. Its potential role as an autocrine growth factor.

Although adrenomedullin (AM) previously has been identified in human tumors, its role has remained elusive. Analysis by reverse transcriptase-polymerase chain reaction (RT-PCR) revealed AM mRNA in 18 of 20 human normal tissues representing major organs, and 55 of 58 (95%) malignant cell lines. Western blot and high performance liquid chromatography analysis showed immunoreactive AM species of 18, 14, and 6 kDa that are consistent with the precursor, intermediate product, and active peptide, respectively. Immunohistochemistry and in situ RT-PCR performed on paraffin-embedded tumor cell lines of various tissue origins exhibited AM cytoplasmic staining. Neutralizing monoclonal antibody to AM inhibits tumor cell growth in a concentration-dependent manner, an effect that was reversed with the addition of exogenous AM. Responding tumor cells were shown to have approximately 50,000 AM receptors per cell by Scatchard analysis with 125I-AM and expressed AM receptor mRNA by RT-PCR. Our data showed 36 of 48 (75%) tumor cell lines expressed AM receptor mRNA by RT-PCR assessment, all of them also expressed AM. In the presence of AM, cAMP levels were shown to increase in tumor cells. Our collective data demonstrate that AM and AM receptor are expressed in numerous human cancer cell lines of diverse origin and constitute a potential autocrine growth mechanism that could drive neoplastic proliferation.

Adrenomedullin↗

PubMed1996

Insulin-like growth factor expression in human cancer cell lines.

The insulin-like growth factors (IGFs), IGF-I and IGF-II, are potent mitogens for human lung and other epithelial cancer cell lines. Previous studies in defined medium lacking added IGF or insulin suggest that an IGF-related ligand can act as an autocrine growth factor for many cancer cell lines through action via the type I IGF receptor (IGF-R). Analysis of RNA isolated from human lung and breast cancer cell lines by reverse transcription of mRNA and polymerase chain reaction reveal that IGF-I and IGF-II mRNAs were co-expressed with IGF-R in the majority of cell lines. IGF-I mRNA was detected in 11/12 small cell lung cancer cell lines (SCLC), 13/14 nonsmall cell lung cancer (NSCLC) cell lines, and 1/2 breast cancer cell lines. IGF-II mRNA was detected in 8/10 SCLC, 11/12 NSCLC cell lines, and 2/2 breast lines. All cell lines expressed IGF-R. For analysis of IGF peptide secretion, cell lines were adapted to growth in serum/hormone-free culture medium (R0), and to avoid interference by IGF-binding proteins, secreted IGF peptides were isolated under acidic conditions and analyzed by Western blotting. Based upon measurement of the sensitivity of the anti-IGF antibodies for detection of recombinant human IGFs, IGF peptides accumulated in conditioned medium at greater than picomolar concentrations should have been readily detected. In three cell lines (two lung and one breast) secreted IGF immunoreactivity was detected as three molecular mass species of 23, 14, and 6 kDa. Isolation and NH2-terminal sequencing of each of these species definitively identified them as differentially processed forms of the IGF-II prohormone. Despite the high frequency of IGF-I gene expression detected by reverse transcription-polymerase chain reaction analysis, only one lung cancer cell line, NCI-N417d, was found that unequivocally secreted IGF-I peptide. This direct sequence determination unambiguously identifies IGF-II as the predominant IGF involved in the autocrine growth stimulation of human lung and breast epithelial tumor cell lines and supports a growing body of literature that implicates IGF-II/IGF-R autocrine loops as a common growth mechanism in epithelial carcinogenesis.

Adenocarcinoma↗

PubMed1996

Monoclonal antibody alpha IR-3 inhibits non-small cell lung cancer growth in vitro and in vivo.

The ability of monoclonal antibody (mAb) alpha IR-3 to interact with non-small cell lung cancer (NSCLC) cells was investigated. MAb alpha IR-3 inhibited specific binding of 125I-IGF-I and 125I-alpha IR-3 to a panel of 8 NSCLC cell lines with high affinity (IC50 = 200 and 50 ng/ml, respectively). 125I-alpha IR-3 bound with high affinity (Kd = 40 ng/ml) to a single class of sites (Bmax = 8,000/cell) using NCI-H838 cells. 125I-alpha IR-3 was internalized when exposed to NCI-H838 or H1299 cells at 37 degrees C but not 4 degrees C. alpha IR-3 immunoprecipitated major 90 and 130 kD proteins. IGF-I stimulated and alpha IR-3 inhibited the clonal growth of NCI-H1299 cells. alpha IR-3 slowed the growth of NCI-H157 and H838 xenografts in nude mice. In a biodistribution study 125I-alpha IR-3 was preferentially localized to the tumor as opposed to other organs. These data suggest that IGF-I may be a regulatory agent in NSCLC.

Animals↗

PubMed1996

Adrenomedullin-like immunoreactivity in the nervous system of the starfish Marthasterias glacialis.

The nervous system of the starfish Marthasterias glacialis was investigated immunocytochemically using an antiserum specific for adrenomedullin (AM), a new regulatory peptide. Immunoreactivity was only found in nerves of the basiepithelial plexus of cardiac and pyloric stomachs and pyloric caeca, while the radial nerve cords and the other digestive organs were negative. The strongest AM-like immunoreactivity was located in the current-producing areas of the cardiac stomach. The distribution of this peptide suggests different functions in echinoderms involving regulation of muscle movement and neurotransmission. The presence of an AM-like substance in echinoderms points to an early phylogenetic origin for this regulatory system.

Adrenomedullin↗

PubMed1996

Biochemical characterization of mouse liver IBE1-amide immunoreactivity: limitations of antibody-based approaches for verification of peptide expression.

A high titer, specific antiserum, raised against a synthetic analogue of a unique peptide region within the human IGF-IB prohormone, detected specific immunoreactivity in extracts of mouse, chicken, sheep, and human liver. Specificity was confirmed by the ablation of immunoreactivity in the presence of excess synthetic immunogen. Here we report the isolation and characterization of one of the immunoreactive species from an extract of mouse liver: amino acid sequencing revealed that the purified product was 78% identical to the NH2-terminus of the alpha-subunit of mouse hemoglobin. Immunoblot analysis of a commercial preparation of mouse hemoglobin confirmed that the antiserum recognized hemoglobin. Addition of excess synthetic peptide to the antiserum eliminated the immunobinding to hemoglobin. The apparent "specificity" of even affinity-purified antiserum for hemoglobin provides a cautionary note for the interpretation of studies concluding antigen expression based solely on the presence of positive immunoreactivity.

Amino Acid Sequence↗

PubMed1996

Detection of adrenomedullin, a hypotensive peptide, in amniotic fluid and fetal membranes.

OBJECTIVE: Our purpose was to determine whether adrenomedullin, a multifunctional regulatory peptide involved in blood flow regulation and growth stimulation and with antimicrobial activity, was a component of amniotic fluid from second-trimester human fetus and to determine the source of this peptide. STUDY DESIGN: A prospective descriptive study was performed on 134 patients undergoing amniocentesis after genetic counseling, ultrasonography, and informed consent. Adrenomedullin expression was determined by immunocytochemical analysis, Western blot analysis, reverse transcriptase-polymerase chain reaction, and in situ reverse transcriptase-polymerase chain reaction in fetal membranes and with radioimmunoassay in amniotic fluids. RESULTS: Radioimmunoassay of the 134 amniotic fluid specimens revealed adrenomedullin-like immunoreactivity in all of them, ranging in concentration from 10 to 300 fmol/25 microliters (170 +/- 62 fmol/25 microliters). Immunocytochemical analysis, Western blot analysis, reverse transcriptase-polymerase chain reaction, and in situ reverse transcriptase-polymerase chain reaction further established the expression of adrenomedullin protein and messenger ribonucleic acid in fetal amniotic membranes, suggesting that this organ is the source of amniotic adrenomedullin. CONCLUSIONS: Our results clearly demonstrate the presence of adrenomedullin in second-trimester human amniotic fluid and adrenomedullin messenger ribonucleic acid and protein in amniotic membranes, suggesting that adrenomedullin is a hormone involved in the maintenance of normal pregnancy. Further studies with these molecular tools are in progress to determine the precise role of this hormone and whether adrenomedullin plays a role in the pathogenesis of various disorders of pregnancy.

Adrenomedullin↗

PubMed1996

Development of Molecular Approaches to Early Lung Cancer Detection.

Lung cancer has been particularly recalcitrant to standard therapeutic interventions. Improvement in the outcome for this disease requires the ability to identify the disease while it is still localized in the airways. Usually, radiation therapy to eliminate this disease fails because of progression of the cancer outside the radiation treatment ports. This is a failure of the diagnostic modalities to detect the extent of the cancer, not a failure of the radiation. With new epithelial-directed diagnostics, the ability to detect the premetastatic phase of lung cancer is emerging. Evolution of the screening infrastructure will be required to allow percolation of this technology out to the medical community, so that a benefit in cancer mortality reduction can be achieved. Some development issues will have to be resolved. Considering our fragmentary understanding of pulmonary carcinogenesis, the heterogeneity of lung cancer suggests that a panel of biomarkers instead of a single marker will be required to clarify the status of an individual's epithelium. However, the data output from multiplexed bioassays performed on large clinical populations will require better integration of information technology to optimize analysis and reporting of test results. An important new area for outcomes research is the systematic analysis of population screening approaches. With population sampling algorithms, selective screening. This type of research requires careful clinical validation. Finally, the general acceptance of population-based screening will be determined by the availability of effective interventions to complement the new diagnostic tools. New measures to prevent the progression of early cancer will be discussed.

Journal Article↗