Polyamine cytochemistry

Summary o-Phtalaldehyde (OPT) reacts with a number of biologically important molecules, including the polyamines, spermidine and spermine. By systematically varying reaction conditions with respect to temperature, pH, concentration and length of exposure to the reagent, using both model systems and tissues, we have succeeded in constructing a cytochemical OPT-method specific for spermidine and spermine. The method detects cell types known to contain these polyamines, including growing and neoplastic cells. The staining pattern obtained with the OPT method is identical to that obtained with the formaldehyde-fluorescamine (FF) technique recently shown to be specific for spermidine and spermine. In contrast to the FF technique, the OPT method can be used for staining suspensions of isolated cells and may hence be employed in studies using fluorescence-activated cell sorting (FACS). Preliminary such studies show a pronounced decrease in cellular OPT-induced fluorescence, paralleled by a decrease in content of polyamines, after treatment with the polyamine biosynthesis inhibitor -difluoromethylornithine (DFMO). In contrast, cells simultaneously treated with DFMO+spermidine show pronounced increases in their spermidine content and parallel increases in their OPT-induced fluorescence. Availability of methods selectively demonstrating polyamines at the cellular and subcellular level is expected to aid our understanding of polyamine functions in normal growth and cancer.     

Localization and possible function of polyamines in protein and peptide secreting cells

Three different cytochemical methods, the formaldehyde-fluorescamine and the ortho-phtalaldehyde fluorescence cytochemical methods as well as immunocytochemistry have been developed for localising the polyamines spermidine and spermine in tissue. All three methods produce identical results and show polyamines to occur inter alia in high concentrations in certain protein- and peptide-secreting cells. Many of these cells also show the capacity to metabolise monoamines and belong to the amine content or amine precursor uptake and decarboxylation (APUD) series. In cell types where fluorescence microscopical resolution allows, most polyamines appear to be localised to secretory granules. Moreover, studies on isolated pancreatic islets reveal active and glucose-dependent polyamine biosynthesis to occur in insulin cells. Possible function of polyamines in secretory granules are discussed in the light of the above findings.     

Towards microfluorometric quantitation of polyamines in situ. Relationship between cellular polyamine concentration and fluorescence yield of the formaldehyde fluorescamine method

Two different fluorescence cytochemical methods, the formaldehyde-fluorescamine (FF) method and the orthophthalaldehyde (OPT) method as well as an immunocytochemical method have been developed for the localization of spermidine and spermine. Of these three methods, the FF-method is the most easy to perform. We have studied the relationship between fluorescence intensity induced by the FF-method and cellular polyamine levels measured by HPLC in MCF-7 cells and HeLa cells. The experiments were designed to obtain different cell concentrations of polyamines. Cells grown on microscope slides in Petri-dishes were partly depleted of spermidine by two days inhibition of their ornithine decarboxylase activity using alpha-difluoromethylornithine. One hr before harvest the cells were exposed to different concentrations (0-30 microM) of spermidine. Microfluorometric results and chemical determinations of spermidine and spermine were obtained from each separate slide. The cellular total polyamine (spermidine + spermine) concentration on the slides varied between 4 and 15 nmol per mg protein (MCF-7 cells) and 5 and 26 nmol per mg protein (HeLa cells) and the corresponding microfluorometric results between 60 and 115 arbitrary units (MCF-7 cells) and 80 and 160 arbitrary units (HeLa cells). Simple regression analysis showed a good linear relationship between cellular polyamine concentration and FF-fluorescence yield. The correlation coefficient for MCF-7 cells was 0.86 and for HeLa cells 0.82, significance of the correlations was p less than or equal to 0.0001. Our results add further credence to the specificity of the FF-method and indicate that the method may be useful for microfluorometric quantitation of polyamines in situ.     

The formaldehyde-fluorescamine method

Summary Formaldehyde reacts with primary amino groups to derivatives which are unable to react with the fluorogenic primary amino group probe, fluorescamine. Paradoxically, however, certain specific cell systems continue to display strong fluorescamine-induced fluorescence after formaldehyde pretreatment. Among such formaldehyde-fluorescamine (FF) positive cell systems are certain peptide- and protein-secreting cells as well as all hitherto investigated types of cancer cells. We have now optimized the cytochemical FF method by using microfluorometry in combination with systematically varied reaction conditions. In addition, the quantitative data indicate that in FF positive cells, formaldehyde pretreatment causes a paradoxical increase in the fluorescence yield with fluorescamine. This has tentatively been ascribed to quenching phenomena, associated with closely spaced primary amino groups. Work with alternative fluorogenic amino group probes (MDPF and OPT) show that these display the same spectrum of tissue selectivity as fluorescamine, but that the latter remains the reagent of choice for the cytochemical FF reaction.     

Polyamines, molecules necessary for cell division, colocalize with peptide growth factors

The naturally occurring polyamines spermidine and spermine are necessary for cell division and growth. By restaining experiments, using three independent polyamine cytochemical methods, together with peptide immunocytochemistry, we show that substantial amounts of polyamines occur in a number of peptide growth factor-producing cell types. These include submandibular granular convoluted duct cells producing epidermal growth factor (EGF), pancreatic islet cells producing insulin and anterior pituitary cells producing growth hormone (GH). Other cell types in these tissues display only weak or no polyamine reactivity. Also blood platelets, known to contain platelet-derived growth factor (PDGF), are strongly stained for polyamines. Moreover, in EGF cells, insulin cells and blood platelets, polyamines are clearly localized in secretory granules. The possibility that polyamines may be coreleased and act in concert with peptide growth factors is discussed.     

Towards microfluorometric quantitation of polyamines in situ

Summary Two different fluorescence cytochemical methods, the formaldehyde-fluorescamine (FF) method and the orthophthalaldehyde (OPT) method as well as an immunocytochemical method have been developed for the localization of spermidine and spermine. Of these three methods, the FF-method is the most easy to perform. We have studied the relationship between fluorescence intensity induced by the FF-method and cellular polyamine levels measured by HPLC in MCF-7 cells and HeLa cells. The experiments were designed to obtain different cell concentrations of polyamines. Cells grown on microscope slides in Petri-dishes were partly depleted of spermidine by two days inhibition of their ornithine decarboxylase activity using -difluoromethylornithine. One hr before harvest the cells were exposed to different concentrations (0–30 M) of spermidine. Microfluorometric results and chemical determinations of spermidine and spermine were obtained from each separate slide. The cellular total polyamine (spermidine + spermine) concentration on the slides varied between 4 and 15 nmol per mg protein (MCF-7 cells) and 5 and 26 nmol per mg protein (HeLa cells) and the corresponding microfluorometric results between 60 and 115 arbitrary units (MCF-7 cells) and 80 and 160 arbitrary units (HeLa cells). Simple regression analysis showed a good linear relationship between cellular polyamine concentration and FF-fluorescence yield. The correlation coefficient for MCF-7 cells was 0.86 and for HeLa cells 0.82, significance of the correlations was p0.0001. Our results add further credence to the specificity of the FF-method and indicate that the method may be useful for microfluorometric quantitation of polyamines in situ.     

Endogenous polyamines are intimately associated with highly condensed chromatin in vivo. A fluorescence cytochemical and immunocytochemical study of spermine and spermidine during the cell cycle and in reactivated nuclei

Polyamines are low molecular weight aliphatic polycations essential for cell proliferation and differentiation. By immunocytochemistry, as well as by two independent fluorescence cytochemical methods, we show that polyamines are associated with highly condensed chromatin in nucleated erythrocytes and in metaphase and anaphase chromosomes. In other cells, polyamines mainly occur in cytoplasm. The association between polyamines and DNA in condensed chromatin is so close that DNase treatment is necessary for making polyamines available for reaction with antibodies. Studies of chick/HeLa cell heterokaryons reveal that polyamines disappear from the chick erythrocyte nuclei concomitantly with DNA decondensation and initiation of RNA synthesis. Our data strongly suggest that polyamines are important for chromatin condensation in vivo.     

Cytochemical and biochemical microanalysis of carcinogenesis

Conclusions While the understanding of early cellular changes gleaned from conventional histopathology alone has been rather limited, modern cytochemical methods at the light and electron microscope level have revealed in a number of experimental models and in some human tumour types important results which indicate that a sequence of qualitatively different cell populations is followed during carcinogenesis Some of the cytochemical and electron microscope findings can be correlated with specific cytopathological phenomena which are readily detectable in routine H & E sections. Thus the evaluation of animal experiments concerned with the testing of chemical compounds for carcinogenicity has been improved. The use of simple cytochemical techniques may considerably increase the reliability of the results. With respect to the further elucidation of the mechanism of neoplastic cell transformation, cytochemistry has broadened research horizons. The identification of putative preneoplastic and early neoplastic cell populations by cytochemical methods allows for the first time the microdissection and subsequent detailed investigation of target cells of the carcinogen which are at a high risk of becoming cancer cells. The combination of cytochemical and biochemical microanalysis seems to be the most useful tool for clarifying a number of important problems of carcinogenesis at present.Dedicated to Professor Ekkehard Grundmann on the occasion of his sixtieth birthday.     

Cellular localization of polyamines: Cytochemical and ultrastructural methods providing new clues to polyamine function in ram spermatozoa

Summary— Polyamine binding sites have been localized in ram spermatozoa using biochemical and cytochemical tools. Incubating the cells with ¹⁴C-spermine and determining its distribution after sonication and differential centrifugation, revealed that 60% of the radioactive spermine was localized in the head, 21.5% in the tail and about 9% in the plasma membrane. A polyamine specific cytochemical staining by the formaldehyde-fluorescamine method, revealed that most of the polyamines were localized in the midpiece, where the cell mitochondria are located, and in the acrosome region. Two additional studies used electron microscopy, employing polycationic colloidal gold and spermine-ferritin as cytochemical markers. The most sensitive and specific method was the staining of the cells with ferritin-spermine whose synthesis is described in this study. The outer acrosomal membrane was the preferential site for spermine binding which was densely distributed in a highly orderly pattern. There was a sparse distribution of spermine binding sites on the plasma membrane surrounding the acrosome and none on the post acrosomal region. The role of spermine in the acrosome reaction and Ca²⁺ fluxes in sperm cells is discussed.     

Endogenous polyamines associate with DNA during its condensation in mammalian tissue. A fluorescence cytochemical and immunocytochemical study of polyamines in fetal rat liver

The polyamines spermidine and spermine are essential for cell proliferation and differentiation. By two independent fluorescence cytochemical methods as well as by immunocytochemistry, we have studied the distribution of these molecules in fetal rat liver. Strong reactions for polyamines were found in highly condensed chromatin, present in chromosomes in mitotic cells, and in condensed nuclei in late erythropoietic cells. Moreover, polyamines were so closely associated with DNA in condensed chromatin that DNase pretreatment was necessary for making them available for reaction with antibodies. In other cells, polyamines were mainly localized to the cytoplasm. Studies of cells at different stages in erythropoiesis revealed that polyamines become associated with DNA during its condensation and inactivation. Our data strongly indicate that polyamines participate in the condensation of DNA.     

Immunocytochemical localization of polyamines in normal and neoplastic cells. Comparisons to the formaldehyde-fluorescamine and o-phthalaldehyde methods

Summary Polyamines are low molecular weight organic cations, necessary for cell proliferation and implicated in numerous biochemical events. Their light microscopical distribution has previously been studied by the use of two fluorescence cytochemical methods. With the aid of an antibody recognizing the two main polyamines, spermidine and spermine, we now report on their immunocytochemical localization in animal tissues. Polyamine immunocytochemistry was found to require very well controlled conditions of fixation in order to prevent diffusion, loss and redistribution of endogenous polyamines. Moreover, in certain cellular compartments, polyamine immunoreactivity was masked by proteins, necessitating proteolytic pretreatment of sections prior to staining. The fluorescence cytochemical methods, employing low molecular weight reagents, did not require such unmasking. The results of the optimized immunocytochemical procedure were in complete agreement with the results obtained by the fluorescence cytochemical methods. Although fluorescence cytochemistry, is simpler and quicker to perform than immunocytochemistry, the latter technique may be extended to studies of polyamines at the ultrastructural level.     

Collagen sandwich culture affects intracellular polyamine levels of human hepatocytes

Abstract. Extracellular matrices, like collagen layers, play an important role in preventing dedifferentiation of hepatocytes in long-term culture experiments. It has also been shown that polyamines are crucial for cell growth and liver differentiation – regeneration. Primary cultured hepatocytes with their low mitotic activity might be a valuable tool in studying the role of polyamines in differentiation. Here, our goal was to investigate whether an extracellular cell culture matrix can influence intracellular polyamine levels in human hepatocytes during long-term culture. Primary human hepatocytes were isolated from surgical tissue resections and were maintained either in single collagen (SG) or double collagen gel (DG) layer (sandwich) culture systems. Cell viability and function were examined and intracellular polyamine levels were measured using a highly sensitive high performance liquid chromatography (HPLC) method. Hepatocytes showed high viability in both culture systems used, but albumin secretion was diminished in SG cultured hepatocytes after 14 days. In general, total intracellular polyamine levels of hepatocytes decreased markedly in both SG and DG within the first days of culture, but remained constant until day 21 with a SG/DG ratio of about 1.4. Individual polyamines levels were dependent on the culture time and system, where spermine decreased and putrescine increased in both SG and DG over time (day 14), but spermidine increased only in DG. Our results suggest that polyamine levels, in particular putrescine, might be important regulators of hepatocyte specific function in vitro and therefore serve as a marker of differentiation for cultivated human hepatocytes.     

Exploring the potential of food forestry to assist in ecological restoration in North America and beyond

Food forests—edible, perennial, polyculture systems—are of increasing interest in North America and the United Kingdom, as reflected in projects ranging from urban food initiatives to integrated conservation and restoration planning. To examine emerging food forestry (FF) against the backdrop of ecological restoration (ER), we conducted semi‐structured interviews with eight experts each from the fields of FF and ER in conjunction with observations of food forests in Canada, the United States, and the United Kingdom. Using content analysis, our study builds a FF model that encompasses the underlying goals of emerging FF—forest function; diversity of yields; education and culture sharing; healthy habitats for people and other species; and sustainability. We argue that FF has potential as an urban restoration tool in terms of enhancing the multifunctionality of heterogeneous landscapes undergoing significant changes. This will require meaningful consideration of ethical issues (e.g. commodification of nature), landscape contexts, ecological integrity, integration of historical knowledge, and resilience for interdependent, dynamic social and ecological systems. Moreover, systematic, long‐term monitoring of different types of food forests will be crucial in order to mindfully apply FF in ER. This research provides one of the first in‐depth analyses of how emerging FF might contribute to restoration in the time of the Anthropocene, especially outside traditional tropic regions where most FF has been practiced.     

Single cell and tissue specific methods for evaluation of radiation and microgravity effects

A discussion of different methods to evaluate dose/response and biological effects of ionizing radiation is given. Confocal scanning laser microscopy (CSLM) is presented as a high performing observation method for evaluating different cytological effects. Standard cytochemical techniques can be used to analyse the cell in situ with minimal disturbance of morphology and structure. If a relatively small number of cells are affected by the treatment, the use of confocal microscope observations is fast and has a better resolution than conventional fluorescence microscopy. The optical sectioning capability of the CSLM makes it possible to analyse stacks of cells on detectors up to a depth of 200 micrometer with a resolution of 0.7 micrometer. This is used to analyse single cell electrophoresis results and nuclear track analysis in poly allyl diglycol carbonate (PADC). Consecutive analysis of cells cultivated on PADC, and analysis of nuclear tracks after chemical etched tracks in the PADC, will make it possible to correlate physical dose with direct cellular effects. This is a promising method for single cell analysis and the study of the effects of ionizing radiation at low particle flux density.     

Preservation and phallotoxin-staining of the microfilament system in Amoeba proteus

The spatial organization of the microfilament system as the main component of the cytoskeleton in Amoeba proteus was preserved by a glutaraldehyde-lysine-fixation and visualized with fluorescent phallotoxins (NBD- phallacidin , R-phalloidin). Results obtained by means of this method coincide exactly with observations gained from immunocytochemical, ultrastructural and molecular cytochemical studies, i.e., the microfilament system is mainly displayed beneath the cell membrane, at the hyalo - granuloplasmic border and around the cell nucleus. The preparation procedure employed is suitable for the rapid demonstration of cytoplasmic microfilaments in cells difficult to preserve by chemical fixation.     

Functional analysis of <Emphasis Type="Italic">OsPUT1</Emphasis>, a rice polyamine uptake transporter

Polyamines are nitrogenous compounds found in all eukaryotic and prokaryotic cells and absolutely essential for cell viability. In plants, they regulate several growth and developmental processes and the levels of polyamines are also correlated with the plant responses to various biotic and abiotic stresses. In plant cells, polyamines are synthesized in plastids and cytosol. This biosynthetic compartmentation indicates that the specific transporters are essential to transport polyamines between the cellular compartments. In the present study, a phylogenetic analysis was used to identify candidate polyamine transporters in rice. A full-length cDNA rice clone AK068055 was heterologously expressed in the Saccharomyces cerevisiae spermidine uptake mutant, agp2∆. Radiological uptake and competitive inhibition studies with putrescine indicated that rice gene encodes a protein that functioned as a spermidine-preferential transporter. In competition experiments with several amino acids at 25-fold higher levels than spermidine, only methionine, asparagine, and glutamine were effective in reducing uptake of spermidine to 60% of control rates. Based on those observations, this rice gene was named polyamine uptake transporter 1 (OsPUT1). Tissue-specific expression of OsPUT1 by semiquantitative RT-PCR showed that the gene was expressed in all tissues except seeds and roots. Transient expression assays in onion epidermal cells and rice protoplasts failed to localize to a cellular compartment. The characterization of the first plant polyamine transporter sets the stage for a systems approach that can be used to build a model to fully define how the biosynthesis, degradation, and transport of polyamines in plants mediate developmental and biotic responses.     

Polyamine metabolism in BHK21/C13 cells : Loss of spermidine from cells following transfer to serum-depleted medium

The growth rate of BHK21/C13 cells in culture was slowed down by transferring growing cells to serum-depleted medium Following deprivation of serum, the intracellular concentration of polyamines decreased. The amount of spermidine relative to spermine decreased, and this change was the result of the spermidine content per cell decreasing more than the spermine content. The decrease in cell content of polyamines was accompanied by release of polyamines from the cells into the culture medium. The polyamines released were examined using cells whose polyamines had been labelled by prior incubation of the cells with radioactive putrescine. Almost all of the radioactivity released into the medium was found in spermidine, even though the cells contained most of their radioactivity in spermine. It is suggested that specific release of spermidine may be an important mechanism by which these cells can regulate their intracellular content of polyamines.     

Polyamines and colon cancer

In colon cancer, the activities of polyamine-synthesizing enzymes and polyamine content are increased 3-4-fold over that found in the equivalent normal colonic mucosa, and polyamines have even been attributed as markers of neoplastic proliferation in the colon. Furthermore, and in contrast with all other cell systems in the body, normal and neoplastic cells in the colon are exposed to high concentrations of putrescine from the lumen, synthesized by colonic microflora. While such a high polyamine supply may be of benefit in non-neoplastic colonic mucosal growth, the role of luminal polyamines in colon cancer is a clear concern. Luminal polyamines are readily taken up by neoplastic colonocytes, they are utilized in full to support neoplastic growth, and their uptake is strongly up-regulated by the mitogens known to play an important role in colonic carcinogenesis. Inhibition of polyamine synthesis and their uptake, impaired utilization of exogenous polyamines, and enhanced catabolism of polyamines in neoplastic colonocytes are therefore logical approaches in the chemoprevention of colorectal cancer.     

Translation of ascites and mengovirus RNA in fractionated cell-free systems from uninfected and mengovirus-infected Ehrlich-ascites-tumor cells.

We have prepared homologous, fractionated, cell-free translational systems from uninfected and mengovirus-infected Ehrlich ascites tumor cells in order to determine what alterations occur following virus infection in the translational machinery of the host cell. Two major differences distinguish the system developed from infected cells. First, it has a 40% lower rate of protein synthesis, primarily a consequence of the rate of chain elongation, which is depressed to 60 amino acids/min from 90 amino acids/min in the system from uninfected cells. Second, at supraoptimal concentrations of Mg2+ and K+ the system from virus-infected cells supports the translation of mengovirus RNA but not host mRNA. These differences between the two systems may reflect specific changes which are responsible for the selective translation of mengovirus RNA in the infected cell. In both systems the optimal concentrations of polyamines, monovalent and divalent cations, mRNA, and ribosomal subunits are the same for the translation of either host or viral RNA. This uniformity is useful in experiments, designed to investigate the selective translation of viral RNA, where various components of the two systems are interchanged.     

The effect of avidin-biotin interactions in detection systems for in situ hybridization.

The effect of avidin-biotin interactions in several detection systems for the non-radioactive in situ hybridization (ISH) technique was studied in a model system using a transitional cell carcinoma line and a biotinylated DNA probe. We performed fluorescence ISH to unravel the individual steps in a sensitive and frequently used amplification method which makes use of the alternating cytochemical detection layers of fluorescein isothiocyanate-conjugated avidin (AvFITC) and biotinylated goat anti-avidin (BioGAA) antibodies to detect the hybridized and biotinylated probe. Our experiments revealed that BioGAA antibodies bind with their antigen binding sites and not with their biotin moieties to avidin molecules that have already interacted with the DNA probe. The probable working mechanism of this amplification method is presented in a model. Furthermore, we used a peroxidase staining technique to compare with each other the sensitivity of several other detection systems in which avidin-biotin interactions play an important role, e.g., the avidin-biotinylated peroxidase complex (ABC) system. The experiments show that avidin molecules can not be efficiently used to interconnect two biotinylated molecular layers, since their introduction leads to firmly closed cytochemical networks. Such a closed network is already formed between the hybridized and biotinylated DNA probe and a first detection layer of avidin molecules, as appears from the finding that biotinylated molecules could hardly be coupled to these avidin molecules in a following detection layer. Therefore, the results presented here provide us with new insight into the molecular basis of cytochemical network formation. This will enable us to choose the proper procedures for increasing the sensitivity of ISH detection systems.