Effect of DGAT loss on hypoxic tumor cells growing under serum-deprived conditions

The metabolic enzyme diglyceride acyltransferase (DGAT) is responsible for the synthesis of triglycerides. Loss of its expression may sensitize cells to conditions of nutrient and oxygen that are commonly present in tumors. This study is designed to identify stress response pathways that may be induced following the shRNA-mediated knockdown of the two genes coding for the DGAT enzymes. In vitro growth conditions lacking serum and oxygen were used to mimic growth conditions commonly found in poorly perfused tumor domains We used a microarray study to identify global transcriptional changes in kidney cancer cells (A498) grown under serum deprived conditions. DGAT was knocked down using doxycycline, which induced DGAT1/2 shRNA expression. The effects of both DGAT loss and oxygen deprivation on the transcriptome were tested. Cells were grown under normoxia and serum replete conditions initially and plated onto all replicate plates. After overnight adherence, media was changed to low serum media and plates were randomly assigned to either vehicle or doxycycline treatment. Following 5 days of treatment, cells were exposed to 2 days of either normoxia or hypoxia (0.5% O2). Cells were collected in Trizol at the end of this 48h period of normoxia or hypoxia.