title: 3-Methylcholanthrene inactivates the p53 gene in Syrian hamster embryo fibroblasts by inducing a specific intronic point mutation.
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.
authors.affiliation: Department of Radiation Medicine, Georgetown University Medical Center, Washington, DC 20007.
authors.forename: A
authors.initials: A
authors.lastname: Albor
authors.forename: D M
authors.initials: DM
authors.lastname: Flessate
authors.forename: T
authors.initials: T
authors.lastname: Soussi
authors.forename: V
authors.initials: V
authors.lastname: Notario
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D006224:Cricetinae; D004622:Embryo, Mammalian; D005347:Fibroblasts / Q000378:metabolism; D017930:Genes, Reporter; D016158:Genes, p53 / Q000187:drug effects* / Q000235:genetics; D007438:Introns / Q000187:drug effects / Q000235:genetics*; D008647:Mesocricetus; D008748:Methylcholanthrene; D008969:Molecular Sequence Data; D017354:Point Mutation / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics* / Q000502:physiology; D012322:RNA Precursors / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
chemical_list: D012322:RNA Precursors; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53; D008748:Methylcholanthrene; D015500:Chloramphenicol O-Acetyltransferase