Cellular and subcellular localization of polyamines cytochemical methods providing new clues to polyamine function in normal and neoplastic cells

Summary Studies on the cellular and subcellular localization of polyamines have previously been hampered by the rapid redistribution of these organic cations during cell fractionation. Cytochemical methods selectively localizing polyamines would, hence, be important for our understanding of where polyamines are stored and where they work. With the aid of chemical characterizations of tissue extracts, biosynthesis experiments and cytochemical model experiments we show that the cytochemical formaldehyde-fluorescamine (FF) technique selectively detects the polyamines spermine and spermidine. Results obtained with the FF method concur with previous chemical observations by showing that growing and neoplastic cell populations are rich in cytochemically detectable polyamines. The FF method also localizes polyamines to certain normal secretory cell systems, including pancreatic exocrine and endocrine (insulin) cells, cells and secretion products in the ventral prostate, pituitary growth hormone cells and neurosecretory nerve endings and leucocytes. In most cell systems studied, polyamines appear to be most concentrated in cytoplasmic structures. However, in HeLa cell mitotic chromosomes and in frog red cell nuclei most polyamines seem to be associated with chromatin. These observations raise important questions concerning both extra- and intracellular functions of polyamines.     

Immunocytochemical localization of polyamines in the gastrointestinal tracts of rats and mice

An immunocytochemical method using a recently produced monoclonal antibody (ASPM-29) with an antibody specificity to spermine (Spm) and spermidine (Spd) fixed in situ, was used to demonstrate an immunocytochemical localization of polyamine (PA) pools in the gastrointestinal tracts of rats and mice. High PA immunoreactivity was always found in the cytoplasm of cells not only at the cell proliferative zone or the precursor cell zone but also at the neighboring non-proliferative premature cell zone of the epithelium, and a gradient of decreasing PA levels was noticed from these cells to the fully mature differentiated gastric surface mucous cells and absorptive cells of the small and large intestines. Also, strong staining for PAs was seen in the cytoplasm of fully differentiated gastric chief cells and neurons of both the myenteric and submucous plexuses, whereas the nuclei of the cells remained virtually unstained. These results may suggest that PAs are closely associated with the high biosynthetic activity in the cells of the gastrointestinal mucosa of normal rats and mice. This seems to be consistent with the PA imunocytochemical results previously obtained for neoplastic cells and active protein- or peptide-secreting cells, including exocrine or endocrine cell types.     

Immunocytochemical localization of polyamines during attachment and spreading of retinal pigment epithelial and intestinal epithelial cells

In order to form and maintain a protective barrier for photoreceptors, the retinal pigment epithelium relies on integrin signaling and related pathways to form adhesion complexes, undergo cell spreading, and establish a confluent cellular monolayer. Polyamines are multifunctional polycations that are essential for cell attachment and spreading, although their exact mechanisms of action are as yet unclear. We report new immunocytochemical evidence suggesting that in the cells of retinal pigment epithelium and also the intestinal epithelium, polyamines are present in a population of intracellular vesicles that appear transiently during initial stages of cell spreading. In newly attached cells with minimal spreading, the vesicles are seen near the nucleus, whereas in more highly spread cells, the vesicles are localized to the plasma membrane, near, but not precisely co-localized with an enzyme marker for adhesion complexes, focal adhesion kinase. We also observe pronounced nuclear staining in newly attached cells that have not spread, whereas this staining is decreased in cells that have spread. Nuclear staining has been previously reported in other cell types and has been attributed to DNA binding of polyamines, which is known to stabilize chromatin structure. We hypothesize that the appearance of polyamine vesicles near focal adhesions of cells undergoing attachment and spreading may reflect the mechanism by which polyamine pools are targeted to appropriate interaction sites necessary for the assembly of adhesion complexes. Alternatively, the vesicles could represent the mechanism by which polyamines are removed from the nucleus and possibly released from the cell.     

Ultrastructural localization of enzymes of the membranes and RNP-structures of the nuclei of normal and neoplastic cells.

Ultrastructural localization of some oxidative and dephosphorylating enzymes was investigated in isolated rat and mouse liver nuclei and in nuclei isolated from various rat hepatomas. In the discussion the significance of changes in the activity of these enzymes in neoplastic cells is considered.     

Ferritin immunohistochemical localization in normal and neoplastic colonic mucosa

High levels of ferritin have been detected in serum and tumoral extracts of gastrointestinal neoplasms. However, its histological localization is not well known. An immunoperoxidase technique (PAP) was used for detecting ferritin in 30 colorectal carcinomas, 20 polyps and 8 cases of non-neoplastic mucosae. Ferritin staining was detected in stromal cells (98%) much more than in epithelial cells (21%). Connective cells were positive in 5 cases of normal mucosae (62%), 19 polyps (95%) and all carcinomas (100%). The number of positive cells gradually rose from normal mucosa to carcinoma with an intermediate score in adenomas. However, no relation could be found between the stromal ferritin score and dysplasia in polyps. Likewise, no relation was found between the stromal ferritin score and the differentiation grade, invasion or metastases in carcinomas. The positive epithelial pattern seen in 12 cases (21%) suggests non-specific staining due to passive diffusion from the stroma. Thus, these immunohistochemical findings suggest that in colonic neoplasms, ferritin could be a tumor marker produced mainly by stromal cell reaction more than by the epithelial cells.     

Immunocytochemical localization of renin in juxtaglomerular cells

The involvement of various organelles in the synthesis, transport, and packaging of renin in the juxtaglomerular cells of newborn mice has been investigated by immunocytochemistry with the protein A-gold technique. Highly specific rabbit antibodies against mouse submandibular renin were used. Mild fixation and embedding in glycol methacrylate allowed enough sensitivity to identify a steep gradient of labeling from rough endoplasmic reticulum to Golgi complex to secretory granules. Routine fixation and embedding in Epon produced labeling differentials that allowed delineation of hitherto ill-defined types of secretory granules and vacuoles. The classical pattern of synthesis, transport, and packaging of secretory proteins involves the rough endoplasmic reticulum and Golgi complex and seems to apply to renin secretion. Immunoreactive renin is packaged as rhomboid crystals at the trans face of the Golgi complex. The limiting membrane of these rhomboids fuses to form coalescing protogranules where the crystals eventually yield their individuality maturing into secretory granules. Vacuoles containing a flocculent material, with or without a dense core, show significant immunocytochemical labeling. These vacuoles are not associated with the Golgi complex but occupy cytoplasmic areas well endowed with rough endoplasmic reticulum. As judged from their morphological features and their immunoreactivity, the vacuoles do not seem to follow the sequence of events typical of protogranules and coalescing protogranules. They possibly represent a parallel pathway of renin synthesis and transport, involving the nuclear envelope and bypassing the Golgi complex.     

Cell cycle control in normal and neoplastic cells.

Recent studies of cell cycle control suggest that cyclin-dependent protein kinases play a central role in the cell's commitment to a new division cycle in late G1. The regulation of these kinases in normal and neoplastic growth is becoming clear.     

Immunocytochemical localization of proteoglycans within normal elastin fibers

Appreciable amounts of glycosaminoglycans have been found by immunocytochemistry within mature elastin fibers of human dermis. On thin sections, elastin fibers showed antigenic sites for monoclonal antibodies recognizing the unsaturated units remaining after digestion of hyaluronic acid with Streptomyces hyaluronidase and after digestion of dermatan and chondroitin sulfates with chondroitinase ABC. Moreover, sectioned elastin fibers were positive towards antibodies raised against synthetic peptides corresponding to amino acid sequences near the N-terminus of the protein core of small matrix proteoglycans PGI and PGII, respectively (Fisher et al., J. Biol. Chem. 262, 9702-9708 (1987)). This is the first demonstration that highly hydrophylic molecules are strictly associated with normally cross-linked elastin. The presence of highly hydrated molecules within the elastin polymer could greatly influence its physiological properties and behavior in pathology.     

Immunocytochemical localization of the elongation factor Tu in E. coli cells

The localization of the elongation factor Tu (EF-Tu) in ultrathin cryosections of E. coli cells was determined with the electron microscope using a highly specific immunological labelling technique. EF-Tu is distributed almost homogeneously throughout the cytoplasm. Although it has often been suggested that EF-Tu could be part of a putative prokaryotic cytoskeleton, we did not find any evidence for supramolecular assemblies, such as fibres or filaments, containing a large amount of EF-Tu. EF-Tu was not observed in association with the outer cell membrane and periplasmic space. A topological relationship with the inner membrane is not apparent in our micrographs. In cells in which the EF-Tu level is raised significantly, the protein piles up in discrete cell regions.     

Immunohistochemical localization of sex hormone-binding globulin in normal and neoplastic breast tissue.

The distribution of sex hormone-binding globulin-like antigens (SHBG-LA) in normal and neoplastic human breast tissues was investigated by immunohistochemistry, employing a monospecific polyclonal antiserum against highly purified human SHBG and an avidin-biotin-peroxidase complex (ABC) method in formalin-fixed paraffin embedded tissue sections. In normal breast tissues the staining of SHBG-LA was present exclusively in the cytoplasm of epithelial cells of ductal and ductular types. Nuclei as well as stromal and lymphatic tissues remained unstained. While the staining was positive in all cases of intraductal carcinoma, only 4 out of 15 infiltrating carcinomas revealed SHBG-LA. The demonstration of a plasma sex steroid binding globulin in the cytoplasm of endocrine target cells is consistent with the hypothesis that steroid-binding globulins are able to enter target cells. The apparent loss of this specific cell function in infiltrating carcinomas may result from dedifferentiation and change of cell membrane properties occurring during the process of neoplastic progression.     

Immunocytochemical identification of myoepithelial cells in normal and neoplastic rat mammary glands with the lectins Griffonia simplicifolia-1 and pokeweed mitogen

Peroxidase-conjugated Griffonia simplicifolia-1 (GS-1) and pokeweed mitogen (PWM) histochemically stain only the myoepithelial cells and not the epithelial or fibroblastic cells of rat mammary glands preserved in methacarn or glutaraldehyde and embedded in paraffin. This pattern of staining occurs in other rat exocrine glands except the pancreas, but is the reverse of that seen in most lining epithelium. The histochemical binding of GS-1 and PWM to myoepithelial cells is inhibited specifically by D-galactose and by polymers of N-acetylglucosamine, respectively. GS-1 and its subcomponent, GS-1-B4, also bind to extracellular structures similar to those stained by anti-laminin serum. At the ultrastructural level, both conjugated GS-1 and PWM bind to the plasma membrane of the myoepithelial cells, as well as to the adjacent basement membrane. Non-metastasizing rat mammary tumors produced by dimethylbenz[a]anthracene, by derivative epithelial stem-cell lines, and by a transplantable tumor all contain more elongated myoepithelium-like cells as well as cuboidal epithelium-like cells; both cell types are neoplastic. The more elongated myoepithelium-like cells are stained by GS-1 and PWM, whereas the cuboidal epithelium-like cells are unstained. Moderately and strongly metastatic rat mammary tumors produced by epithelial cell lines and by transplantable tumors, respectively, contain no such neoplastic cells that bind either lectin. We suggest that the carbohydrate receptors for GS-1 and PWM are consistent markers for the presence of the myoepithelial cell in normal and tumorous rat mammary glands.     

Differentiation and the neoplastic transformation of normal cells

It is suggested that blastomogenic factors can be considered as the means isolating normal cells from each other. Carcinogenic substances caused cell functions to disturb, to inhibit or to intensify the synthesis of specific substances, to change the cell surface disrupted contacts of interactions between cells, pressing cells to become on the way of evolution, and to create heterogenic populations of cells.     

Immunocytochemical localization of the extracellular calcium-sensing receptor in normal and malignant human large intestinal mucosa.

We identified the parathyroid type Ca(2+)-sensing receptor (CaR) in normal human colon mucosa and in cancerous lesions at the mRNA and protein level. Polymerase chain reaction produced an amplification product from reverse-transcribed large intestinal RNA which corresponded in size and length to a 537-bp sequence from exon 7 of the CaR gene. With a specific antiserum against its extracellular domain, the CaR could be detected by immunostaining in normal human colon mucosa in cells preferentially located at the crypt base. The CaR protein was also expressed in tumors of the large bowel in all 20 patients examined. However, the great majority of CaR-positive cells in the adenocarcinomas inspected were confined to more differentiated areas exhibiting glandular-tubular structures. Poorly or undifferentiated regions were either devoid of specific immunoreactivity or contained only isolated CaR-positive cells. In the normal mucosa and in glandular-tubular structures of cancerous lesions, the CaR was exclusively expressed in chromogranin A-positive enteroendocrine cells and in only a small fraction of PCNA-positive cells.     

Immunocytochemical localization of fodrin and ankyrin in bovine chromaffin cells in vitro.

We raised antibodies to brain fodrin and erythrocyte ankyrin and examined the distribution of the antigens in cultured bovine chromaffin cells by immunocytochemical techniques. Immunofluorescence microscopy of whole cells showed intense labeling for both proteins, but fine localization could not be determined. In contrast, in cell specimens mechanically unroofed before fixation, the distribution of the two proteins revealed an apparent difference in the ventral plasma membrane: immunofluorescence for fodrin was dense and mostly even, whereas that for ankyrin appeared as scattered dots. Immunogold electron microscopy of the unroofed cells showed that labeling for fodrin was localized in a network of thin filaments, the diameter of which was 2-3 nm at the thinnest portion. Ankyrin labeling was mostly associated with filaments 5-10 nm in diameter. Notably, labeling for both fodrin and ankyrin was found over the coated membrane. The present results indicate that fodrin and ankyrin in the chromaffin cell do not constitute a submembranous network as spectrin and ankyrin do in the erythrocyte; whereas fodrin is closely associated with the plasma membrane, ankyrin is mostly linked to the cytoskeleton. The existence of both proteins in the coated region implies that they are functionally related to exocytosis and/or to ensuing membrane retrieval in the chromaffin cell.     

Immunocytochemical demonstration of polyamines in nucleoli and nuclei

Although biochemical studies have shown that polyamines (PAs) occur in the nucleus, only few studies have examined the intranuclear distribution of these organic cations. By immunocytochemistry, we have previously demonstrated that PAs are located in ribosomes. We now show that PAs also are present in both nucleoli and nuclei of a variety of cell types. Detection of nucleolar and nuclear PAs required novel pretreatment procedures involving protease and/or DNase digestion of specimens prior to immunoreaction. Double fluorescence staining confirmed the localizations. This suggests that PAs may be important to the formation of ribosomes in nucleoli, as well as adds support to biochemical studies suggesting that PAs are involved in many biological events in the nucleus. Further biochemical studies will be needed to substantiate this hypothesis.     

Immunocytochemical localization of endothelin in cultured bovine endothelial cells

Summary To investigate the intracellular localization of endothelin in cultured endothelial cells, an immunocytochemical study was carried out by the post-embedding protein A-gold technique with endothelin-specific antiserum. Gold particles were seen on the rough endoplasmic reticulum, the Golgi cisternae, the Golgi vesicles, small vesicles beneath the cell membrane, and the lysosomes. By contrast, no secretory granules were observed. These results suggest that endothelin is secreted by a constitutive pathway and that the lysosome may play an important role in regulating the biological activity of endothelin.     

Immunocytochemical localization of ornithine decarboxylase in cultured murine cells

Summary Antiserum elicited to ornithine decarboxylase (ODC) purified from murine RAW 264 macrophage-like cells has been employed to localize ODC in cultured murine cells. The antiserum immunoprecipitated 100% of the ODC activity from the cultured cells. The specificity of the antiserum was demonstrated by the immunoprecipitation from ³⁵S-methionine metabolically-labeled cell extracts of a single protein which migrated upon SDS-gel electrophoresis coincident with authentic ODC. Indirect immunofluorescence experiments were performed on paraformaldehyde-fixed RAW 264 cells and JB6 epidermal cells using the rabbit anti-ODC antiserum and FITC-conjugated goat anti-rabbit IgG. Little immunofluorescence was apparent in non-stimulated cells. Intense immunofluorescence was detectable in stimulated cells at times of peak cellular ODC activity. Antigenically-reactive ODC was localized diffusely in the cytoplasm and was absent in the nuclei of RAW 264 cells, whereas in the JB6 cells the immunodetectable enzyme protein was localized in a punctate pattern in both the cytoplasm and nucleoplasm and was absent in the nucleolus. The appearance and disappearance of immunoreactive ODC in both cell types after stimulation was consistent with the alterations in ODC activity.     

Immunocytochemical localization of endothelin in cultured bovine endothelial cells

To investigate the intracellular localization of endothelin in cultured endothelial cells, an immunocytochemical study was carried out by the post-embedding protein A-gold technique with endothelin-specific antiserum. Gold particles were seen on the rough endoplasmic reticulum, the Golgi cisternae, the Golgi vesicles, small vesicles beneath the cell membrane, and the lysosomes. By contrast, no secretory granules were observed. These results suggest that endothelin is secreted by a constitutive pathway and that the lysosome may play an important role in regulating the biological activity of endothelin.     

Transgenic mouse models for studies of the role of polyamines in normal,hypertrophic and neoplastic growth

Transgenic mice expressing proteins altering polyamine levels in a tissue-specific manner have considerable promise for evaluation of the roles of polyamines in normal, hypertrophic and neoplastic growth. This short review summarizes the available transgenic models. Mice with large increases in ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase or antizyme, a protein regulating polyamine synthesis by reducing polyamine transport and ODC in the heart, have been produced using constructs in which the protein is expressed from the alpha -myosin heavy-chain promoter. These mice are useful in studies of the role of polyamines in hypertrophic growth. Expression from keratin promoters has been used to target increased synthesis of ODC, spermidine/spermine-N(1)-acetyltransferase (SSAT) and antizyme in the skin. Such expression of ODC leads to an increased sensitivity to chemical and UV carcinogenesis. Expression of antizyme inhibits carcinogenesis in skin and forestomach. Expression of SSAT increases the incidence of skin papillomas and their progression to carcinomas in response to a two-stage carcinogenesis protocol. These results establish the importance of polyamines in carcinogenesis and neoplastic growth and these transgenic mice will be valuable experimental tools to evaluate the importance of polyamines in mediating responses to oncogenes and studies of cancer chemoprevention.