We have selected a series of cell lines from the parental Syrian hamster 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 competitive inhibitor of the multidrug transporter p-glycoprotein (pgp). Since TG is a known substrate for pgp, selection for TG resistance was carried out in the presence of CSA in an attempt to minimize the contribution of pgp, and to identify the potential range of adaptive responses of the SERCA pump itself, during the development of the TG-resistant phenotype. Irrespective of whether the selection is carried out in the presence or absence of CSA, pgp is overexpressed in the TG-resistant DDT1-MF2 cells. SERCA protein is also overproduced in the TG-resistant cell lines, which occurs through one of several mechanisms. Included among these, is amplification of the SERCA gene and enhanced transcription of the gene. Enhanced transcription is observed only upon long-term selection and occurs through the SERCA gene proximal promoter elements. Although SERCA transcription in wild-type cells is dependent upon the -284 to -72 bp region of the SERCA promoter, the TG-resistant cells utilize both the -284 to -72 bp and the -72 to +80 bp promoter regions for enhanced SERCA transcription. That is, additional elements within the -72 to +80 bp region are recruited in the TG-resistant cells to allow for increased SERCA expression. A post-transcriptional step may also be recruited by the TG-resistant cells in their overall strategy to produce increased amounts of the SERCA protein. These studies demonstrate that the DDT1-MF2 cells can utilize different mechanisms which lead to increased levels of SERCA protein as the cells adapt to inhibition of the ATPase by TG.
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