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3-Methylcholanthrene inactivates the p53 gene in Syrian hamster embryo fibroblasts by inducing a specific intronic
A Albor1, D M Flessate, T Soussi
1Department of Radiation Medicine, Georgetown University Medical Center, Washington, DC 20007.
Abstract:
The expression of the tumor suppressor gene p53 was studied in Syrian hamster embryo cells neoplastically initiated with a single dose of 3-methylcholanthrene. Ten randomly selected individual 3-methylcholanthrene-transformed colonies were established in culture independently. Eight of these cell lines contained levels of p53 mRNA similar to those in primary embryo cells (p53+ cell lines), as measured by Northern blot analysis of total RNA, whereas two of them (81C43 and 81C47) showed no detectable levels of p53 mRNA (p53- cell lines). However, Southern blot and karyotype analyses did not reveal any significant changes in copy number or gross rearrangements of the p53 gene in any of the p53- cell lines. A 3-kilobase genomic fragment cloned from p53- cells (81C47) containing both upstream and downstream promoters of the p53 gene was able to drive the expression of a CAT reporter gene when transfected into either p53+ or p53- cells. Furthermore, run-on assays performed on nuclei of p53- cells showed that the p53 gene was transcriptionally active, demonstrating that the genetic defect leading to the lack of p53 expression was not due to alterations in the promoter region. Detection of mRNA species corresponding to p53 mRNA precursors in Northern blot analysis of polyadenylated RNA from both p53+ and p53- cells indicated that the lack of p53 expression was not caused by mutations in the 3' regulatory region of the p53 gene affecting transcription termination and/or polyadenylation of p53 precursor mRNA. PCR amplification and nucleotide sequence analysis of extensive internal regions of the gene revealed that both p53- cell lines were homozygous for the same unique point mutation on the splice acceptor site of the fifth intron, a G to C transversion in the last nucleotide of the intron. The presence of this mutation in both p53- cell lines strongly suggests that it was induced specifically by 3-methyl-cholanthrene treatment and indicates that the resulting splicing malfunction may account for the lack of p53 gene expression.
Insights
A specific mutation in the p53 gene, induced by 3-methylcholanthrene, prevents its expression in hamster cells. This splicing defect, not promoter or regulatory region issues, explains the loss of tumor suppressor function.
Area of Science:
- Molecular Biology
- Cancer Research
- Genetics
Background:
- The tumor suppressor gene p53 plays a critical role in preventing cancer.
- Understanding how p53 expression is regulated and lost is crucial for cancer research.
Purpose of the Study:
- To investigate the molecular mechanisms behind the loss of p53 gene expression in 3-methylcholanthrene-transformed Syrian hamster embryo cells.
Main Methods:
- Cell culture and transformation using 3-methylcholanthrene.
- Northern and Southern blot analyses for gene expression and structure.
- Reporter gene assays (CAT) and run-on assays for transcriptional activity.
- Polymerase Chain Reaction (PCR) and nucleotide sequencing for mutation analysis.
Main Results:
- Two out of ten transformed cell lines (p53- cells) showed no detectable p53 mRNA, while eight retained normal levels (p53+ cells).
- No significant gene copy number changes or gross rearrangements were found in p53- cells.
- The p53 gene remained transcriptionally active, and promoter/3' regulatory regions were intact.
- A specific homozygous point mutation (G to C transversion) was identified at the splice acceptor site of the fifth intron in both p53- cell lines.
Conclusions:
- The loss of p53 expression in these cells is caused by a splicing defect due to a specific point mutation in the p53 gene's fifth intron.
- This mutation was likely induced by 3-methylcholanthrene treatment.
- The identified splicing malfunction provides a clear mechanism for the loss of p53 tumor suppressor function.