Reversibility and apoptosis in rat urinary bladder papillomatosis induced by uracil

Transcription

1 Carcinogenesis vol.18 no.8 pp , 1997 Reversibility and apoptosis in rat urinary bladder papillomatosis induced by uracil Kazuhiko Otori 1, Yoshihisa Yano 2, Nobuyasu Takada, conditions. Originally apoptosis was characterized by a marked Chyi Chia R.Lee, Shuji Hayashi, Shuzo Otani 2 and reduction in cell volume and increase in density (1 3). Apop- Shoji Fukushima totic bodies are histologically characterized by their small size, nuclear chromatin condensation, DNA fragmentation and First Department of Pathology and 2 Second Department of Biochemistry, Osaka City University Medical School, Asahi-machi, Abeno-ku, compactness of cytoplasmic organelles (4,5). Recent evidence Osaka 545, Japan indicated that specific glycosylation on the cell surface is 1 To whom correspondence should be addressed associated with apoptosis (6). These morphological changes have been observed in cultured cells after exposure to chemo- Apoptosis is a morphologically and biochemically distinct therapeutic agents, γ irradiation or glucocorticoid hormones form of cell death which determines specific patterns of in vitro (7 14). It plays a controlling role in counteracting cell tissue size and shape and balances cell proliferation. In proliferation in determining the size and shape of developing the present study, the sequence of cellular proliferative organs and tissues and in defining the balance of cell populaalterations in urinary bladder epithelium associated with tions (3,8). uracil-induced reversible urinary calculi was investigated Lalich (15) first reported induction of stone formation in in male F344 rats. Group 1 consisted of 45 rats, 6 weeks the urinary bladder of rats orally given uracil, a component of old at commencement of the experiment, which were given RNA. Shirai et al. (16,17) reported that p.o. administration of a diet containing 3% uracil for 8 weeks and were then 3% uracil to rats induced reversible mucosal papillomatosis, returned to basal diet until week 20. Five rats were killed diffuse papillary proliferation of the epithelium of the urinary at each of weeks 2, 4, 8, 9, 10, 11, 12, 14 and 20. Group 2 bladder associated with urolithiasis. Fukushima et al. (18) consisted of 15 rats which were given basal diet for 20 demonstrated induction of urinary bladder carcinomas at high weeks. Five rats were killed at each of weeks 0, 8 and incidence following uracil administration to rats and mice for 20. Microscopic, reversible papillomatosis, which showed 2 years and the incidences were related to the presence of papillary projections of epithelial proliferation, was seen calculi in the urinary bladder. However, when uracil treatment in the urinary bladder of all rats in group 1 through week was stopped at least by week 15 from starting, calculi immedi- 8. No epithelial lesions were apparent in any of rats ately disappeared and uracil-induced papillomatosis regressed in group 2. Anti-Le y (BM-1/JIMRO)-positive areas of the to normal epithelium (16). This phenomenon is quite interesting urinary bladder epithelia were immunohistochemically and may be the key to understanding the general mechanism seen in all rats of group 1 at weeks At week 9 the of reversible epithelial proliferation. We speculate whether the percentage of anti-le y -positive areas reached a maximum. key mechanism may be apoptosis. In the present study we Nick-end labeling stained nuclei of cells in the urinary have evaluated whether apoptosis occurs during the period of bladder epithelium were observed in all rats of group 1 at appearance and disappearance of papillomatosis in the urinary weeks At week 10 the labeling index was at a bladder of rats induced by p.o. administration of 3% uracil maximum. Proliferating cell nuclear antigen (PCNA)- and and its cessation. 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 Materials and methods cells. In urinary bladder papillomatosis the simultaneous Test chemicals existence of apoptotic cells and proliferating cells was Uracil (2,4-dioxypyrimidine) was obtained from Yamasa Shoyu Co. (Chiba, shown by double staining with anti-le y (BM-1/JIMRO) Japan). The purity of the preparation was 100%. and for PCNA. At week 10 apoptosis, stained by BM-1 Animals and their maintenance and nick-end labeling, occurred extensively in regressing A total of 60 male F344 rats, 5 weeks old, were purchased from Charles urinary bladder papillomatosis. Agarose gel electrophoresis River Japan Inc. (Shiga, Japan) and quarantined for 1 week before the start of DNA in regressing papillomatosis at week 9 showed DNA of the experiment. Animals were housed five to a plastic cage with hard wood fragmentation. Thus, these results indicate that apoptosis chips for bedding in an air-conditioned room. The animals had free access to occurs in the process of papilloma regression following feed (Oriental MF powdered diet; Oriental Yeast Co., Tokyo, Japan) and tap water. Bedding and cages were changed twice per week. The room temperature withdrawal of uracil treatment. was controlled at 22 2 C and the relative humidity at 55 10%. Fluorescent lighting provided a 12 h light/dark cycle. Animals were weighed every week. Introduction Diet preparation Uracil was added to the basal diet, Oriental MF powdered diet, at a Apoptosis is a particular process of cell death and a major concentration of 3.0%. mechanism of maintenance of homeostasis in organs and Experimental procedure tissues under a variety of physiological and pathological A group of 45 male F344 rats (group 1) were given the diet containing uracil (weeks 1 8) and were then given basal diet without uracil (weeks 9 20). The *Abbreviations: ABC, avidin biotin peroxidase complex; NEL, nick-end remaining 15 rats (group 2) were given diets without uracil (weeks 1 20) as labeling; PCNA, proliferating cell nuclear antigen. controls. Five rats in group 1 were killed at each of weeks 2, 4, 8, 9, 10, 11, Oxford University Press 1485

2 K.Otori et al. Fig. 1. Sequential changes in the lengths of rat urinary bladder papillary projections in papillomatosis. Data presented are mean SD values. 12, 14 and 20 after the beginning of experiment. Five non-treated control rats (group 2) were killed at each of weeks 0, 8 and 20. Histological examination and immunohistochemical analyses For histological examination the urinary bladder was fixed in Carnoy solution and embedded in paraffin and a series of tissue sections (3 4 µm thickness) were prepared from each sample. The sections were stained with hematoxylin and eosin. The lengths of lesions were analyzed with a light microscope connected to an IPAP image analysis system (Sumika Technos Corp., Osaka, Japan). To assess levels of apoptosis, sections were treated immunohistochemically using the avidin biotin peroxidase complex (ABC*) method (19) with a monoclonal antibody against Le y (BM-1/JIMRO; Ohtsuka Seiyaku Co., Tokushima, Japan) (20,21). BM-1-positive areas of urinary bladder epithelium were measured by a color video image analyzer (VIP-21CH; Olympus- Ikegami, Tokyo, Japan) and we calculated a BM-1-positive index (BM-1- positive area/entire area of mucosa). To detect DNA fragmentation in situ associated with apoptosis, the tissue sections were stained by nick-end labeling (NEL) (transfer of biotinylated nucleotide to the 3 -OH end) according to the method of Gavrieli et al. (22). To assess the levels of proliferating cell nuclear antigen (PCNA) and the expression of cyclin D1 in the nuclei of the epithelial cells, PCNA and cyclin D1 in nuclei were stained by an ABC method with a monoclonal antibody against PCNA (PC-10, IgG2a; DAKO, Glostrup, Denmark) and a polyclonal antibody against cyclin D1 (rabbit polyclonal IgG; UBI, Lake Placid, NY) (23). At week 8 the tissue sections of group 1 and group 2 were double stained with BM-1 and anti-pcna and at week 10 they were doubly stained for BM-1 and by NEL. Positive cell indices (NEL, PCNA and cyclin D1) were obtained by counting the number of positive cells among at least 1000 epithelial cells per urinary bladder and expressed as percentages. Detection of DNA fragmentation in an agarose gel Mucosa samples of urinary bladders, with different histological changes at week 9, were scraped off with glass slides to obtain exfoliated cells for analysis of DNA fragmentation by 1.9% agarose gel electrophoresis. The method described by Ishida et al. (24) was adopted for this purpose. Statistical analysis Body weights and food and water consumptions were analyzed with a StatView system (Abacus Concepts Inc., Berkeley, CA) using Student s t-test. The difference was considered significant at P Fig. 2. At week 8 double staining of PCNA (red) and BM-1 (brown) in group 1. (a) PCNA-positive nuclei are located in the top to middle portion, while BM-1-positive areas are confined to the bottom to middle area of papillary hyperplasia in urinary bladder papillomatosis (magnification 130). (b) Arrows show typical PCNA stained nuclei cells (magnification 260). Results completely disappeared. Microscopically, at week 2 simple The mean body weights of rats given uracil (group 1) for hyperplasia and diffuse papillary hyperplasia (papillomatosis) 8 weeks were less than those of controls during the first 14 were seen in the urinary bladder epithelium of all rats treated weeks (maximum ~77%, P 0.05, data not shown) and then with uracil (group 1), increasing in severity with uracil treat- returned to control (group 2) ranges. Food consumption was ment. The lengths of papillary projections in papillomatosis at not significantly different between groups. Water consumption several time points are shown in Figure 1. From week 9 the was not significantly different except at week 4 (P 0.05, papillomatosis regressed to a significant extent. At week 20 that of group 2 were ~65% of that of group 1). the urinary bladder in group 1 had returned to normal and the At week 2 all rats (group 1) given uracil already had calculi mucosa of the urinary bladder showed a normal histology. No in the urinary bladder. The urinary bladders gradually enlarged neoplastic lesions were encountered in this study. and were completely filled with numerous yellowish-white Periodic changes in the appearance of PCNA-, cyclin D1- calculi at week 8. But at week 9, 1 week after the rats were and BM-1-positive areas and NEL in the urinary bladder returned to the uracil-free diet, the urinary bladder calculi had epithelium of rats in group 1 are shown in Figure 1. PCNA- 1486

3 Apoptosis in rat bladder papillomatosis Fig. 3. At week 4 staining of papillomas in group 1 by cyclin D1. There are many cyclin D1-positive cells diffusely distributed throughout the hyperplastic epithelium in an area of urinary bladder papillomatosis (magnification 300). Fig. 5. Sequential change of PCNA-positive index, cyclin D1-positive index, BM-1-positive area index and NEL-positive index in urinary bladder epithelium of group 1. Data presented are mean SD values. Fig. 6. DNA fragmentation pattern at week 9 in group 1. This reveals a ladder pattern. Fig. 4. At week 10 double staining of BM-1 (brown) and by NEL (arrows; red) in group 1. BM-1-positive areas and NEL-positive cells are observed in Almost no PCNA-, cyclin D1- or BM-1-positive cells or the surface layer of reduced hyperplastic epithelium in urinary bladder NEL were observed in the urinary bladders of the controls, papillomatosis (magnification 400). group 2. The papillomatosis of group 1 at week 8, when epithelial proliferation was most severe, was double stained with BM-1 and cyclin D1-positive cells in the urinary bladder epithelium and anti-pcna (Figure 2). The results of the double staining were observed in all rats in group 1 at weeks 2, 4 and 8 showed a tendency for most BM-1-positive areas to be located (Figures 2 and 3). There were many PCNA labeled cells in in the middle to bottom of the epithelial papillary projections the top to middle portion of the papillary projections. Cyclin D1-positive cells were observed diffusely in the epithelium of and PCNA-positive cells were mainly observed at the top to urinary bladder papillomatosis (Figure 3). middle. At week 10, regressing papillomas in group 1 stained BM-1-positive areas were seen in all rats treated with uracil for BM-1 and NEL (Figure 4). Cell nuclei stained by NEL (group 1) at weeks 2 12 (Figures 2 and 4). At weeks 2 8 were abundant in the surface layer of epithelial cells in BM-1-positive areas gradually increased and at week 9 the papillomatosis and there were weakly BM-1-positive areas in index, expressed as the percentage of BM-1-positive areas, the same layer. reached a maximum ( %). The areas showed a Agarose gel electrophoresis of DNA fragmentation showed tendency to locate in surface layers of the bottoms to middle a ladder pattern in group 1 at week 9 (Figure 6). of the papillary projections during weeks 2 8 (Figure 2). At week 9 labeled areas were located in the surface layer of the Discussion entire papillary projection, from the top to bottom. NEL stained In this study we have confirmed that administration of 3% nuclei of cells in the urinary bladder epithelium were observed uracil induces urinary calculi which cause epithelial cell in all rats given uracil at weeks 4 14 in group 1 (Figure 5). proliferation in the urinary bladder of male rats, resulting in The indices increased rapidly from week 8 to 10 and the papillomatosis (15 17). These lesions were reversible, as maximum labeling index was % on week 10. At reported previously (16,17). When the uracil treatment was weeks 11 and 12 the labeling index was still maintained at stopped, urinary calculi disappeared immediately and papil- high values. lomatosis also regressed to normal epithelium. The striking 1487

4 K.Otori et al. result in the present study was that the mechanism of drastic lower than in their reversible counterparts in the urinary disappearance of the papillomatosis of the urinary bladder was bladders of rats initiated with N-butyl-N-(4-hydroxybutyl)- intensive development of apoptosis. nitrosamine followed by uracil treatment. Therefore, they At week 8, when there were many urinary calculi and welldeveloped speculated that an increase in apoptosis may play an important papillomatosis, the length of papillary projections role in preventing carcinogenesis. We found no irreversible was very high and PCNA-positive cells were mainly present lesions in the rat urinary bladders in this study. Alterations in at the top to middle of the papillary projection (Figure 2). On the balance between cell proliferation and apoptosis may reflect the other hand, BM-1-positive areas were seen mainly at the an important mechanism of carcinogenesis, which in turn may bottom to middle of the papillary proliferations (Figure 2). It be caused by various other factors, e.g. chemical carcinogens. is speculated that the areas of PCNA-positive cells could be in The present uracil model is useful for examining how some direct contact with urinary calculi and responded to mechanical reversible hyperplastic lesions prevent carcinogenesis and how irritation of the calculi. However, we speculate that BM-1- tumors develop from some hyperplastic lesions. positive areas were not irritated by calculi directly and many proliferating cells quickly became apoptotic. The apoptotic cells controlled the size and shape of the urinary bladder Acknowledgements papillomas, which were enlarged by irritation by the calculi This work was sponsored by grants for Cancer Research from the Ministry of Education, Science Sports and Culture and the Ministry of Health and and expressed Le y on the surfaces of the cells. These results Welfare of Japan and the Osaka City University Medical Research Foundation imply that irritation by the calculi increased cell proliferation Fund for Medical Research. and apoptosis in urinary bladder papillomatosis at the same time. Since at weeks 2, 4 and 8, when uracil treatment was continued, expression of apoptosis was weaker than that of References proliferation and papillomas of the urinary bladder grew, with 1. Wyllie,A.H., Kerr,J.F.R. and Currie,A.R. (1980) Cell death: the significance the length of the papillary projections becoming very high. of apoptosis. Int. Rev. Cytol., 68, On the other hand, at week 9, 1 week after uracil-containing 2. Arends,M.J. and Wyllie,A.H. (1991) Apoptosis: mechanism and roles in pathology. Int. Rev. Exp. Pathol., 32, diet was stopped and urinary calculi quickly disappeared, there 3. Wyllie,A.H. (1987) Apoptosis: cell death in tissue regulation. J. Pathol., was no irritation due to calculi and PCNA-positive cells almost 153, disappeared from the urinary bladder epithelium. Moreover, 4. Kerr,J.F.R., Wyllie,A.H. and Currie,A.R. (1982) Apoptosis: a basic expression of apoptosis increased and was stronger than that biological phenomenon with wide-ranging implications in tissue kinetics. of proliferation. As a result, the length of the papillary Br. J. Cancer, 26, Arends,M.J., Morris,R.G. and Wyllie,A.H. (1980) Apoptosis: the role of projections became smaller and eventually returned to normal the endonuclease. Am. J. Pathol., 136, epithelium. In the regressing papillomatosis at week 10 the 6. Hiraishi,K., Suzuki,K., Hakomori,S. and Adachi,M. (1993) Le y antigen epithelium was clearly stained with BM-1 and by NEL, as expression is correlated with apoptosis (programmed cell death). shown in Figure 4. There were many cells stained by NEL in Glycobiology, 3, Bursch,W., Kleine,L. and Tenniswood,M. (1993) The biochemistry of cell the surface layers of the papillomas and BM-1-positive areas death by apoptosis. Biochem. Cell Biol., 68, in the same layers. In addition, Figure 6 shows agarose gel 8. Searle,J., Lawson,T.A., Abbott,P.J., Harmon,B. and Kerr,J.F.R. (1975) An electrophoresis of DNA fragmentation. This revealed a ladder electron microscope study of the mode of cell death induced by cancer- pattern which is a key molecular feature reported for chemotherapeutic agents in populations of proliferating normal and neoplastic cells. Pathology, 116, apoptosis (4,5). 9. Yamada,T. and Ohyama,H. (1988) Radiation-induced interphase death of Comparing the peak of BM-1-positive areas with that of thymocytes is internally programmed (apoptosis). Int. J. Radiat. Biol., 53, NEL index, there is a time lag of ~1 week. BM-1-positive results indicate the expression of Le y, a specific membrane 10. Wyllie,A.H. (1980) Glucocorticoid-induced thymocyte apoptosis is antigen, which is an important predisposing marker for associated with endogenous endonuclease activation. Nature (Lond.), 284, apoptosis (6). On the other hand, the nick-end labeling method 11. Galili,U., Leizerowitz,R., Moreb,J., Gamliel,H., Gurfel,D. and Polliack,A. detects DNA fragmentation associated with apoptosis. Thus, (1982) Metabolic and ultrastructural aspects of the in vitro lysis of early stage apoptotic cells show increased Le y expression on chronic lymphocytic leukemia cells by glucocorticoids. Cancer Res., 42, their membranes (6) and in the second stage, when expression Waiker,P.R., Smith,C., Yaudale,T., Leblanc,J., Whitfield,J.F. and of Le y becomes more severe, DNA fragmentation occurs in Sikorska,M. (1991) Topoisomerase II-reactive chemotherapeutic drugs the apoptotic process by unknown mechanisms. At this time induce apoptosis in thymocytes. Cancer Res., 51, the functional mechanism of Le y expression associated with 13. McConkey,D.J., Hartzell,P., Nicotera,P. and Orrenius,S. (1989) Calcium apoptosis is totally unknown. activated DNA fragmentation kills immature thymocytes. FASEB J., 3, Prolonged stimulation of excessive proliferation of urinary Cohen,J.J. and Duke,R.C. (1984) Glucocorticoid activation of a calciumbladder epithelial cells in rats results in carcinoma formation dependent endonuclease in thymocyte nuclei leads to cell death. (18). In this study cyclin D1-positive cells were observed J. Immunol., 132, simultaneously in urinary bladder papillomatosis in group 1 at 15. Lalich,J.J. (1966) Experimentally induced uracil urolithiasis in rats. J. Urol., weeks 2, 4 and 8, when PCNA-positive cells were seen. 95, Overexpression of cyclin D1 has been reported in several 16. Shirai,T., Ikawa,E., Fukushima,S., Masui,T. and Ito,N. (1986) Uracilinduced urolithiasis and the development of reversible papillomatosis in human carcinomas (25 30). Increased cell proliferation can the urinary bladder of F344 rats. Cancer Res., 46, account for the carcinogenicity of non-genotoxic chemicals 17. Shirai,T., Fukushima,S., Tagawa,Y., Okumura,M. and Ito,N. (1989) Cell (18,31). Recently, Magnuson et al. (32) reported that aberrant proliferation induced by uracil-calculi and subsequent development of crypt foci, putative preneoplastic lesions of colonic cancer, reversible papillomatosis in the rat urinary bladder. Cancer Res., 49, displayed resistance to the induction of apoptosis by a colonic 18. Fukushima,S., Tanaka,H., Asakawa,E., Kagawa,M., Yamamoto,A. and carcinogen, azoxymethane. Shirai et al. (33) described the Shirai,T. (1992) Carcinogenicity of uracil, a nongenotoxic chemical, in rat frequency of apoptotic bodies in irreversible lesions to be and mice and its rationale. Cancer Res., 52,

5 Apoptosis in rat bladder papillomatosis 19. Hsu,S.M., Raine,L. and Fanger,H. (1981) Use of avidin biotin peroxidase complexes (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J. Histochem. Cytochem., 29, Lloyd,K.O., Kabat,E.A., Layug,E.J. and Gruezo,F. (1966) Immunochemical studies on blood groups: XXXIV, Structures of some oligosaccharides produced by alkaline degradation of blood group A,B and H substances. Biochemistry, 5, Hakomori,S. and Kobata,A. (1974) Blood group antigens. In Sela,M. (ed.), The Antigens. Academic Press, New York, NY, Vol. II, pp Gavrieli,Y., Sherman,Y. and Ben-Sasson,S.A. (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell Biol., 119, Hall,P.A. et al. (1990) Proliferating cell nuclear antigen (PCNA) immunolocalization in paraffin sections: an index of cell proliferation with evidence of deregulated expression in some neoplasms. J. Pathol., 162, Ishida,Y., Agata,Y., Shibahara,K. and Honjo,T. (1992) Induced expression of PD-1, a novel member of the immunoglobulin gene superfamily, upon programmed cell death. EMBO J., 11, Naitoh,H., Shibata,J., Kawaguchi,A., Kodama,M. and Hattori,T. (1995) Overexpression and localization of cyclin D1 mrna and antigen in esophageal cancer. Am. J. Pathol., 146, Zhang,S.Y., Caamano,J., Cooper,F., Guo,X. and Klein-Szanto,A.J.P. (1994) Immunohistochemistry of cyclin D1 in human breast cancer. Am. J. Clin. Pathol., 102, McIntosh,G.G. et al. (1995) Determination of the prognostic value of cyclin D1 overexpression in breast cancer. Oncogene, 11, Nishida,N. et al. (1994) Amplification and overexpression of the cyclin D1 gene in aggressive human hepatocellular carcinoma. Cancer Res., 54, Betticher,D.C., Heighway,J., Hasleton,P.S., Altermatt,H.J., Ryder,W.D.J., Cerny,T. and Thatcher,N. (1996) Prognostic significance of CCND1 (cyclin D1) overexpression in primary resected non-small-cell lung cancer. Br. J. Cancer, 73, Michalides,R., Van Veelen,N., Hart,A., Loftus,B., Wientjens,E. and Balm,A. (1995) Overexpression of cyclin D1 correlates with recurrence in a group of forty-seven operable squamous cell carcinoma of the head and neck. Cancer Res., 55, Cohen,S.M. and Ellwein,L.B. (1990) Cell proliferation in carcinogenesis. Science (Wash.), 249, Magnuson,B.A., Shirtliff,N. and Bird,R.P. (1994) Resistance of aberrant crypt foci to apoptosis induced by azoxymethane in rats chronically fed cholic acid. Carcinogenesis, 15, Shirai,T., Shibata,M., Takahashi,S., Tagawa,Y., Imaeda,K. and Hirose,M. (1995) Differences in cell proliferation and apoptosis between reversible and irreversible mocosal lesions associated with uracil-induced urolithasis in N-butyl-N-(4-hydroxybutyl) nitrosamine-pretreated rats. Carcinogenesis, 161, Received on December 19, 1996; revised on April 21, 1997; accepted on April 30,