Remodelling of store-operated Ca²⁺ entry and G0/G1 cell cycle arrest

Abstract

The concentration of intracellular free Ca2+ ([Ca2+]i) plays an essential role in cell cycle progression. Understanding the Ca2+ signalling mechanisms that drive and maintain cell cycle arrest in the quiescent state, or stimulate quiescent cells to re-enter the cell cycle and proliferate, will be crucial for developing therapeutic potentials for highly detrimental diseases such as cancer and aging disorders. The work in this thesis investigates remodelling of the Store-Operated Ca2+ Entry (SOCE) signalling pathway that is associated with cell cycle arrest in the quiescent G0/G1 phase. Serum starvation was used to induce cell cycle arrest in quiescent G0/G1 phase to allow investigation of remodelling of SOCE when cells exit the cell cycle. Cancer HeLa, precancerous NIH 3T3 and immortal non-cancerous hTERT RPE-1 cell lines were used in this comparative study. Serum starvation induced cell cycle arrest in G0/G1 phase in HeLa and NIH 3T3 cells that was accompanied by a marked down regulation in SOCE. In hTERT RPE-1 cells, serum starvation induced a modest down regulation in SOCE that was not accompanied by cell cycle arrest. Further experiments revealed that SOCE downregulation was attributed to changes in expression and localisation of the Ca2+ sensing protein STIM1 and the Ca2+ release-activated channel (CRAC) protein Orai1. Serum was added-back in order to induce arrested cells to re-enter the cell cycle. Cell cycle re-entry was associated with restoration of SOCE, STIM1 and Orai1 expression in NIH 3T3 cells, and the restoration of only Orai1 expression in HeLa cells. By comparing these changes to that of hTERT RPE-1 cells, which were not arrested in G0/G1 cells, it appears that there may be a significant role for cell cycle arrest in quiescent G0/G1 phase in SOCE remodelling. In addition, these results suggest an additional role for Orai1 as a positive regulator of cell cycle progression in HeLa cells. In NIH 3T3 cells, SOCE and its proteins, STIM1 and Orai1, were remodelled with cell cycle arrest in quiescent G0/G1 phase and restored with cell cycle re-entry, indicating that each of STIM1 and Orai1 appears to have a role in SOCE and cell cycle progression. SOCE seems to be coupled to cell proliferation in NIH 3T3 cells but not in HeLa cells. This study provides evidence that there are significant differences in SOCE remodelling with cell cycle arrest in quiescent G0/G1 phase in cancerous and pre-cancerous cells, and identifies potential targets for drug therapy aimed at regulating the cell cycle.