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Published on: August 4, 2019
Characterization of p53 expression in rainbow trout
Michelle Liu1, Catherine Tee, Fanxing Zeng
1Department of Biology, University of Waterloo, Waterloo, Ontario, N2L 3G1, Canada.
Abstract:
The tumour suppressor protein p53 is a critical component of cell cycle checkpoint responses. It upregulates the expression of cyclin-dependent kinase inhibitors in response to DNA damage and other cellular perturbations, and promotes apoptosis when DNA repair pathways are overwhelmed. Given the high incidence of p53 mutations in human cancers, it has been extensively studied, though only a small fraction of these investigations have been in non-mammalian systems. For the present study, an anti-rainbow trout p53 polyclonal antibody was generated. A variety of rainbow trout (Oncorhynchus mykiss) tissues and cell lines were examined through western blot analysis of cellular protein extracts, which revealed relatively high p53 levels in brain and gills. To evaluate the checkpoint response of rainbow trout p53, RTbrain-W1 and RTgill-W1 cell lines were exposed to varying concentrations of the DNA damaging agent bleomycin and ribonucleotide reductase inhibitor hydroxyurea. In contrast to mammals, these checkpoint-inducing agents provoked no apparent increase in rainbow trout p53 levels. These results infer the presence of alternate DNA damage checkpoint mechanisms in rainbow trout cells.
Insights
Rainbow trout (Oncorhynchus mykiss) p53 protein levels did not increase after DNA damage, unlike in mammals. This suggests alternative DNA damage response pathways exist in trout.
Area of Science:
- Aquatic biology
- Molecular biology
- Cellular biology
Background:
- The tumor suppressor protein p53 is vital for cell cycle checkpoints, responding to DNA damage by upregulating inhibitors or promoting apoptosis.
- p53 mutations are frequent in human cancers, leading to extensive research, primarily in mammalian systems.
- Investigating p53 in non-mammalian vertebrates like rainbow trout can reveal conserved and divergent cellular mechanisms.
Purpose of the Study:
- To generate a specific antibody for rainbow trout p53.
- To investigate the expression and DNA damage response of p53 in rainbow trout (Oncorhynchus mykiss).
- To compare rainbow trout p53's response to DNA damage with that of mammals.
Main Methods:
- Generation of a custom polyclonal antibody against rainbow trout p53.
- Western blot analysis of p53 protein levels in various rainbow trout tissues and cell lines.
- Exposure of rainbow trout cell lines (RTbrain-W1, RTgill-W1) to DNA damaging agents (bleomycin, hydroxyurea) to assess p53 response.
Main Results:
- High basal levels of p53 were detected in rainbow trout brain and gill tissues.
- Exposure to bleomycin and hydroxyurea did not induce an increase in p53 levels in rainbow trout cell lines.
- This contrasts with the known mammalian response where p53 levels typically rise upon DNA damage.
Conclusions:
- Rainbow trout p53 does not appear to be upregulated by common DNA damaging agents in a manner analogous to mammals.
- These findings suggest the existence of alternative or distinct DNA damage checkpoint mechanisms in rainbow trout.
- Further research is needed to elucidate these alternate pathways in fish.
