Differential utilization of multiple transcription start points accompanies the overexpression of the P-glycoprotein-encoding gene in Chinese hamster lung cells

Abstract

The overproduction of P-glycoprotein (Pgp) has been associated with the development and maintenance of the multidrug resistant (MDR) phenotype, although the regulatory events responsible have not yet been elucidated. We have analyzed the overexpression of the TATA-less hamster class-I Pgp-encoding gene ( Pgpl) in several MDR Chinese hamster cell lines. The MDR lung cell line DC-3F/VCRd5L, as well as the MDR ovary cell line CH RC5, express a level of Pgpl RNA commensurate with the increase in Pgpl dosage; in contrast, the actinomycin D (ActD)-selected sublines of DC-3F overexpress Pgpl mRNA without a concomitant increase in Pgpl gene-copy number. Analysis of Pgpl transcription start point ( tsp) utilization revealed that drug-sensitive DC-3F cells, as well as DC-3F/VCRd5L and CH RC5 cells, utilize one major tsp; in contrast, the ActD-resistant sublines ‘switch’ to a more complex pattern, using four additional Pgpl tsp 32, 42, 52, and 67 bp downstream from the major parental tsp (+ 1). This observation of a difference in the regulation of transcription of Pgp in MDR vs. drug-sensitive cells suggests that the ‘switch’ in tsp selection may be involved in the increased expression of Pgpl mRNA. Interestingly, despite the existence of several hundred MDR cell lines, very few have been analyzed with respect to tsp selection; it is therefore possible that alternate tsp selection is a relatively common yet heretofore unobserved component of the MDR phenotype. Moreover, these cells provide an excellent system in which to evaluate the sequence elements and protein factors that govern the selection of tsp in TATA-less promoters.