Induction of P-glycoprotein mRNA by protein synthesis inhibition is not controlled by a transcriptional repressor protein in rat and human liver cells.

Recent studies have suggested that a labile transcriptional repressor protein is important in the regulation of pgp mRNA expression. However, cycloheximide (CHX) the protein synthesis inhibitor used, can increase mRNAs by either stabilizing the mRNA transcript or directly activating gene transcription. To determine whether CHX posttranscriptionally increased pgp mRNA, we compared the effect of CHX, which inhibits protein synthesis by stabilizing polysomes, with puromycin (PURO), which inhibits protein synthesis by polysome destabilization. In rat hepatocytes, CHX induced pgp2 mRNA, and the increase was proportional to the degree of protein synthesis inhibition. In contrast, despite almost complete inhibition of protein synthesis, PURO did not induce pgp2 mRNA. Further studies demonstrated that PURO pretreatment could block pgp2 mRNA induction by CHX. Likewise, in cultures of primary human hepatocytes CHX, but not PURO, induced MDR1 mRNA. A polymerase chain reaction assay was developed to assess whether CHX treatment altered the length of the 3'-untranslated region (UTR) of pgp2. CHX treatment time dependently increased the length of the pgp2 3'-UTR. To determine whether CHX acts as a transcriptional agonist, we performed nuclear run-off analysis and found no increase in pgp2 gene transcription compared to untreated control. Further, transcription studies were performed by transiently transfecting HepG2 cells with plasmids containing 5' segments of human MDR1 fused with the reporter chloramphenicol acetyltransferase (CAT). These plasmids were not transcriptionally activated by CHX. In summary, our results cast doubt on the existence of a labile transcriptional repressor protein for pgp. Furthermore, these are the first studies to demonstrate that polysomal destabilization by PURO can block CHX induction of pgp.