Targeting Intracellular Calcium Signaling ([Ca2+]i) to Overcome Acquired Multidrug Resistance of Cancer Cells: A Mini-Overview.

Cancer is a main public health problem all over the world. It affects millions of humans no matter their age, gender, education, or social status. Although chemotherapy is the main strategy for the treatment of cancer, a major problem limiting its success is the intrinsic or acquired drug resistance. Therefore, cancer drug resistance is a major impediment in medical oncology resulting in a failure of a successful cancer treatment. This mini-overview focuses on the interdependent relationship between intracellular calcium ([Ca2+]i) signaling and multidrug resistance of cancer cells, acquired upon treatment of tumors with anticancer drugs. We propose that [Ca2+]i signaling modulates gene expression of multidrug resistant (MDR) genes which in turn can be modulated by epigenetic factors which in turn leads to modified protein expression in drug resistant tumor cells. A precise knowledge of these mechanisms will help to develop new therapeutic strategies for drug resistant tumors and will improve current chemotherapy.

Overcoming chemotherapy drug resistance by targeting inhibitors of apoptosis proteins (IAPs).

Inhibitors of apoptosis (IAPs) are a family of proteins that play a significant role in the control of programmed cell death (PCD). PCD is essential to maintain healthy cell turnover within tissue but also to fight disease or infection. Uninhibited, IAPs can suppress apoptosis and promote cell cycle progression. Therefore, it is unsurprising that cancer cells demonstrate significantly elevated expression levels of IAPs, resulting in improved cell survival, enhanced tumor growth and subsequent metastasis. Therapies to target IAPs in cancer has garnered substantial scientific interest and as resistance to anti-cancer agents becomes more prevalent, targeting IAPs has become an increasingly attractive strategy to re-sensitize cancer cells to chemotherapies, antibody based-therapies and TRAIL therapy. Antagonism strategies to modulate the actions of XIAP, cIAP1/2 and survivin are the central focus of current research and this review highlights advances within this field with particular emphasis upon the development and specificity of second mitochondria-derived activator of caspase (SMAC) mimetics (synthetic analogs of endogenously expressed inhibitors of IAPs SMAC/DIABLO). While we highlight the potential of SMAC mimetics as effective single agent or combinatory therapies to treat cancer we also discuss the likely clinical implications of resistance to SMAC mimetic therapy, occasionally observed in cancer cell lines.

Calcium-regulatory proteins as modulators of chemotherapy in human neuroblastoma.

Neuroblastoma (NB) is a pediatric cancer treated with poly-chemotherapy including platinum complexes (e.g. cisplatin (CDDP), carboplatin), DNA alkylating agents, and topoisomerase I inhibitors (e.g. topotecan (TOPO)). Despite aggressive treatment, NB may become resistant to chemotherapy. We investigated whether CDDP and TOPO treatment of NB cells interacts with the expression and function of proteins involved in regulating calcium signaling. Human neuroblastoma cell lines SH-SY5Y, IMR-32 and NLF were used to investigate the effects of CDDP and TOPO on cell viability, apoptosis, calcium homeostasis, and expression of selected proteins regulating intracellular calcium concentration ([Ca2+]i). In addition, the impact of pharmacological inhibition of [Ca2+]i-regulating proteins on neuroblastoma cell survival was studied. Treatment of neuroblastoma cells with increasing concentrations of CDDP (0.1-10 µM) or TOPO (0.1 nM-1 µM) induced cytotoxicity and increased apoptosis in a concentration- and time-dependent manner. Both drugs increased [Ca2+]i over time. Treatment with CDDP or TOPO also modified mRNA expression of selected genes encoding [Ca2+]i-regulating proteins. Differentially regulated genes included S100A6, ITPR1, ITPR3, RYR1 and RYR3. With FACS and confocal laser scanning microscopy experiments we validated their differential expression at the protein level. Importantly, treatment of neuroblastoma cells with pharmacological modulators of [Ca2+]i-regulating proteins in combination with CDDP or TOPO increased cytotoxicity. Thus, our results confirm an important role of calcium signaling in the response of neuroblastoma cells to chemotherapy and suggest [Ca2+]i modulation as a promising strategy for adjunctive treatment.

MicroRNA-138 promotes acquired alkylator resistance in glioblastoma by targeting the Bcl-2-interacting mediator BIM.

Glioblastoma is the most aggressive brain tumor in adults with a median survival below 12 months in population-based studies. The main reason for tumor recurrence and progression is constitutive or acquired resistance to the standard of care of surgical resection followed by radiotherapy with concomitant and adjuvant temozolomide (TMZ/RT→TMZ). Here, we investigated the role of microRNA (miRNA) alterations as mediators of alkylator resistance in glioblastoma cells. Using microarray-based miRNA expression profiling of parental and TMZ-resistant cultures of three human glioma cell lines, we identified a set of differentially expressed miRNA candidates. From these, we selected miR-138 for further functional analyses as this miRNA was not only upregulated in TMZ-resistant versus parental cells, but also showed increased expression in vivo in recurrent glioblastoma tissue samples after TMZ/RT→TMZ treatment. Transient transfection of miR-138 mimics in glioma cells with low basal miR-138 expression increased glioma cell proliferation. Moreover, miR-138 overexpression increased TMZ resistance in long-term glioblastoma cell lines and glioma initiating cell cultures. The apoptosis regulator BIM was identified as a direct target of miR-138, and its silencing mediated the induced TMZ resistance phenotype. Altered sensitivity to apoptosis played only a minor role in this resistance mechanism. Instead, we identified the induction of autophagy to be regulated downstream of the miR-138/BIM axis and to promote cell survival following TMZ exposure. Our data thus define miR-138 as a glioblastoma cell survival-promoting miRNA associated with resistance to TMZ therapy in vitro and with tumor progression in vivo.

Interferon-ß Modulates the Innate Immune Response against Glioblastoma Initiating Cells.

Immunotherapy targeting glioblastoma initiating cells (GIC) is considered a promising strategy. However, GIC are prone to evade immune response and there is a need for potent adjuvants. IFN-ß might enhance the immune response and here we define its net effect on the innate immunogenicity of GIC. The transcriptomes of GIC treated with IFN-ß and controls were assessed by microarray-based expression profiling for altered expression of immune regulatory genes. Several genes involved in adaptive and innate immune responses were regulated by IFN-ß. We validated these results using reverse transcription (RT)-PCR and flow cytometry for corresponding protein levels. The up-regulation of the NK cell inhibitory molecules HLA-E and MHC class I was balanced by immune stimulating effects including the up-regulation of nectin-2. In 3 out of 5 GIC lines tested we found a net immune stimulating effect of IFN-ß in cytotoxicity assays using NKL cells as effectors. IFN-ß therefore warrants further investigation as an adjuvant for immunotherapy targeting GIC.

miR-23a-3p causes cellular senescence by targeting hyaluronan synthase 2: possible implication for skin aging.

Even though aging and cellular senescence appear to be linked, the biological mechanisms interconnecting these two processes remain to be unravelled. Therefore, microRNA (miRNA/miR) profiles were analyzed ex vivo by means of gene array in fibroblasts isolated from young and old human donors. Expression of several miRNAs was positively correlated with donor age. Among them, miR-23a-3p was shown to target hyaluronan synthase 2 (HAS2). HA is a polysaccharide of the extracellular matrix that critically regulates the phenotype of fibroblasts. Indeed, both aged and senescent fibroblasts showed increased miR-23a-3p expression and secreted significantly lower amounts of HA compared with young and non-senescent fibroblasts. Ectopic overexpression of miR-23a-3p in non-senescent fibroblasts led to decreased HAS2-mediated HA synthesis, upregulation of senescence-associated markers, and decreased proliferation. In addition, siRNA-mediated downregulation of HAS2 and pharmacological inhibition of HA synthesis by 4-methylumbelliferone mimicked the effects of miR-23a-3p. In vivo, miR-23a-3p was upregulated and HAS2 was downregulated in the skin of old mice compared with young mice. Inhibition of HA synthesis by 4-methylumbelliferone in mice reduced dermal hydration and viscoelasticity, thereby mimicking an aged skin phenotype. Taken together, these findings appear to link miR-23a-3p and the HA microenvironment as effector mechanisms in both dermal aging and senescence.

Proteome-wide analysis reveals an age-associated cellular phenotype of in situ aged human fibroblasts.

We analyzed an ex vivo model of in situ aged human dermal fibroblasts, obtained from 15 adult healthy donors from three different age groups using an unbiased quantitative proteome-wide approach applying label-free mass spectrometry. Thereby, we identified 2409 proteins, including 43 proteins with an age-associated abundance change. Most of the differentially abundant proteins have not been described in the context of fibroblasts' aging before, but the deduced biological processes confirmed known hallmarks of aging and led to a consistent picture of eight biological categories involved in fibroblast aging, namely proteostasis, cell cycle and proliferation, development and differentiation, cell death, cell organization and cytoskeleton, response to stress, cell communication and signal transduction, as well as RNA metabolism and translation. The exhaustive analysis of protein and mRNA data revealed that 77 % of the age-associated proteins were not linked to expression changes of the corresponding transcripts. This is in line with an associated miRNA study and led us to the conclusion that most of the age-associated alterations detected at the proteome level are likely caused post-transcriptionally rather than by differential gene expression. In summary, our findings led to the characterization of novel proteins potentially associated with fibroblast aging and revealed that primary cultures of in situ aged fibroblasts are characterized by moderate age-related proteomic changes comprising the multifactorial process of aging.

Cisplatin (CDDP) triggers cell death of MCF-7 cells following disruption of intracellular calcium ([Ca(2+)]i) homeostasis.

Breast cancer (BC) is a public health problem all over the world. Cisplatin (CDDP) is an antineoplastic agent with high rate of success in treating cancers. The down side of CDDP treatment is the development of chemo-resistance. Beside DNA damage and activation of p53 signaling pathway, CDDP induces tumor-cell death due to elevation in the intracellular calcium concentration ([Ca(2+)]i).However, the role of [Ca(2+)]i in CDDP induced apoptosis of breast cancer cells (MCF-7) is not well understood. Here we investigate the cytotoxic effects of CDDP in relation to [Ca(2+)]i homeostasis in MCF-7-sensitive and -resistant cell lines. Live-cell imaging using [Ca(2+)]i sensitive fluorescent dyes was employed to monitor [Ca(2+)]i CDDP treated MCF-7 cells (0.001-10 µM) and [Ca(2+)]i modulators i.e. Caffeine (10 mM); Nimodipine (10 µM); Ionomycin (10 µM); Thapsigargin (500 nM). A concentration-dependent increase of[Ca(2+)]i was observed in CDDP MCF-7 treated cells. From the concentration range tested 100 nM CDDP triggered the highest [Ca(2+)]i increase (120%; n = 19)while in drug resistant MCF-7 cells the effects of CDDP on [Ca(2+)]i were reduced as compared with the drug sensitive MCF-7 cells. Furthermore, the CDDP induced cell death correlates with the increase of [Ca(2+)]i, and thus, significantly lower in the CDDP desensitized cells (p < 0.05). Pre-application of the calcium channel blocker, Nimodipine reduced [Ca(2+)]i elevation significantly (46.6% increase; n = 26) as well as when a pre-application of Caffeine, Ionomycin or Thapsigargin occurred followed by the subsequent application of CDDP (n = 15; 37.8%, n = 32; 34.9%, n = 21; 53.7% increase respectively).

Interferon-ß induces loss of spherogenicity and overcomes therapy resistance of glioblastoma stem cells.

Glioblastoma is the most common malignant brain tumor in adults and characterized by a poor prognosis. Glioma cells expressing O(6)-methylguanine DNA methyltransferase (MGMT) exhibit a higher level of resistance toward alkylating agents, including the standard of care chemotherapeutic agent temozolomide. Here, we demonstrate that long-term glioma cell lines (LTL) as well as glioma-initiating cell lines (GIC) express receptors for the immune modulatory cytokine IFN-ß and respond to IFN-ß with induction of STAT-3 phosphorylation. Exposure to IFN-ß induces a minor loss of viability, but strongly interferes with sphere formation in GIC cultures. Furthermore, IFN-ß sensitizes LTL and GIC to temozolomide and irradiation. RNA interference confirmed that both IFN-ß receptors, R1 and R2, are required for IFN-ß-mediated sensitization, but that sensitization is independent of MGMT or TP53. Most GIC lines are highly temozolomide-resistant, mediated by MGMT expression, but nevertheless susceptible to IFN-ß sensitization. Gene expression profiling following IFN-ß treatment revealed strong upregulation of IFN-ß-associated genes, including a proapoptotic gene cluster, but did not alter stemness-associated expression signatures. Caspase activity and inhibition studies revealed the proapoptotic genes to mediate glioma cell sensitization to exogenous death ligands by IFN-ß, but not to temozolomide or irradiation, indicating distinct pathways of death sensitization mediated by IFN-ß. Thus, IFN-ß is a potential adjunct to glioblastoma treatment that may target the GIC population. IFN-ß operates independently of MGMT-mediated resistance, classical apoptosis-regulatory networks, and stemness-associated gene clusters.

Thymosin ß 4 gene silencing decreases stemness and invasiveness in glioblastoma.

Thymosin beta 4 is a pleiotropic actin-sequestering polypeptide that is involved in wound healing and developmental processes. Thymosin beta 4 gene silencing promotes differentiation of neural stem cells whereas thymosin beta 4 overexpression initiates cortical folding of developing brain hemispheres. A role of thymosin beta 4 in malignant gliomas has not yet been investigated. We analysed thymosin beta 4 staining on tissue microarrays and performed interrogations of the REMBRANDT and the Cancer Genome Atlas databases. We investigated thymosin beta 4 expression in seven established glioma cell lines and seven glioma-initiating cell lines and induced or silenced thymosin beta 4 expression by lentiviral transduction in LNT-229, U87MG and GS-2 cells to study the effects of altered thymosin beta 4 expression on gene expression, growth, clonogenicity, migration, invasion, self-renewal and differentiation capacity in vitro, and tumorigenicity in vivo. Thymosin beta 4 expression increased with grade of malignancy in gliomas. Thymosin beta 4 gene silencing in LNT-229 and U87MG glioma cells inhibited migration and invasion, promoted starvation-induced cell death in vitro and enhanced survival of glioma-bearing mice. Thymosin beta 4 gene silencing in GS-2 cells inhibited self-renewal and promoted differentiation in vitro and decreased tumorigenicity in vivo. Gene expression analysis suggested a thymosin beta 4-dependent regulation of mesenchymal signature genes and modulation of TGFß and p53 signalling networks. We conclude that thymosin beta 4 should be explored as a novel molecular target for anti-glioma therapy.

DNA methylation pyrosequencing assay is applicable for the assessment of epigenetic active environmental or clinical relevant chemicals.

Exposure of cells and organisms to stressors might result in epigenetic changes. Here it is shown that investigation of DNA methylation using pyrosequencing is an alternative for in vitro and in vivo toxicological testing of epigenetic effects induced by chemicals and drugs. An in vitro evaluation of global and CpG site specific DNA methylation upon treatment of cells with chemicals/drugs is shown. Bisulfite genomic sequencing of methylation controls showed high methylation of LINE1 in methylation positive control and low methylation in the negative controls. The CpG sites within the LINE1 element are methylated at different levels. In vitro cell cultures show a methylation level ranging from 56% to 49%. Cultures of drug resistant tumor cells show significant hypomethylation as compared with the originating nonresistant tumor cells. The in vitro testing of epigenetically active chemicals (5-methyl-2'-deoxycytidine and trichostatin A) revealed a significant change of LINE1 methylation status upon treatment, while specific CpG sites were more prone to demethylation than others (focal methylation). In conclusion, DNA methylation using pyrosequencing might be used not only for testing epigenetic toxins/drugs but also in risk assessment of drugs, food, and environmental relevant pollutants.

Distinct molecular mechanisms of acquired resistance to temozolomide in glioblastoma cells.

Temozolomide (TMZ) is an alkylating chemotherapeutic agent that prolongs the survival of patients with glioblastoma. Clinical benefit is more prominent in patients with methylation of the O(6) -methyl-guanine DNA methyltransferase (MGMT) promoter. However, all patients eventually suffer from tumor progression because their tumors become resistant to TMZ. Here, we modeled acquired TMZ resistance in glioma cells in vitro to identify underlying molecular mechanisms. To this end, the glioma cell lines LNT-229, LN-308, and LN-18 were exposed repetitively to increasing concentrations of TMZ to induce a stable resistant phenotype (R) defined by clonogenic survival assays. The molecular mechanisms mediating acquired resistance were assessed by immunoblot, PCR, and flow cytometry. Rescue experiments were performed with siRNA-mediated candidate gene silencing. We found in LN-18 cells constitutively expressing MGMT a strong up-regulation of MGMT levels in TMZ-resistant cells. TMZ resistance in the MGMT-negative cell lines LNT-229 and LN-308 was not associated with de novo expression of MGMT. Instead, we found a down-regulation of several DNA mismatch-repair proteins in resistant LNT-229 cells. A TMZ-resistant phenotype was also achieved by silencing selected DNA mismatch repair proteins in parental LNT-229 cells. No obvious mechanism of resistance was identified in the third cell line, LN-308, except for reduced methylation of LINE-1 repetitive elements. In conclusion, we demonstrate that different molecular mechanisms may contribute to the development of acquired TMZ resistance in glioma cells, indicating the need to develop distinct strategies to overcome resistance.

Metals and breast cancer: risk factors or healing agents?

Metals and metal compounds are part of our environment. Several metals are essential for physiological functions (e.g., zinc or magnesium); while the beneficial effects of others are uncertain (e.g., manganese), some metals are proven to be toxic (e.g., mercury, lead). Additionally there are organic metal compounds; some of them are extremely toxic (e.g., trimethyltin, methylmercury), but there is very little knowledge available how they are handled by organisms. Scientific evidence indicates that long-term exposure to (some) metallic compounds induces different forms of cancer, including breast cancer. On the other side, several metal compounds have clinical use in treating life-threatening diseases such as cancer. In this paper we discuss the recent literature that shows a correlation between metal exposure and breast cancer.

Cisplatin as an anti-tumor drug: cellular mechanisms of activity, drug resistance and induced side effects.

Platinum complexes are clinically used as adjuvant therapy of cancers aiming to induce tumor cell death. Depending on cell type and concentration, cisplatin induces cytotoxicity, e.g., by interference with transcription and/or DNA replication mechanisms. Additionally, cisplatin damages tumors via induction of apoptosis, mediated by the activation of various signal transduction pathways, including calcium signaling, death receptor signaling, and the activation of mitochondrial pathways. Unfortunately, neither cytotoxicity nor apoptosis are exclusively induced in cancer cells, thus, cisplatin might also lead to diverse side-effects such as neuro- and/or renal-toxicity or bone marrow-suppression. Moreover, the binding of cisplatin to proteins and enzymes may modulate its biochemical mechanism of action. While a combination-chemotherapy with cisplatin is a cornerstone for the treatment of multiple cancers, the challenge is that cancer cells could become cisplatin-resistant. Numerous mechanisms of cisplatin resistance were described including changes in cellular uptake, drug efflux, increased detoxification, inhibition of apoptosis and increased DNA repair. To minimize cisplatin resistance, combinatorial therapies were developed and have proven more effective to defeat cancers. Thus, understanding of the biochemical mechanisms triggered by cisplatin in tumor cells may lead to the design of more efficient platinum derivates (or other drugs) and might provide new therapeutic strategies and reduce side effects.

Anti-cancer drugs interfere with intracellular calcium signaling.

(Neuro-)toxicity of metal and metal compounds is frequently highlighted. While specific metals or metal compounds are essential for cellular function, other metals are toxic and/or carcinogens. Metals can trigger accidental cell death in the form of necrosis, or activate programmed cell death in the form of apoptosis. The aim of anti-cancer therapy is induction of apoptosis in tumor cells. Therefore, there is an interesting twist in the toxicity of metals and metal compounds (e.g., arsenic trioxide, cisplatin); since they have a higher specificity to induce apoptosis in cancer cells (possibly due to the high turnover in these cells) they are used to cure some forms of cancer. A body of evidence suggests that second messengers, such as modulations in the intracellular calcium concentration, could be involved in metals induced toxicity as well as in the beneficial effects shown by anti-cancer drugs. Here we review the influence on calcium homeostasis induced by some metallic compounds: cisplatin, arsenic trioxide and trimethyltin chloride.

Binding of PDZ proteins to HPV E6 proteins does neither correlate with epidemiological risk classification nor with the immortalization of foreskin keratinocytes.

There is compelling evidence that high-risk human papillomaviruses (HPV) can cause cervical cancer. Strikingly, HPV16 and 18 account for approximately 70% of all cervical cancers, whereas phylogenetically related types are found at much lower frequencies. Most likely, differences in the activities of the viral E6 and E7 oncoproteins account for the in vivo carcinogenicity. We demonstrate here that E6 proteins from low-risk HPV70 and possibly high-risk HPV82 interact and degrade PDZ proteins hDlg and Magi1 identical to HPV16E6 and HPV18E6. In contrast high-risk HPV66E6 did not bind or degrade hDlg or Magi1. We also show that low-risk HPV70 E6/E7 immortalizes normal human keratinocytes. Together with our previous analysis concerning p53 degradation, this shows that neither binding of E6 to p53, to E6AP, to Magi1 and hDlg, the degradation of hDlg and Magi1, nor immortalization of normal human keratinocytes seems to be a reliable predictor for carcinogenic behavior of HPV in the cervix.

Co-application of arsenic trioxide (As2O3) and cisplatin (CDDP) on human SY-5Y neuroblastoma cells has differential effects on the intracellular calcium concentration ([Ca2+]i) and cytotoxicity.

Arsenic trioxide (As(2)O(3)) and cisplatin (CDDP) are clinically relevant chemotherapeutics which modulate the intracellular calcium concentration ([Ca(2+)](i)) by different mechanisms: As(2)O(3) depletes intracellular calcium stores while CDDP triggers an influx of Ca(2+). We investigate whether co-application of As(2)O(3) and CDDP has an effect on [Ca(2+)](i) homeostasis, resulting in an increase of cytotoxicity/apoptosis in human SY-5Y neuroblastoma cells. Confocal imaging with Ca(2+)-sensitive dye (fluo-4) was used for investigating [Ca(2+)](i) dynamics. The induction of cell death was assayed using Trypan blue exclusion and Hoechst 33347 staining. Application of As(2)O(3) (1microM) or CDDP (1microM) increased [Ca(2+)](i). The largest elevation was observed when the basic [Ca(2+)](i) concentration was low. Both, transient and sustained [Ca(2+)](i)-increases were observed in response to a single application of As(2)O(3) or CDDP. Sustained increase showed clear additive effects when both drugs were co-applied. The magnitude of the [Ca(2+)](i)-increase depends on the order of application; the most pronounced effect occurred when the cells were preincubated with CDDP followed by a co-application with As(2)O(3). The sustained [Ca(2+)](i) elevations resulted in increased cytotoxicity and apoptosis. Therefore, co-treatment with CDDP and As(2)O(3) may be a more effective anti-cancer therapy then either agent alone.

Arsenic trioxide in environmentally and clinically relevant concentrations interacts with calcium homeostasis and induces cell type specific cell death in tumor and non-tumor cells.

While arsenic compounds are known as environmental toxicants (especially in drinking water) and as carcinogens, some arsenic compounds, like arsenic trioxide (As2O3), are clinically used in humans to treat some forms of cancer (e.g. leukemia). Although arsenic compounds have been studied intensively, their interactions with living cells are still not fully elucidated. We have previously proposed that modulation of intracellular calcium ([Ca2+]i) homeostasis induced by As2O3 could be an important mechanism to induce cytotoxicity. Here we demonstrate, using human cell models (neuroblastoma (SY-5Y) or embryonic kidney cells (HEK)) and confocal microscopy in combination with the calcium sensitive dye fluo 4-AM, that As2O3 interferes with calcium signaling at low (environmentally and clinically relevant concentrations of 100 pM to 1 microM). Within this concentration range, As2O3 had cell type specific cytotoxic effects, with neuroblastoma cells being more sensitive to As2O3 than HEK 293. In addition, by staining with Hoechst 33347 and counting micronucleated cells as well as apoptotic nuclei, As2O3 was found to increase the rate of apoptosis and DNA damage, which was also cell type specific. These results indicate that the As2O3-induced cell death could be triggered or mediated by [Ca2+]i signals and suggest that low concentrations of As2O3 are able to interfere with specific physiological processes in diverse cell models.

Arsenic trioxide (As2O3) induced calcium signals and cytotoxicity in two human cell lines: SY-5Y neuroblastoma and 293 embryonic kidney (HEK).

Arsenic trioxide (As(2)O(3)) has anticancer properties; however, its use also leads to neuro-, hepato- or nephro-toxicity, and therefore, it is important to understand the mechanism of As(2)O(3) toxicity. We studied As(2)O(3) influence on intracellular calcium ([Ca(2+)](i)) homeostasis of human neuroblastoma SY-5Y and embryonic kidney cells (HEK 293). We also relate the As(2)O(3) induced [Ca(2+)](i) modifications with cytotoxicity. We used Ca(2+) sensitive dyes (fluo-4 and rhod-2) combined with laser scanning microscopy or fluorescence activated cell sorting to measure Ca(2+) changes during the application of As(2)O(3) and we approach evaluation of cytotoxicity. As(2)O(3) (1 microM) increased [Ca(2+)](i) in SY-5Y and HEK 293 cells. Three forms of [Ca(2+)](i)-elevations were found: (1) steady-state increases, (2) transient [Ca(2+)](i)-elevations and (3) Ca(2+)-spikes. [Ca(2+)](i) modifications were independent from extracellular Ca(2+) but dependent on internal calcium stores. The effect was not reversible. Inositol triphosphate (IP(3)) and ryanodine (Ry) receptors are involved in regulation of signals induced by As(2)O(3). 2-APB and dantrolene significantly reduced the [Ca(2+)](i)-rise (p<0.001, t-test) but did not completely abolish [Ca(2+)](i)-elevation or spiking. This indicates that other Ca(2+) regulating mechanisms are involved. In cytotoxicity tests As(2)O(3) significantly reduced cell viability in both cell types. Staining with Hoechst 33342 showed occurrence of apoptosis and DNA damage. Our data suggest that [Ca(2+)](i) is an important messenger in As(2)O(3) induced cell death.