Immunohistochemical demonstration of nonspecific cross-reacting antigen in normal and neoplastic human tissues using a monoclonal antibody. Comparison with carcinoembryonic antigen localization

Nonspecific cross-reacting antigen (NCA) immunoreactivity was localized in normal and neoplastic human tissues using a monoclonal antibody to 55, 90 and 95 kDa molecules of NCA. This was compared to the localization of immunoreactive carcinoembryonic antigen (CEA) as demonstrated by polyclonal and monoclonal antibodies. In frozen sections, CEA was localized in normal surface epithelium of the stomach and colon where NCA was only weakly detected. Type 1 and type 2-like pneumocytes were positive for NCA, while CEA was localized only in type 2-like pneumocytes. CEA and NCA were both demonstrated in ductal cells of frozen pancreatobiliary and mammary tissues. The antigenicity of CEA and NCA in normal tissues was significantly lost after paraffin embedding as compared to frozen sections. NCA was consistently demonstrated in eccrine sweat glands embedded in paraffin. In various tumor tissues, CEA and NCA were colocalized and expression increased sufficiently to be detected in paraffin sections. Adenocarcinomas of the stomach and colon and cystadenocarcinoma of the pancreas, as well as neuroendocrine carcinomas of the lung and thyroid, showed a CEA predominance over NCA. In ductal adenocarcinomas of the pancreas and breast and in cholangiocarcinoma, NCA reactivity was greater than CEA. Keratinizing foci of most squamous cell carcinomas of mucosal origin and some adenocarcinomas equally expressed both. Hepatocellular carcinoma, lobular mammary carcinoma and papillary thyroid carcinoma were positive only with unabsorbed polyclonal antibody which widely recognizes CEA-related substances. Renal cell carcinoma, prostatic adenocarcinoma, transitional cell carcinoma, anaplastic carcinomas, choriocarcinoma and basal cell carcinomas showed little or no immunoreactivity. Hence the relative ratio of CEA/NCA expression in tumors was dependent on the tissue of origin and histologic type. The cytoplasmic granular staining of NCA in cancer cells was a noteworthy difference from the plasma membrane-associated localization of CEA.     

Immunoelectron microscopic localization of carcinoembryonic antigen in gastric adenocarcinoma cell lines

The distribution of carcinoembryonic antigen (CEA) in human gastric adenocarcinoma cell lines (HPE-GAC-3 cells and HPE-GAC-2 cells) was determined immunohistochemically by indirect peroxidase-labeled antibody method at the light and electron microscopic levels. In GAC-3 cells that proliferated as non-adherent single cells, CEA was located in the perinuclear spaces, the endoplasmic reticulum, Golgi apparatus, vesicles, multivesicular body (MVB) and entire plasma membrane. Membrane CEA was shown to be internalized into MVB in GAC-3 cells. In GAC-2 cells that form an acinus, CEA was predominantly present along the microvilli of the lumina) surface and in glycocalyceal bodies, the vesicles which bud from the microvilli into the lumen. These results suggest that in poorly differentiated cancer cells CEA is transported over the entire cell surface, retained on the membrane and accumulated Into the cell by way of the MVB, but in well differentiated cancer cells the newly synthesized CEA is rapidly and predominantly transported to the luminal surface and rapidly released from the membrane into the lumen by way of the glycocalyceal body.     

Role of nonspecific cross-reacting antigen, a CD66 cluster antigen, in activation of human granulocytes.

Nonspecific cross-reacting antigen (NCA) is the name of a family of highly glycosylated bacterial-binding receptors found on human granulocytes and other tissues. These glycoproteins are members of the immunoglobulin supergene family and are related structurally to carcinoembryonic antigen. In this study, we demonstrate that ligation of granulocyte NCA results in the activation of the cells, as measured by degranulation and the flux of intracellular calcium. These studies further the proposition that NCA has a function in the immune response of granulocytes against bacterial infections.     

Localization of thrombomodulin antigen in rabbit endothelial cells in culture. An immunofluorescence and immunoelectron microscope study

A total of 13 rabbit endothelial cell lines were examined for the presence, distribution, and localization of the thrombomodulin (TM) antigen. Three endothelial cell lines were derived from aorta, 5 lines from brain microvessels, and 5 lines from fat microvessels. Immunofluorescence demonstrated the presence of the TM antigen in all cell endothelial lines examined, at all passage levels, and at all stages of growth i.e., log phase, confluent, postconfluent stationary. The percentage of positive cells in a given culture and the distribution of the TM antigen within the cells, as well as on their surfaces, was a cell line characteristic. The intercellular variation in the expression of the TM antigen was insignificant in some cell lines but was striking in others. These characteristics were clonable. Immunofluorescence indicated and immunoperoxidase electron microscopy confirmed the localization of the TM antigen to the outer cell surface and in the Golgi-endoplasmic reticulum (GERL) complex.     

Proteomic analysis of ubiquitination-associated proteins in a cisplatin-resistant human lung adenocarcinoma cell line

The objective of this study was to screen for ubiquitination-associated proteins involved in cisplatin resistance in a human lung adenocarcinoma cell strain using a comparative proteomic strategy. We employed 1D SDS-PAGE to separate ubiquitinated proteins isolated and enriched from A549 and A549/CDDP lysates via affinity chromatography. The differentially expressed bands between 45-85 kDa were subsequently hydrolyzed by trypsin and subjected to HPLC-CHIP-MS/MS analysis. Of the 11 proteins identified, 7 proteins were monoubiquitinated or polyubiquitinated substrates and 4 proteins were E3 ubiquitin ligase-associated proteins. The results of western blotting and confocal laser scanning microscopy indicated that the expression levels of the E3 ubiquitin ligases RNF6, LRSAM1 and TRIM25 in A549 cells were significantly lower than those in the A549/CDDP cell line. The expression levels of the above three ubiquitin ligases in both cell lines were significantly decreased upon treatment with cis-diamminedichloroplatinum (CDDP), and the expression in the A549/CDDP cell after the treatment with CDDP decreased to a lesser extent. The expression of the substrate PKM2 in the A549 cell was higher than that in the A549/CDDP cells. Moreover, the expression of PKM2 increased in the A549 cell line and decreased in the A549/CDDP cell line upon CDDP treatment. This study suggests that drug resistance is closely correlated with changes in the ubiquitination process at the protein level in a human lung adenocarcinoma cell line.     

Elastin in alcoholic liver disease. An immunohistochemical and immunoelectron microscopic study

Increased elastic stained material has been described in fibrotic and cirrhotic liver processes. The aim of this work was to follow the development and distribution of elastic fibers from 48 chronic alcoholic patients. Patients were scored for fibrosis as 0, without fibrosis or minimal (n = 5); 1, incipient or early fibrosis (n = 9); 2, fibrosis or incomplete cirrhosis (n = 12); and 3, cirrhosis (n = 22). Elastica staining was performed by orcein, resorcin-fuchsin and iron hematoxylin and confirmed by immunofluorescence staining with an anti-human elastin antibody (Institut Pasteur). Electron microscopy of representative cases of each group and electron microscopy of immunolabelled elastin (n = 5) were also performed. In early alcoholic fibrosis, oxytalan fibers were pointed out in terminal hepatic veins and in Disse space. In fibrous portal extensions and cirrhotic internodular septa, oxytalan and elaunin fibers represented the major elastin components in association with the alcoholic liver fibroplasia. Immunostaining with anti-elastin Ab exhibits the same distribution as with histochemical methods in portal and septal zones. Electron microscopy confirmed abundant microfibrillar bundles between collagen fibers that mesh and are in continuity with elaunin fibers. Immunoelectron microscopy confirmed elastin deposits in the amorphous material and in association with the microfibrillar material in the portal and septal zones and disclosed elastin even in the thin strands of fibrotic tissue. In conclusion, elastogenesis, mainly represented by oxytalan and elaunin fibers, develops in alcoholic disease and takes part, with collagen deposits, in the fibrotic process.     

Atypical endocrine granules in atypical endocrine tumor (AET) of the lung. An immunoelectron microscopic study

Highly dense granules are a hallmark for recognizing atypical endocrine tumor (AET) of the lung. We report a case of AET with many atypical neurosecretory-type granules: moderately dense granules (mean size 373.7 nm) and "target" granules with a central dense core (425.1 nm), both apparently larger than the highly dense granules (223.3 nm). Immunoelectron microscopical studies demonstrated that all three types of granule were positive for gastrin-releasing peptide (GRP), human chorionic gonadotropin alpha-subunit (hCG alpha), calcitonin or serotonin. Although the size profiles of positive granules were similar for calcitonin and hCG alpha, they were different from those of GRP or serotonin granules. The presence of atypical granules and the different size profiles of hormonal products in AET indicate that caution is required in ultrastructural evaluation of granules in lung carcinomas.     

Chromosomal localization of amplified c-myc in a human colon adenocarcinoma cell line with a biotinylated probe

A c-myc amplified sequence has been localized on a chromosome marker 19q+ with a biotin-labeled probe in the human colon adenocarcinoma SW480 cell line. The advantages of the technique for the localization of amplified DNA sequences are discussed.     

Subcellular localization of HMFG2 in breast carcinomas: an immunohistochemical and immunoelectron microscopic study.

Malignant transformation of human cells is associated with morphological and biochemical alterations. We have studied the distribution and pattern of staining of HMFG2 (human milk fat globulin) in normal breast, benign breast lesions, and 137 primary and metastatic breast carcinomas. Immunohistochemical staining was performed with an antibody to HMFG2 using the indirect peroxidase technique. Three patterns of staining were noted: 1) secretion and luminal staining (in normal breast, most benign breast lesions and some breast carcinomas); 2) plasma membrane staining (in breast carcinomas); 3) intracytoplasmic staining (in breast carcinomas). Immunoelectron microscopy was also performed on normal breast, infiltrating duct, and lobular carcinomas. Immunoelectron microscopy showed localization of the gold particles on the electron dense granules of the HMFG2 protein. These were localized along the surface of the extracytoplasmic lumina in normal breast ducts/acini and breast carcinomas, whereas localization was also noted within the intracytoplasmic lumina in cancer cells only. These results show that there is altered localization of milk fat globulin in breast cancer cells associated with membrane internalization and formation of intracytoplasmic lumina. This contributes to the understanding of the phenotypic alterations associated with malignant transformation in breast cancer.     

Establishment and characterization of a serous papillary adenocarcinoma cell line of the human ovary in a serum-free culture.

In order to clarify the biologic characteristics of serous papillary adenocarcinoma of the human ovary, we tried to establish a continuous cell line using four primary tumor specimens derived from four patients with such tumors. We also evaluated c-myc, MYCN and c-erbB2 gene amplification in the cultured cells, and the xenografts, as well as in these four primary tissue specimens by a Southern blot analysis. One continuous cell line, named as FU-OV-1, was established in a serum-free culture system and this line propagated continuously for 96 serial passages over a 2-year-period in vitro. FU-OV-1 reproduced and still maintained the characteristics of the original tumor. C-myc gene amplification was found in the FU-OV-1 cells, and the xenografts, as well as in only the primary tumor of FU-OV-1 out of the four primary serous papillary adenocarcinomas. However, neither MYCN amplification nor c-erbB2 amplification was observed in any tumor specimens including FU-OV-1 cells. In conclusion, FU-OV-1 is thus considered to be a useful system for studying the biological behavior of serous papillary adenocarcinoma in the human ovary. The results of this study suggest that c-myc gene amplification might thus be associated with the progression of this tumor both in vitro and in vivo.     

Neuronal uptake of plasma proteins in cryogenic brain lesions. An immunoelectron microscopic study.

A previous light microscopic study on cryogenic brain lesions in rats demonstrated uptake of plasma proteins into damaged neurons within a few minutes after the lesion. The protein concentration was much higher inside the nerve cell bodies than in the surrounding neuropil. This is puzzling since the neuropil to a large extent consists of damaged neuronal processes. The present investigation describes the intracellular localization of albumin in this model using a post-embedding immunoelectron microscopic technique. The distribution of albumin in the lesions was studied after 1, 6 and 12 h survival periods. The intraneuronal albumin was mainly bound to the particulate elements of the cytoplasm and nuclei, while the watery parts of the cells showed no immunoreactivity. The intracellular organelles contained very little albumin, indicating that their membranes may be more resistant to freezing than those of the cells. Most of the neuronal and glial processes in the neuropil were swollen and contained almost no albumin. This explains the contrast between the strong immunoreactivity of the neurons and the vague reactivity of the neuropil in light microscopy.     

Scar adenocarcinoma of the lung: a light and electron microscopic study.

Five well differentiated peripheral adenocarcinomas of the lung were investigated, using light and electron microscopy. Each tumour contained a central nidus of fibrous tissue and fulfilled the criteria for "scar cancer." One tumour also had a focus of lamellated collagenous tissue, suggestive of an old tuberculous granuloma. Electron microscopy showed the features of Clara cells, with characteristic dense bodies in the apical cytoplasm and scattered microvilli on the luminal surface. It was concluded that this variant of scar cancer was a carcinoma of Clara cells, which was sufficiently distinctive in appearance to be recognised on light microscopy alone. It remains uncertain, however, whether the central fibrous area is a desmoplastic response to tumour growth or a pre-existing scar.     

Actin in the extracellular matrix of smooth muscle cells. An immunoelectron microscopic study

Actin has been specifically detected in the intercellular matrix of mouse smooth muscle cells and along the vascular internal elastic membrane by means of immunoferritin-electron microscopy, employing human antibodies to smooth muscle actin. The presence of actin in the smooth muscle matrix suggests that this protein may have relevance in controlling cell-to-cell adhesion and the sliding of one smooth muscle cell over the other, both in contracted and expanded status. The association of actin with the elastic membrane may represent the anatomical basis of a functional link between elastic membrane, adjacent fibronectin and smooth muscle cells. Thus the transmission of movement from the smooth muscle cells to the elastic membrane can be achieved.     

Elastin in human,baboon, and mouse liver: An immunohistochemical and immunoelectron microscopic study

Light microscope histochemistry and immunohistochemistry, and routine electron microscopy techniques were performed to analyse elastin distribution and structure in the human liver compared with that in baboon and mouse. In man and baboon, elastic fibers stained by iron hematoxylin or orcinolnew fuchsin seemed to be solitary and were few in number; in the mouse they were thinner but abundant, both in the portal tract and in hepatic veins. Orcein or resorcin-fuchsin stains, employed after oxidation of tissue sections, revealed a network comprising elastic, elaunin, and oxytalan fibers, which was also demonstrated by immunofluorescence with anti-elastin antibody in man and baboon. At the ultrastructural level, the elastic fibers of the human portal tract corresponded to discontinuous patches of amorphous material intermingled with few microfibrils. These contrasted with the thinner elastic fibers of baboon and mouse liver which had a core of amorphous material. In man and baboon, these fibers meshed into slender bundles of microfibrils often exhibiting small spots of amorphous material (elaunin fibers) and terminated as isolated microfibrils (oxytalan fibers). Immunoelectron microscopy of elastin carried out on baboon liver tissue labelled the amorphous material and also its microfibrillar component. Immunoperoxidase deposits were also associated with isolated bundles of microfibrils in the baboon portal stroma. Immunolabelling and elastic stains disclosed an important elastin portal network located around vascular, biliary structures and interspaced with collagen bundles. The structural polymorphism of elastin, assembling different relative amounts of amorphous material and microfibrils, might have a relationship with the required elasticity in a given species.     

Surface and cytoplasmic expression of CD2, CD13, and CD25 antigens in human T lymphotropic virus type I infected cell line. An immunoelectron microscopic study

Immunoperoxidase electron microscopy revealed intracellular expression of CD2, CD13, and CD25 antigens in a unique human T lymphotropic virus type I infected cell line, TI-CL. This cell line is thought to be derived from cord mononuclear cells in the bifurcation stage of T cell and myelomonocytic lineage, based on cytochemical, immunologic, and molecular genetic analyses. Each antigen expression was observed on the plasma membranes, the cisternae of rough endoplasmic reticulum, and the perinuclear cisternae. Surface and cytoplasmic expression of CD25 antigen in adult T cell leukemia/lymphoma cells have been reported, but similar events concerning CD2 and CD13 antigens have not been described previously. CD13 antigen is a marker of myelomonocytic lineage, and biochemical analyses have demonstrated that monoclonal antibodies belonging to CD13 precipitated two glycoprotein molecules of 130,000 and 150,000 molecular weight and that the former molecule (gp130) is the precursor protein of the latter (gp150). In our findings, CD13-positive reaction in the cisternae of rough endoplasmic reticulum and the perinuclear cisternae may correspond to the precursor protein of CD13. CD2 antigen, a 50 kilodalton sheep erythrocyte rosette receptor protein, is the early T lineage antigen and plays an important role in T cell activation. The detection of CD2 antigen on the cell surface and in the cytoplasm may morphologically confirm that this antigen is synthesized in the cytoplasm and inserted into the plasma membrane.     

Immunoelectron microscopic localization of DF 3 cancer-associated antigen in human breast cancer.

Breast cancer cells are characterized by frequent occurrence of the DF 3 breast-cancer associated antigen in the cytoplasmic area. This study elucidated the details of this cytoplasmic reactivity by immunoelectron microscopy. In infiltrating breast carcinomas, the DF 3 antigen was localized in rough endoplasmic reticulum (rER), perinuclear space, Golgi complex, membrane-bound granules and along the outer surface of cell membrane. The antigen was also accumulated in the cytosol. In contrast to this, benign lesions showed localization of the DF 3 antigen mainly along the outer surface of apical cell membrane and rarely in rER or cytosol. The present study indicated that the DF 3 antigen was of secretory type and commonly localized on the outer surface of apical cell membrane in both malignant and benign lesions. Carcinoma tissues were further characterized by frequent occurrence of immunoreactivity related to the process of antigen synthesis. The diffuse cytosolic reactivity may be associated with disordered transportation of the antigen in the cytoplasm.     

Calcitonin, katacalcin, and calcitonin gene-related peptide in the human prostate. An immunocytochemical and immunoelectron microscopic study

We recently described for the first time the presence of calcitonin immunoreactivity (CTIR) in a subpopulation of prostatic and urethral endocrine-paracrine (EP) cells. We now further evaluate the distribution of the CTIR cell, characterize the coexistence of serotonin and calcitonin, and for the first time describe the coexpression of calcitonin and other calcitonin gene family peptides (calcitonin gene-related peptide and katacalcin) in the CTIR cell. Finally, the morphological ultrastructure of the secretory granule of the CTIR cell is analyzed. The finding of multiple calcitonin gene family peptides in prostatic and urethral EP cells and the specific localization of calcitonin to secretory granules strongly suggest that the calcitonin gene is expressed in this region and the products stored in the EP cells. The relatively high levels of calcitonin reported in the semen and the dendritic and nondendritic morphological features of the CTIR cell, respectively, suggest a lumencrine (exocrine), paracrine, and possibly endocrine role for calcitonin. The production of calcitonin and related peptides by the prostate may have implications in various pathologic processes of the prostate.     

Three-dimensional reorganization of a cell line of papilla Vateri adenocarcinoma in various culture conditions.

SAV-I, a cell line derived from a well differentiated adenocarcinoma of Vater's papilla, was cultured under four different conditions using collagen gel matrices (type I collagen): 1) double-layered, 2) floating double-layered, 3) embedded, and 4) floating embedded, then observed by light and electron microscopy and immunohistochemistry. Under all four conditions, three-dimensional growth with tubules occurred. In particular, the floating double-layered condition, where the cells were cultured between two collagen gel layers, then floated onto the medium, was useful for showing cellular reorganization. The three-dimensional growth patterns observed in vitro closely resembled the in vivo growth of SAV-I cells transplanted into nude mice. Therefore, we conclude that the floating double-layered condition is useful for demonstrating the morphological characteristics of the parent cells of established cell lines, and should be advantageous for studies of the relationship between cellular morphology and function in vitro.     

Incorporation of human chorionic gonadotropin alpha-subunit into different types of granule in stomach endocrine cells. An immunoelectron microscopic study

In order to demonstrate the ultrastructural localization of the alpha-subunit of human chorionic gonadotropin (hCG) in the stomach mucosa, an immunoelectron microscopic study was performed using formalin-fixed specimens. In pyloric glands, alpha hCG-positive granules were irregular in outline, with a mean area and maximum diameter of 2,857 x 10(4) nm2 and 218.4 nm, respectively. In fundic glands, the granules had a smoother outline and were larger in both area and maximum diameter (3,943 x 10(4) nm2, 246.8 nm) than those of pyloric glands (p less than 0.001). In the atrophic fundic glands of non-antral gastritis, the alpha hCG granules showed a difference in shape; more elliptical granules appeared to be increased, as indicated by the higher value of the axial ratio (1,452) and lower value of D (1,862) (log10 area alpha D.log10 perimeter) compared with those in pyloric (1,231, 1,968) and fundic glands (1,148, 1,979) (p less than 0.001). The alpha-subunit of hCG is thus incorporated into different types of endocrine cell in pyloric and fundic glands, and the granule morphology appears to differ in hyperplastic alpha hCG cells of non-antral gastritis.