Chyi Chia R. Lee

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.

Urinary Bladder Lesions after the Chernobyl Accident: Immunohistochemical Assessment of p53, Proliferating Cell Nuclear Antigen, Cyclin D1 and p21WAF1/Cip1

During the 11‐year period subsequent to the Chernobyl accident, the incidence of urinary bladder cancer in Ukraine has increased from 26.2 to 36.1 per 100,000 population. Cesium‐137 (137Cs) accounts for 80–90% of the incorporated radioactivity in this population, which has been exposed to long‐term, low‐dose ionizing radiation, and 80% of the more labile pool of cesium is excreted via the urine. The present study was performed to evaluate the histopathological features and the immunohistochemical status of p53, p21WAF1/Cip1, cyclin D1 and PCNA (proliferating cell nuclear antigen) in urinary bladder mucos a of 55 males (49‐92 years old) with benign prostatic hyperplasia who underwent surgery in Kiev, Ukraine, in 1995 and 1996. Group I (28 patients) inhabiting radiocontaminated areas of the country, group II (17 patients) from Kiev city with less radiocontamination and a control group III (10 patients) living in so‐called “clean” areas of Ukraine were compared. In groups I and II, an increase in multiple areas of moderate or severe dysplasia or carcinoma in situ was seen in 42 (93%) of 45 cases. In addi tion, two small transitional cell carcinomas were found in one patient in each of groups I and II. Nuclear accumulation of p53, PCNA, cyclin D1, and to a lesser extent p21WAF1/Cip1, was significantly increased in both groups I and II as compared with the control group III, indicating possible transformation events or enhancement of repair activities, that may precede the defect in the regulatory pathway itself, at least in the G1 phase of the cell cycle. Our results suggest that early malignant transformation is taking place in the bladder urothelium of people in the radiocontaminated areas of Ukraine and that this could possibly lead sometime in the future to an increased incidence of urinary bladder cancer.

p53 status in multiple human urothelial cancers: assessment for clonality by the yeast p53 functional assay in combination with p53 immunohistochemistry.

Multifocal synchronous or metachronous tumor development is a common observation in human urothelial cancer cases. However, the underlying mechanism has remained obscure. We have employed a new tool to investigate the p53 gene status, the yeast p53 functional assay, in combination with immunohistochemistry in a total of 50 tumor samples from 32 cases with urothelial cancers, including 8 with multiple synchronous tumor development and 2 demonstrating metachronous tumors. p53 mutations were found in 13 cases (9 with missense mutations, 3 with deletion, 1 with splicing mutation) by the yeast p53 functional assay. p53 protein overexpression was seen in all 9 cases with missense mutations, but in only one of the 4 cases with nonsense mutations. Two tumors without p53 mutation also showed positive p53 immunoreactivity. Overall, p53 abnormalities including mutations and/or protein overexpression were found in 15 (47%) cases. p53 abnormalities were significantly more frequent in non‐papillary and in high grade tumors. Loss of the wild type allele in addition to a p53 mutation was suggested in 8 of the 15 (53%) cases. All 4 cases with mutations in multiple synchronous tumors had identical p53 mutations in the separate urothelial cancers, strongly suggestive of monoclonality. The one case with multiple metachronous tumors, in contrast, was characterized by variation in the p53 status, indicative of different clonal origins. In conclusion, combined assessment for p53 status as used here (yeast p53 functional assay plus immunohistochemistry) may provide insights into the molecular mechanisms of urothelial carcinogenesis.

Review article Alterations in cyclin D1, p53, and the cell cycle related elements: Implications for distinct genetic pathways of urinary bladder carcinogenesis

Data from recent studies support the hypothesis that overexpression of cyclin D1 acts as an oncogenic event during development of transitional cell carcinomas of urinary bladder, possibly modifying the evolution of a particular subset of these tumors. We recently described overexpression of cyclin D1 and alteration in other cell cycle related elements in chemical carcinogenesis of the rat urinary bladder. Here, we discuss in more detail the implications of these changes for pathways of urinary bladder neoplasia.

Loss of Heterozygosity in (Lewis×F344)F1 Rat Urinary Bladder Tumors Induced with N‐Butyl‐N‐(4‐hydroxybutyl)nitrosamine Followed by Dimethylarsinic Acid or Sodium L‐Ascorbate

Dimethylarsinic acid (DMA), a main metabolite of arsenicals which are carcinogenic in man, exerts tumor‐promoting activity on rat urinary bladder carcinogenesis initiated with N‐butyl‐N‐(4‐hydroxybutyl)nitrosamine (BBN). Sodium L‐ascorbate (Na‐AsA) is also a strong tumor promoter in this animal model. In this study, we used (Lewis×F344)F1 rats to compare molecular alterations in urinary bladder tumors caused by BBN followed by DMA or Na‐AsA. Male, 6‐week‐old rats were given 0.05% BBN in their drinking water for 4 weeks, and then the rats in group 1 were maintained with no further treatment for 40 weeks. The animals of groups 2 and 3 were administered 0.01% DMA in their drinking water (group 2) or 5% Na‐AsA in the powder diet (group 3) after the BBN treatment. Group 4 rats were given 0.05% BBN continuously for 36 weeks. At weeks 12, 20, 36 and 44, subgroups of rats were killed. Histopathological examination revealed promoting activity for DMA and, to a greater extent, Na‐AsA on urinary bladder carcinogenesis. Loss of heterozygosity (LOH), detected with the polymerase chain reaction using 36 microsatellite markers, was found to be present in 2 of 9 (22%) urinary bladder tumors after treatment with DMA and 3 of 22 (14%) induced by continuous administration with BBN. No LOH was, however, detected in urinary bladder tumors after treatment with Na‐AsA. The results thus suggest that the mechanisms of action of these two promoters, DMA and Na‐AsA, may differ in rat urinary bladder carcinogenesis.

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.

Differences of Promoting Activity and Loss of Heterozygosity Between Dimethylarsinic Acid and Sodium L-ascorbate in F1 Rat Urinary Bladder Carcinogenesis

Publisher Summary Dimethylarsinic acid (DMA) is known to have promoting activity on rat urinary bladder carcinogenesis in F344 rats initiated with N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). Sodium L-ascorbate is also a strong promoter in this animal model. (LewisxF344) F1 rats were used to compare the promoting activity between DMA and sodium L-ascorbate and to find molecular alterations in the urinary bladder tumors. Male, 6-week-old rats were given 0.05% BBN in drinking water for 4 weeks, and then the rats were kept with no treatment for group 1, administered 0.01% DMA in drinking water (group 2) or 5% sodium L-ascorbate in the powdered diet (group 3). Group 4 rats were continuously given BBN alone. At weeks 36 and 44, the rats were sacrificed and the urinary bladders were fixed in 10% phosphate buffered formalin and embedded in paraffin. HE however, DMA revealed weaker promotion activity than that of sodium L-ascorbate, although doses were different. LOH existed in the urinary bladder tumors treated with DMA, whereas no LOH was detected in the urinary bladder tumors treated with sodium L-ascorbate.

Significance of Cyclin D1 Overexpression in Transitional Cell Carcinomas of the Urinary Bladder and its Correlation With Histopathologic Features

BACKGROUND Genetic alterations leading to neoplastic transformation of the urothelium are likely to involve the activation of oncogenes and loss of functional tumor suppressor genes. Cyclin D1 has been implicated as a putative protooncogene whereas mutations of the p53 gene occur frequently in invasive transitional cell carcinomas (TCCs) of the urinary bladder. In this study, cyclin D1 overexpression and nuclear accumulation of p53 were evaluated and the results correlated with histopathologic features. METHODS TCCs of the urinary bladder from 161 surgical procedures were evaluated for cyclin D1 overexpression and nuclear accumulation of p53. Results were correlated with tumor grade, T classification, and papillary status. Topologic distributions of cyclin D1, p53, and proliferating cellular nuclear antigen (PCNA) were evaluated. Northern blot analysis was performed on selected specimens. RESULTS Overexpression of cyclin D1 was observed in 47% (24 of 51) of Grade 1 TCCs and 20% (13 of 65) of Grade 2 TCCs but in no Grade 3 TCCs. Approximately 34% (14 of 41) of Ta classified TCCs and 21% (13 of 63) of T1 classified TCCs were immunoreactive for cyclin D1 whereas none of the TCCs beyond T1 was immunoreactive. Overexpression of cyclin D1 was observed only in papillary type TCCs. Results of Northern blot analysis for cyclin D1 were comparable to those of immunohistochemistry. CONCLUSIONS The observed significant relation between cyclin D1 overexpression and tumor grade/T classification suggests that cyclin D1 may be a useful biologic marker for biopsied materials or urine cytology specimens. The prognostic significance of cyclin D1 overexpression in TCCs remains to be determined.