Optimizing simple calreticulin upregulation strategies in Caenorhabditis elegans

Abstract

Calreticulin (CALR) is a multifunctional calcium-binding protein whose expression levels have been correlated with detection, clinical phase of disease, metastasis, and survival of various types of cancer. Therefore, the study of the regulation of the cellular levels of calreticulin may be important to understand the neoplastic process. Caenorhabditis elegans, which has a CALR ortholog (CRT-1), has been used as a model organism for the characterization of calreticulin, and several conditions promoting the upregulation of crt-1 have been studied and established to understand the molecular control of crt-1 transcription and assess the function of the protein. Here, we propose several modifications of previously published crt-1 upregulation strategies that improve the reproducibility of the assay and allow to achieve higher levels of overexpression. Firstly, the manipulation of synchronized populations of worms instead of mixed-stage animals and the use of solid culture medium in all experimental conditions are proposed. Likewise, we evaluate four new experimental approaches that attempt to promote a higher crt-1 upregulation (short-term exposure to 30 µg/mL tunicamycin at 25 °C, short-term exposure to 7% ethanol at 25 °C, short-term exposure to 30 °C of worms grown at 25 °C, and a long-term exposure to 7% ethanol). Our results not only validate previously published methods, but also point to a new experimental approach that increases previously achieved levels of crt-1 upregulation. More specifically, a 6-hour exposure of synchronized worms grown at 25 °C to 7% ethanol on solid medium promotes almost a 7-fold upregulation of crt-1.