Investigating the effects of human Carbonic Anhydrase 1 expression in mammalian cells

Abstract

Amyotrophic Lateral Sclerosis (ALS) is one of the most common motor neuron diseases with a crude annual incidence rate of ~2 cases per 100,000 in European countries, Japan, United States and Canada. The role of Carbonic Anhydrase 1 (CA1) in ALS pathogenesis is completely unknown. Previous unpublished results from Dr. Jian Liu have shown in the spinal cords of patients with sporadic amyotrophic lateral sclerosis (SALS) there is a significant increased expression of CA1 proteins. The purpose of this study is to examine the effect of CA1 expression in mammalian cells, specifically, whether CA1 expression will affect cellular viability and induce apoptosis. To further understand whether such effect is dependent upon CA1 enzymatic activity, three CA1 mutants (Thr199Val, Glu106Ile and Glu106Gln) were generated using two-step PCR mutagenesis. Also, a fluorescence-based assay using the pH-sensitive fluorophore Pyranine (8-hydroxypyrene-1,3,6-trisulfonic acid) to measure the anhydrase activity was developed. The assay has been able to circumvent the requirement of the specialized equipment by utilizing a sensitive and fast microplate reader and demonstrated that three mutants are enzymatically inactive under the physiologically relevant HCO3- dehydration reaction which has not been tested before by others. The data show that transient expression of CA1 in Human Embryonic Kidney 293 (HEK293), African Green Monkey Kidney Fibroblast (COS7) and Human Breast Adenocarcinoma (MCF7) cell lines did not induce significant changes to the cell viability at 36hrs using the Water Soluble Tetrazolium-8 (WST8) assay. Wild-type CA1 significantly reduced cell viability in HEK293 using a virally transduced inducible stable expression system at 96hrs and 144hrs of protein induction whereas out of the two mutants used only Thr199Val induced significant toxicity at 144hrs. Wild-type CA1 has also been found to protect COS7 cells against doxycycline-induced toxicity at 96hrs and 144hrs of protein induction whereas no protective effect was seen by the mutants. Using flow cytometry analysis the results has shown wild-type CA1 expression significantly increased Caspase-3 activation and its downstream molecule Poly (ADP-Ribose) Polymerase 1 (PARP-1) cleavage at 96hrs whereas Glu106Ile only significantly increased Caspase-3 activation. In conclusion, this study marks the first time where CA1 expression has shown to directly cause significant apoptotic toxicity in HEK293 cells and protect against doxycycline-induced toxicity in COS7 cells. Although the implication of this study in ALS requires further investigation, the results here suggest in healthy cells increased levels of CA1 expression may cause onset of toxicity, whereas when cells undergo stress, increased CA1 expression can be protective to prevent further loss in cell viabilities. Despite numerous previous studies that have examined CA1 as potential diseases marker, these results represent for the first time in understanding the effect of CA1 in mammalian cells.