Gene, 156 (1995) 287 290 © 1995 Elsevier Science B.V. All rights reserved. 0378-1119/95/$09.50 287 GENE 08696 Differential utilization of multiple transcription start points accompanies the overexpression of the P-glycoprotein-encoding gene in Chinese hamster lung cells (Multidrug resistance; TATA-Iess promoter; gene expression; actinomycin D) Tan A. Ince and Kathleen W. Scotto Memorial Sloan-Kettering Cancer Center, New York, NYIO021, USA Received by F. Barany: 14 August 1994; Revised/Accepted: 22 November/5 December 1994; Received at publishers: 8 December 1994 SUMMARY 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 CHRC5, 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 CHRC5 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-Iess promoters. INTRODUCTION P-glycoprotein (Pgp) is a membrane protein overex- pressed in both tissue culture cells and human MDR Correspondence to: Dr. K.W. Scotto, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA. Tel. (1-212) 639-8972; Fax (1-212) 639-2767; e-mail: k-scotto@mskcc.org Abbreviations: ActD, actinomycin D; bp, base pair(s); eDNA, DNA complementary to RNA; CHO, Chinese hamster ovary; dNTP, deoxyri- bonucleoside triphosphate(s); MDR, multidrug resistant; nt, nueleo- tide(s); oligo, oligodeoxyribonucleotide; Pgp, P-glycoprotein(s); Pgp, Pgp-encoding gene(s) (DNA, RNA); Pgpl, hamster Pgp (class-I homo- logue); R, resistant/resistance; tsp, transcription start point(s); u, unit(s). tumors (Veinott-Drebot and Ling, 1990; Germann et al., 1993). Despite considerable effort, the transcriptional and/or post-transcriptional mechanism(s) whose modula- tion results in the overexpression of the Pgp gene in MDR cells have not been defined. Although in some instances overexpression has been attributed to an increase in the rate of Pgp transcription (Madden et al., 1993); no differences have been observed in MDR cells relative to drug-sensitive cells in the DNA elements and trans-acting factors which have been suggested to be involved in the regulation of Pgp transcription. In some MDR sublines, such as DC-3F/VCRd5L, the increase in Pgp mRNA is accomplished by an increase in the Pgp gene copy number. However, in other cell SSD1 0378-1119(94)00907-4