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PubMed1999

Comparative localization of heme oxygenase-2 and nitric oxide synthase in the autonomic innervation to the human ductus deferens and seminal vesicle.

PURPOSE: The aim of present study was to determine the topographic relationship between heme oxygenase-2 (HO-2), which synthesizes carbon monoxide (CO), and neuronal nitric oxide synthase (nNOS), which generates nitric oxide (NO), in the autonomic nerves of the human ductus deferens and seminal vesicle. MATERIALS AND METHODS: Specimens of the ductus deferens and seminal vesicle were obtained during cancer surgery or vasectomy. HO-2 and nNOS were localized by indirect immunofluorescence. Additionally, the histochemical NADPH-diaphorase (NADPH-d) activity of NOS was demonstrated using a standard staining method and some modifications. RESULTS: Anti-HO-2 labeling stained virtually all nerve cell bodies in local ganglia of the pelvic plexus, which is composed of a mixed population of postganglionic sympathetic and parasympathetic neurons supplying the pelvic viscera. Furthermore, nerve cell bodies in the wall of the seminal vesicle, which are considered an extension of the pelvic plexus, were also found to stain positively for HO-2. Some of the HO-2-immunoreactive ganglion cells were also nNOS-positive, their proportion varying between individual ganglia but generally not exceeding 20%. Both enzymes were present in large adventitial nerve trunks. Only nNOS but no HO-2 was found in small intramuscular and mucosal nerve fibers. In both the ductus deferens and seminal vesicle, the highest density of nNOS-containing nerve fibers was in the lamina propria of the mucosa. A well-developed plexus of nNOS-positive nerve fibers was also observed in the muscular layer of the seminal vesicle. By contrast, there was a very sparse innervation by nNOS-positive nerve fibers in the muscle coat of the ductus deferens. In addition, a population of epithelial cells in the seminal vesicle may contain an isoform of NOS, as revealed by a resistant NADPH-d activity. CONCLUSIONS: These findings set the scene for functional studies which will hopefully clarify the biological role of CO and NO in the control of the ductus deferens and seminal vesicle.

Aged↗
PubMed2001

Apoptosis and allograft rejection in the absence of CD8+ T cells.

BACKGROUND: The requirement for cytotoxic T lymphocytes during allograft rejection is controversial. We previously demonstrated that CD8+ T cells are not necessary for allograft rejection or for the induction of apoptosis in rat small intestinal transplantation. In this study, we examined the mechanisms of apoptosis and rejection after liver transplantation in the absence of CD8+ T cells. METHODS: Either Lewis or dark agouti rat liver grafts were transplanted into Lewis recipients to create syngeneic and allogeneic combinations. CD8+ T cells were depleted in an additional allogeneic group by treatment with OX-8 mAb on day -1 and day 1 after liver transplant. RESULTS: Apoptosis and rejection were observed in both the CD8+ T cell-depleted allogeneic and allogeneic grafts by hematoxylin and eosin staining, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining, and radiolabeled-annexin V in vivo imaging. Granzyme B and FasL were expressed in all allogeneic transplants, including those depleted of CD8+ T cells, indicating that a mononuclear cell other than a CD8+ T cell can be the source of these molecules during allograft rejection. Activation of the caspase cascade was detected in all rejecting allografts. Caspases 3, 8, and 9 were activated at similar significantly elevated levels in both allogeneic and CD8+ T cell-depleted liver grafts. CONCLUSION: These data indicate that in the absence of CD8+ T cells an alternative pathway, associated with granzyme B and FasL expression and activation of the caspase cascade, can mediate apoptosis and graft rejection.

Animals↗
PubMed1991

Binding and endocytosis of thrombospondin and thrombospondin fragments in endothelial cell cultures analyzed by cuprolinic blue staining, colloidal gold labeling, and silver enhancement techniques.

We investigated the distribution of thrombospondin-specific binding sites and the uptake of thrombospondin-gold conjugates in cultured porcine endothelial cells by light and electron microscopy. Colloidal gold marker and silver enhancement techniques were applied for cytochemical detection of monomeric thrombospondin and fragments of thrombospondin. Thrombospondin binds to granular and fibrillar structures and to sites of cell-cell contact on the cell surface, as indicated by many proteoglycan-cuprolinic blue precipitates. Cell migration tracks on the culture dish bottom are most heavily stained. Labeling of intact thrombospondin and of proteolytic fragments of thrombospondin with colloidal gold followed by silver intensification enables one to detect its binding and uptake in endothelial cells. Binding to the cell surface and uptake of thrombospondin-gold particles was inhibited by heparin but not by hyaluronic acid or chondroitin sulfate. The heparin binding region at the N-terminal end of the thrombospondin molecule proved to be essential for cell surface binding. Gold-conjugated thrombospondin fragments devoid of the heparin binding region were not internalized. After 60 min incubation at 37 degrees C, thrombospondin-gold particles accumulated in the lysosomal compartment close to the nucleus. In the presence of monensin and ammonium chloride, vesicles in this area are swollen and the concentration of particulate marker is reduced. Binding and uptake of thrombospondin by vascular endothelial cells appears to require linkage of the heparin binding region of the thrombospondin molecule to coated pits and heparan sulfate-rich molecules as receptors. Colloidal gold conjugation of thrombospondin fragments proved to be useful for cytochemical characterization of molecular domains.

Ammonium Chloride↗
PubMed1988

New high-resolution 2-deoxyglucose method featuring double labeling and automated data collection.

A new approach to high-resolution 2-deoxy-D-glucose (2DG) emulsion-autoradiography which combines improved retention of 2DG labeling, staining with immunohistochemical and other specific markers, and automated data collection and analysis of local silver grain and stain densities is described. The Durham et al. (J. Neurosci. 1:519-526, '81) procedure for fixation of 2DG with periodate-lysine-paraformaldehyde (PLP, McLean and Nakane: J. Histochem. Cytochem. 22:1077-1083, '74) was adapted to increase retained label roughly tenfold. Phenobarbital anesthesia is induced 45 minutes after 2DG injection. Barbiturate anesthesia increases brain glycogen (Nelson et al.: J. Neurochem. 15:1271-1279, '68) and presumably increases the incorporation of intracellular 2DG from 2DG-6P into brain glycogen and other molecules (Nelson et al.: J. Neurochem. 43:949-956, '84; Pentreath et al.: Neuroscience 7:759-767, '82). Iodoacetate is added to cold fixative to prevent glycogen breakdown (Cammermeyer and Fenton: Histochemistry 76:339-356, '82). This high-resolution 2DG protocol is directly compatible with many other neuroanatomical techniques. We demonstrate 2DG emulsion autoradiography combined with cytochrome oxidase (CO) histochemistry, markers for axonal pathway tracing, plastic embedding for semithin sections, and immunohistochemical staining for glutamate decarboxylase (GAD). The method should be compatible with antibodies for other antigens and with other neuroanatomical stains. To collect the data directly from microscope slides, a computer-controlled microscope was integrated with image-processing software to eliminate the need for manual counting and scoring of autoradiograms. Regions of interest are scanned automatically at high resolution to map regional labeling and/or stain density. There is excellent correspondence between computer-enhanced two-dimensional maps of the data and the original autoradiograms. Automated counts for five specimens were compared to counts of labeled cells by trained observer. The correlation between the two sets of measurements is high (r = .93). Automated data collection has been generalized to measure regional stain densities on the autoradiographed sections for direct comparison with silver grain density. The method is extremely flexible, especially since new image-processing strategies can be developed in software to extract the desired information from materials labeled by other methods (e.g., HRP).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1986

Analysis of the length of S-phase required to show sister chromatid differential staining.

In vitro studies of BrdU-dependent sister chromatid differential staining typically employ two cycles of BrdU incorporation. Experiments are described which determined the actual fraction of both S-phases that the rat embryonic fibroblasts (Rat-1) cells had to traverse in order to show distinctive differential staining. Following synchronization of cells by a combination of serum deprivation and hydroxyurea blockage, sister chromatid differential staining, labelling index, mitotic index, and per cent DNA replication are determined. Results indicate that only approximately 50% of the first S-phase is necessary in order to show distinctive differential staining. The importance of this finding to studies of cellular proliferation using BrdU incorporation is discussed.

Animals↗
PubMed2005

Olanzapine attenuates the okadaic acid-induced spatial memory impairment and hippocampal cell death in rats.

It is hypothesized that olanzapine, an atypical antipsychotic drug, has beneficial effects on cognitive impairment and neuropathological changes in treating neurodegenerative diseases. In the present study, we investigated the effects of chronic administration of olanzapine on the spatial memory impairment and hippocampal cell death induced by the direct injection of okadaic acid (OA), a potent neurotoxin, into the rat hippocampus. After being pretreated with olanzapine (0.5 or 2 mg/kg/day, i.p.) for 2 weeks, the rats were unilaterally microinjected with OA (100 ng) into the hippocampus, and then were continuously administrated with olanzapine for an additional week The rats were trained on a spatial memory task in an eight-arm radial maze before OA administration, and tested on the same task 18 h after the last olanzapine injection. After the behavioral test, the rats were killed for Nissl staining and terminal deoxynucleutidyl transferase-mediated biotinylated UTP nick end labeling staining. OA significantly induced spatial memory impairment, and caused pyramidal cell loss in the CAI and apoptotic cell death in the hippocampus. Olanzapine significantly attenuated OA-induced spatial memory impairment and the OA-induced neuropathological changes in the hippocampus. These findings suggest that olanzapine may have therapeutic effects in treatment of cognitive impairment and neuropathological changes of schizophrenia and other neurodegenerative diseases.

Analysis of Variance↗
PubMed2006

Effect of altering dietary omega-6/omega-3 fatty acid ratios on prostate cancer membrane composition, cyclooxygenase-2, and prostaglandin E2.

PURPOSE: To determine whether altering the dietary content of omega-6 (n-6) and omega-3 (n-3) polyunsaturated fatty acids affects the growth of androgen-sensitive prostate cancer xenografts, tumor membrane fatty acid composition, and tumor cyclooxygenase-2 and prostaglandin E(2) (PGE(2)) levels. EXPERIMENTAL DESIGN: Individually caged male severe combined immunodeficiency mice were fed isocaloric 20% kcal fat diets with the fat derived either primarily from n-6 fatty acids (n-6 group) or with the fat consisting of n-6 and n-3 fatty acids in a ratio of 1:1 (n-3 group), and injected s.c. with Los Angeles Prostate Cancer 4 (LAPC-4) cells. Tumor volumes and mouse weights were measured weekly, caloric intake was measured 3 days per week, and tumors and serum were harvested at 8 weeks postinjection. RESULTS: Tumor growth rates, final tumor volumes, and serum prostate-specific antigen levels were reduced in the n-3 group relative to the n-6 group. The n-3 group tumors had decreased proliferation (Ki67 staining) and increased apoptosis (terminal nucleotidyl transferase-mediated nick end labeling staining). In vitro proliferation of LAPC-4 cells in medium containing n-3 group serum was reduced by 22% relative to LAPC-4 cells cultured in medium containing serum from the n-6 group. The n-6/n-3 fatty acid ratios in serum and tumor membranes were lower in the n-3 group relative to the n-6 group. In addition, n-3 group tumors had decreased cyclooxygenase-2 protein and mRNA levels, an 83% reduction in PGE(2) levels, and decreased vascular endothelial growth factor expression. CONCLUSION: These results provide a sound basis for clinical trials evaluating the effect of dietary n-3 fatty acids from fish oil on tumor PGE(2) and membrane fatty acid composition, and serum and tumor biomarkers of progression in men with prostate cancer.

Administration, Oral↗
PubMed2004

Evidence that transfer of functional p53 protein results in increased apoptosis in prostate cancer.

PURPOSE: INGN 201 (Ad-p53) is a replication-defective adenoviral vector that encodes a wild-type p53 gene driven by the cytomegalovirus promoter. INGN 201 has been shown to have antitumoral activity against human prostate cancer cell lines. This study was undertaken to determine the safety of INGN 201 in patients with locally advanced prostate cancer, to assess transgene expression, and to evaluate antitumoral activity. EXPERIMENTAL DESIGN: Our study included patients with clinical stage T3, T1c-T2a with Gleason score 8-10 disease, or T2a-T2b with Gleason score 7 disease and a prostate-specific antigen level >10 ng/ml. INGN 201 was administered by intraprostatic injection under ultrasonographic guidance. One course of INGN 201 was defined as three separate INGN 201 administrations 2 weeks apart. Biopsies at baseline and 24 h after the first administration were assessed for p53 protein by immunohistochemical staining and for apoptosis by terminal deoxynucleotidyl transferase-mediated nick end labeling assay. RESULTS: A total of 38 courses of INGN 201 gene therapy were administered to 30 patients, of whom 26 underwent radical prostatectomy. There were no grade 3 or 4 adverse events related to INGN 201 administration. Of the 11 patients with negative baseline immunostaining for p53 protein, 10 had positive p53 immunostaining after the first administration of INGN 201, and 8 had an increase in apoptotic cells by terminal deoxynucleotidyl transferase-mediated nick end labeling staining. All 26 of the patients who underwent radical prostatectomy had significant residual viable prostate cancer, and 12 have experienced biochemical failure (median follow-up, 42 months). CONCLUSION: Intraprostatic INGN 201 gene therapy is safe and can reliably result in p53 protein production and apoptosis.

Adenoviridae↗
PubMed2005

Differential effects of orbital and laminar shear stress on endothelial cells.

OBJECTIVE: Laminar shear stress is atheroprotective for endothelial cells (ECs), whereas nonlaminar, disturbed, or oscillatory shear stress correlates with development of atherosclerosis and neointimal hyperplasia. The effects of orbital and laminar shear stress on EC morphology, proliferation, and apoptosis were compared. METHODS: ECs were exposed to orbital shear stress with an orbital shaker (210 rpm) or laminar shear stress (14 dyne/cm 2) with a parallel plate. Shear stress in the orbital shaker was measured with optical velocimetry. Cell proliferation was assessed with direct counting and proliferating cell nuclear antigen staining; apoptosis was assessed with transferase-mediated deoxyuridine triphosphate nick end labeling staining. Cell surface E-selectin and intercellular adhesion molecule expression were assessed with fluorescence-activated cell sorting. Akt phosphorylation was assessed with Western blotting. RESULTS: Orbital shear stress increased EC proliferation by 29% and 3 [H]thymidine incorporation two-fold compared to 16% and 38% decreases, respectively, in ECs treated with laminar shear stress (P < .0001 and P = .03, analysis of variance). Cells in the periphery of the culture well aligned to the direction of shear stress similar to the shape change seen with laminar shear stress, whereas ECs in the center of the well appeared unaligned similar to ECs not exposed to shear stress. Shear stress at the bottom surface of the culture well was reduced in the center of the well (5 dyne/cm 2) compared to the periphery (11 dyne/cm 2); the Reynolds' number was 2066. ECs were seeded differentially in the center and periphery of the wells. ECs in the center of the well had increased proliferation, increased apoptosis, reduced Akt phosphorylation, increased intercelluar adhesion molecule expression, and reduced E-selectin down-regulation, compared with ECs in the periphery of the well. CONCLUSION: Although the orbital shaker does not apply uniform shear stress throughout the culture well, arterial magnitudes of shear stress are present in the periphery of the well. ECs cultured in the center of the well exposed to low magnitudes of orbital shear stress might be a model of the "activated" EC phenotype. CLINICAL RELEVANCE: The perfect in vitro model to study and assess treatments for atherosclerosis and neointimal hyperplasia does not exists. An extensive body of literature describing effects of laminar shear stress on endothelial cells has contributed to our understanding of the interactions between shear stress and blood vessels. Laminar shear stress is atheroprotective, whereas oscillatory or disturbed shear stress correlates with areas of atherosclerosis and neointimal hyperplasia in vivo. This study describes the orbital shear stress model, its effects on endothelial cell proliferation and apoptosis, and suggests that activation of the intracellular Akt pathway is associated with these differing effects of laminar and orbital shear stress on endothelial cells.

Animals↗
PubMed2003

Microtubule depolymerization rapidly collapses capillary tube networks in vitro and angiogenic vessels in vivo through the small GTPase Rho.

Maintenance of endothelial cell tube integrity is dependent on an intact cytoskeleton. We present data indicating that rapid collapse of endothelial tubular networks in vitro occurs in a dose-dependent manner after administration of microtubule-depolymerizing reagents but not after actin depolymerization. Pretreatment of endothelial cell networks with C3 exoenzyme or recombinant adenoviruses expressing dominant negative RhoA resulted in complete blockade of tube collapse, indicating a role for RhoA in these events. Microtubule depolymerization also resulted in activation of RhoA, whereas increased expression of constitutively active RhoA induced cell rounding and apoptosis of endothelial cells. Furthermore, following treatment with the chemotherapeutic agent vinblastine, rapid capillary tube network collapse occurred followed by endothelial cell apoptosis. Vinblastine, but not control agents, induced cleavage of procaspase-3, procaspase-9, and procaspase-8, along with the known caspase targets p21-activated kinase-2 and gelsolin, indicating that tube collapse caused a defined apoptotic response. Using a model of vascular endothelial growth factor-stimulated angiogenesis in vivo, vinblastine treatment also resulted in collapse and apoptosis of angiogenic blood vessels. Apoptotic endothelial cells stained strongly for cleaved caspase-3, and terminal dUTP nick-end labeling staining revealed fragmented nuclei in vinblastine-treated but not control angiogenic areas. Together, these findings indicate that microtubule-depolymerizing agents directly induce endothelial network collapse in vitro and in vivo leading to endothelial cell apoptosis in a manner dependent on the small GTPase, RhoA. In addition, these findings reveal a novel function for microtubule disrupting chemotherapeutic agents, namely their ability to rapidly collapse newly formed angiogenic vessels, which may contribute to their effectiveness in limiting angiogenesis and tumor growth.

Animals↗
PubMedDate not supplied

Immunoperoxidase labelling of previously stained tissue sections: application in oral histopathology.

We have investigated by the avidin-biotin peroxidase technique a wide variety of recognized cellular antigens in paraffin sections of both normal and pathological material which have been previously stained with hematoxylin and eosin. The intensity of peroxidase reaction was adequate to permit the diagnosis and the background of the immunoreaction minimal. This technique therefore, appears useful either for retrospective analysis of cellular antigens or when limited unstained material is available.

Acetates↗
PubMedDate not supplied

[Bengal rose labeled with short-living cyclotron iodine 123 in the radionuclide study of the hepatobiliary system].

Bengal rose stain labeled with short-lived 123I (123I-BR) was used in diagnostic examinations of the hepatobiliary system of 201 patients with various diseases. This agent was found superior in many respects to other hepatobiliary compounds. Due to an essential reduction of the radiation load of the critical organs use of higher activities of 123I-BR becomes possible (120 MBc and higher), this permitting an effective study of rapidly coursing hemodynamic processes. A single complex radionuclide examination permits obtaining complete information on the hepatobiliary system structure and function. The presence of hyperbilirubinemia is not a factor reducing the investigation efficacy.

Adult↗
PubMed1994

Immunolocalization of albumin and transferrin in germ cells and Sertoli cells during rat gonadal morphogenesis and postnatal development of the testis.

The localization of albumin and transferrin was examined immunohistochemically in germ cells and Sertoli cells during rat gonadal morphogenesis and postnatal development of the testis. These proteins appeared as early as the 13th day of gestation in migrating primordial germ cells before Sertoli cell differentiation. In the fetal testis, strong immunoreactivity was only detected in the gonocytes. In the prepubertal testis, spermatogonia, primary spermatocytes, and some Sertoli cells accumulate albumin and transferrin. At puberty, different patterns of immunostaining of the germ cells were observed at the various stages of the cycle of the seminiferous epithelium. Diplotene spermatocytes at stage XIII, spermatocytes in division at stage XIV, and round spermatids at stages IV-VIII showed maximal staining. Labeling was evident in the cytoplasm of adult Sertoli cells. Albumin and transferrin staining patterns paralleled each other during ontogenesis.

Animals↗
PubMed2007

Kurozu moromimatsu inhibits tumor growth of Lovo cells in a mouse model in vivo.

OBJECTIVE: In Japan, rice vinegar that has been matured and fermented for years in earthenware jars is considered a health food with anticolon cancer action. It is divided into the liquid component (Kurozu) and the sediment (Kurozu moromimatsu), which contains large amounts of organic materials and minerals. The effect of Kurozu moromimatsu (Kurozu-M) on cancer has not yet been examined. In this study, we examined the activity of Kurozu-M on colon cancer and investigated the mechanisms involved, focusing on active oxygen generation, apoptosis, and metalloproteinases (MMPs). METHODS: We used Lovo cells transplanted into nude mice as an experimental model. We measured the tumor volume and MMP levels and conducted hematoxylin-eosin staining (for polymorphonuclear leukocytes), terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining (for apoptosis), and immunostaining for nitrotyrosine (a marker of active oxygen generation) in control, Kurozu-treated, and Kurozu-M--treated groups. RESULTS: The tumor volume was the same in the control group (231 +/- 36 mm(3)) and Kurozu group (238 +/- 52 mm(3)), but was significantly reduced in the Kurozu-M group (152 +/- 28 mm(3), P < 0.001 versus control). Apoptosis of tumor cells and accumulation of polymorphonuclear leukocytes were not observed. Nitrotyrosine production, total MMP levels, and MMP activation were significantly reduced in the Kurozu-M group. CONCLUSION: The administration of Kurozu-M prolonged the lifespan of cancer cell-transplanted mice, inhibited tumor progression, and reduced nitrotyrosine production and MMP activation, but did not induce apoptosis.

Animals↗
PubMed1998

Inhibition of murine bladder tumorigenesis by soy isoflavones via alterations in the cell cycle, apoptosis, and angiogenesis.

Soy isoflavones exhibit a number of biological effects, suggesting that they may have a role in cancer prevention. Our objectives are to determine whether components of soy products or purified soy isoflavones can inhibit the progression of bladder cancer. We compared the in vitro effects of pure soy isoflavones and soy phytochemical concentrate on growth curves, cell cycle progression, and apoptosis in murine and human bladder cancer cell lines. Pure soy isoflavones (genistein, genistin, daidzein, and biochanin A) and soy phytochemical concentrate exhibit dose-dependent growth inhibition of murine (MB49 and MBT-2) and human (HT-1376, UM-UC-3, RT-4, J82, and TCCSUP) bladder cancer cell lines, although the degree of inhibition varies among lines. Soy isoflavones induce a G2-M cell cycle arrest in all human and murine lines evaluated by flow cytometry. In addition, some bladder cancer lines show DNA fragmentation consistent with apoptosis. We next evaluated the ability of genistein, soy phytochemical concentrate, and soy protein isolate, respectively, to inhibit the growth of transplantable murine bladder cancer in vivo. C57BL/6 mice were randomly assigned to treatment groups (n = 12/group): (a) AIN-76A diet; (b) AIN-76A diet plus genistein, i.p., 50 mg/kg body weight/day; (c) AIN-76 diet with soy phytochemical concentrate at 0.2% of the diet; (d) AIN-76 diet with soy phytochemical concentrate at 1.0% of the diet; and (e) AIN-76A diet with soy protein isolate, 20% by weight. Mice were inoculated s.c. with 5 x 10(4) syngeneic MB49 bladder carcinoma cells, and tumor growth was quantitated. Neither genistein nor soy products reduced body weight gain. Tumor volumes from mice treated with genistein, dietary soy phytochemical concentrate at 1%, or dietary soy protein isolate were reduced by 40% (P < 0.007), 48% (P < 0.001), or 37% (P < 0.01), respectively, compared with controls. We characterized the effects of treatment on several biomarkers in tumor tissue: proliferation index by proliferating cell nuclear antigen staining, apoptotic index by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling staining, and angiogenesis by microvessel quantitation. Soy products reduced angiogenesis, increased apoptosis, and slightly reduced proliferation while showing no histopathological effects on the normal bladder mucosa. Our data suggest that soy isoflavones can inhibit bladder tumor growth through a combination of direct effects on tumor cells and indirect effects on the tumor neovasculature. Soy products warrant further investigation in bladder cancer prevention and treatment programs or as antiangiogenic agents.

Animals↗
PubMed1997

Reversibility and apoptosis in rat urinary bladder papillomatosis induced by uracil.

Apoptosis is a morphologically and biochemically distinct form of cell death which determines specific patterns of tissue size and shape and balances cell proliferation. In the present study, the sequence of cellular proliferative alterations in urinary bladder epithelium associated with uracil-induced reversible urinary calculi was investigated in male F344 rats. Group 1 consisted of 45 rats, 6 weeks old at commencement of the experiment, which were given a diet containing 3% uracil for 8 weeks and were then returned to basal diet until week 20. Five rats were killed at each of weeks 2, 4, 8, 9, 10, 11, 12, 14 and 20. Group 2 consisted of 15 rats which were given basal diet for 20 weeks. Five rats were killed at each of weeks 0, 8 and 20. Microscopic, reversible papillomatosis, which showed papillary projections of epithelial proliferation, was seen in the urinary bladder of all rats in group 1 through week 8. No epithelial lesions were apparent in any of rats in group 2. Anti-Le(y)(BM-1/JIMRO)-positive areas of the urinary bladder epithelia were immunohistochemically seen in all rats of group 1 at weeks 2-12. At week 9 the percentage of anti-Le(y)-positive areas reached a maximum. Nick-end labeling stained nuclei of cells in the urinary bladder epithelium were observed in all rats of group 1 at weeks 4-14. At week 10 the labeling index was at a maximum. Proliferating cell nuclear antigen (PCNA)- and cyclin D1-positive cells of the urinary bladder epithelium were observed in group 1 at weeks 2, 4 and 8, however, at week 9 there were no PCNA- and cyclin D1-positive cells. In urinary bladder papillomatosis the simultaneous existence of apoptotic cells and proliferating cells was shown by double staining with anti-Le(y) (BM-1/JIMRO) and for PCNA. At week 10 apoptosis, stained by BM-1 and nick-end labeling, occurred extensively in regressing urinary bladder papillomatosis. Agarose gel electrophoresis of DNA in regressing papillomatosis at week 9 showed DNA fragmentation. Thus, these results indicate that apoptosis occurs in the process of papilloma regression following withdrawal of uracil treatment.

Animals↗