GENE AMPLIFICATION AND TRANSCRIPTIONAL UP-REGULATION OF THE SARCO ENDOPLASMIC RETICULUM CA2+ TRANSPORT ATPASE IN THAPSIGARGIN-RESISTANT HAMSTER SMOOTH-MUSCLE CELLS/

We have selected a series of cell lines from the parental Syrian hamst er smooth muscle cell line DDT1-MF2 that are resistant to thapsigargin (TG), a specific inhibitor of the sarcoplasmic/endoplasmic reticulum Ca2+ transport ATPases (SERCAs), Cells were selected for resistance to TG in the presence or absence of cyclosporin (CSA), which is a compet itive inhibitor of the multidrug transporter p-glycoprotein (pgp), Sin ce TG is a known substrate for pgp, selection for TG resistance was ca rried out in the presence of CSA in an attempt to minimize the contrib ution of pgp, and to identify the potential range of adaptive response s of the SERCA pump itself, during the development of the TG-resistant phenotype, Irrespective of whether the selection is carried out in th e presence or absence of CSA, pgp is overexpressed in the TO-resistant DDT1-MF2 cells. SERCA protein is also overproduced in the TO-resistan t cell lines, which occurs through one of several mechanisms. Included among these, is amplification of the SERCA gene and enhanced transcri ption of the gene. Enhanced transcription is observed only upon long-t erm selection and occurs through the SERCA gene proximal promoter elem ents, Although SERCA transcription in wild-type cells is dependent upo n the -284 to -72 bp region of the SERCA promoter, the TO-resistant ce lls utilize both the -284 to -72 bp and the -72 to +80 bp promoter reg ions for enhanced SERCA transcription. That is, additional elements wi thin the -72 to +80 bp region are recruited in the TO-resistant cells to allow for increased SERCA expression. A post-transcriptional step m ay also be recruited by the TG-resistant cells in their overall strate gy to produce increased amounts of the SERCA protein, These studies de monstrate that the DDT1-MF2 cells can utilize different mechanisms whi ch lead to increased levels of SERCA protein as the cells adapt to inh ibition of the ATPase by TG.