The calcium-binding protein S100P in normal and malignant human tissues

Background  

S100P is a Ca²⁺ binding protein overexpressed in a variety of cancers, and thus, has been considered a potential tumor biomarker. Very little has been studied about its normal expression and functions.     

人正常脑组织和脑胶质瘤中S-100、CD83分子的检测

目的：研究S-100、CD83 在人正常脑 组织和脑胶质瘤中的表达。方法：应用免疫组织化学方法检测30例脑胶 质瘤、10例脑胶质瘤瘤旁组织、10例正常脑组织切片中S-100、CD83的表达水平，并与10例 肝炎合并肝硬化组织切片进行比较。结果:S-100蛋白在脑胶质瘤、瘤旁 组织和正常脑组织的阳性率均为100%，用HPISA-1000图像分析测得其不同的平均阳性灰度值 （AGV）分别为72.20±12.30、35.60±9.50和28.60±10.20,脑胶质瘤组织与正常脑 组织比较，差别显著（P＜0.05)，而瘤旁组织与正常脑组织比较，差别无显著性（P ＞0.05）；脑胶质瘤及其瘤旁组织、正常脑组织标本中无CD83阳性细胞表达；CD83在肝 炎 并肝硬化中的阳性率为100%。结论:正常脑组织和脑胶质瘤中无成熟、 有活性的树突状细胞存在;在脑组织和脑疾病的树突状细胞研究中，S-100不能作为DCs的标 志物。     

Differential distribution of immunoreactive S100-alpha and S100-beta proteins in normal nonnervous human tissues

Differential distribution of alpha-subunit (S100-alpha) and beta-subunit (S100-beta) of S100 protein in nonnervous tissues was studied by employing the indirect peroxidase-labeled antibody method with monospecific antibodies to human S100-alpha or bovine S100-beta. It became clear that S100-alpha and S100-beta were more widespread than previously reported. In light of this study, together with the previous report describing quantitative differential distribution of S100-alpha and S100-beta (Kato K, Kimura S: Biochim Biophys Acta, 842:146, 1985), S100-alpha was localized at much higher levels in myocardial cells and slow-twitch muscle fibers, at high levels in renal tubules, chondrocytes, follicular cells of thyroid, exocrine cells of salivary, mammary, and eccrine sweat glands, and centroacinar and acinar cells of the pancreas. On the other hand, S100-beta was mainly localized in Schwann cells, chondrocytes, and adipocytes. There were several tissues showing a remarkable differential localization of S100-alpha and S100-beta; heart, skeletal muscle, exocrine tissues such as mammary gland, salivary gland, and pancreas, liver, kidney, testis, epididymidis, and lymphoreticular system. Based on the wide distribution and the characteristic differential localization, the possible biologic significance of S100 proteins and their application for diagnostic pathology were discussed.     

Ret protein expression in adrenal medullary hyperplasia and pheochromocytoma

Ret is a developmentally regulated tyrosine kinase involved in formation and maintenance of the nervous system. Ret mutations predisposing to pheochromocytomas and medullary thyroid carcinomas occur in multiple endocrine neoplasia (MEN) syndromes 2A and 2B. Biochemical studies have demonstrated overexpression of Ret mRNA and protein in pheochromocytomas compared to normal adrenal medulla. However, the cellular distribution of Ret in the normal human adrenal and in hyperplastic lesions that antecede pheochromocytomas are unclear. The present investigation was undertaken to resolve the histological distribution of Ret in the normal human adrenal, in pheochromocytomas evolving from adrenal medullary hyperplasia in MEN2A and in sporadic pheochromocytomas. Ret expression was studied by immunohistochemistry using both a polyclonal and a monoclonal antibody, with confirmation by immunoblotting of representative cases. Only occasional cells stained for Ret in the normal adrenal, consistent with the distribution in adult adrenals of other species. Heterogeneous, progressively increased Ret expression was observed during the evolution of pheochromocytomas. In both normal and neoplastic adrenal, the most intense immunoreactivity was observed in cells with neuron-like features. Our finding that Ret is not expressed at high levels in the early stages of disease suggests that elucidation of mechanisms that regulate Ret expression is required for understanding the pathobiology of MEN2A. The association of high-level Ret expression with neuronal morphology suggests that the variable overexpression of Ret in pheochromocytomas might in part be an epiphenomenon, reflecting the known phenotypic plasticity of these tumors.     

Expression of S100 proteins in normal human tissues and common cancers using tissue microarrays: S100A6, S100A8, S100A9 and S100A11 are all overexpressed in common cancers

AIMS: To survey the expression of members of the S100 family of calcium-binding proteins in normal human tissues and common cancers using tissue microarrays. S100A6, S100A8, S100A9 and S100A11 have all been suggested to have potential roles in carcinogenesis and tumour progression but their expression has not been described in a wide range of human tissues and tumours. METHODS AND RESULTS: A custom-made tissue array, containing 291 tissue cores representing 28 tissue types and 21 tumour types, was used to produce sections that were immunostained for S100A2, S100A6, S100A8, S100A9, S100A11, calbindin 1, calbindin 2, S100B and parvalbumin. S100A6, S100A8 and S100A9 were expressed in 32%, 12% and 28% of breast cancers, respectively. There was a translocation of S100A11 expression from exclusively nuclear in normal tissues to cytoplasmic and nuclear in all common cancers. CONCLUSIONS: S100A6, S100A8, S100A9 and S100A11 are all expressed in common cancers, especially breast cancer. In addition, S100A11 undergoes a nucleocytoplasmic translocation which may have a direct influence on the proliferation of the cancer cells.     

Immunohistochemical study of chromogranin in 100 cases of pheochromocytoma,carotid body tumour,medullary thyroid carcinoma and carcinoid tumour

Summary Neuroendocrine cells have histologically common features represented by argyrophilic cytoplasm containing neuroendocrine granules. Neuroendocrine granules are composed of various kinds of peptide hormones, amines, carrier proteins and ATP. Although various kinds of peptide hormones have been detected in neuroendocrine tumours, a peptide hormone has not been required as a standard marker for these tumours. Chromogranin is a purified protein which binds catecholamines specifically and is recognized as a carrier protein. We carried out an immunohistochemical study of chromogranin immunoreactivity in 100 neuroendocrine tumours including pheochromocytomas, carotid body tumours, medullary thyroid carcinomas and carcinoid tumours. Marked immunoreactivity was observed in 85% of carcinoid tumours and 100% of the other tumour types. A non-functioning paraganglioma and a malignant carcinoid tumour without any other detectable marker also showed strong immunoreactivity to chromogranin. Chromogranin immunoreactivity is a useful tool for neuroendocrine tumours.     

S-100 protein immunostaining in teratomatous well-differentiated tissues

The presence and distribution of S-100 protein were studied in a case of ovarian teratoma using avidin-biotin complex immunoperoxidase method. S-100 immunoreactivity within the well-differentiated teratomatous tissues appeared to mirror that of their normally developed human counterparts, although we could unexpectedly detect S-100 immunoreactivity in bone osteoblasts and osteocytes, and in tooth germ odontoblasts. Choroid plexus-like formations present in the teratoma were also positive. Our results suggest that the presence of S-100 protein might be associated with a basic cell function regardless of the presence of a physiologically end-developed human organism.     

Immunohistochemical localization of chromostatin and pancreastatin,chromogranin A-Derived bioactive peptides,in normal and neoplastic neuroendocrine tissues

Despite the widespread distribution of chromogranin A (CgA) in neuroendocrine tissues, the biological function of CgA has not yet been elucidated. The primary amino acid sequence of CgA, elucidated by cDNA analysis, has been revealed to include several pairs of basic amino acid residues that are homologous to the bioactive peptides, such as pancreastatin (PST) and chromostatin (CST). Using antibodies for human PST and CST, the immunohistochemical localization of these peptides was investigated in neuroendocrine tissues, including human pituitary glands, pancreas, adrenal medulla, various types of neuroendocrine neoplasms (13 pheochromocytomas, 10 medullary thyroid carcinomas, 11 pancreatic endocrine tumors, and 19 carcinoid tumors), and the cell line QGP-1N derived from human somatostatin-producing pancreatic endocrine tumor. Variable immunoreactive intensities of PST and CST were seen, but both peptides were detectable in all neuroendocrine tissues and in most of the neoplasms. Immunoreactivity for both PST and CST was observed in 100 and 73%, respectively, of pancreatic endocrine tumors, all pheochromocytomas, and 80 and 40%, respectively, of medullary thyroid carcinomas, as well as all nonrectal carcinoid tumors. In rectal carcinoids, cells immunoreactive for PST and CST were sparse. The distribution of PST and CST was similar to that of CgA, and it is considered that these peptides are simultaneously processed from CgA, and may play roles in autocrine and paracrine regulation on various hormones in addition to their previously known functions.     

Proinflammatory properties of the human S100 protein S100A12

S100 proteins represent a new class of chemoattractants. Here we extend earlier evidence for the proinflammatory properties of human S100A12. A12 induced migration of monocytoid cells, with optimal activity at 10(-10) M and potency of >10(-9) M C5a. Neutrophils were poorly responsive, and lymphocyte migration was not affected. Actin polymerization in monocytoid cells was accompanied by a sustained [Ca(2+)]i flux of a magnitude comparable with C5a. A12 elicited a transient infiltration of neutrophils (4-8 h) and more delayed recruitment of monocytes (8-24 h) in vivo. A12 (approximately 70 nM) was present in synovial fluid (SF) from rheumatoid arthritis patients, and synovium contained A12-positive neutrophils in the sublining and interstitial region, often surrounding the perivasculature but rarely in the synovial lining layer, although some macrophages were positive. The A12 gene was transiently up-regulated in monocytes by tumor necrosis factor alpha (6 h); induction by lipopolysaccharide (LPS) was sustained (12-48 h). A12 may contribute to leukocyte migration in chronic inflammatory responses.     

Localization of S100C immunoreactivity in various human tissues

Using 2-dimensional gel electrophoresis, we previously demonstrated that the S100C protein remarkably decreased after immortalization of normal human fibroblasts, and that this protein caused growth inhibition of human tumor cells when forcibly expressed in these cells, suggesting that S100C plays a significant role in tumor suppression. The present study was carried out to determine what type of human tissues express S100C protein, and, subsequently, whether the S100C content in these tissues changes after normal cells have been transformed into cancer cells. We found that ductal cells in various tissues were positively stained with the S100C protein. In comparison, epithelial cells in digestive organs such as the stomach, small intestine, and colon were not stained as strongly. When 14 pairs of human normal and cancerous tissues were stained with the antibody, decreases in the staining levels of S100C were observed in 6 kinds of cancerous tissues--from the bronchus, mammary duct, renal tubule, prostate, uterus, and testis--in comparison with staining in their normal counterparts. These results suggest that S100C is a new tumor marker protein, the expression of which significantly decreases after malignant transformation of human tissues.     

Functioning and nonfunctioning adrenal medullary tumors

Recent development of imaging tools such as computed tomography, ultrasonography, and magnetic resonance imaging have incidentally discovered hormonally functioning or nonfunctioning adrenal tumors. Most adrenal medullary tumors are pheochromocytomas and neuroblastoma group tumors. They are representative of neuroendocrine tumors and can be diagnosed using neuroendocrine markers such as chromogranin A, synaptophysin, and neurofilament proteins. Catecholamine-synthesizing enzymes are also useful markers for these catecholamine-producing tumors. Both pheochromocytoma and neuroblastoma group tumors have cells that are immunohistochemicaJly positive for many peptide hormones including m-enkephalin, neuropeptide Y, somatostatin, vasoactive intestinal peptide, corticotropinreleasing hormone, adrenocorticotropic hormone, calcitonin, and calcitonin gene-related peptide, among others. The evidence for production of these hormones is confirmed by mRNA analysis using in situ hybridization or Northern blot hybridization and by measuring protein levels with radioimmunoassay. Only a limited number of patients, however, complain of clinical symptoms associated with excessive peptide hormone production such as watery diarrhea, hypokalemia, and achlorhydria syndrome or Cushing’s syndrome. The monoclonal human neuroblastoma cell line (NB-1) is a good model by which to understand the mechanism of excessive hormone production. NB-1 cells are usually nonfunctioning, but when they are stimulated by cyclic adenosine monophosphate and phorbol ester, they become capable of production and release of many peptide hormones and undergo morphological changes in their endocrine features. Thus, microenvironmental change seems to be one of the factors regulating gene expression and hormone production. Some molecular studies of oncogenes and growth factors are reviewed to gain an understanding of cell differentiation and proliferation. Finally, several chromosomal abnormalities reported in multiple endocrine neoplasia are introduced as potential tumorigenic factors.     

S100 protein-positive sustentacular cells in malignant and locally aggressive adrenal pheochromocytomas.

The absence or presence of S100-positive sustentacular cells has been previously shown to be correlated with benign and malignant pheochromocytomas and paragangliomas. We evaluated a total of 17 malignant and recurrent or locally aggressive adrenal pheochromocytomas for their quantity of sustentacular cells. An absence of sustentacular cells was demonstrated in the majority of malignant cases, while the locally aggressive or recurrent group usually contained an abundance of these cells. However, in one malignant case a constant moderate number of sustentacular cells in the primary site and in two sequential metastases was found. We conclude that the absence of sustentacular cells in pheochromocytomas may indicate a lesion with a greater potential for metastasis and that sustentacular cells, when they are present in a malignant pheochromocytoma, are an integral part of the tumor.     

Enkephalin-like immunoreactivity in human adrenal medullary cultures

Normal adult human chromaffin cells in culture contain abundant immunoreactive enkephalin and exhibit spontaneous and nerve growth factor-induced outgrowth of processes. In cultures of adrenals from two age-matched white males, approximately 70 per cent of chromaffin cells from one case and 50 per cent from the other contained immunocytochemically demonstrable leuenkephalin-like material. There was no apparent correlation between enkephalin-like immunoreactivity and capacity for process outgrowth. Cells with and without processes stained for enkephalin, and the percentages of stained cells with processes appeared to change in proportion of overall process outgrowth. Numerous cells with and without processes also failed to stain for enkephalin. Marked variations in sizes of stained cells and in distribution and intensity of staining were observed in all of the cultures. These findings suggest that adult chromaffin cells are heterogeneous at a number of levels.     

Discordant secretion of calcitonin and chromogranin in the human medullary thyroid carcinoma cell line

A cell fine of human medullary carcinoma of the thyroid, abundantly producing calcitonin (Ct) and related hormones, has proved remarkably useful as an endocrine tumor model for the study of the secretion mechanism. This cell line (TT cell) was used in studies to elucidate the dynamics of the release of Ct and chromogranin (Cg) to culture medium. The studies evaluated the intracellular concentration of Ct and Cg and the concentration changes elicited by the protein kinase C activator, phorbol ester (TPA); the adenylate cyclase-associated protein kinase A activator, forskolin; and the calcium ionophore, A23187. In addition, immunogold labeling of Ct and Cg was carried out to investigate the ultrastructural changes resulting from the stimulations. All these secretagogues effected the release of Ct and Cg into the medium in a dose-dependent manner, and the rate of the increase in the Ct secretion was consistently and markedly higher than that of Cg in more than certain dosages of the secretagogues. Most cells contained secretory granules immunolabeled for both Ct and Cg, and a considerable decrease was noted in the poststimulation count of the granules containing both substances, with the cells retaining more Cg than Ct. The discordance may be explained by different secretory pathways of the two proteins or different rates of synthesis.     

人S100蛋白的表达及其抗血清的制备和脑脊液中S100含量测定方法的建立

目的：建立定量检测脑脊液（CSF）及血清中S100蛋白的方法，探讨S100蛋白的检测在辅助诊断克－雅病（CJD）中的应用。方法：利用脑cDNA文库，经PCR获得了S100基因并克隆至原核表达载体pGEX-2T上，在大肠埃希菌中表达了谷胱甘肽－S－转移酶（GST）－S100融合蛋白；融合蛋白经亲和纯化后，免疫家兔，制备抗体；抗体经纯化后，用生物素（BNHS）标记，建立了可定量检测S100蛋白的生物素－亲和素系统ELISA方法，并初步用于临床脑脊液的检测中。结果：所表达的GST－S100蛋白相对分子质量约为35000，以其为抗原制备的S100特异性抗血清具有良好的免疫反应性。建立了定量检测脑脊液中S100蛋白的双抗体夹心ELISA方法，对3例“可能性的CJD”患者（14－3－3蛋白阳性）和15例无痴呆症状患者脑脊液进行检测，结果显示，3例CJD患者脑脊液S100含量均超过2．900μg/L,而在无痴呆症状患者组中14例患者脑脊液S100含量都低于0．180μg/L。对正常人和CJD患者血清进行检测，显示S100蛋白含量个体间差异很大。结论：所建立的方法可用于脑脊液中S100蛋白的检测，进一步扩大标本量有助于明确脑脊液中S100蛋白的检测在辅助诊断CJD中的价值。     

Distribution of chromogranin A and secretogranin I (chromogranin B) in neuroendocrine cells and tumors.

The distribution of chromogranin A and secretogranin I (chromogranin B) in normal and neoplastic human endocrine tissues was analyzed with two human monoclonal antibodies against chromogranin A, anti-bovine antiserum against chromogranin A, and an anti-rat antiserum against secretogranin I. Western blotting analyses showed both chromogranin A and secretogranin I in normal adrenals, pheochromocytomas, a pituitary adenoma, and in normal pituitary glands, but not in a bladder carcinoma. Rat adrenal medullary and anterior pituitary tissues reacted with the polyclonal chromogranin A and secretogranin I antisera, but not with the two monoclonal chromogranin A antibodies. All antibodies reacted with most of the neuroendocrine cells and tumors examined. Pituitary prolactinomas contained immunoreactive secretogranin I, but not chromogranin A. Analysis of the distribution of chromogranin A and secretogranin I in pancreatic islet cells showed that chromogranin A was found predominantly in the glucagon-producing A cells, whereas secretogranin I was present in less than 5% of islet cells. These results indicate that chromogranin A and secretogranin I are both useful in the characterization of some neuroendocrine cells and neoplasms.