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Updated: Aug 23, 2026
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Published on: April 22, 2019
High frequency methylation of p16INK4A gene during 4-nitroquinoline 1-oxide-induced rat tongue carcinogenesis
Yuuji Nakahara1, Satoru Shintani, Mariko Mihara
1Department of Oral and Maxillofacial Surgery, Ehime University School of Medicine, Ehime, Japan. nakahara@m.ehime-u.ac.jp
Abstract:
The p16INK4A tumor suppressor gene can be inactivated by hypermethylation of the promoter region in many type of tumors including oral cancer. We recently studied the relationship of inactivation of p16INK4A and tumorigenesis in oral cancer. The aim of the present study was to describe the relationship between macroscopic changes of rat oral mucosa treated with 4-nitroquinoline 1-oxide (4NQO) and an inactivation of p16INK4A. We analyzed the relation of p16INK4A inactivation by hypermethylation of the promoter region of p16INK4A gene using polymerase chain reaction (PCR), PCR-single-strand confirmation polymorphism (PCR-SSCP), and methylation-specific-PCR (MSP). We observed that methylation of p16INK4A genes were rare in mild and moderate dysplasia, but inactivation was observed at high frequency even in severe dysplasia and SCCs in rat carcinogenesis. The expression pattern of p16INK4A protein, detected by western blotting and immunohistochemistry, were similar to the hypermethylation status of the p16INK4A promoter region. Inactivation by hypermethylation of the promoter region of p16INK4A gene was related to carcinogenesis of oral cancer.
Insights
Promoter hypermethylation inactivates the p16INK4A tumor suppressor gene in oral cancer. This inactivation, particularly in severe dysplasia and oral squamous cell carcinoma (OSCC), is linked to oral carcinogenesis in rats.
Area of Science:
- Oncology
- Molecular Biology
- Cancer Genetics
Background:
- The p16INK4A tumor suppressor gene plays a crucial role in cell cycle regulation.
- Hypermethylation of the p16INK4A promoter is a known mechanism of gene inactivation in various cancers, including oral cancer.
- Understanding the role of p16INK4A inactivation in oral carcinogenesis is vital for developing targeted therapies.
Purpose of the Study:
- To investigate the relationship between macroscopic changes in rat oral mucosa induced by 4-nitroquinoline 1-oxide (4NQO) and the inactivation of the p16INK4A gene.
- To determine the frequency and pattern of p16INK4A promoter hypermethylation during oral carcinogenesis in a rat model.
- To correlate p16INK4A gene inactivation with the progression of oral dysplasia and oral squamous cell carcinoma (OSCC).
Main Methods:
- Induction of oral carcinogenesis in rats using 4-nitroquinoline 1-oxide (4NQO).
- Analysis of p16INK4A promoter methylation status using methylation-specific PCR (MSP).
- Assessment of p16INK4A gene inactivation and protein expression via PCR-single-strand confirmation polymorphism (PCR-SSCP), Western blotting, and immunohistochemistry.
Main Results:
- p16INK4A promoter hypermethylation and subsequent gene inactivation were infrequent in mild and moderate oral dysplasia.
- High frequencies of p16INK4A inactivation were observed in severe dysplasia and oral squamous cell carcinoma (OSCC) in the 4NQO-treated rats.
- The expression patterns of p16INK4A protein correlated with the methylation status of its promoter region, indicating functional inactivation.
Conclusions:
- Inactivation of the p16INK4A gene through promoter hypermethylation is a significant event in the carcinogenesis of oral cancer.
- The findings suggest that p16INK4A plays a critical role in the progression from dysplasia to OSCC in this rat model.
- Targeting p16INK4A reactivation could be a potential therapeutic strategy for oral cancer.
