Abstract

Clones of the mouse hepatoma cell line Hepa1c1c7 (Hepa-1) with lesions in the Cyp1a1 gene were isolated previously. A subset of these clones fails to express CYP1A1 mRNA even when treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin, which induces this mRNA in wild-type Hepa-1 cells. The current investigation sought an explanation for this phenotype in one of these clones, c33. Loss of mRNA expression in c33 was shown to be caused by mutational changes in the Cyp1a1 gene rather than by its epigenetic silencing. No mutations were identified in the 5' flanking region of the Cyp1a1 gene, containing the promoter and dioxin-responsive enhancer sequences. A single nucleotide insertion occurred at nucleotide 418 in the coding region of one Cyp1a1 allele, and a single nucleotide insertion occurred at nucleotide 465 in the other allele in c33. These sequence alterations were confirmed in the genomic DNA of the clone. Both insertions generate a premature termination codon at codon 172. This termination codon occurs in a position within the intron/exon structure of the Cyp1a1 gene such that the encoded mRNA should be subject to "nonsense-mediated decay" (NMD). Inhibition of protein synthesis is known to reverse NMD. The protein synthesis inhibitors cycloheximide and puromycin fully restored CYP1A1 mRNA expression to c33 cells, supporting the notion that NMD degrades CYP1A1 mRNA in this strain. The mutations identified in the coding region of c33 provide an explanation, therefore, for its loss of both CYP1A1 enzymatic activity and inducible CYP1A1 mRNA expression.