The p.E152K-STIM1 mutation deregulates Ca2+ signaling contributing to chronic pancreatitis.

Since deregulation of intracellular Ca2+ can lead to intracellular trypsin activation, and stromal interaction molecule-1 (STIM1) protein is the main regulator of Ca2+ homeostasis in pancreatic acinar cells, we explored the Ca2+ signaling in 37 STIM1 variants found in three pancreatitis patient cohorts. Extensive functional analysis of one particular variant, p.E152K, identified in three patients, provided a plausible link between dysregulated Ca2+ signaling within pancreatic acinar cells and chronic pancreatitis susceptibility. Specifically, p.E152K, located within the STIM1 EF-hand and sterile α-motif domain, increased the release of Ca2+ from the endoplasmic reticulum in patient-derived fibroblasts and transfected HEK293T cells. This event was mediated by altered STIM1-sarco/endoplasmic reticulum calcium transport ATPase (SERCA) conformational change and enhanced SERCA pump activity leading to increased store-operated Ca2+ entry (SOCE). In pancreatic AR42J cells expressing the p.E152K variant, Ca2+ signaling perturbations correlated with defects in trypsin activation and secretion, and increased cytotoxicity after cholecystokinin stimulation.This article has an associated First Person interview with the first author of the paper.

Bi-allelic loss-of-function OBSCN variants predispose individuals to severe recurrent rhabdomyolysis.

Rhabdomyolysis is the acute breakdown of skeletal myofibres in response to an initiating factor, most commonly toxins and over exertion. A variety of genetic disorders predispose to rhabdomyolysis through different pathogenic mechanisms, particularly in patients with recurrent episodes. However, most cases remain without a genetic diagnosis. Here we present six patients who presented with severe and recurrent rhabdomyolysis, usually with onset in the teenage years; other features included a history of myalgia and muscle cramps. We identified 10 bi-allelic loss-of-function variants in the gene encoding obscurin (OBSCN) predisposing individuals to recurrent rhabdomyolysis. We show reduced expression of OBSCN and loss of obscurin protein in patient muscle. Obscurin is proposed to be involved in sarcoplasmic reticulum function and Ca2+ handling. Patient cultured myoblasts appear more susceptible to starvation as evidenced by a greater decreased in sarcoplasmic reticulum Ca2+ content compared to control myoblasts. This likely reflects a lower efficiency when pumping Ca2+ back into the sarcoplasmic reticulum and/or a decrease in Ca2+ sarcoplasmic reticulum storage ability when metabolism is diminished. OSBCN variants have previously been associated with cardiomyopathies. None of the patients presented with a cardiomyopathy and cardiac examinations were normal in all cases in which cardiac function was assessed. There was also no history of cardiomyopathy in first degree relatives, in particular in any of the carrier parents. This cohort is relatively young, thus follow-up studies and the identification of additional cases with bi-allelic null OBSCN variants will further delineate OBSCN-related disease and the clinical course of disease.

Extracellular Calcium Has Multiple Targets to Control Cell Proliferation.

Calcium channels and the two G-protein coupled receptors sensing extracellular calcium, calcium-sensing receptor (CaSR) and GPRC6a, are the two main means by which extracellular calcium can signal to cells and regulate many cellular processes including cell proliferation, migration and invasion of tumoral cells. Many intracellular signaling pathways are sensitive to cytosolic calcium rises and conversely intracellular signaling pathways can modulate calcium channel expression and activity. Calcium channels are undoubtedly involved in the former while the CaSR and GPRC6a are most likely to interfere with the latter. As for neurotransmitters, calcium ions use plasma membrane channels and GPCR to trigger cytosolic free calcium concentration rises and intracellular signaling and regulatory pathways activation. Calcium sensing GPCR, CaSR and GPRC6a, allow a supplemental degree of control and as for metabotropic receptors, they not only modulate calcium channel expression but they may also control calcium-dependent K+ channels. The multiplicity of intracellular signaling pathways involved, their sensitivity to local and global intracellular calcium increase and to CaSR and GPRC6a stimulation, the presence of membrane signalplex, all this confers the cells the plasticity they need to convert the effects of extracellular calcium into complex physiological responses and therefore determine their fate.

Are Orai1 and Orai3 channels more important than calcium influx for cell proliferation?

Transformed and tumoral cells share the characteristic of being able to proliferate even when external calcium concentration is very low. We have investigated whether Human Embryonic Kidney 293 cells, human hepatoma cell Huh-7 and HeLa cells were able to proliferate when kept 72h in complete culture medium without external calcium. Our data showed that cell proliferation rate was similar over a range of external calcium concentration (2µM to 1.8mM). Incubation in the absence of external calcium for 72h had no significant effect on endoplasmic reticulum (ER) Ca(2+) contents but resulted in a significant decrease in cytosolic free calcium concentration in all 3 cell types. Cell proliferation rates were dependent on Orai1 and Orai3 expression levels in HEK293 and HeLa cells. Silencing Orai1 or Orai3 resulted in a 50% reduction in cell proliferation rate. Flow cytometry analysis showed that Orai3 induced a small but significant increase in cell number in G2/M phase. RO-3306, a cdk-1 inhibitor, induced a 90% arrest in G2/M reversible in less than 15min. Our data showed that progression through G2/M phase after release from RO-3306-induced cell cycle arrest was slower in both Orai1 and Orai3 knock-downs. Overexpressing Orai1, Orai3 and the dominant negative non-permeant mutants E106Q-Orai1 and E81Q-Orai3 induced a 50% increase in cell proliferation rate in HEK293 cells. Our data clearly demonstrated that Orai1 and Orai3 proteins are more important than calcium influx to control cell proliferation in some cell lines and that this process is probably independent of ICRAC and Iarc.

Pivotal role of the ORAI3-STIM2 complex in the control of mitotic death and prostate cancer cell cycle progression.

Prostate cancer (PCa) represents one of the most frequent diagnosed cancer in males worldwide. Due to routine screening tests and the efficiency of available treatments, PCa-related deaths have significantly decreased over the past decades. However, PCa remains a critical threat if detected at a late stage in which, cancer cells would have already detached from the primary tumor to spread and invade other parts of the body. Calcium (Ca2+) channels and their protein regulators are now considered as hallmarks of cancer and some of them have been well examined in PCa. Among these Ca2+ channels, isoform 3 of the ORAI channel family has been shown to regulate the proliferation of PCa cells via the Arachidonic Acid-mediated Ca2+ entry, requiring the involvement of STIM1 (Stromal Interaction Molecule 1). Still, no study has yet demonstrated a role of the "neglected" STIM2 isoform in PCa or if it may interact with ORAI3 to promote an oncogenic behavior. In this study, we demonstrate that ORAI3 and STIM2 are upregulated in human PCa tissues. In old KIMAP (Knock-In Mouse Prostate Adenocarcinoma) mice, ORAI3 and STIM2 mRNA levels were significantly higher than ORAI1 and STIM1. In vitro, we show that ORAI3-STIM2 interact under basal conditions in PC-3 cells. ORAI3 silencing increased Store Operated Ca2+ Entry (SOCE) and induced a significant increase of the cell population in G2/M phase of the cell cycle, consistent with the role of ORAI3 as a negative regulator of SOCE. Higher expression levels of CDK1-Y15/Cyclin B1 were detected and mitotic arrest-related death occurred after ORAI3 silencing, which resulted in activating Bax/Bcl-2-mediated apoptotic pathway and caspase-8 activation and cleavage. STIM2 and ORAI3 expression increased in M phase while STIM1 expression and SOCE amplitude significantly decreased. Taken together, ORAI3 -STIM2 complex allows a successful progression through mitosis of PCa cells by evading mitotic catastrophe.

[Immunodeficiencies and pathologies associated with mutations in STIM/ORAI, a membrane complex in the heart of calcium signalling]. / Immunodéficiences et pathologies associées aux mutations dans STIM/ORAI - Un complexe membranaire au cœur de la signalisation calcique.

Six years ago, STIM1 (stromal interaction molecule 1) was identified as an essential component of store-operated calcium channels and in less than one year teamed up with its first partner ORAI1 in immune cells to reconstitute CRAC (calcium-release activated current) channel function. Since then, STIM1 and ORAI1 have developed an ever increasing social network and to date are now linked to nine families of proteins involved in calcium signalling. As a result of this, STIM1 and ORAI1 are now involved in three separate calcium entry pathways, Icrac, Iarc (arachidonic regulated calcium current) and voltage-dependent channels. Physiopathological roles of STIM1 and ORAI1 were first described in the immunological system but, as main actors at the central node in the calcium signalling network, there are now clear evidences that mutations in genes coding STIM1 or ORAI1 interfere with several other diseases.

Capacitative calcium entry and transient receptor potential canonical 6 expression control human hepatoma cell proliferation.

UNLABELLED: Store-operated calcium entry (SOCE) is the main Ca(2+) influx pathway involved in controlling proliferation of the human hepatoma cell lines Huh-7 and HepG2. However, the molecular nature of the calcium channels involved in this process remains unknown. Huh-7 and HepG2 cells express transient receptor potential canonical 1 (TRPC1) and TRPC6, as well as STIM1 and Orai1, and these 4 channels are the most likely candidates to account for the SOCE in these cells. We generated stable TRPC6-overexpressing or TRPC6-knockdown Huh-7 clones, in which we investigated correlations between the presence of the protein, the rate of cell proliferation, and SOCE amplitude. TRPC6-overexpressing Huh-7 cells proliferated 80% faster than did untransfected cells and their SOCE amplitude was 160% higher. By contrast, proliferation rate was 50% lower and SOCE amplitude 85% lower in TRPC6-knockdown clones than in untransfected cells. OAG (olyl acetyl glycerol)-induced calcium entry was similar in all cells, and small interfering RNA (siRNA) against TRPC1 had no effect on SOCE amplitude, highlighting the relationship among SOCE, TRPC6 and cell proliferation in Huh-7 cells. SOCE amplitude was reduced by STIM1 and Orai1 knockdowns, suggesting possible cooperation between these proteins and TRPC6 in these cells. Endothelial growth factor and hepatocyte growth factor increased TRPC6 expression and SOCE amplitude in Huh-7 cells, and cyclin D1 expression was decreased by STIM1, Orai1, and TRPC6 knockdowns. CONCLUSION: TRPC6 was very weakly expressed in isolated hepatocytes from healthy patients and expressed more strongly in tumoral samples from the liver of a cancer patient, strongly supporting a role for these calcium channels in liver oncogenesis.

Microcalcifications, calcium-sensing receptor, and cancer.

Calcium stones and calculi are observed in numerous human tissues. They are the result of deposition of calcium salts and are due to high local calcium concentrations. Prostatic calculi are usually classified as endogenous or extrinsic stones. Endogenous stones are commonly caused by obstruction of the prostatic ducts around an enlarged prostate resulting from benign prostatic hyperplasia or from chronic inflammation. The latter occurs mainly around the urethra and is generally caused by reflux of urine into the prostate. Calcium concentrations higher than in the plasma at sites of infection may induce the chemotactic response that eventually leads to recruitment of inflammatory cells. The calcium sensing receptor (CaSR) may be crucial for this recruitment as its expression and activity are increased by cytokines such as IL-6 and high extracellular calcium concentrations, respectively. The links between calcium calculi, inflammation, calcium supplementation, and CaSR functions in prostate cancer patients will be discussed in this review.

Differential role of transient receptor potential channels in Ca2+ entry and proliferation of prostate cancer epithelial cells.

One major clinical problem with prostate cancer is the cells' ability to survive and proliferate upon androgen withdrawal. Because Ca2+ is central to growth control, understanding the mechanisms of Ca2+ homeostasis involved in prostate cancer cell proliferation is imperative for new therapeutic strategies. Here, we show that agonist-mediated stimulation of alpha1-adrenergic receptors (alpha1-AR) promotes proliferation of the primary human prostate cancer epithelial (hPCE) cells by inducing store-independent Ca2+ entry and subsequent activation of nuclear factor of activated T cells (NFAT) transcription factor. Such an agonist-induced Ca2+ entry (ACE) relied mostly on transient receptor potential canonical 6 (TRPC6) channels, whose silencing by antisense hybrid depletion decreased both hPCE cell proliferation and ACE. In contrast, ACE and related growth arrest associated with purinergic receptors (P2Y-R) stimulation involved neither TRPC6 nor NFAT. Our findings show that alpha1-AR signaling requires the coupled activation of TRPC6 channels and NFAT to promote proliferation of hPCE cells and thereby suggest TRPC6 as a novel potential therapeutic target.

Do dietary calcium and vitamin D matter in men with prostate cancer?

Active surveillance (AS) is an attractive alternative to immediate treatment for men with low-risk prostate cancer. Thus, the identification of environmental factors that promote the progression of indolent disease towards aggressive stages is critical to optimize clinical management. Epidemiological studies suggest that calcium-rich diets contribute to an increased risk of developing prostate cancer and that vitamin D reduces this risk. However, the potential effect of these nutrients on the progression of early-stage prostate tumours is uncertain, as studies in this setting are scarce and have not provided unambiguous conclusions. By contrast, the results of a preclinical study from our own group demonstrate that a diet high in calcium dose-dependently accelerated the progression of early-stage prostate tumours and that dietary vitamin D prevented this effect. The extent to which the conclusions of preclinical and epidemiological studies support a role for calcium and vitamin D and the relevance of monitoring and adjustment of calcium and/or vitamin D intake in patients on AS require further investigation.

Sarco/endoplasmic reticulum Ca2+-ATPase gene transfer reduces vascular smooth muscle cell proliferation and neointima formation in the rat.

Proliferation of vascular smooth muscle cells (VSMC) is a primary cause of vascular disorders and is associated with major alterations in Ca2+ handling supported by loss of the sarco/endoplasmic reticulum calcium ATPase, SERCA2a. To determine the importance of SERCA2a in neointima formation, we have prevented loss of its expression by adenoviral gene transfer in a model of balloon injury of the rat carotid artery. Two weeks after injury, the intima/media ratio was significantly lower in SERCA2a-infected than in injured noninfected or injured beta-galactosidase-infected carotids (0.29+/-0.04 versus 0.89+/-0.19 and 0.72+/-0.14, respectively; P<0.05), and was comparable to that observed in control carotids (0.21+/-0.03). The pathways leading to proliferation were analyzed in serum-stimulated VSMC. Forced expression of SERCA2a arrested cell cycle at the G1 phase and prevented apoptosis. SERCA2a inhibits proliferation through inactivation of calcineurin (PP2B) and its target transcription factor NFAT (nuclear factor of activated T-cells) resulting in lowering of cyclin D1 and pRb levels. By using NFAT-competing peptide VIVIT, we showed that NFAT activity is strongly required to promote VSMC proliferation. In conclusion, we provide the first evidence that increasing SERCA2a activity inhibits VSMC proliferation and balloon injury-induced neointima formation.

Technical Insights into Highly Sensitive Isolation and Molecular Characterization of Fixed and Live Circulating Tumor Cells for Early Detection of Tumor Invasion.

Circulating Tumor Cells (CTC) and Circulating Tumor Microemboli (CTM) are Circulating Rare Cells (CRC) which herald tumor invasion and are expected to provide an opportunity to improve the management of cancer patients. An unsolved technical issue in the CTC field is how to obtain highly sensitive and unbiased collection of these fragile and heterogeneous cells, in both live and fixed form, for their molecular study when they are extremely rare, particularly at the beginning of the invasion process. We report on a new protocol to enrich from blood live CTC using ISET® (Isolation by SizE of Tumor/Trophoblastic Cells), an open system originally developed for marker-independent isolation of fixed tumor cells. We have assessed the impact of our new enrichment method on live tumor cells antigen expression, cytoskeleton structure, cell viability and ability to expand in culture. We have also explored the ISET® in vitro performance to collect intact fixed and live cancer cells by using spiking analyses with extremely low number of fluorescent cultured cells. We describe results consistently showing the feasibility of isolating fixed and live tumor cells with a Lower Limit of Detection (LLOD) of one cancer cell per 10 mL of blood and a sensitivity at LLOD ranging from 83 to 100%. This very high sensitivity threshold can be maintained when plasma is collected before tumor cells isolation. Finally, we have performed a comparative next generation sequencing (NGS) analysis of tumor cells before and after isolation from blood and culture. We established the feasibility of NGS analysis of single live and fixed tumor cells enriched from blood by our system. This study provides new protocols for detection and characterization of CTC collected from blood at the very early steps of tumor invasion.

Vitamin D3 Prevents Calcium-Induced Progression of Early-Stage Prostate Tumors by Counteracting TRPC6 and Calcium Sensing Receptor Upregulation.

Active surveillance has emerged as an alternative to immediate treatment for men with low-risk prostate cancer. Accordingly, identification of environmental factors that facilitate progression to more aggressive stages is critical for disease prevention. Although calcium-enriched diets have been speculated to increase prostate cancer risk, their impact on early-stage tumors remains unexplored. In this study, we addressed this issue with a large interventional animal study. Mouse models of fully penetrant and slowly evolving prostate tumorigenesis showed that a high calcium diet dramatically accelerated the progression of prostate intraepithelial neoplasia, by promoting cell proliferation, micro-invasion, tissue inflammation, and expression of acknowledged prostate cancer markers. Strikingly, dietary vitamin D prevented these calcium-triggered tumorigenic effects. Expression profiling and in vitro mechanistic studies showed that stimulation of PC-3 cells with extracellular Ca2+ resulted in an increase in cell proliferation rate, store-operated calcium entry (SOCE) amplitude, cationic channel TRPC6, and calcium sensing receptor (CaSR) expression. Notably, administration of the active vitamin D metabolite calcitriol reversed all these effects. Silencing CaSR or TRPC6 expression in calcium-stimulated PC3 cells decreased cell proliferation and SOCE. Overall, our results demonstrate the protective effects of vitamin D supplementation in blocking the progression of early-stage prostate lesions induced by a calcium-rich diet. Cancer Res; 77(2); 355-65. ©2016 AACR.

UNC93B1 interacts with the calcium sensor STIM1 for efficient antigen cross-presentation in dendritic cells.

Dendritic cells (DC) have the unique ability to present exogenous antigens via the major histocompatibility complex class I pathway to stimulate naive CD8+ T cells. In DCs with a non-functional mutation in Unc93b1 (3d mutation), endosomal acidification, phagosomal maturation, antigen degradation, antigen export to the cytosol and the function of the store-operated-Ca2+-entry regulator STIM1 are impaired. These defects result in compromised antigen cross-presentation and anti-tumor responses in 3d-mutated mice. Here, we show that UNC93B1 interacts with the calcium sensor STIM1 in the endoplasmic reticulum, a critical step for STIM1 oligomerization and activation. Expression of a constitutively active STIM1 mutant, which no longer binds UNC93B1, restores antigen degradation and cross-presentation in 3d-mutated DCs. Furthermore, ablation of STIM1 in mouse and human cells leads to a decrease in cross-presentation. Our data indicate that the UNC93B1 and STIM1 cooperation is important for calcium flux and antigen cross-presentation in DCs.

High glucose-induced apoptosis through store-operated calcium entry and calcineurin in human umbilical vein endothelial cells.

Diabetes mellitus causes multiple cardiovascular complications. Previous studies have shown that prolonged exposure (96 h) of human umbilical vein endothelial cells (HUVECs) to hyperglycemia causes a significant increase in apoptosis. We report here that this increase in apoptosis is associated with an increase in Ca(2+) current (whole cell patch-clamp recorded) resulting from Ca(2+) entry mediated by store-operated channels (SOCs). The number of apoptotic cells after prolonged high glucose (HG, 30 mmol/L) exposure was significantly reduced in the presence of the SOC inhibitor 2-APB or of La(3+). A marked increase (approximately 80%) in Ca(2+)-dependent calcineurin (CN-A) phosphatase activity also occurred after prolonged HG exposure. Prolonged HG exposure-induced increase in CN-A activity was prevented by 2-APB, and selective CN-A phosphatase inhibition by FK506 or calmodulin inhibition by calmidazolium decreased HG-induced apoptosis. Blocking hydrogen peroxide production using catalase or inhibiting the tyrosine kinase pp60(src) during prolonged exposure to HG, resulted in a marked decrease in apoptosis and was further associated with a significant reduction in CN-A phosphatase activity. The results demonstrate a significant role for Ca(2+) entry in HG-induced apoptosis in HUVECs, and suggest that this role is mediated via H(2)O(2) generation and the action of the Ca(2+)-activated protein phosphatase calcineurin.

Phosphatidylinositol 3-kinase and calcium-activated transcription pathways are required for VLDL-induced smooth muscle cell proliferation.

Little is known regarding the molecular mechanisms of atherogenicity of triglyceride-rich lipoproteins such as very low-density lipoproteins (VLDLs). We examined the effect of VLDL on proliferation of rat aortic smooth muscle cells, intracellular Ca2+ handling, and activity of cAMP-responsive element binding protein (CREB) and nuclear factor of activated T cells (NFAT) transcription factors. VLDL, isolated from human serum, dose- and time-dependently promoted proliferation. After 4 hours of exposure to VLDL (0.15 g/L proteins), the caffeine-induced Ca2+ release was inhibited and the IP3-sensitive Ca2+ release induced by ATP (10 micromol/L) was markedly prolonged. In quiescent cells, CREB was phosphorylated (pCREB) and NFAT was present in the cytosol, whereas in cells exposed to VLDL for 4 to 24 hours, pCREB disappeared and NFAT was translocated to the nucleus. VLDL-induced NFAT translocation and proliferation were blocked by cyclosporin A and LY294002 involving calcineurin and phosphatidylinositol 3-kinase (PI3K) pathways. Indeed, VLDLs rapidly phosphorylate protein kinase B and glycogen synthase kinase-3beta in a PI3K-dependent way. These results provide the first evidence that VLDLs induce smooth muscle cell proliferation by activating the PI3K pathway and nuclear NFAT translocation. Blockade of the Ca2+-induced Ca2+ release mechanism and dephosphorylation of pCREB contribute but were not sufficient to induce a proliferating phenotype.

Primary-cilium-dependent autophagy controls epithelial cell volume in response to fluid flow.

Autophagy is an adaptation mechanism that is vital for cellular homeostasis in response to various stress conditions. Previous reports indicate that there is a functional interaction between the primary cilium (PC) and autophagy. The PC, a microtubule-based structure present at the surface of numerous cell types, is a mechanical sensor. Here we show that autophagy induced by fluid flow regulates kidney epithelial cell volume in vitro and in vivo. PC ablation blocked autophagy induction and cell-volume regulation. In addition, inhibition of autophagy in ciliated cells impaired the flow-dependent regulation of cell volume. PC-dependent autophagy can be triggered either by mTOR inhibition or a mechanism dependent on the polycystin 2 channel. Only the LKB1-AMPK-mTOR signalling pathway was required for the flow-dependent regulation of cell volume by autophagy. These findings suggest that therapies regulating autophagy should be considered in developing treatments for PC-related diseases.

High milk consumption does not affect prostate tumor progression in two mouse models of benign and neoplastic lesions.

Epidemiological studies that have investigated whether dairy (mainly milk) diets are associated with prostate cancer risk have led to controversial conclusions. In addition, no existing study clearly evaluated the effects of dairy/milk diets on prostate tumor progression, which is clinically highly relevant in view of the millions of men presenting with prostate pathologies worldwide, including benign prostate hyperplasia (BPH) or high-grade prostatic intraepithelial neoplasia (HGPIN). We report here a unique interventional animal study to address this issue. We used two mouse models of fully penetrant genetically-induced prostate tumorigenesis that were investigated at the stages of benign hyperplasia (probasin-Prl mice, Pb-Prl) or pre-cancerous PIN lesions (KIMAP mice). Mice were fed high milk diets (skim or whole) for 15 to 27 weeks of time depending on the kinetics of prostate tumor development in each model. Prostate tumor progression was assessed by tissue histopathology examination, epithelial proliferation, stromal inflammation and fibrosis, tumor invasiveness potency and expression of various tumor markers relevant for each model (c-Fes, Gprc6a, activated Stat5 and p63). Our results show that high milk consumption (either skim or whole) did not promote progression of existing prostate tumors when assessed at early stages of tumorigenesis (hyperplasia and neoplasia). For some parameters, and depending on milk type, milk regimen could even exhibit slight protective effects towards prostate tumor progression by decreasing the expression of tumor-related markers like Ki-67 and Gprc6a. In conclusion, our study suggests that regular milk consumption should not be considered detrimental for patients presenting with early-stage prostate tumors.

The blocking of capacitative calcium entry by 2-aminoethyl diphenylborate (2-APB) and carboxyamidotriazole (CAI) inhibits proliferation in Hep G2 and Huh-7 human hepatoma cells.

Calcium entry is a component of the processes regulating the proliferative phenotype of some types of cancer. In non-excitable cells, capacitative calcium entry (CCE) and non-capacitative calcium entry (NCCE) are thought to be the main pathways of Ca2+ influx into cells. Thus, blocking calcium entry may prevent normal and pathological cell proliferation and there is evidence to suggest that molecules blocking calcium entry also have antiproliferative properties. Carboxyamidotriazole (CAI), a novel inhibitor of the non-voltage-dependent calcium entry has been shown to have such properties in model systems in vitro and in vivo. We used Hep G2 and Huh-7 human hepatoma cells to investigate the effects of calcium entry blockers on cell proliferation. CAI (10 microM) and 2-APB (20 microM) completely blocked CCE in thapsigargin-treated Huh-7, and CAI and 2-APB inhibited cell proliferation with IC50 of 4.5 and 43 microM, respectively. The plateau phase of the [Ca2+]i increases triggered by 10% FCS were abolished in the absence of external Ca2+ and in the presence of CAI or 2-APB. We, therefore, suggest that CCE is the main pathway involved in regulation of the processes leading to cell proliferation.