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PubMed1988

[Demonstration of an intact complex of epidermal growth factor and its receptor in the endosomes of A-431 cells].

The compartmentalization of the epidermal growth factor (EGF) receptors in A-431 cells was studied using centrifugation of the microsomal fraction of these cells in continuous Percoll gradient. The existence of an intact (non-degraded) EGF receptor in plasma membrane and endosome fraction was demonstrated by electrophoretic analysis of in vitro phosphorylated Percoll fractions. No phosphorylated receptor was revealed in lysosomal fraction by this method. The existence of non dissociated EGF-receptor complexes in intracellular compartments 30 minutes after the start of internalization was proven using a synthesized photoreactive labeled EGF derivative (125I-EGF-SANAH). The removing of pH gradient in organellar membranes by 10 mkM of monensin did not affect dissociation from its receptor. The data obtained proved the existence of non-dissociated and non-degraded EGF-receptor complexes in the endosomal compartment of A-431 cells.

Carcinoma, Squamous Cell↗

PubMedDate not supplied

Nucleotide-gated KATP channels integrated with creatine and adenylate kinases: amplification, tuning and sensing of energetic signals in the compartmentalized cellular environment.

Transmission of energetic signals to membrane sensors, such as the ATP-sensitive K+ (KATP) channel, is vital for cellular adaptation to stress. Yet, cell compartmentation implies diffusional hindrances that hamper direct reception of cytosolic energetic signals. With high intracellular ATP levels, KATP channels may sense not bulk cytosolic, but rather local submembrane nucleotide concentrations set by membrane ATPases and phosphotransfer enzymes. Here, we analyzed the role of adenylate kinase and creatine kinase phosphotransfer reactions in energetic signal transmission over the strong diffusional barrier in the submembrane compartment, and translation of such signals into a nucleotide response detectable by KATP channels. Facilitated diffusion provided by creatine kinase and adenylate kinase phosphotransfer dissipated nucleotide gradients imposed by membrane ATPases, and shunted diffusional restrictions. Energetic signals, simulated as deviation of bulk ATP from its basal level, were amplified into an augmented nucleotide response in the submembrane space due to failure under stress of creatine kinase to facilitate nucleotide diffusion. Tuning of creatine kinase-dependent amplification of the nucleotide response was provided by adenylate kinase capable of adjusting the ATP/ADP ratio in the submembrane compartment securing adequate KATP channel response in accord with cellular metabolic demand. Thus, complementation between creatine kinase and adenylate kinase systems, here predicted by modeling and further supported experimentally, provides a mechanistic basis for metabolic sensor function governed by alterations in intracellular phosphotransfer fluxes.

Adenylate Kinase↗

PubMed1987

Rabies virus infection of cultured rat sensory neurons.

The axonal transport of rabies virus (challenge virus strain of fixed virus) was studied in differentiated rat embryonic dorsal root ganglion cells. In addition, we observed the attachment of rabies virus to neuronal extensions and virus production by infected neurons. A compartmentalized cell culture system was used, allowing infection and manipulation of neuronal extensions without exposing the neural soma to the virus. The cultures consisted of 60% large neuronal cells whose extensions exhibited neurofilament structures. Rabies virus demonstrated high binding affinity to unmyelinated neurites, as suggested by assays of virus adsorption and immunofluorescence studies. The rate of axoplasmic transport of virus was 12 to 24 mm/day, including the time required for internalization of the virus into neurites. The virus transport could be blocked by cytochalasin B, vinblastine, and colchicine, none of which negatively affected the production of virus in cells once the infection was established. It was concluded that, for the retrograde transfer of rabies virus by neurites from the periphery to the neuronal soma, the integrity of tubulin- and actin-containing structures is essential. The rat sensory neurons were characterized as permissive, moderately susceptible, but low producers of rabies virus. These neurons were capable of harboring rabies virus for long periods of time and able to release virus into the culture medium without showing any morphological alterations. The involvement of sensory neurons in rabies virus pathogenesis, both in viral transport and as a site for persistent viral infection, is discussed.

Adhesiveness↗

PubMed2000

Ultrastructure and distribution of interstitial glandular cells and associated elements in human fetal ovaries.

In order to understand the fine structure and distribution of the interstitial glandular cells (IGCs) and associated elements in the human fetal ovary, we studied human fetal ovaries at 16 weeks post fertilization (p. f.) by transmission electron microscopy. Semithin sections revealed voluminous typical IGCs usually grouped in clusters, located in the interstitium among the ovigerous cords. Isolated primordial follicles were seen in the cords located close to the interstitium in which IGCs were present. Besides the main ultrastructural characteristics of steroid secreting cells, the IGCs showed lipofuscin granules and stacks of annulate lamellae in their cytoplasm. Fibrocytes, macrophages and mast cells were detected close to the IGCs. In particular, the fibrocytes were located around the IGCs, with which they occasionally formed focal cell contacts. Fibrocytes issued numerous long projections, which, together with collagen fibers, surrounded the clusters of IGCs and small vessels (mainly capillaries), often extending into the intercellular spaces among IGCs. These data indicated that, already at the initiation of folliculogenesis, the IGCs are present numerously in a close association with the ovigerous cords. The morphological aspects of IGCs were comparable to that of fetal testis interstitial (Leydig) cells and hilar cells in adult ovary, and suggest that fetal IGCs may be source of adult ovary hilar cells. In addition, we have here demonstrated for the first time that IGCs are associated with stromal cells whose distribution seems to support IGCs microtopography. Fetal ovarian fibrocytes revealed a structural arrangement similar to that of the "compartmentalizing cells" previously described in the adult testis. Macrophages and mast cells presumably have a role as local modulators of steroid synthesis. Mast cells may also affect fibrocyte organization and vascular permeability. We thus suggest that IGCs and associated cells may form a glandular unit in the human fetal ovary similar to that in the adult testis, and this structure is likely involved in early steroid secretion during gonadal differentiation.

Female↗

PubMed1997

Compartmentalization of cAMP signaling in mesangial cells by phosphodiesterase isozymes PDE3 and PDE4. Regulation of superoxidation and mitogenesis.

Some major pathobiologic processes in renal mesangial cells, elicited in response to immunoinflammatory stimuli, are modulated via cAMP-protein kinase A (PKA) signaling pathways; namely, generation of reactive oxygen metabolites (ROM) and accelerated proliferation of mesangial cells. We investigated the role of cAMP phosphodiesterase (PDE) isozymes in these regulatory mechanisms. Generation of ROM in cultured rat mesangial cells was inhibited by selective inhibitors of PDE4, rolipram and denbufylline, whereas PDE3 inhibitors, cilostamide and lixazinone, had no effect. Conversely, cilostamide or lixazinone suppressed mitogenic synthesis of DNA in mesangial cells, but 1 microM rolipram or 1 microM denbufylline showed no inhibitory effect. The efficacy of PDE isozyme inhibitors (IC50) to suppress [3H]thymidine incorporation or ROM generation paralleled IC50 values for inhibition of cAMP PDE. Incubation of mesangial cells with either rolipram alone or with cilostamide alone increased significantly in situ activity of PKA in mesangial cells, assessed by (-cAMP/+cAMP) PKA activity ratio, and the stimulatory effects were additive. Results indicate that in mesangial cells a cAMP pool that is metabolized by PDE4 activates PKA and thereby inhibits ROM generation; another cAMP pool that is metabolized by PDE3 activates another PKA (isozyme or pool) which suppresses proliferation of mesangial cells. We propose that in mesangial cells, a cAMP-PKA pathway that regulates mitogenesis is determined by activity of PDE3, whereas another cAMP-PKA pathway is directed by activity of PDE4 and controls ROM generation. Therefore, two PDE isozymes within one cell type compartmentalize distinct cAMP signaling pathways.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1998

Nuclear antigens in Trichinella spiralis infected muscle cells: nuclear extraction, compartmentalization and complex formation.

Infection of mammalian skeletal muscle cells by Trichinella spiralis induces a series of changes that include: reentry of the terminally differentiated host cell into the cell cycle; suspension of infected cells in apparent G2/M; and transcriptional inactivation of the differentiated skeletal muscle gene program. Cell cycle repositioning and genetic reprogramming are chronic characteristics of host cells that can remain infected for years. Nuclear antigens (NA, 79, 86 and 97 kDa) that localize to host cell nuclei have been detected with antibodies against T. spiralis proteins. Since NA may play a role in regulating the infected cell phenotype, their origin, nuclear compartmentalization, and biochemical properties were investigated. We show that a monoclonal antibody to a defined epitope of T. spiralis glycans binds these NA, which indicates the parasite origin of these proteins. NA were not extracted under conditions that solubilized chromatin from infected cell nuclei. In contrast, NA were coextracted with B lamins (nuclear envelope) by 4 M urea. Urea extraction was pH dependent (8.0), suggesting ionic interaction of NA in protein complexes. Nevertheless, confocal microscopy demonstrated colocalization of NA with host chromatin, and not B lamins. Nuclear protein complexes containing NA were observed under non-reducing conditions, and NA were readily cross-linked in isolated nuclei by succinimidyl protein conjugating reagents. The results establish methods to extract NA from infected cell nuclei for further biochemical analysis, establish the existence of nuclear protein complexes containing NA and demonstrate colocalization of NA with host chromatin. Collectively, the results provide a foundation from which to investigate the role of NA in regulating the T. spiralis infected skeletal muscle cell phenotype.

Animals↗

PubMed2004

Spatial coordination of cytokinetic events by compartmentalization of the cell cortex.

During cytokinesis, furrow ingression and plasma membrane fission irreversibly separate daughter cells. How actomyosin ring assembly and contraction, vesicle fusion, and abscission are spatially coordinated was unknown. We found that during cytokinesis septin rings, located on both sides of the actomyosin ring, acted as barriers to compartmentalize the cortex around the cleavage site. Compartmentalization maintained diffusible cortical factors, such as the exocyst and the polarizome, to the site of cleavage. In turn, such factors were required for actomyosin ring function and membrane abscission. Thus, a specialized cortical compartment ensures the spatial coordination of cytokinetic events.

Actomyosin↗

PubMed1991

Biological membrane deterioration and associated quality losses in food tissues.

Biological membranes are rarely considered by food scientists when the deteriorative reactions that take place during the processing or storage of food tissues are studied. Yet, membranes and their deterioration play a major but underestimated role in food losses, and recent biochemical information indicates that at least some of these reactions can be controlled by procedures suited to food materials. Much of the present information available on membrane degradation in food systems is incomplete and speculative. It is known, however, that in order to accomplish their many indispensable functions in cells, membranes are constituted mainly of phospholipids, protein, and some carbohydrates arranged in thin, bimolecular sheet-like structures that serve to compartmentalize cells and their organelles. Membranes have embedded in their asymmetric surfaces complements of catalytic and cytoskeletal proteins that serve permeability and structural functions. Membrane surfaces exhibit fluidity, due partially to the continuous lateral diffusion of lipids and some proteins. Two important consequences of fluidity are the ability of membrane phospholipids to exist in different interconvertible conformational phase structures and the formation of heterogenous lipid domains on the membrane surface. Cellular death leads unavoidably to the initiation of membrane deterioration. While the time course of this series of reactions differs in animal and plant tissue, they are damaged by generally similar mechanisms. These include an initial peroxidative attack on polyunsaturated fatty acids, with the concomitant production of free radicals. Many biological agents can act as accelerating agents in these reactions, including transition metal ions, heme compounds, radiation, illuminated chlorophyll, calcium, and ethylene. Once formed, free radicals catalyze further reactions that can affect all aspects of membrane function and cellular metabolism, and lead ultimately to significant losses in food quality through defects such as chilling injury and cold shortening. These are aggravated by many food-processing steps, especially those that involve tissue disruption. Control of membrane breakdown by exogenous chemical intervention has been practiced, but, at best, this only slows the rate of these reactions. Newer approaches to this problem include dietary treatment of meat animals, modified storage and packaging conditions, and genetic interventions. This review advances the proposition that membrane deterioration can be considered a "universal mechanism" that leads to significant quality losses in food. Perhaps because the study of biological membranes and the biochemical and physiological properties has only begun recently, not much progress has been made in finding practical control mechanisms for these reactions in food systems.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed2003

Fluorescence bleaching reveals asymmetric compartment formation prior to cell division in Caulobacter.

Asymmetric cell division in Caulobacter crescentus yields daughter cells that have different cell fates. Compartmentalization of the predivisional cell is a critical event in the establishment of the differential distribution of regulatory factors that specify cell fate. To determine when during the cell cycle the cytoplasm is compartmentalized so that cytoplasmic proteins can no longer diffuse between the two nascent progeny cell compartments, we designed a fluorescence loss in photobleaching assay. Individual cells containing enhanced GFP were exposed to a bleaching laser pulse tightly focused at one cell pole. In compartmentalized cells, fluorescence disappears only in the compartment receiving the bleaching beam; in noncompartmentalized cells, fluorescence disappears from the entire cell. In a 135-min cell cycle, the cells were compartmentalized 18 +/- 5 min before the progeny cells separated. Clearance of the 22000 CtrA master transcriptional regulator molecules from the stalked portion of the predivisional cell is a controlling element of Caulobacter asymmetry. Monitoring of a fluorescent marker for CtrA showed that the differential degradation of CtrA in the nascent stalk cell compartment occurs only after the cytoplasm is compartmentalized.

Bacterial Proteins↗

PubMed1991

Phenotypic and functional characterization of human TCR gamma delta+ intestinal intraepithelial lymphocytes.

Intestinal intraepithelial lymphocytes (IEL) appear to represent a peculiar set of immune cells compartmentalized at the interface between the organism and the external environment. In previous studies we observed that within human IEL TCR-tau/delta T cells represent a major fraction that predominantly express the CD8 molecule and preferentially uses the V-delta-1 gene segment. Thus these data suggested a preferential accumulation/homing of CD8+ V-delta-1+ IEL within the human intestinal epithelium. However, to date the functional role of these cells with regard to immune regulation at this most critical immunological site is poorly understood. In this study, the cytotoxic potential and proliferative capacity of human IEL in response to mitogenic stimuli has been characterized with respect to IEL T cell receptor type and TCR-tau/delta variable gene segment usage as determined by flowmetry. The frequency of TCR-1+ IEL expressing both CD56 and CD16 which are considered to be NK-cell markers was found to be much higher (38.9 +/- 12.4%) than within intestinal lamina propria lymphocytes (LPL) (9.1 +/- 4.8%) or peripheral blood lymphocytes (PBL) (6.4 +/- 3.3%). In contrast, the fractions of CD16-CD56+ cells within IEL, LPL and PBL were comparable. Surprisingly, IEL mediated NK-cell activity (K562 lysis) was virtually absent whereas within PBL it was within the normal range. Furthermore, in cytotoxicity assays employing 51Cr-labeled OKT3 hybridoma cells and P815 cells as targets, the cytotoxic potential of IEL was much lower than that of PBL.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1978

Mechanism of densensitization in DNCB-contact sensitive guinea pigs.

Contact sensitivity to dinitrochlorobenzene (DNCB) in guinea pigs could be rapidly suppressed by intravenous injection of dinitrobenzene sulfonic acid sodium salt (DNBSO3). This suppression is transient and antigen-specific. Macrophages from desensitized animals are not inactivated as shown by their ability to react, both in vivo and in vitro to lymphokines produced in a separate system. Therefore, effector lymphocytes are considered the target for the desensitizing antigen. Using an adoptive transfer system it was demonstrated that effector lymphocytes are inactivated by a direct effect of the hapten. Since this inactivation can be reversed by trypsin treatment, a receptor blockade of effector lymphocytes is proposed as the mechanism of desensitization of DNCB-contact sensitive guinea pigs. This does not exclude the possibility that additional mechanisms such as suppressor cells, compartmentalization or endogenous proliferation of lymph node lymphocytes may play an additional role.

Animals↗

PubMed1975

On the physiological functions of teichoic acids.

The choline-containing teichoic acids of pneumococci can be modified by biosynthetic replacement of the choline residues with certain structural analogues, such as ethanolamine (EA) or the N-monomethyl-(MEA) and N-dimethyl-(DEA) amino derivatives of ethanolamine. Cells containing such analogues in their teichoic acids develop pleiomorphic alterations in several physiological properties, which include resistance to detergent-induced lysis and inhibition of cell separation (chain formation). We report here the results of physiological studies on the mechanism of these two phenomena. Our results are summarized in the following: (a) Pneumococci grown on various amino alcohols produce cell walls of identical amino sugar and amino acid composition. (b) Both choline- and EA-containing teichoic acids seem to follow the same conservative pattern of segregation during growth and cell division.(c)Lysis sensitivity of pneumococci requires the juxtaposition oflysissensitive (choline-containing) cell walls and endogenous autolysin at the cell wall growth zone. (d) Upon readdition of choline to ethanolamine-containing cells, lysis sensitivity and catalytically active (C-type) autolysin reappear in the bacteria with the same kinetics. (e) The chains of EA-grown pneumococci contain fully compartmentalized cells and normal cross walls.

Amino Alcohols↗

PubMed1990

Fine structure of the retinal photoreceptors of the domestic cat (Felis catus).

The morphology of the retinal photoreceptors of the domestic cat (Felis catus) has been investigated by light and electron microscopy. In this species the photoreceptors can be readily differentiated and adequately described by the classical terminology of rods and cones, with rods being the more numerous. Rods are long slender cells with inner and outer segments of much the same diameter. Cones are shorter cells with a conical outer segment that tapers distally and an inner segment which is wider than the outer segment. Both rods and cones are highly polarized and compartmentalized cells and consist of an outer segment, a connecting cilium, an inner segment, a nuclear region and a synaptic process leading to an expanded synaptic ending. Morphological similarities and differences are noted between the rods and cones for the various regions of these cells.

Animals↗

PubMed2000

Functional relationships between capacitation-dependent cell signaling and compartmentalized metabolic pathways in murine spermatozoa.

Spermatozoa are highly polarized cells with specific metabolic pathways compartmentalized in different regions. Previously, we hypothesized that glycolysis is organized in the fibrous sheath of the flagellum to provide ATP to dynein ATPases that generate motility and to protein kinases that regulate motility. Although a recent report suggested that glucose is not essential for murine sperm capacitation, we demonstrated that glucose (but not lactate or pyruvate) was necessary and sufficient to support the protein tyrosine phosphorylation events associated with capacitation. The effect of glucose on this signaling pathway was downstream of cAMP, and appeared to arise indirectly as a consequence of metabolism as opposed to a direct signaling effect. Moreover, the phosphorylation events were not affected by uncouplers of oxidative respiration, inhibitors of electron transfer, or by a lack of substrates for oxidative respiration in the medium. Further experiments aimed at identifying potential regulators of sperm glycolysis focused on a germ cell-specific isoform of hexokinase, HK1-SC, which localizes to the fibrous sheath. HK1-SC activity and biochemical localization did not change during sperm capacitation, suggesting that glycolysis in sperm is regulated either at the level of substrate availability or by downstream enzymes. These data support the hypothesis that ATP specifically produced by a compartmentalized glycolytic pathway in the principal piece of the flagellum, as opposed to ATP generated by mitochondria in the mid-piece, is strictly required for protein tyrosine phosphorylation events that take place during sperm capacitation. The relationship between these pathways suggests that spermatozoa offer a model system for the study of integration of compartmentalized metabolic and signaling pathways.

Adenosine Triphosphate↗

PubMed2002

Coupling of cell energetics with membrane metabolic sensing. Integrative signaling through creatine kinase phosphotransfer disrupted by M-CK gene knock-out.

Transduction of metabolic signals is essential in preserving cellular homeostasis. Yet, principles governing integration and synchronization of membrane metabolic sensors with cell metabolism remain elusive. Here, analysis of cellular nucleotide fluxes and nucleotide-dependent gating of the ATP-sensitive K+ (K(ATP)) channel, a prototypic metabolic sensor, revealed a diffusional barrier within the submembrane space, preventing direct reception of cytosolic signals. Creatine kinase phosphotransfer, captured by 18O-assisted 31P NMR, coordinated tightly with ATP turnover, reflecting the cellular energetic status. The dynamics of high energy phosphoryl transfer through the creatine kinase relay permitted a high fidelity transmission of energetic signals into the submembrane compartment synchronizing K(ATP) channel activity with cell metabolism. Knock-out of the creatine kinase M-CK gene disrupted signal delivery to K(ATP) channels and generated a cellular phenotype with increased electrical vulnerability. Thus, in the compartmentalized cell environment, phosphotransfer systems shunt diffusional barriers and secure regimented signal transduction integrating metabolic sensors with the cellular energetic network.

Adenosine Diphosphate↗

PubMed2002

PRCC-TFE3 renal carcinomas: morphologic, immunohistochemical, ultrastructural, and molecular analysis of an entity associated with the t(X;1)(p11.2;q21).

The reappraisal of genetically defined subsets of renal tumors can help to highlight the key pathologic features of specific neoplastic entities. We report the morphologic, immunophenotypic, ultrastructural, and molecular features of 11 renal carcinomas bearing a t(X;1)(p11.2;q21) and/or the resulting PRCC-TFE3 gene fusion. The male/female ratio was 4:7. Ten patients were in the age range of 9-29 years and one was 64 years old (mean 21.3 years, median 15 years). The predominant histologic pattern was nested, with islands of tumor cells compartmentalized by thin-walled capillary vasculature. Minor variations on this pattern yielded solid, acinar, alveolar, and tubular architecture. Papillary architecture was seen in nine cases, usually as a minor component. Neoplastic cells were typically characterized by irregularly shaped nuclei with vesicular chromatin and small nucleoli not visible with a 10x objective, and cytoplasm that ranged from clear to densely granular and eosinophilic. Mitoses were extremely rare; 5 were found in 900 high power fields examined from the 11 neoplasms. The most distinctive immunohistochemical feature of these neoplasms was moderate to intense nuclear labeling for TFE3 protein. These tumors were also consistently immunoreactive for the RCC antigen (10 of 11) and CD10 (9 of 9), whereas cytokeratin and epithelial membrane antigen were negative in four cases and were positive focally in the others. Ultrastructurally, all of the six neoplasms examined showed features consistent with conventional-type (clear cell) renal carcinoma, although two demonstrated distinctive intracisternal microtubules. Both tumors tested contained PRCC-TFE3 fusion transcripts. The differential diagnosis includes conventional-type papillary renal cell carcinoma, conventional-type (clear cell) renal carcinoma, and the ASPL-TFE3 renal carcinomas associated with the t(X;17)(p11.2;q25), with the latter two being morphologically the most similar to the t(X;1) renal carcinomas. Aside from their distinctive clinicopathologic features described here, there is experimental evidence suggesting that these tumors may show differential sensitivity to certain chemotherapeutic agents.

Adolescent↗

PubMed2004

Morphoproteomic and pharmacoproteomic correlates in hormone-receptor-negative breast carcinoma cell lines.

The aim of this study was to elucidate protein circuitry in breast cancer based on the profiling of hormone-receptor-negative breast carcinomas using morphoproteomic and pharmacoproteomic techniques. Three human breast carcinoma cell lines (SKBR-3, MDA-175, MDA-231) were reacted by immunohistochemical (IHC) procedures to detect several categories of protein analytes. Immunoreactivities and cell compartmentalizations were scored from 0 to 3+ positivity using bright-field microscopy. An automated cellular imaging system (ACIS) was used to obtain a final combined score of staining intensity and positive cells in the IHC reactions, to enable comparisons with the visual scores and the rates of inhibition by pharmaceutical agents. FDA-approved inhibitors that target the protein circuitry were added to the cultures for 2-4 days. Proliferation assays were conducted, and in vitro inhibition rates were calculated as (control - treated)/control. Western blot analyses of whole cell lysates assessed the effects of the pharmaceutical agents on selected aspects of protein circuitry. Good to excellent correlation was observed between visual scores and ACIS scores (r values from 0.732 to 0.996 in 10 of 11 trials). Gleevec produced growth inhibition that correlated with the composite expressions of the platelet-derived growth factor (PDGF) family of ligands and receptors; captopril inhibited only MDA-175, consistent with its unique expression of plasmalemmal angiotensin-converting enzyme (ACE); and interferon (IFN)-alpha effected growth inhibition in accordance with the degree of conventional (c) protein kinase C (PKC)-alpha and phosphorylated (p)-PKCalpha/betaII expressions. Western blot analyses revealed correlative changes of several intracellular signals following incubation with these inhibitors. This study shows (a) a close association between the immunohistochemical expression of signal transduction markers and in vitro inhibition by pharmaceutical agents, and (b) correlations between the sites of action of the pharmaceutical agents and the downstream expression of proteins in hormone-receptor-negative breast cancer cell lines. Such morphoproteomic and pharmacoproteomic profiling of individual tumors may enable the pathologist and oncologist to design antitumor therapy that is customized for an individual patient.

Antineoplastic Agents↗

PubMed1990

Immunocytology with microwave-fixed fibroblasts shows 1 alpha,25-dihydroxyvitamin D3-dependent rapid and estrogen-dependent slow reorganization of vitamin D receptors.

Prior studies have given no evidence for regulation of vitamin D receptor (VDR) compartmentalization or subcellular organization. Microwave fixation (9-15 s) and an indirect immunodetection system of avidin-biotin enhancement and phycoerythrin fluorophore resulted in sufficient spatial and temporal resolution to allow analysis of these processes. We studied cultured fibroblasts from normals or from patients with four different types of hereditary defect compromising VDR function (mutant cells). Compartmentalization of VDRs in the absence of 1,25-dihydroxyvitamin D3 (calcitriol) was regulated by serum or estrogen. VDRs were mainly cytoplasmic in cells cultured without serum and phenol red, but VDRs were mainly intranuclear after addition of serum or an estrogen to cells for at least 18 h (slow regulation). Calcitriol initiated a rapid and multistep process (rapid regulation) of reorganization in a portion of VDRs: clumping within 15-45 s, alignment of clumps along fibrils within 30-45 s, perinuclear accumulation of clumps within 45-90 s, and intranuclear accumulation of clumps within 1-3 min. We found similar rapid effects of calcitriol on VDRs in various other types of cultured cells. These sequential VDR pattern changes showed calcitriol dose dependency and calcitriol analogue specificity characteristic for the VDR. In mutant fibroblasts VDR pattern changes after calcitriol were absent or severely disturbed at selected steps. Treatment of normal cells with wheat germ agglutinin, which blocks protein transport through nuclear pores, also blocked calcitriol-dependent translocation of VDRs. We conclude that immunocytology after microwave fixation provides evidence for regulation of VDR organization and localization.

Androstenediols↗