Culture conditions affect photoreactivating enzyme levels in human fibroblasts

Abstract

Photoreactivation of pyrimidine dimers in mammalian cells occurs under our experimental conditions but has not been observed under conditions used by others. We have tested three possible differences in experimental procedures including dimer separation and analysis methods, illumination conditions and cell culture techniques. We show that out methods of dimer separation and analysis indeed measure cis-syn pyrimidine dimers and give results in quantitative agreement with the methods of others. We find that while light pre-illumination of fibroblasts from the xeroderma pigmentosum line XP12BE or of normal cells does not affect the cellular capacity for dimer photoreactivation. However, we show that cell culture conditions can affect photoreactivating enzyme levels and thus, cellular dimer photoreactivation capacity. Cells grown in Eagle's minimal essential medium (supplemented with 15% fetal bovine serum) contain very low levels of photoreactivating enzyme and cannot photoreactivate dimers in their DNA; however, companion cultures maintained in Dulbecco's modified Eagle's minimal medium do contain photoreactivating enzyme and can photoreactivate cellular dimers.