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mRNA and protein expression of p53 mutations in human bladder cancer cell lines
T Kawasaki1, Y Tomita, R Watanabe
1Department of Urology, School of Medicine, Niigata University, Japan.
Abstract:
We investigated mRNA and protein expression in p53 gene mutations in four human bladder cancer cell lines using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and Northern blot and Western blot analyses. The following mutations were identified in three of the four cell lines: a missense transversion at codon 110, a missense transition at codon 250 and a non-sense transversion at codon 126. These mutations were located outside previously identified hot spot codons and have rarely been reported in bladder cancer tissues or other neoplasms. Positive intranuclear p53 immunostaining in neoplastic cells in the two missense mutations and the premature stop codon in the non-sense mutation suggested the presence of structural and functional alterations in the p53 protein. Northern and Western blot analyses revealed either an intense or a weak p53 mRNA band together with an intense p53 protein band in the missense mutations, but no p53 mRNA or protein band in the non-sense mutation. A weak p53 mRNA band, but no distinct p53 protein band was observed in the cell line without a mutation and in normal control bladder cells. Our findings suggest that regulation of p53 expression in these cell lines differs at the post-transcriptional and/or post-translational level between the wildtype and the mutant p53 genes and also among different mutant p53 genes. The three cell lines with mutations were derived from high-grade carcinomas; the cell line without mutation was derived from a low-grade carcinoma.
Insights
This study identified novel p53 gene mutations in bladder cancer cell lines, revealing altered p53 mRNA and protein expression. These findings suggest complex post-transcriptional and post-translational regulation in bladder cancer development.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- The p53 tumor suppressor gene plays a critical role in preventing cancer.
- Mutations in the p53 gene are common in various human cancers, including bladder cancer.
- Understanding p53 alterations is crucial for developing targeted cancer therapies.
Purpose of the Study:
- To investigate the impact of p53 gene mutations on mRNA and protein expression in human bladder cancer cell lines.
- To identify novel p53 mutations and their locations within the gene.
- To explore the post-transcriptional and post-translational regulation of p53 in bladder cancer.
Main Methods:
- Utilized polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) to detect p53 gene mutations.
- Employed Northern blot analysis to assess p53 mRNA expression.
- Applied Western blot analysis and p53 immunostaining to evaluate p53 protein expression and localization.
Main Results:
- Identified three novel p53 mutations (missense and non-sense) in three out of four bladder cancer cell lines, located outside known hot spots.
- Observed positive intranuclear p53 immunostaining in cells with missense mutations and a premature stop codon, indicating altered protein structure and function.
- Detected distinct patterns of p53 mRNA and protein expression: intense mRNA/protein for missense mutations, absent for non-sense mutations, and weak mRNA/no distinct protein in wild-type cells.
Conclusions:
- p53 gene mutations in bladder cancer can occur at rare sites and lead to altered p53 protein.
- Regulation of p53 expression differs significantly at post-transcriptional and/or post-translational levels between wild-type and mutant p53.
- The identified p53 mutations were associated with high-grade bladder carcinomas, suggesting a role in tumor progression.