Aberrant expression of p27Kip1 is associated with malignant transformation of the rat urinary bladder epithelium
Kumiko Ogawa1,3,4,
Naoya Kimoto1,
Makoto Asamoto1,
Makoto Nakanishi2,
Satoru Takahashi1 and
Tomoyuki Shirai1
1 First Department of Pathology and
2 Second Department of Biochemistry, Nagoya City University, Medical School, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601,
3 Nagoya-shi Kohseiin Geriatric Hospital, 2-1501 Sekobou, Meito-ku, Nagoya 465-0055, Japan
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Abstract
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Alteration in cell cycle regulators is considered to play an important role in carcinogenesis. In order to cast light on changes in reversible hyperplastic and irreversible tumorigenic lesions in the rat urinary bladder, expression of p27Kip1, cyclin D1 and cyclin E proteins was sequentially compared. In the first study, 3% uracil was fed for 4 weeks to cause urinary calculi and consequent hyperplasia and papillomatosis, both regressing after withdrawal of the insult. Compared with normal bladder epithelium, in papillomatosis at week 4, the BrdU index and immunohistochemical positivities for cyclin D1 and cyclin E were significantly elevated, whereas values for p27Kip1 tended to be reduced. One week after withdrawal of uracil, the BrdU index and positivities for cyclin D1 and cyclin E were decreased to below the control levels, while positivity for p27Kip1 was dramatically increased, with a strong staining intensity. In a second study, rats were initiated with a bladder carcinogen, N-butyl-N-(4-hydroxybutyl)nitrosamine for 4 weeks, then fed 3% uracil for 8 weeks. During this latter period, expression of cyclin D1, cyclin E and p27Kip1 in hyperplastic urothelium were comparable with those in the first study. One week after withdrawal of uracil, most urothelial lesions regressed, showing high p27Kip1 and low cyclin D1 and cyclin E staining. Two weeks after uracil withdrawal, transitional cell carcinomas, with a low p27Kip1 and high cyclin D1 and cyclin E staining pattern, could be easily distinguished from surrounding regressing epithelium. These data indicate that during regression of papillomatosis after cessation of a proliferative stimulus, expression of p27Kip1is elevated, accompanied by a lowering of cyclin D1 and cyclin E. In irreversible tumorous bladder lesions, on the other hand, persistent low expression of p27Kip1 and elevated cyclin D1 and cyclin E are characteristic.
Abbreviations: BBN, N-butyl-N-(4-hydroxybutyl)nitrosamine; BrdU, bromodeoxyuridine.
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Introduction
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p27Kip1, a member of the protein family that includes p21Waf1/Cip1 and p57Kip2, is a universal cdk inhibitor that negatively regulates cyclin D-, E- and A-dependent kinases, thereby inhibiting progression from the G1 to the S phase (1,2). It is present in large amounts in quiescent cells and declines when cells proliferate in response to mitogenic stimulation by growth factors and cytokines (3). Although mutations in the p27Kip1 gene are rare in human tumors (46), a decrease in or loss of p27Kip1 protein correlates with a poor prognosis in breast (79), stomach (10), colon (11) and prostate cancers (12).
Animal models allow determination of sequential changes in p27Kip1 protein levels in carcinogenesis. In the rat urinary bladder, uracil in the diet is reported to induce calculi (13) and papillomatosis (14) which are completely reversible after withdrawal of the insult (1416). Our previous work demonstrated that papillomatosis results from mechanical irritation by calculi (17). It could further be shown that uracil can strongly promote N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN)-induced rat urinary bladder carcinogenesis (15). In this case, background hyperplasia quickly disappeared with withdrawal, while carcinomas continued to grow (15). The reversibility of uracil-induced papillomatosis is clearly correlated with a decrease in cell proliferation and increased apoptosis in the urothelium (14,16,18). Clear differences in this respect exist between reversible and irreversible mucosal lesions in rat bladder (18,19). This might be related to overexpression of cyclin D1, one of the G1 cyclins associated with p27Kip1, as seen in human papillary type transitional cell carcinomas (20) and in rat bladder lesions (21).
In the present study, to cast light on the mechanisms of reversibility of papillomatosis, expression of p27Kip1 as well as cyclins D1 and E were sequentially analyzed in the urinary bladders of rats treated with uracil, with and without BBN pretreatment.
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Materials and methods
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Study 1
A total of 30 male 7-week-old Fisher 344 rats (Charles River Japan Inc., Atsugi, Japan) were housed five per plastic cage on hardwood chip bedding in an environment-controlled room, maintained at 23 ± 2°C at 50% relative humidity and a 12 h light/dark cycle. Diet (Oriental MF; Oriental Yeast Co., Tokyo, Japan) and water were given ad libitum. Five non-treated rats served as controls. Uracil obtained from Yamasa Shoyu Co. (Choshi, Japan) was mixed in the diet at a final concentration of 3% and given to 25 rats for 4 weeks. They were maintained for a further 2 weeks on diet not containing uracil (Figure 1
). Sub-groups of five rats each were killed at the end of weeks 1 and 4 and 2 days, 1 week and 2 weeks after withdrawal of uracil. For assessment of DNA synthesis in the urothelium, 100 mg/kg body wt bromodeoxyuridine (BrdU) (Sigma Chemical Co., St Louis, MO) was injected i.p. 1 h before death. Since uracil-induced mucosal lesions are diffuse, one randomly chosen part of each bladder was frozen for western blotting analysis and the rest was fixed in 4% buffered formalin.
For immunohistochemical analysis, formalin-fixed tissues were cut into two or three strips, then paraffin sections were made and incubated with anti-BrdU (Dako Japan, Kyoto, Japan), anti-p27Kip1 (C-19; Santa Cruz Biotechnology, Santa Cruz, CA), anti-cyclin D1 (Immunobiological Laboratories, Gunma, Japan) and anti-cyclin E (M-20; Santa Cruz Biotechnology) antibodies at dilutions of 1000, 500, 100 and 500, respectively. A Vectastain ABC Elite Kit (Vector Laboratories Inc.) and DAB were used for visualization. Since the BrdU labeling index in the normal urothelium is known to be low with a large standard deviation, 6000 or all urothelial cells for each rat were counted for all cell cycle-related proteins. Both strongly and weakly stained cells were counted as positive.
For western analysis, frozen materials were lysed in cell lysis buffer (20 mM Tris, pH 7.5, 250 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 2 µg/ml aprotinin, 2 µg/ml leupeptin, 20 µg/ml soybean trypsin inhibitor, 0.1 µM phenylmethylsulfonyl fluoride, 0.1 mM sodium orthovanadate and 1 mM sodium fluoride). Total lysates (100 µg each) were separated by SDSPAGE and transferred to nitrocellulose membrane. p27Kip1 and cyclin D1 proteins were detected with anti-p27Kip1 and anti-cyclin D1 antibodies (the same antibodies as used for immunohistochemistry), respectively, and protein signals were enhanced by chemiluminescence (ECL; Amersham-Pharmacia).
Study 2
Paraffin sections obtained in a former study (18) were also examined for comparative immunohistochemical analysis of tumorous lesions. Briefly, the experimental procedure was as follows. Male 6-week-old F344 rats (Charles River Japan Inc., Atsugi, Japan) were given drinking water containing 0.05% BBN for 4 weeks as a pretreatment. They were then fed 3% uracil-containing diet for 8 weeks followed by a return to basal diet for another 8 weeks. Bladder samples at the end of weeks 1, 2, 4, 8, 9, 10 and 16 were routinely processed for pathological observation and typical hyperplastic and tumorous lesions were separately analyzed for expression of p27Kip1, cyclin D1 and cyclin E. Positive immunostaining of sections for these factors was analyzed with the aid of an image processor (Image Processor for Analytical Pathology; Sumika Technoservise, Osaka, Japan) (22) as areas of positive nuclei per total area of nuclei in ~25 fields of urothelium at a magnification of x60.
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Results
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Study 1
As we expected, the urinary bladders of all rats killed at weeks 1 and 4 were filled with small uracil calculi and demonstrated papillomatosis. Two weeks after withdrawal of uracil, all calculi had disappeared and papillomatosis was reduced with the appearance of numerous apoptoses. Data for sequential changes in BrdU labeling indices and in positivities for cyclin D1, cyclin E and p27Kip1 are summarized in Figure 1
and Table I
. In non-treated control bladder (indicated as week 0 in Figure 1
), positive staining for BrdU, cyclin D1 and cyclin E were very low. During uracil administration, they were all clearly increased, while positivity for p27Kip1 was at almost the control level or slightly decreased. One week after withdrawal of uracil, staining for BrdU, cyclin D1 and cyclin E had significantly decreased to below those in the controls, while the percentage of p27Kip1-positive cells was significantly increased.
Staining intensity for cyclin D1 (Figure 2a
) and cyclin E (Figure 2b
) was heterogeneous, basal cells showing strong intensity and intermediate cells also being positive. Increase was evident in uracil-induced papillomatosis (Figure 2a and b
) as compared with the control case. Some cyclin E expression was also seen in apoptotic cells in the uracil withdrawal stage (Figure 2e
). Expression of p27Kip1 was very weak in control tissue and papillomatosis during uracil stimulus (Figure 2c
), but extremely strong in the basal layer of the bladder epithelium after the uracil diet was discontinued (Figure 2f
).

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Fig. 2. Immunohistochemical staining of a uracil-treated bladder. (ac) Hyperplastic urothelium at day 28; (df) hyperplastic urothelium in the reversing stage at day 35. (a and d) cyclin D1; (b and e) cyclin E; (c and f) p27kip1.
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Western blotting analysis of cyclin D1 and p27Kip1 (Figure 3
) confirmed the immunohistochemistry results. The amount of cyclin D1 protein was increased at week 4, then decreased at 1 week after withdrawal of uracil. Total amounts of p27Kip1 were low at week 4 and extremely high after cessation of the insult, even after 2 weeks.
Study 2
Lesions observed in this BBN/uracil study were described in our previous paper (18). Briefly, moderate to marked papillomatosis was seen at weeks 4 and 8, which gradually disappeared after withdrawal of uracil. Papillomas and/or TCCs were also observed from week 4, persisting thereafter.
Positive nuclear staining for cyclin D1, cyclin E and p27Kip1 is summarized in Figure 4
and Table II
. The data obtained with the image analyzer in this study were similar to those gained by counting manually in Study 1. In apparently benign hyperplastic urothelium during uracil administration, positivities for cyclin D1 and cyclin E were 31.153.4 and 39.147.1, respectively. They were significantly decreased after withdrawal of the insult. Positivity for p27Kip1 was, however, significantly increased. Tumorous urothelium, distinguished from benign lesions by the presence of cellular atypia, showed high positivities for cyclin D1 (Figure 5a
) and cyclin E (Figure 5b
) and low expression of p27Kip1 (Figure 5c
). One week after withdrawal of uracil, several lesions, which were not yet unequivocally tumors, showed mixed expression with negative and positive for cyclin D1, cyclin E and p27Kip1 (Figure 5df
).

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Fig. 5. Immunohistochemical staining of a BBN/uracil-treated bladder. (ac) Transitional cell carcinoma and underlying hyperplastic urothelium at week 10 (2 weeks after withdrawal of uracil). (a) Cyclin D1; (b) cyclin E; (c) p27kip1. The carcinoma seen has strong staining for cyclin D1, but is weak for p27kip1, the opposite being the case for regressing epithelium. (de) Papilloma at week 9 (1 week after withdrawal of uracil). (d) Cyclin D1; (e) cyclin E; (f) p27kip1.
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Discussion
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There is increasing evidence that cell cycle regulation is disrupted during human and experimental animal carcinogenesis. Even though cell cycle pathways are not fully understood, positive regulators, such as cyclins and cyclin-dependent kinases, and negative regulators, such as INK4 family members (p15INK4B, p16INK4A, p18 and p19) and CIP/KIP family members (p21Waf1/Cip1, p27Kip1 and p57Kip2), are considered to be important elements of cell cycle checkpoints.
From the results of the present Study 1, stimulation by uracil calculi causes an increase in total protein level and number of cells positive for BrdU incorporation, cyclin D1 and cyclin E, while the negative regulator p27Kip1 is slightly suppressed. After withdrawal, expression of cyclin D1 and cyclin E is immediately decreased and that of p27Kip1 is clearly increased. This is not a passive return to normal levels but rather an active response, suggesting that dynamic changes in expression levels of these cell cycle regulators are associated with regression of hyperplastic lesions. Cyclins D1 and E are known to effect the G1 and late G1 phases, respectively (23). Thus their increase during uracil administration and the presence of cyclin E-positive apoptotic bodies in the urothelium after withdrawal indicate that the G1 to S phase may be critical for the appearance and regression of hyperplastic urothelial cells.
In Study 2, both initiated and non-initiated cells strongly proliferated in response to uracil caliculi stimulation, resulting in early development of carcinomas from the former and papillomatosis from the latter. On cessation of the cell proliferation stimulus, papillomatosis disappeared but carcinomas persisted with reduction in the cyclins and increased p27Kip1 only in the former.
Loss of or decrease in p27Kip1 is related to prognosis in several human cancers (712). p27Kip1 itself inhibits a variety of cyclinCdk complexes and shares a sequence encoding an inhibitory motif with p21Waf1/Cip1 (24). When phosphorylated on Thr187, it is far more stable than the wild-type, this resulting from cyclin E/Cdk2 association in human fibroblasts (25). Overexpression of cyclin D1 and/or cyclin E has also been observed in several human carcinomas (2631) including bladder lesions (20). A critical point for carcinogenesis may be an imbalance in expression of such positive and negative regulators. These alterations can be the consequence and/or the cause of transformation. Recently, p27Kip1 has been reported as haplo-insufficient for tumor suppression from the fact that both p27Kip1 nullizygous and heterozygous mice were predisposed to tumors in multiple tissues when challenged with
-irradiation or a chemical carcinogen (32). Thus, a decreased expression level of p27Kip1 itself could be a predisposing factor for carcinogenesis.
There are a number of data suggesting that elevation of p27Kip1 expression or mimicking its activity might be useful for cancer therapy, because of inhibitory effects on the growth of cancer cells (3335). It has been reported that mRNA levels of p27Kip1 remain constant throughout the cell cycle, while protein amounts are controlled post-transcriptionally (36). For a better understanding of the altered homeostatic network of the cell cycle in carcinogenesis, further analysis covering not only the factors upstream of p27Kip1 protein, such as transforming growth factor-ß, IFN-
and IFN-ß, but also those related to its turnover or stabilization are required.
In conclusion, the present study has shown that there are clearly different responses of cell cycle regulators to discontinuance of a proliferating stimulus between papillomatosis and carcinomas in the rat urinary bladder. These differences may have a direct bearing on reversibility.
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Acknowledgments
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This work was supported in part by a Grant-in-Aid for Cancer Research from the Ministry of Health and Welfare and the Ministry of Education, Sports, Science and Culture of Japan, a Grant-in-Aid from the Ministry of Health and Welfare for the Second Term Comprehensive 10-Year Strategy for Cancer Control, Japan and a grant from the Society for Promotion of Toxicologic Pathology of Nagoya, Japan.
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Notes
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4 To whom correspondence should be addressed at: First Department of Pathology, Nagoya City University, Medical School, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan. Email: kogawa{at}med.nagoya-cu.ac.jp 
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Received July 23, 1999;
revised September 16, 1999;
accepted September 21, 1999.