Genome-wide profiling of non-smoking-related lung cancer cells reveals common RB1 rearrangements associated with histopathologic transformation in EGFR-mutant tumors.

BACKGROUND: The etiology and the molecular basis of lung adenocarcinomas (LuADs) in nonsmokers are currently unknown. Furthermore, the scarcity of available primary cultures continues to hamper our biological understanding of non-smoking-related lung adenocarcinomas (NSK-LuADs). PATIENTS AND METHODS: We established patient-derived cancer cell (PDC) cultures from metastatic NSK-LuADs, including two pairs of matched EGFR-mutant PDCs before and after resistance to tyrosine kinase inhibitors (TKIs), and then performed whole-exome and RNA sequencing to delineate their genomic architecture. For validation, we analyzed independent cohorts of primary LuADs. RESULTS: In addition to known non-smoker-associated alterations (e.g. RET, ALK, EGFR, and ERBB2), we discovered novel fusions and recurrently mutated genes, including ATF7IP, a regulator of gene expression, that was inactivated in 5% of primary LuAD cases. We also found germline mutations at dominant familiar-cancer genes, highlighting the importance of genetic predisposition in the origin of a subset of NSK-LuADs. Furthermore, there was an over-representation of inactivating alterations at RB1, mostly through complex intragenic rearrangements, in treatment-naive EGFR-mutant LuADs. Three EGFR-mutant and one EGFR-wild-type tumors acquired resistance to EGFR-TKIs and chemotherapy, respectively, and histology on re-biopsies revealed the development of small-cell lung cancer/squamous cell carcinoma (SCLC/LuSCC) transformation. These features were consistent with RB1 inactivation and acquired EGFR-T790M mutation or FGFR3-TACC3 fusion in EGFR-mutant tumors. CONCLUSIONS: We found recurrent alterations in LuADs that deserve further exploration. Our work also demonstrates that a subset of NSK-LuADs arises within cancer-predisposition syndromes. The preferential occurrence of RB1 inactivation, via complex rearrangements, found in EGFR-mutant tumors appears to favor SCLC/LuSCC transformation under growth-inhibition pressures. Thus RB1 inactivation may predict the risk of LuAD transformation to a more aggressive type of lung cancer, and may need to be considered as a part of the clinical management of NSK-LuADs patients.

Complement activation product C4d in oral and oropharyngeal squamous cell carcinoma.

OBJECTIVE: Complement C4d-containing fragments have been proposed as diagnostic markers for lung cancer. The purpose of this study was to evaluate the presence of C4d in oropharyngeal (OPSCC) and oral (OSCC) squamous cell carcinomas. SUBJECTS AND METHODS: C4d staining was analyzed by immunohistochemistry in 244 OPSCC surgical specimens. C4d levels were quantified by ELISA in resting saliva samples from 48 patients with oral leukoplakia and 62 with OSCC. Plasma samples from 21 patients with leukoplakia and 30 with oral carcinoma were also studied. RESULTS: C4d staining in OPSCC specimens was associated with nodal invasion (P = 0.001), histopathologic grade (P = 0.014), disease stage (P = 0.040), and focal-adhesion kinase expression (P < 0.001). No association was found between C4d and prognosis. Saliva C4d levels were higher in patients with oral cancer than in subjects with leukoplakia (0.07 ± 0.07 vs 0.04 ± 0.03 µg ml(-1) , P = 0.003). The area under the ROC curve was 0.63 (95%CI: 0.55-0.71). Salivary C4d levels in stage IV patients were higher than in patients with earlier stages (P = 0.028) and correlated with tumor size (P = 0.045). Plasma C4d levels also correlated with salivary C4d levels (P = 0.041), but differences between patients with oral cancer and subjects with leukoplakia were not significant (1.26 ± 0.59 vs 1.09 ± 0.39 µg ml(-1) , P = 0.232). CONCLUSION: C4d-containing fragments are detected in oral primary tumors and are increased in saliva from patients with OSCC.

TGFBI expression is an independent predictor of survival in adjuvant-treated lung squamous cell carcinoma patients.

BACKGROUND: Transforming growth factor ß-induced protein (TGFBI) is a secreted protein that mediates cell anchoring to the extracellular matrix. This protein is downregulated in lung cancer, and when overexpressed, contributes to apoptotic cell death. Using a small series of stage IV non-small cell lung cancer (NSCLC) patients, we previously suggested the usefulness of TGFBI as a prognostic and predictive factor in chemotherapy-treated late-stage NSCLC. In order to validate and extend these results, we broaden the analysis and studied TGFBI expression in a large series of samples obtained from stage I-IV NSCLC patients. METHODS: TGFBI expression was assessed by immunohistochemistry in 364 completely resected primary NSCLC samples: 242 adenocarcinomas (ADCs) and 122 squamous cell carcinomas (SCCs). Kaplan-Meier curves, log-rank tests and the Cox proportional hazards model were used to analyse the association between TGFBI expression and survival. RESULTS: High TGFBI levels were associated with longer overall survival (OS, P<0.001) and progression-free survival (PFS, P<0.001) in SCC patients who received adjuvant platinium-based chemotherapy. Moreover, multivariate analysis demonstrated that high TGFBI expression is an independent predictor of better survival in patients (OS: P=0.030 and PFS: P=0.026). CONCLUSIONS: TGFBI may be useful for the identification of a subset of NSCLC who may benefit from adjuvant therapy.

TMPRSS4 regulates levels of integrin α5 in NSCLC through miR-205 activity to promote metastasis.

BACKGROUND: TMPRSS4 is a membrane-anchored protease involved in cell migration and invasion in different cancer types including lung cancer. TMPRSS4 expression is increased in NSCLC and its inhibition through shRNA reduces lung metastasis. However, molecular mechanisms leading to the protumorigenic regulation of TMPRSS4 in lung cancer are unknown. METHODS: miR-205 was identified as an overexpressed gene upon TMPRSS4 downregulation through microarray analysis. Cell migration and invasion assays and in vivo lung primary tumour and metastasis models were used for functional analysis of miR-205 overexpression in H2170 and H441 cell lines. Luciferase assays were used to identify a new miR-205 direct target in NSCLC. RESULTS: miR-205 overexpression promoted an epithelial phenotype with increased E-cadherin and reduced fibronectin. Furthermore, miR-205 expression caused a G0/G1 cell cycle arrest and inhibition of cell growth, migration, attachment to fibronectin, primary tumour growth and metastasis formation in vivo. Integrin α5 (a proinvasive protein) was identified as a new miR-205 direct target in NSCLC. Integrin α5 downregulation in lung cancer cells resulted in complete abrogation of cell migration, a decreased capacity to adhere to fibronectin and reduced in vivo tumour growth, compared with control cells. TMPRSS4 silencing resulted in a concomitant reduction of integrin α5 levels. CONCLUSION: We have demonstrated for the first time a new molecular pathway that connects TMPRSS4 and integrin α5 through miR-205 to regulate cancer cell invasion and metastasis. Our results will help designing new therapeutic strategies to inhibit this novel pathway in NSCLC.

Overexpression of TMPRSS4 in non-small cell lung cancer is associated with poor prognosis in patients with squamous histology.

BACKGROUND: Mortality rates in lung cancer patients have not decreased significantly in recent years, even with the implementation of new therapeutic regimens. One of the main problems is that a large proportion of patients present local or distant metastasis at the time of diagnosis. The need for identification of novel biomarkers and therapeutic targets for a more effective management of lung cancer led us to investigate TMPRSS4, a protease reported to promote tumour growth and metastasis. MATERIAL AND METHODS: In all, 34 lung cancer cell lines were used to evaluate the TMPRSS4 expression. Cell migration and clonogenic assays, and an in-vivo lung metastasis model were used for functional analysis of the TMPRSS4 downregulation in H358, H441 and H2170 cell lines. The TMPRSS4 expression analysis in normal and malignant lung tissue samples was performed by qPCR. Five different microarray-based publicly available expression databases were used to validate our results and to study prognosis. RESULTS: The TMPRSS4 knock down in H358, H441 and H2170 cells resulted in a significant reduction in proliferation, clonogenic capacity and invasion. A significant (P<0.05) decrease in the lung colonisation and growth was found when mice were injected with TMPRSS4-depleated H358-derived clones, as compared with controls. Expression of TMPRSS4 showed a >30-fold increase (P<0.001) in tumours in comparison with non-malignant samples. Levels in tumours with squamous cell carcinoma (SCC) histology were found to be significantly higher (P<0.001) than those with adenocarcinoma (AC) histology, which was confirmed in data retrieved from the microarrays. Kaplan-Meier curves demonstrated that high levels of TMPRSS4 were significantly associated (P=0.017) with reduced overall survival in the patients with SCC histology, whereas no correlation was found for the AC histology. CONCLUSION: Our results demonstrate that TMPRSS4 has a role in the lung cancer development. The potential use of TMPRSS4 as a biomarker for lung cancer detection or as a predictor of patient's outcome warrants further investigation.

Surgical Outcomes in a Lung Cancer-Screening Program Using Low Dose Computed Tomography.

OBJECTIVE: Lung cancer (LC) is the leading cause of death from cancer worldwide. More than 27,000 LCs are diagnosed annually in Spain, and most are unresectable. Early detection and treatment reduce LC mortality. This study describes surgical outcomes in a longstanding LC screening cohort in Spain. METHODS: We conducted a retrospective study of surgical outcomes in a LC screening (LCS) program using low dose computed tomography (LDCT) since the year 2000. A descriptive analysis of clinical and radiological parameters, presence or absence of a preoperative diagnosis, pathological staging, morbidity, mortality, and survival was performed. RESULTS: Ninety-seven (2.5%) LC were diagnosed in 3825 screened. Twenty individuals with LC had no surgery due to advanced stage or small cell histology. Eighty-seven surgical procedures were carried out for suspected or biopsy proven LC, detected by LDCT. Most operated patients were male (57[85%]) aged 64±9.1 years. Nine patients underwent a second operation for a metachronous primary lung cancer. Mean tumor size was 15.2±7.6mm. Eight nodules were benign (9.2%). Lobectomy was performed in 56 cases (83.6%). Adenocarcinoma (n=39; 58.2%) was the most frequent histological type followed by squamous cell carcinoma (n=17; 25.4%). Fifty-nine (88%) tumors were in Stage I. Thirteen patients (15.4%) had 16 complications. The estimated survival rates at 5 and 10 years for stage I were 93% (95% CI: 79%-98%) and 83% (95% CI: 65%-92%), respectively. CONCLUSION: Lung cancer screening was associated with excellent surgical outcomes with 5 and 10-year survival rates exceeding 90 and 80%, respectively.

EUELC project: a multi-centre, multipurpose study to investigate early stage NSCLC, and to establish a biobank for ongoing collaboration.

The European Early Lung Cancer (EUELC) project aims to determine if specific genetic alterations occurring in lung carcinogenesis are detectable in the respiratory epithelium. In order to pursue this objective, nonsmall cell lung cancer (NSCLC) patients with a very high risk of developing progressive lung cancer were recruited from 12 centres in eight European countries: France, Germany, southern Ireland, Italy, the Netherlands, Poland, Spain and the UK. In addition, NSCLC patients were followed up every 6 months for 36 months. A European Bronchial Tissue Bank was set up at the University of Liverpool (Liverpool, UK) to optimise the use of biological specimens. The molecular-pathological investigations were subdivided into specific work packages that were delivered by EUELC Partners. The work packages encompassed mutational analysis, genetic instability, methylation profiling, expression profiling utilising immunohistochemistry and chip-based technologies, as well as in-depth analysis of FHIT and RARbeta genes, the telomerase catalytic subunit hTERT and genotyping of susceptibility genes in specific pathways. The EUELC project engendered a tremendous collaborative effort, and it enabled the EUELC Partners to establish protocols for assessing molecular biomarkers in early lung cancer with the view to using such biomarkers for early diagnosis and as intermediate end-points in future chemopreventive programmes.

Hypoxia-inducible factor-1 (HIF-1) up-regulates adrenomedullin expression in human tumor cell lines during oxygen deprivation: a possible promotion mechanism of carcinogenesis.

Little is known about the molecular mechanisms that control adrenomedullin (AM) production in human cancers. We demonstrate here that the expression of AM mRNA in a variety of human tumor cell lines is highly induced in a time-dependent manner by reduced oxygen tension (1% O2) or exposure to hypoxia mimetics such as desferrioxamine mesylate (DFX) or CoCl2. This AM expression seems to be under hypoxia-inducible factor-1 (HIF-1) transcriptional regulation, since HIF-1alpha and HIF-1beta knockout mouse cell lines had an ablated or greatly reduced hypoxia AM mRNA induction. Similarly, inhibition or enhancement of HIF-1 activity in human tumor cells showed an analogous modulation of AM mRNA. Under hypoxic conditions, immunohistochemical analysis of tumor cell lines revealed elevated levels of AM and HIF-1alpha as compared with normoxia, and we also found an increase of immunoreactive AM in the conditioned medium of tumor cells analyzed by RIA. AM mRNA stabilization was shown to be partially responsible for the hypoxic up-regulated expression of AM. In addition, we have identified several putative hypoxia response elements (HREs) in the human AM gene, and reporter studies with selected HREs were capable of enhancing luciferase expression after exposure to DFX. Furthermore, transient coexpression of HIF-1alpha resulted in an augmented transactivation of the reporter gene after DFX treatment. Given that most solid human tumors have focal hypoxic areas and that AM functions as a mitogen, angiogenic factor, and apoptosis-survival factor, our findings implicate the HIF-1/AM link as a possible promotion mechanism of carcinogenesis.

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.

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.

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 binding protein in the plasma of multiple species: characterization by radioligand blotting.

Frequently, peptide hormones circulate in plasma associated with specific binding proteins that modify the clearance and biochemical activities of the peptide. Our experimental approach was to use 125I-ligand blotting procedures to probe for the presence of specific adrenomedullin (AM) binding proteins (AMBPs). Plasma proteins from chick, calf, dog, goat, guinea pig, human, mouse, pig, rabbit and sheep blood were separated electrophoretically in 10% nonreducing SDS-polyacrylamide gels and transferred to nitrocellulose. Nonspecific binding of tracer was blocked on the nitrocellulose with a hydrolyzed casein matrix. Blots were probed with synthetic human 125I-AM. Autoradiogram scanning of blots revealed a mixture of 140- and/or 120- kD protein complexes that bound 125I-AM in all species tested. Binding of the ligand was specific as judged by a linear competitive displacement of the tracer binding from human, bovine and pig plasma AMBP bands with increasing concentrations of nonlabelled AM in the binding buffer. A series of peptide fragments of AM representing amino- and carboxyterminal regions of the hormone, or amylin, calcitonin gene-related peptide (CGRP), or insulin failed to displace intact 125I-AM from ligand blot bands. Bovine plasma proteins from healthy and parasitized calves with an infection-related stunting syndrome were separated electrophoretically, transferred to nitrocellulose and probed with 125I-AM; phosphoimage densitometry analysis revealed a 67% decrease in AMBP band intensity in the 120 and 140 kD proteins from infected calves. We conclude that a specific binding protein(s) for AM exists in mammalian and avian blood that might impact on the bioactivity and function of AM in health and disease.

Underlying disease stress augments plasma and tissue adrenomedullin (AM) responses to endotoxin: colocalized increases in AM and inducible nitric oxide synthase within pancreatic islets.

Rapid onset metabolic impairments accompany the initiation of the acute phase response to many disease stresses, whereas more chronic metabolic perturbations may prolong the recovery period. In the present experiment the application of a mild endotoxin challenge [lipopolysaccharide (LPS)] alone or additive to a chronic subclinical parasitic infection (Sarcocystis cruzi; LPS + PI) in calves was used as a model to investigate and define a dynamic axis coordinated between adrenomedullin (AM) and nitric oxide in response to immune challenge. Plasma AM and NO2/NO3 concentration responses after LPS (0.45 microg/kg, iv) were rapid in onset and of higher magnitude and longer duration in PI + LPS calves than in those challenged with LPS alone. The post-LPS increase in plasma insulin was significantly greater in PI + LPS than in LPS; following refeeding of calves, insulin secretion was most blunted in PI + LPS calves, consistent with the inhibitory effects of NO and AM on insulin secretion. A more chronic response to the immune challenge (organ specific) was in evidence in tissues harvested 24 h after LPS challenge. Where lung and liver showed no immunostaining for inducible nitric oxide (iNOS), iNOS immunostaining was present in the pancreas, localized to islets only. The percentages of iNOS-immunopositive cells in islets were 1.7%, 21%, 6.7%, and 24% for control (C; saline infused), PI, LPS, and PI + LPS calves, respectively. AM immunostaining was not evident in the liver and was present, but not differentially affected by treatment, in airway epithelium in the lung. The number of islet cells with positive immunostaining for AM was increased in LPS, PI, and PI + LPS calves. The percentages ofAM-immunopositive cells in islets were 8%, 27%, 20%, and 33% for C, PI, LPS, and PI + LPS, respectively. Immunostaining for AM and iNOS was colocalized with cells positive for pancreatic polypeptide. By triple label confocal fluorescence immunocytochemistry, colocalization of intense AM and iNOS immunostaining was confirmed in peripheral islet cells. A weaker, more diffuse iNOS signal was also apparent in insulin-containing cells in PI + LPS. We conclude that chronic low level infection potentiates the severity of metabolic perturbations that arise with additive sudden onset immune challenge, as can occur with bacterial toxins. These metabolic disturbances are reflected in and possibly mediated by early onset increases in plasma tumor necrosis factor-alpha, insulin, and AM and up-regulated iNOS activity. These acute complications rapidly progress into a more chronic state characterized by diminished insulin response to feeding stimulus and colocalized increases in pancreatic islet AM and iNOS. The pancreatic responses in AM and iNOS may play a major role in mediating prolonged disturbances in nutrient use by tissues through their influences on temporal patterns of pancreatic hormone secretion during chronic illness.

Inducible nitric oxide synthase in human lymphomononuclear cells activated by synthetic peptides derived from extracellular matrix proteins.

Synthetic peptides with sequences present in extracellular matrix proteins are capable of causing the expression of the inducible form of nitric oxide synthase (iNOS), detected by immunocytochemistry, and the release of NO by human lymphomononuclear cells incubated in their presence. Active peptides are 15-mers containing a characteristic 2-6-11 motif in which the amino acid residue at position 2 is Leu, Ile, Val, Gly, Ala or Lys; the residue at position 6 is always Pro; and residue 11 is Glu or Asp. The induction of iNOS in human monocytes and macrophages could be involved in the cytotoxicity against tumor cell lines also elicited by these peptides.

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.

Detection of nitric oxide synthase (NOS) in somatostatin-producing cells of human and murine stomach and pancreas.

The aim of this study was to identify by immunocytochemistry the distribution of nitric oxide synthase (NOS) in human and murine gastric epithelium. Using two different antisera specific for neuronal NOS (nNOS), we detected nNOS immunoreactivity in endocrine cells of the epithelium of the body and pyloric regions as well as in ganglion cells of the intrinsic plexi of the stomach of the three species studied. Both immunocytochemistry of contiguous sections and double immunolabeling methods showed that the nNOS-immunoreactive cells were also immunoreactive for somatostatin. Co-localization of nNOS and somatostatin has also been found in the pancreatic islets, where strong nNOS immunoreactivity appeared in scattered cells, which were peripheral in rat and mouse islets and more randomly distributed in human. The possibility of crossreactivity between the antisera against nNOS and somatostatin was ruled out by means of absorption controls. Immunocytochemical techniques were also applied to thin sections, confirming the immunostaining of gastric D-cells, which was restricted principally to the secretory granules. The possible functional implications of these findings for gastric and pancreatic physiology are discussed.

Simultaneous immunostaining method for localization of bromodeoxyuridine and calcitonin gene-related peptide.

A new simultaneous double immunostaining method has been optimized to localize the DNA synthesis marker bromodeoxyuridine (BrdU) and calcitonin gene-related peptide (CGRP) in endocrine cells of Bouin's-fixed, paraffin-embedded rat lung. Nuclease pre-treatment before immunostaining is compatible with optimal tissue morphology and CGRP antigenicity preservation. Nickel-enhanced development of avidin-biotin-peroxidase staining is used to show CGRP immunoreactivity in black and alkaline phosphatase-anti-alkaline phosphatase is applied to demonstrate incorporated BrdU in red. The present methodology could be useful for studies requiring detection of incorporated BrdU in cells producing regulatory peptides or other labile antigens.

Immunocytochemical localization of peptidylglycine alpha-amidating monooxygenase enzymes (PAM) in human endocrine pancreas.

We studied the distribution of the enzymes that are involved in the post-translational alpha-amidation of regulatory peptides in human endocrine pancreas, using immunocytochemical methods for light and electron microscopy. Immunoreactivity for the two enzymes involved, peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL), was located in the periphery of the islets of Langerhans and in ductal endocrine cells. Staining of reverse-face serial sections demonstrated that these immunoreactivities co-localize with glucagon but not with pancreatic polypeptide (PP), insulin, or somatostatin. Double immunogold staining for electron microscopy confirmed the previous results and revealed a different localization for each enzyme inside the secretory granule: PHM is present in the central core of the glucagon-containing granules, whereas PAL is predominantly located near the granule membrane. The existence of an amidated peptide, GLP1, in the A-cells explains the presence of peptidylglycine alpha-amidating monooxygenase enzymes (PAM) in these cells. The absence of the enzymes in the PR-cells raises the possibility that a different form of amidating enzyme may be involved in the post-translational processing of this peptide.

Expression of adrenomedullin and proadrenomedullin N-terminal 20 peptide in human and rat prostate.

Adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP) are two recently discovered hypotensive peptides translated from the same message transcript (preproAM mRNA). In this article we report the presence of AM, PAMP, and their mRNA in human and rat prostate and of AM receptor mRNA in rat prostate. PreproAM mRNA was found in the epithelium of normal human and rat prostate glands by in situ hybridization. In humans, it was mainly expressed in the basal cells. In rat, its expression was higher in the ducts than in the acini of all the prostate lobes. Immunocytochemistry identified a similar distribution pattern for AM compared with its mRNA but showed different locations for AM and PAMP immunoreactivity. The former was widespread in the epithelia, whereas the latter was almost exclusively found in neuroendocrine cells. In rat, Western blot analysis confirmed the presence of high levels of AM peptide in the ventral lobe and of its precursor in the ventral and dorsolateral lobes. Immunoreactivity for serotonin, chromogranin A, PAMP, and AM defined four subpopulations of prostate neuroendocrine-like cells in rat, a cell type that has not been previously described.