Related Experiment Videos
Induction of p16/INK4a gene expression and cellular senescence by toyocamycin
Yosuke Kurihara1, Kiyoshi Egawa, Setsuko Kunimoto
1Department of Microbiology, Showa University School of Pharmaceutical Sciences Tokyo, Japan.
Abstract:
We constructed an assay system of a luciferase reporter with p16/lNK4a gene transcriptional regulatory domain to identify p16-inducing substances, and found toyocamycin to induce gene expression from the screening of culture fluids of Streptomyces. Toyocamycin is a nucleoside analog, and it increased the p16 mRNA level in human normal fibroblasts or synovial cells as assessed by Northern blot hybridization or real time RT-PCR. It also induced cellular senescence in normal human fibroblasts. The transcriptional regulatory regions of human p16 gene that were responsible for the induction were analyzed using deletion mutants of the transcriptional regulatory region of p16 linked to the luciferase gene. The DNA fragment -111 to +1 bp from the cap site was sufficient to drive toyocamycin-activated transcription of p16/luciferase reporter. Nucleotide sequences within this domain contained the Sp1- and Ets-binding sequences. Mutations were introduced into these sequences, and the Sp1 sequence was found to be critical for the induction, and this notion was confirmed from gel-mobility shift assay.
Insights
Toyocamycin, a nucleoside analog, was identified as a substance that induces p16INK4a gene expression. This compound also triggers cellular senescence in human fibroblasts, highlighting its potential role in cell cycle regulation.
Area of Science:
- Molecular Biology
- Cell Biology
- Pharmacology
Background:
- The p16INK4a gene plays a crucial role in cell cycle regulation and tumor suppression.
- Identifying novel inducers of p16INK4a expression is important for understanding cellular senescence and developing therapeutic strategies.
Purpose of the Study:
- To develop an assay system for identifying p16INK4a-inducing substances.
- To screen for novel compounds that modulate p16INK4a gene expression.
- To elucidate the regulatory mechanisms underlying toyocamycin-induced p16INK4a transcription.
Main Methods:
- Construction of a luciferase reporter assay system linked to the p16INK4a transcriptional regulatory domain.
- Screening of microbial culture fluids (Streptomyces) to identify gene-inducing substances.
- Analysis of p16 mRNA levels using Northern blot hybridization and real-time RT-PCR.
- Investigation of regulatory regions using deletion mutants and site-directed mutagenesis.
- Gel-mobility shift assays to confirm transcription factor binding.
Main Results:
- Toyocamycin, a nucleoside analog, was identified as a potent inducer of p16INK4a gene expression.
- Toyocamycin increased p16 mRNA levels in human fibroblasts and synovial cells.
- The DNA fragment from -111 to +1 bp relative to the cap site of the p16 gene was sufficient for toyocamycin-induced transcription.
- The Sp1-binding sequence within this regulatory region was found to be critical for toyocamycin-mediated induction.
Conclusions:
- Toyocamycin effectively induces p16INK4a gene expression and cellular senescence in human fibroblasts.
- The Sp1 transcription factor binding site is essential for the induction of p16INK4a by toyocamycin.
- This study provides a novel reporter system for identifying p16INK4a inducers and reveals toyocamycin as a potential modulator of cell cycle control.