Correlating Ca2+ Concentrations With Cell Proliferations In Myeloid Cell Lines Using Ca2+ Channel Blockers

Calcium (Ca2+) is a molecule that acts as an intracellular signal which regulates numerous cellular processes such as cell differentiation, proliferation, and apoptosis. Cell proliferation is an intricate procedure organised by numerous proteins related to Ca2+ signalling in different parts of the cell. In resting cells, the Ca2+ concentration in cytosol is fairly low – usually around 100 nM whereas the concentration in the organelles like mitochondria, endoplasmic reticulum and Golgi apparatus is in millimolar range (Patel and Docampo, 2010). In addition, cytosolic concentration can increase in response to numerous stressors and hormones like Inositol-1,4,5-trisphosphate (IP3) that binds to its receptor (IP3R) to elicit the release of Ca2+ from intracellular stores like endoplasmic reticulum into the cytosol (Figure. 1; Resende et al., 2013). Another set of Ca2+ channels called ryanodine receptors (RyR) is also used to release the Ca2+ from endoplasmic and sarcoplasmic reticulum and to convert different external stimuli to intracellular Ca2+ signals. This mechanism of release and entry of Ca2+ gives rise to highly localised Ca2+ signalling and interorganellar exchange whilst ensuring that the changes in Ca2+ concentration is not prolonged or in extreme which could be harmful for the cell (Cunha Xavier Pinto et al., 2015).

Figure 1. When a hormone or a signal binds to transmembrane receptors like tyrosine kinase receptor (RTK) or G-protein coupled receptor (GPR) and activates it, it recruits and activates secondary messengers like phospholipase C (PLC) which produces inositol-1,4,5-trisphosphate (IP3) and diacylglycerol by cleaving phosphatidylinositol 4,5-biphosphate (PIP2). The IP3 binds to its receptor IP3R and activates it which releases Ca2+ from the endoplasmic reticulum. Ca2+ released in the cytosol can participate in different intracellular cascades and further trigger release of Ca2+ by activating another class of Ca2+ channels called ryanodine receptor (RyR).

[bookmark: _Hlk3793247]In non-excitable cells like haematopoietic stem cells the major entry of Ca2+ is through store-operated calcium entries (SOCE) which activates external Ca2+ entry through plasma membranes when intracellular Ca2+ store is reduced. These Ca2+ channels are expressed in cells like lymphocytes, cancer stem cells, breast cancer cells, hepatocytes and mesenchymal cells. Moreover, these channels play a significant role in cell activation, cell proliferation, gene regulation and exocytosis to name a few (Taylor et al., 2008). When agonists like IP3 commence intracellular Ca2+ influx through SOCE in cells like lymphocytes and cancer stem cells, these cells initiate proliferation by allowing re-entry into the cell cycle (Amaya et al., 2013). The nucleus of a cell is separated from the cytosol by nuclear membrane which has pores that allow molecules up to 60 kDa to pass through (Malhas, Goulbourne and Vaux 2011). This allows some of the IP3 triggered Ca2+ molecules in the cytosol to enter the nucleus through diffusion to maintain the equilibrium between the cytosol and the nucleus where Ca2+ can bind directly to DNA structure or regulate transcription factors involved in cell proliferation like NF-κB (Resende et al., 2013).

The cell cycle is mainly controlled by cyclin-dependant protein kinases (CDKs) that only show kinase activity when they’re bound to a cyclin. Four CDKs (CDK 1 in M, CDK 2 in G1 and S, CDK 4 and 6 in G1) play a significant role during cell cycle with their respective cyclins (cyclin A with CDK1/2, cyclin B with CDK1, cyclin E with CDK2, and cyclin D with CDK4/6). Ca2+ controls cell cycle mostly via Ca2+- Calmodulin (CaM) complex where it modulates the expression of CDK1, CDK2 and cyclin B in human T lymphocytes. Ca2+ signalling affects both the early and late G1 phase where Ca2+/CaM complex activates the CDK4/cyclin D1 complex, eliciting a response that promoted G1 transition to S phase (Kahl and Means, 2004). Moreover, Ca2+ also accompanies G1, G2 and spindle assembly checkpoints as they modulate CDK activity.

Differentiation of stem cells has several stages, where eventually new cell types with different characteristics arise (Figure 2). A series of physical and chemical factors can stimulate the differentiation process which include primary chemical messengers, (like growth factors and steroid hormones) and secondary messengers like cyclic nucleotides (Pchelintseva and Djamgoz, 2017). Although commitment, proliferation and migration may occur at any given stage of differentiation, studies show that Ca2+ and pH plays an imperative role in stem cell differentiation and its downstream effects. This occurs especially in progenitor stage where Ca2+ acts as an intracellular coordinator of diverse cytokine-induced effects by activating different proteins like calmodulin-dependant protein kinase (CaMK) and Phospholipase Cγ (PLCγ), Protein kinase C (PKC). These are further involved in the phosphorylation of transcription factors and therefore, in the regulation of expression of responding genes (Paredes-Gamero, Barbosa and Ferreira, 2012).

Figure 2. Differentiation of Stem cells. The undifferentiated cell (unDiC) may either proliferate or give rise to progenitor cells. The progenitor cells then either give rise to populations of progenitor cells or differentiate into DiCs of different lineages (yellow or green) either individually or as a population. Cell to cell interaction and feedback effects may occur at all stages, further refining the whole process (Chen, et al., 2016).

Acute Promyelocytic Leukaemia (APL) is a subtype of acute myelogenous lymphoma (AML) where there is an irregular accumulation of promyelocytes where the cells cannot differentiate past promyelocytic phase of haematopoiesis. It is the first haematological malignancy in which therapeutic approach specifically targeting the underlying molecular lesion has been successfully introduced into clinical practice by all-trans retinoic acid therapy (ATRA).

Taking all the above observation in consideration, the aim of this study is to observe how various levels of Ca2+ concentration elicit different responses in numerous stages of cells especially in those differentiating into progenitor stage, and to see if certain concentration of Ca2+ will elicit such a response where the unDiC stem cells would differentiate directly into DiC cells, skipping the progenitor stage altogether.

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