Activation of p53 in anoxic freshwater crayfish, Faxonius virilis.

Abstract

Tumor suppressing transcription factor p53 regulates multiple pathways including DNA repair, cell survival, apoptosis, and autophagy. The current work studies stress-induced activation of p53 in anoxic crayfish (Faxonius virilis). Relative levels of target proteins and mRNAs involved in the DNA damage response was measured in normoxic control and anoxic hepatopancreas and tail muscle. Phosphorylation levels of p53 was assessed using immunoblotting at sites known to be phosphorylated (Serine 15 and 37) in response to DNA damage or reduced oxygen signaling. The capacity for DNA binding by phospho-p53 was also measured, followed by transcript analysis of a potentially pro-apoptotic downstream target, the etoposide induced (ei24) gene. Following this, both inhibitor (MDM2) and activator (p19-ARF) protein levels in response to low oxygen stress were studied. The results showed an increase in p53 levels during anoxia in both hepatopancreases and tail muscle. Increased transcript levels of ei24, a downstream target of p53, support the activation of p53 under anoxic stress. Cytoplasmic accumulation of Ser-15 p-p53 was observed during anoxia when proteins from cytoplasmic and nuclear fractions were measured. Increased cytoplasmic concentration is known to initiate an apoptotic response, which can be assumed as a preparatory step to prevent autophagy. The results suggest that p53 might play a protective role in crayfish defense against low oxygen stress. Understanding how anoxia-tolerant organisms are able to protect against DNA damage could provide important clues towards survival under metabolic rate depression and preparation for recovery to minimize damage.