Biomedical subjects

S Fukushima

Publications and source records attributed to S Fukushima.

At least 91 records · Page 5Linked to original sources

PubMed1999

Specific p53 gene mutations in urinary bladder epithelium after the Chernobyl accident.

After the Chernobyl accident, the incidence of urinary bladder cancers in the Ukraine population increased gradually from 26.2 to 36.1 per 100,000 between 1986 and 1996. Urinary bladder epithelium biopsied from 45 male patients with benign prostatic hyperplasia living in radiocontaminated areas of Ukraine demonstrated frequent severe urothelial dysplasia, carcinoma in situ, and a single invasive transitional cell carcinoma, combined with irradiation cystitis in 42 cases (93%). No neoplastic changes (carcinoma in situ or transitional cell carcinoma) were found in 10 patients from clean areas (areas without radiocontamination). DNA was extracted from the altered urothelium of selected paraffin-embedded specimens that showed obviously abnormal histology (3 cases) or intense p53 immunoreactivity (15 cases), and mutational analysis of exons 5-8 of the p53 gene was performed by PCR-single-strand conformational polymorphism analysis followed by DNA sequencing. Nine of 17 patients (53%) had one or more mutations in the altered urothelium. Urine sediment samples were also collected from the patients at 4-27 months after biopsy and analyzed by PCR-single-strand conformational polymorphism analysis or yeast functional assay, and identical or additional p53 mutations were found in four of five cases. Interestingly, a relative hot spot at codon 245 was found in five of nine (56%) cases with mutations, and 11 of the 13 mutations determined (73%) were G:C to A:T transitions at CpG dinucleotides, reported to be relatively infrequent (approximately 18%) in human urinary bladder cancers. Therefore, the frequent and specific p53 mutations found in these male patients may alert us to a future elevated occurrence of urinary bladder cancers in the radiocontaminated areas.

Aged↗

PubMed1999

Inhibition of liver glutathione S-transferase placental form-positive foci development in the rat hepatocarcinogenesis by Porphyra tenera (Asakusa-nori).

The effects of Asakusa-nori, Porphyra tenera (PT), a popular edible seaweed in Japan, on the development of putative preneoplastic lesions, glutathione S-transferase placental form (GST-P)-positive foci, in the male F344 rat liver were examined using a medium-term bioassay system. PT significantly decreased both the number and area of GST-P-positive foci in rat livers initiated with diethylnitrosamine (DEN). To investigate possible mechanisms of inhibition, effects of PT on 5-bromo-2'-deoxyuridine (BrdU) labeling in GST-P-positive foci and the surrounding area of hepatocytes were studied. The ratio of the GST-P-positive foci to surrounding tissue labeling indices was decreased in the PT-treated group as compared with the DEN alone group. Ornithine decarboxylase activity in the liver was slightly increased and spermidine/spermine N'-acetyltransferase activity was slightly decreased in the PT-treated animals. These results suggest that PT possesses chemopreventive effects against DEN-induced hepatocarcinogenesis.

Acetyltransferases↗

PubMed1999

Matrix metalloproteinases and tissue inhibitors of metalloproteinases in joint fluid of the patients with loose artificial hip joints.

The pseudojoint cavity formed in patients undergoing total hip arthroplasty (THA) is later remodeled to synovial membrane-like tissue, which produces pseudosynovial fluid. This pseudosynovium also is an important source of matrix metalloproteinases (MMPs). As it is widely speculated that synovial fluid MMPs may contribute to local tissue degradation in rheumatoid arthritis (RA) and osteoarthritis (OA), we hypothesize that locally produced MMPs are found in the pseudosynovial fluid, via which they have access to the implant-host interface, and that if they retain their proteolytic potential, they might contribute to aseptic loosening. Enzyme-linked immunosorbent assay (ELISA), immunoblotting, and zymography were used to analyze MMPs and tissue inhibitors of metalloproteinases (TIMPs) in synovial fluid in aseptic loosening, which was compared to RA and OA. Pseudosynovial THA fluid was characterized using low levels of MMP-1 but moderate levels of MMP-13 and MT1-MMP (MMP-14). Due to the lack of an appropriate assay, MMP-13 and MT1-MMP were not similarly assessed, but the immunoblotting indicated that they were in the 56 kD intermediate proteolytically processed forms. The MMP-9 level was intermediate between RA and OA. MMP-2 was on a significant level, but there were no differences among study groups. The THA group also was characterized using relatively high levels of TIMP-1 and TIMP-2. Accordingly, MMP-9 and MMP-2 were found to occur in the 92 kD and 72 kD proenzyme form, respectively, with full activity retained in all study groups. The data suggest that proMMP-2-TIMP-2 and proMMP-9-TIMP-1 complexes are formed in the pseudosynovial fluid due to the excess of TIMPs over MMPs in aseptic loosening of THA. TIMP-complexed MMPs are resistant to MMP-mediated proteolytic activation, which may explain their latency and proenzyme zymogen form. Thus, formation of stabilizing proMMP-TIMP complexes enable transportation of proMMPs far from their original site of production. Due to motion-associated cyclic changes of the intra-articular pressure, fluid-phase MMPs stabilized by TIMPs might be absorbed to implant surfaces and interface tissues and help to dissect the implant/cement-to-bone interface in situ. Consequently, they may contribute to local proteolytic/tissue destructive events and aseptic loosening.

Aged↗

PubMed1999

Expression of the cyclin D1 gene in rat colorectal aberrant crypt foci and tumors induced by azoxymethane.

Cyclin D1 is a cell cycle regulator which is overexpressed in a variety of human cancers. We examined overexpression of cyclin D1 in several stages of rat colorectal carcinogenesis induced by azoxymethane (AOM) treatment. The level of cyclin D1 in 13 aberrant crypt foci (ACF) (atypical hyperplasias), 22 colorectal tumors (14 non-invasive adenocarcinomas and eight invasive adenocarcinomas) was assessed by immunostaining using a polyclonal antibody. Cell proliferation of these samples was investigated by measurement of 5-bromo-2'-deoxyuridine-labeling index. Indices of cyclin D1-positive cells in adenocarcinomas and atypical hyperplasias were significantly higher than that in normal crypts (P < 0.05). Moreover, cyclin D1-positive rates in the two types of adenocarcinomas were significantly higher than that in atypical hyperplasias (P < 0.05). Staining of nuclear cyclin D1 was very strong in almost all adenocarcinomas and four ACF. Comparisons of BrdU-positive indices in colorectal lesions showed similar results to the cyclin D1-positive indices. These results suggested that overexpession of cyclin D1 occurs early in the multistep carcinogenesis, and plays an important role in rat colorectal carcinogenesis.

Adenocarcinoma↗

PubMed1999

Dose-dependent induction of aberrant crypt foci in the colons but no neoplastic lesions in the livers of heterozygous p53-deficient mice treated with low dose 2-amino-3-methylimidazo [4,5-f]quinoline.

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) is a food derived heterocyclic amine which induces aberrant crypt foci (ACF) and tumors in the livers in mice. However, most previous studies of carcinogenicity were carried out with high dose treatments, so the practical risk associated with the low dose exposure is unclear. We, therefore, assessed whether low dose IQ causes ACF formation in the colons of mice constitutively hemizygous for functional p53. Simultaneously, we screened for development of preneoplastic foci in the liver. A total of 60 heterozygous p53-deficient mice as well as 60 wild-type mice were divided into five groups and administered IQ in the diet at concentrations of 50, 10, 2, 0.4 and 0 ppm until the end of the experiment. ACF were detected in the 50, 10 and 2 ppm-treated groups and the numbers of those comprising one aberrant crypt (AC) in p53-deficient mice treated with 10 or 2 ppm were significantly increased, compared to counterpart wild-type values. A dose-dependent increase of ACF was also observed in transgenic mice groups but no large ACF developed. In spite of extensive examination, no preneoplastic foci could be detected in either transgenic or wild-type mice. The results suggested that germline p53 deficiency may slightly enhance the development of ACF in colons but not in the liver. The fact that no ACF were detected in the lowest, 0.4 ppm, treated groups may imply a practical non-effective level of IQ for tumor induction.

Animals↗

PubMed1999

Stereoselective analyses of selegiline metabolites: possible urinary markers for selegiline therapy.

The stereoselective analysis of selegiline metabolites in human urine and plasma by gas chromatography using the chiral column with the non-chiral reagent was investigated for the differentiation of selegiline therapy from the methamphetamine (MA) abuse. This method gave clear separations of MA and amphetamine (AM) isomers without any artifactual optical-opposite peaks due to the reagent. After the administration of selegiline tablets, desmethylselegiline (DMS), MA and AM were observed as (-)-isomers in the urine and plasma. Within the first 48 h after dosing, approximately 40% of selegiline administered was excreted in urine as these three metabolites. The parent drug, selegiline, was not detected in any urine or plasma samples. On the other hand, MA and AM were observed only as (+)-isomers in the urine of MA abusers. For the distinction of selegiline users from street MA abusers in urinalysis, (-)-DMS, a specific metabolite of selegiline, was not a suitable marker. (-)-DMS rapidly disappeared from urine and was excreted only 1% of the given dose. By the moment analysis with the trapezoidal integration, the mean residence times of (-)-DMS in plasma and urine were 2.7 and 3.8 h, respectively, which were 5-20 times shorter than those of (-)-MA or (-)-AM. The values of AM/MA in the urine increased from 0.24 to 0.67 (r = 0.857) along with time after the selegiline administration. This ratio was not a sufficient marker to differentiate selegiline users from MA abusers, although the values of AM/MA in 74% of MA abusers were less than 0.24. The present GC technique improved the chiral analyses of MA and AM. This chiral analysis is the most useful technique to avoid the misinterpretation in the discrimination between clinical selegiline therapy and illicit MA use.

Adult↗

PubMed1999

Cytotoxic activity of pierisin, from the cabbage butterfly, Pieris rapae, in various human cancer cell lines.

Pierisin, a protein purified from pupae of the cabbage butterfly, Pieris rapae, exhibits cytotoxic effects against the human gastric cancer TMK-1 cell line, inducing apoptosis. The present study was performed to determine whether pierisin might exert a similar influence on nine other human cancer cell lines and human umbilical vein endothelial cells (HUVECs). Pierisin showed cytotoxic effects in all the human cells tested, with IC50 values ranging from 0.043 ng/ml to 150 ng/ml. Among the target cells, the cervical carcinoma cell line, HeLa, was the most sensitive to pierisin, showing a 1000-fold less IC50 value than that of HUVECs. While pierisin clearly induced apoptotic cell death in most cancer cell lines and HUVECs, the pathway appeared to be probably different from that involving anti-Fas, TNF-alpha and p53. Pierisin may thus be a promising new candidate for cancer therapy.

ADP Ribose Transferases↗

PubMed1999

Postoperative irradiation of pterygium with 90Sr eye applicator.

PURPOSE: Pterygium are triangular growths of fibrovascular tissue of the bulbar conjunctiva that ultimately cause visual disturbances. Surgical removal is the initial treatment of choice for pterygium, but the recurrence rate after excision alone is 20-40%. The purpose of this paper is to reaffirm the effectiveness of postoperative irradiation with a 90Sr eye applicator. METHODS AND MATERIALS: From 1993 to 1996, 361 patients (393 lesions) were treated with 90Sr postoperative radiation therapy. Forty-five lesions were recurrent cases. All cases had been excised using the "bare sclera" method. Postoperative radiation performed within 48 h after surgery received radiation doses of 30 Gy, the others received doses of 35 Gy, using the 90Sr eye applicator. RESULTS: Thirty-four (8.6%) of the 393 lesions recurred. The overall 1-year local control rate was 93.7%. Seventy-one percent of the recurrent cases were noticed within 1.5 years after treatment. The multivariate analysis for total cases demonstrated that sex, age, prior treatment, and duration of pterygium affected the overall local control rate. No complications from this treatment have been observed. CONCLUSION: It was reconfirmed that postoperative irradiation was effective in preventing local recurrence after surgical removal of pterygium.

Adolescent↗

PubMed1999

Enhancement of urinary bladder carcinogenesis in nullizygous p53-deficient mice by N-butyl-N-(4-hydroxybutyl)nitrosamine.

We recently reported p53 mutations to be frequent in mouse invasive urinary bladder carcinomas, with and without metastasis. However, the role of p53 dysfunctions during carcinogenesis remains unclear. In the present study, heterozygous and nullizygous p53-deficient mice and their littermates were treated with the urinary bladder carcinogen, N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN), at a concentration of 0.01% in the drinking water throughout the experiment. This markedly accelerated urinary bladder carcinogenesis but not development of other tumors in the nullizygous p53-deficient mice. Thus the appearance of neoplastic urothelial lesions in nullizygotes (at day 60 of the experiment) was earlier than in wild-type mice and heterozygotes (at day 125). Moreover, malignant vascular tumors (hemangiosarcomas (HS)) were found in all four nullizygotes killed later than day 108. Mutational inactivation of the wild-type allele was not apparent in either the single transitional cell carcinoma observed in a wild-type mouse and a hemangiosarcoma in a heterozygote. Overall, it can be concluded that the number of normal p53 alleles is a significant determining factor in the susceptibility of urothelial cells to carcinogens. The role of the p53 defect in mouse urinary bladder carcinogenesis may thus be to diminish the threshold for occurrence of additional genetic alterations.

Animals↗

PubMed1999

Hepatocarcinogenic activity of N-butyl-N-(4-hydroxybutyl)nitrosamine in rats is not modified by sodium L-ascorbate.

Male F344 and Wistar Shionogi (WS) rats were treated with 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) for 20 weeks and then killed at week 36 (experiment 1). Although reduction of body weight increase was found, no effects on liver weights were noted. Formalin-fixed and paraffin-embedded liver tissues from rats killed terminally were cut and stained for glutathione S-transferase placental form (GST-P) immunohistochemically. Marked elevation of quantitative values of small GST-P positive (GST-P+) foci were apparent in both strains of rat administered BBN. In experiment 2, both sexes of F344 rats were given 0.05% BBN in the drinking water for 4 weeks and then fed diet containing 0 or 5.0% sodium L-ascorbate (SA) for 32 weeks. No body and liver weight changes were evident in any group. Quantitative values for small GST-P+ foci were increased in both sexes of rats exposed to BBN but were not modified by additional SA treatment. Thus, it was confirmed that the selective bladder carcinogen BBN also acts as a liver carcinogen. These results, from the quantitative analysis of small GST-P+ foci as end point marker lesions, indicate that the liver tumor modifying potential of test chemicals can be evaluated in rats by using an initiation/promotion protocol for urinary bladder carcinogenesis.

Animals↗

PubMed1999

Effects of uracil calculi on cell growth and apoptosis in the BBN-initiated Wistar rat urinary bladder mucosa.

The different potential of initiated and non-initiated urinary bladder mucosa (UBM) to develop neoplasia was quantitatively evaluated in the male Wistar rat. Initiation of carcinogenesis was accomplished with N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN). Stimuli for cell proliferation and apoptosis were obtained by exposure followed by withdrawal of 3% Uracil in the diet. The proliferation index (PI) was estimated in UBM immunostained for the proliferating nuclear cell antigen (PCNA). The apoptotic index (AI) and the density of papillary/nodular hyperplasia (PNH) were estimated in hematoxilin-eosin stained sections. PNH was the main proliferative response to the mechanical irritation by uracil, irrespective of previous initiation with BBN. Uracil exposure induced higher PI and PNH density in the initiated rats. After uracil withdrawal, there was a significant increase of the AI in both uracil-treated groups, which correlated well to the respective PNH density. However, at the end of the experiment, PNH incidence and density were significantly higher in the BBN-initiated mucosa, which also presented 18% incidence of papillomas and 27% of carcinomas. Therefore, under prolonged uracil calculi trauma, the UBM of BBN-initiated Wistar rats gives rise to epithelial proliferative lesions that progress to neoplasia through acquired resistance to apoptosis.

Animals↗

PubMed1999

Induction of smooth muscle cells in the fibrous capsule of human hepatocellular carcinoma but not in the septa of hepatic cirrhosis.

We examined the expression of smooth muscle cytoskeleton in spindle-shaped cells in the capsule of hepatocellular carcinoma (HCC) and the septa of liver cirrhosis (LC). Serial sections of livers resected from 11 patients were stained with monoclonal antibodies against vimentin, desmin, smooth muscle actin (1A4, HHF35, CGA7) and smooth muscle myosin heavy chain isoforms (SM1, SM2). Capsular spindle-shaped cells exhibited a cytoskeletal feature indicative of intermediately differentiated smooth muscle cells. Computer-assisted morphometry revealed that the proportions of 1A4-, HHF35-, CGA7- and SM1- positive areas to vimentin-positive area were 88.0+/-11.0%, 50.8+/-17.4%, 25.3+/-16.4% and 19.4+/-12.4% (n=11) in main tumours and 86.6+/-9.4%, 50.9+/-18.7%, 21.1+/-12.3% and 17.6+/-9.7% (n=12) in daughter tumours, indicating that spindle-shaped cells are heterogeneous in cytoskeletal expression. Septal spindle-shaped cells in LC lacked the cytoskeletal proteins specific to differentiated smooth muscle cells (CGA7, SM1, SM2 and desmin). Electron microscopically, capsular spindle-shaped cells contained more microfilaments and less rough endoplasmic reticulum than do septal cells. Intermediately differentiated smooth muscle cells are induced in the capsule of HCC but not in the septa of LC, suggesting a role for stromal interaction by tumour cells in the induction of smooth muscle cells.

Actins↗

PubMed1999

Non-carcinogenicity, but dose-related increase in preneoplastic hepatocellular lesions, in a two-year feeding study of phenobarbital sodium in male F344 rats.

Phenobarbital sodium (PB) was administered at dietary levels of 0 (control), 8, 30, 125 and 500 ppm to groups of 20 male F344/DuCrj rats for 104 weeks. There were no treatment-related clinical signs or adverse effects on survival rate, body weights, food consumption, and haematology or blood biochemistry data. Statistically significant increases of relative liver weights were found in the 500 and 125 ppm, but not the 30 and 8 ppm groups. Quantitative analysis of glutathione S-transferase placental form positive (GST-P+) hepatocyte foci/areas revealed clear increases limited to the 500 and 125 ppm groups. Western blotting revealed CYP2B1, 2C6 and 3A2 proteins to be also increased only with these high doses. In addition, significant increase of regenerative hepatocellular hyperplasias was noted in the 500 ppm group. No hepatocellular adenomas were observed, but a hepatocellular carcinoma arose in single rats of the 8 ppm and 125 ppm groups. No treatment-related changes were found in any other organs or tissues. Thus, under the experimental conditions used, the highest dose of PB (500 ppm) was not carcinogenic in male F344 rats. Furthermore, increase in putative preneoplastic proliferative hepatocytic lesions was only noted with 500 and 125 ppm.

Administration, Oral↗

PubMed1999

Rhythmic changes in the stomach movement of the human fetus with congenital duodenal obstruction during the third trimester of pregnancy.

In order to reveal whether or not rhythmic changes exist in fetal stomach movement (FSM), in utero FSM was assessed in three fetuses between 27 and 33 weeks' gestation with congenital duodenal obstruction. A total of four observations, one each at 27, 29, 31 and 33 weeks' gestation, was obtained. The longitudinal transection of each fetal stomach was continuously observed for 60 min using real-time ultrasound. The configuration and the area of the stomach were analyzed for each 15-s epoch. The complexity of the stomach configuration was quantified and defined as stomach complexity. For each case, the chronological changes of the stomach complexity were analyzed using the least median square of regression. The correlation between changes of the stomach complexity and the area of the stomach was analyzed using the cross-correlation method. (1) For gestational ages of 27, 29, 31 and 33 weeks, the 240 sequential measurements of the stomach complexity were significantly stratified into outlying and non-outlying points. The outlying points were 13.3% (32/240), 30.8% (74/240), 32.9% (79/240) and 36.3% (87/240) of the total observation points, respectively. (2) The percentages in which outlying points lasted 3 min (12 points) or more were 0% (0/240), 5.0% (12/240), 28.3% (68/240) and 30.4% (73/240) of the total observation points, respectively. (3) For each gestational age, no significant time series correlation was found between the stomach complexity and the area of the stomach. These findings suggest that: (1) two different conditions emerge in the FSM, at the latest at 27 weeks' gestation, and begin manifesting from 29 weeks' gestation onwards. (2) These chronological changes cluster into 'active' and 'quiet' phases from 31 weeks' gestation onwards. (3) FSMs are not related to the changes in the stomach area throughout the observation periods. The underlying mechanism of this rhythmicity may represent the development of ultradian rhythm of the stomach movement, generated by the central nervous system.

Duodenal Obstruction↗

PubMed1999

Molecular cytogenetic identification of cyclin D1 gene amplification in a renal pelvic tumor attributed to phenacetin abuse.

Despite extensive epidemiologic evidence of phenacetin abuse as a risk factor for renal pelvic carcinomas, genetic alterations in the resultant tumors remain largely unclear. In this report, a phenacetin-associated renal pelvic carcinoma (histologically a transitional-cell carcinoma) from an 80-year-old female patient was evaluated by molecular cytogenetic methods. Fluorescence in situ hybridization was used to identify chromosome gains or losses for the cyclin D1, p53, Rb and c-myc genes and the ploidy of their respective chromosomes. Cyclin D1 gene amplification, but normal copy numbers of p53, Rb and c-myc, and normal ploidy of chromosomes 8, 11, 13 and 17 were observed. Expression of cyclin D1 protein was confirmed by immunohistochemistry. In the absence of p53, Rb or c-myc abnormalities, the results suggested that cyclin D1 gene amplification and its protein overexpression may be involved in the genesis of renal pelvic carcinomas associated with phenacetin abuse.

Aged↗

PubMed1999

LOH and mutational analysis of p53 alleles in mouse urinary bladder carcinomas induced by N-butyl-N-(4-hydroxybutyl) nitrosamine.

In human urinary bladder carcinogenesis, alterations in the p53 tumor suppressor gene are common events. We have previously reported that they are also frequent in invasive urinary bladder carcinomas induced by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in NON/Shi mice. To further investigate the significance of the p53 gene status for mouse urinary bladder carcinogenesis, we examined both allele loss and mutational alterations in urinary bladder cancers of (NON/Shi x C3H/He/Shi) F1 hybrid mice exposed to the carcinogen for 12 weeks and then maintained for a further 9 weeks without treatment. An intragenic silent polymorphism within exon 7 of the p53 gene between NON/Shi and C3H/He/Shi mice allows assessment of allele loss of the p53 gene and determination of the parental origin of mutated and/or lost alleles. A tissue microdissection method was employed to obtain carcinoma samples without excessive contamination with normal tissue. Allele losses were detected in one of 14 tumors (7.1%) and nine mutations in eight of 14 (57%) tumors were found in exons 5-8 by polymerase chain reaction-single strand conformation polymorphism followed by DNA direct sequencing analysis. All mutations involved one base substitution with an amino acid change, although the types of base substitution were random. In conclusion, the high incidence of p53 alterations suggests a significant role in the genesis of invasive urinary bladder tumors in BBN-treated mice.

Alleles↗

PubMed1999

Elevation of urinary enzyme levels in rat bladder carcinogenesis.

Urinary enzyme levels were investigated in rats administered different promoters in their diet for 32 weeks after being initiated by treatment with 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine in their drinking water for 4 weeks. All groups were composed of 10 rats each. Group 1: females treated with 3% uracil (100% carcinoma incidence). Group 2: control females kept on basal diet only (0% carcinoma incidence). Group 3: males treated with 5% sodium L-ascorbate (100% carcinoma incidence). Group 4: control males (0% carcinoma incidence). Urine was collected at the end of weeks 12, 24 and 36 and tested for lactate dehydrogenase (LDH), alkaline phosphatase, N-acetyl-beta-D-glucosaminase and aspartate aminotransferase activity. To facilitate comparison, data were related to the corresponding excreted creatinine levels. All measurements were made using a centrifugal automatic analyzer. The urine of rats with cancer lesions (groups 1 and 3) showed significant elevation in all enzyme activities at weeks 24 and/or 36 except for LDH in females (group 1). The M/H ratio of the LDH isozymes was reversed (1.10 +/- 0.10) in the tested rats with carcinomas at week 36. This study thus provides evidence of a correlation between high urinary enzyme levels and cancer development in the rat bladder. Measurement of the tested enzymes might thus provide a method to detect malignant changes in bladder epithelium by direct urine analysis.

Acetylglucosaminidase↗

PubMed1999

Reduced expression of the CDK inhibitor p27(KIP1) in rat two-stage bladder carcinogenesis and its association with expression profiles of p21(WAF1/Cip1) and p53.

The cyclin-dependent kinase (CDK) inhibitor p27(KIP1) exerts its growth suppressive effects by targeting the cyclin-CDK complexes. Reduced protein levels of p27(KIP1) have been reported in numerous human cancers and this has been attributed to increased degradation. However, few reports have addressed the significance of p27(KIP1) expression in chemical carcinogenesis of rodents. In a rat two-stage urinary bladder carcinogenesis model, with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) initiation followed by promotion with sodium L-ascorbate (Na-AsA), we evaluated the expression of p27(KIP1) protein using immunohistochemistry during various stages of urinary bladder carcinogenesis. In addition, we evaluated the mRNA expression profiles for p27(KIP1), p21(WAF1/Cip1) and p53 in tumors. Fisher 344 rats were initiated with 0.05% BBN in the drinking water for 4 weeks and then administered 5% Na-AsA in the diet. Immunohistochemical examination revealed p27(KIP1) protein to be constitutively expressed in normal urothelium, simple hyperplasia and in most papillary and nodular (PN) hyperplasias and small papillomas, but diminished or absent in large papillomas and in transitional cell carcinomas. An inverse correlation between expression of p27(KIP1) and cell proliferation was generally observed. Quantitation of mRNA by multiplex reverse transcription-PCR showed a significant downregulaton of p27(KIP1), p21(WAF1/Cip1) and p53 mRNA in tumors. More than 50% reduction in p27(KIP1) mRNA expression was observed in 42 and 47% of tumors at weeks 18 and 24, respectively; similar reduction in p21(WAF1/Cip1) mRNA expression was observed in 58 and 73% of tumors at weeks 18 and 24, and in p53 mRNA expression in 50 and 73% of tumors at weeks 18 and 24, respectively. None of the 25 tumors we examined by PCR-single-strand conformational polymorphism analysis had p53 mutations. These data imply that abnormal down-regulation of p27(KIP1), p21(WAF1/Cip1) and/or p53 in tumor cells may contribute to the malignant progression of tumors during rat two-stage bladder carcinogenesis.

Animals↗