Aim: Dysfunction of the cardiac ryanodine receptor (RyR2) is an almost ubiquitous finding in animal models of heart failure (HF) and results in abnormal Ca2+ release in cardiomyocytes that contributes to contractile impairment and arrhythmias. We tested whether exercise training (ET), as recommended by current guidelines, had the potential to stabilize RyR2-dependent Ca2+ release in rats with post-myocardial infarction HF.Materials and Methods: We subjected male Wistar rats to left coronary artery ligation or sham operations. After 1 week, animals were characterized by echocardiography and randomized to high-intensity interval ET on treadmills or to sedentary behavior (SED). Running speed was adjusted based on a weekly VO2max test. We repeated echocardiography after 5 weeks of ET and harvested left ventricular cardiomyocytes for analysis of RyR2-dependent systolic and spontaneous Ca2+ release. Phosphoproteins were analyzed by Western blotting, and beta-adrenoceptor density was quantified by radioligand binding.Results: ET increased VO2max in HF-ET rats to 127% of HF-SED (P < 0.05). This coincided with attenuated spontaneous SR Ca2+ release in left ventricular cardiomyocytes from HF-ET but also reduced Ca2+ transient amplitude and slowed Ca2+ reuptake during adrenoceptor activation. However, ventricular diameter and fractional shortening were unaffected by ET. Analysis of Ca2+ homeostasis and major proteins involved in the regulation of SR Ca2+ release and reuptake could not explain the attenuated spontaneous SR Ca2+ release or reduced Ca2+ transient amplitude. Importantly, measurements of beta-adrenoceptors showed a normalization of beta1-adrenoceptor density and beta1:beta2-adrenoceptor ratio in HF-ET.Conclusion: ET increased aerobic capacity in post-myocardial infarction HF rats and stabilized RyR2-dependent Ca2+ release. Our data show that these effects of ET can be gained without major alterations in SR Ca2+ regulatory proteins and indicate that future studies should include upstream parts of the sympathetic signaling pathway. Current guidelines recommend exercise training (ET) as part of rehabilitation programs after myocardial infarction (MI) and for patients with heart failure (HF) , 2. ET h2+ homeostasis (2+ release channel of the sarcoplasmic reticulum (SR)  , 12. Thicoupling . RyR2 dyhythmias . Inhibitstrategy , 16. Howstrategy , 18. HowThis investigation was approved by the Norwegian National Committee for Animal Welfare under the Norwegian Animal Welfare Act (FOTS ID: 4173 and 6577). It conformed to the National Institutes of Health guidelines .2O, 32% O2, and 2.5% isoflurane through an endotracheal tube, as previously described  , respectively. Three days before the first VO2max-test, rats were habituated to the treadmill with daily 15-min exercise bouts at walking pace.ET was initiated 1 week after coronary artery ligation. Separate treadmill and metabolic chambers were used for exercise training and for weekly measurements of maximal oxygen uptake  in rats that were anesthetized by a mixture of 65% N2, 0.4 NaH2PO4, adjusted to pH 7.4 with NaOH), as previously described . The hearts were perfused for 20 min before the left ventricular tissue was excised rapidly, and the infarcted area was removed. The ventricular tissue was then cut into small pieces and was gently mixed with a cutoff Pasteur pipette for about 1 min in a buffer that contained 1% bovine serum albumin (BSA) (Sigma Aldrich) and 0.02 U/ml deoxyribonuclease 1 . The solution with the ventricular tissue was then filtered through a nylon mesh . After sedimentation, the cells were resuspended three times in 1% BSA solutions with increasing Ca2+ concentration . The cells were used for experiments within 10 h.Ventricular cardiomyocytes were isolated using constant-pressure perfusion of the coronary arteries with collagenase-containing solution . Left-ventricular cardiomyocytes were field stimulated at 1, 2, and 4 Hz by a 3-ms symmetrical bipolar pulse, which was ~20% above the voltage threshold for contraction of the individual cardiomyocytes. The experimental superfusate was based on modified HEPES–Tyrode's solution, and contained (in mM) HEPES 5, NaCl 140, KCl 5.4, MgCl2 0.5, glucose 5.5, NaH2PO4 0.4, and CaCl2 1. pH was adjusted to 7.4 with NaOH. Cytosolic Ca2+ was visualized the by use of 5 μM fluo-4 acetomethyl (AM) ester , with 10-min loading before experiments. All experiments were performed at 37°C. For beta-adrenoceptor activation, 10 nM isoprenaline sulfate (ISO)  was added to the modified HEPES–Tyrode's solution. After addition of ISO, the Ca2+ transient amplitude reached steady state after ~1 min. The cardiomyocytes were stimulated for another 30 s before measurements were made of the Ca2+ transients. Ca2+ waves were measured in a 10-s pause after the stimulation period. SR Ca2+ removal rate was estimated as the average rate of decay (k = 1/tau) that was taken from the last three Ca2+ transients before a pause. SR Ca2+ content was measured as peak fluorescence after rapid application of 10 mM caffeine at 1 Hz in the presence and absence of ISO. SERCA2-dependent Ca2+ removal was measured as the difference between the decay rate of Ca2+ transients and the decay rate of SR Ca2+ release after exposure to 10 mM caffeine. Ca2+ removal rate by the sodium–calcium exchanger (NCX) and plasma membrane Ca2+ ATPase (PMCA) was measured as the decay rate of the caffeine-induced Ca2+ release.Whole-cell Ca2+ during pacing was measured as the difference in mean F0 between 1, 2, and 4 Hz for three Ca2+ transients after 30 s stimulation.The relative increase in diastolic Ca2+ transients after 30 s stimulation divided with F for caffeine release.Fractional release was measured as mean F of 3 Ca2+ imaging measurements of Ca2+ waves, but with a 6-s post-stimulation rest period in which Ca2+ sparks were recorded.Confocal microscopy line-scan imaging was performed using a Zeiss LSM 7 Live confocal microscope . Cardiomyocytes were stimulated at 1 and 4 Hz in a protocol that was similar to the one described for whole-cell Ca125I]-(–)iodocyanopincolol (0.066 nM) and the indicated concentration of either CGP20712A or ICI118551 for 2 h at 23°C. Data were fitted to a two-site binding model, and high and low binding affinities (pKi) were calculated in GraphPad Prism 8.0.1 using a Kd of 0.04 nM (affinity of [125I]-(–)iodocyanopincolol was determined in the left ventricular membranes). Beta1-adrenoceptor density was determined as an average of high-affinity CGP20712A and low-affinity ICI118551 binding in the same heart. Similarly, beta2-adrenoceptor density was determined as an average of high-affinity ICI118551 and low-affinity CGP20712A binding in the same heart.To assess beta-adrenoceptor density, a radioligand binding assay was performed on left ventricles snap frozen in liquid nitrogen. Crude cell membrane fractions were prepared as described by Krobert et al. . RadioliWestern blots were performed on total protein homogenates from the left ventricles that had been stored at −70°C. Homogenates were denatured at 100°C for 5 min or at 37°C for 10 min in a sample buffer that contained 50% sucrose, 7.5% sodium dodecyl sulfate (SDS), 62.5 mM Tris–HCl, 2 mM ethylenediaminetetraacetic acid (EDTA), 0.2 M dithiothreitol (DTT), and 0.01% bromophenol blue. Proteins were then fractionated according to size on 4–15% Criterion TGX gels  and blotted on 0.45 μM polyvinylidene fluoride (PVDF) membranes . The examination of phospholamban (Plb) was performed through use of 18% Criterion TGX gels . Membranes were blocked in 5% non-fat milk in Tris-buffered saline with 0.1% polysorbate 20 for 1 h at room temperature. Then, they were incubated with primary antibody overnight at 4°C and with secondary antibody for 1 h at room temperature. Blots were developed by application of enhanced chemiluminescence , and signals were quantified using ImageQuant software .1 adrenergic receptor antibody , and monoclonal antivinculin antibody produced in mouse . Secondary antibodies were antirabbit or antimouse immunoglobulin G (IgG) horseradish peroxidase (HRP)-linked whole antibodies, Cat no. NA934/NA931 , diluted in the ratio 1:5,000. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)  was used as the loading control for all Western blots, except for phosholamban, and for beta1 adrenergic receptor, for which vinculin was used. The membrane was reprobed with the specified antibodies with stripping of the membrane between each antibody when GAPDH was used as the loading control. HeLa Whole-cell lysate  was used as a positive control for Western blots of beta1-adrenoceptor.The primary antibodies for protein detection were anti-RYR2 phosphoserine-2814 , anti-RYR2 phosphoserine-2808 , ryanodine receptor , anti-CaMKII (phospho-threonine286) , CaMKIIδ , anti-pht-test was used to compare the effect of exercise training on VO2max at baseline and after 5 weeks within each group. Unpaired nested ANOVA was used to compare ET and SED for all results from whole-cell Ca2+ imaging. In addition, two-way ANOVA was used to compare difference between ET and SED in the presence or absence of ISO at all frequencies for Ca2+ waves, Ca2+ transient amplitude, and Ca2+ decay rate. A unpaird Student's t-test was used to compare differences in Ca2+ waves frequency across frequencies and in absense and presence of ISO for HF cardiomyocytes. Ca2+ spark frequency was analyzed by application of a Poisson test to adjust for skewed distribution. One-way ANOVA was used when comparing beta-adrenoceptors. Bonferroni, Holm–Sidak, or Tukey's correction was performed when appropriate. Normal distribution was assessed with Shapiro–Wilk test. All statistics were performed by using IBM SPSS statistics 27, Sigmaplot 12.5 or R software . P < 0.05 was considered statistically significant. Results are reported as the mean ± standard error of mean (SEM).All experiments and analyses were performed by investigators who were blinded to the phenotype and group identity of each cell and animal. Statistical tests were selected based on advice from an external statistical advisor. A paired Student's We performed left coronary artery ligation on 23 male Wistar rats to induce large left ventricular myocardial infarctions. Sham operations were performed on 29 rats. One week after surgery, all rats were examined by echocardiography before randomization to ET or standard sedentary conditions (SED) .P < 0.05) but did not differ from that of the HF-SED rats , while VO2max in the sham-ET group was 119% of that of the sham-SED group .One week after surgery, the aerobic capacity of HF-ET rats was 86% of that of the sham-ET rats , both in the presence and absence of ISO (P < 0.05), and was clearly lower in the HF-ET than in the HF-SED cardiomyocytes ..2+ relea2+ release that was observed after ET, phosphoproteins levels were quantified in HF-ET and HF-SED .To explore other potential explanations for the stabilization of RyR2-dependent spontaneous Cad HF-SED . We focuobserved . The Plb2+ wave frequency even in sham-ET compared to sham-SED both in the presence and absence of ISO when compared across all frequencies with two-way ANOVA -dependent phosphorylation. Although previous studies have clearly shown that ET can exert beneficial effects on SR Ca2+ handling through attenuated phosphorylation by CaMKII especially, our results show that other mechanisms may also contribute to a similar result. Interestingly, we observed that Ca2+ transient amplitude and reuptake of Ca2+ was lower in HF-ET rats during adrenergic stress. It was therefore somewhat surprising that this coincided with increased beta1-adrenoceptor density compared to HF-SED. Our radioligand binding experiments show that the number of beta1-adrenoceptors was reduced in HF rats and increased to normal levels by ET  in our analysis, and such effects should therefore not be excluded. The reader should be aware that a one-sided test of CaMKII-dependent phosphorylation of RyR2 would have resulted in a P < 0.05. Based on previous data, the a priori assumption that ET would decrease (not increase) CaMKII activity could be claimed, thus supporting a one-sided test. Furthermore, our analyses of phosphoproteins were only performed in the absence of sympathetic stimulation. Analysis performed after f.ex. exposure to ISO could potentially have clarified some of the non-significant observations.We investigated CaAnother potentially contributing factor that should be included in future studies is altered cell ultrastructure. ET can effect cell size and has been shown to alter t-tubule organization . These ain vivo arrhythmias. This limits the potential for extrapolation to propensity for arrhythmias in vivo. However, other mechanisms are also important for antiarrhythmic effects of ET, such as alterations in repolarization reserve (Our study is limited to cellular phenomena and does not include measurements of  reserve  or reduc reserve , althoug reserve . As thesOur model of HF is based on induction of large MIs, which leads to clear mechanical and structural alterations in the left ventricle at an early time point after the MI. Any extrapolation to HF that develops after smaller MIs, which involves less remodeling, is unclear. Especially, we did not separate cardiomyocytes according to relative localization or distance from the MI, although the effects of ET could vary between regions , 47.We chose to start high-intensity ET at an early time point after MI. High-intensity ET was chosen based on previous studies in rats . Moderat2+ release in post-MI HF. Our data indicate that the protective mechanism involves regulation of both SR Ca2+ release and reuptake, and that effects of ET at several levels of the sympathetic signaling pathways controlling SR Ca2+ release should be explored in future studies.ET can stabilize RyR2-dependent CaThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Norwegian National Committee for Animal Welfare.TD, MS, RM, JA, MF, MH, KA, KH, and IS conceived and designed the analysis, collected data, contributed to data analysis, performed the analysis, and wrote the paper. FL, WL, OS, and MS conceived and designed the analysis, contributed to data analysis, and wrote the paper. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Several selective and non-selective Ca2+-permeable ion channels are implicated in mediating Ca2+ signaling in cancer cells. In this review, we are focusing on TRPC1, a member of the TRP protein superfamily and a potential modulator of store-operated Ca2+ entry (SOCE) pathways. While TRPC1 is ubiquitously expressed in most tissues, its dysregulated activity may contribute to the hallmarks of various types of cancers, including breast cancer, pancreatic cancer, glioblastoma multiforme, lung cancer, hepatic cancer, multiple myeloma, and thyroid cancer. A range of pharmacological and genetic tools have been developed to address the functional role of TRPC1 in cancer. Interestingly, the unique role of TRPC1 has elevated this channel as a promising target for modulation both in terms of pharmacological inhibition leading to suppression of tumor growth and metastasis, as well as for agonistic strategies eliciting Ca2+ overload and cell death in aggressive metastatic tumor cells.Calcium ions (Ca Changes in intracellular Ca2+ concentration include fast processes that require milliseconds of intracellular Ca2+ spikes necessary for exocytosis and muscle contraction, to processes requiring minutes to hours of Ca2+ flux that affect cellular proliferation, cell cycle control, migration, gene expression, and cell death [2+-dependent signaling pathways are involved in augmenting tumor proliferation, differentiation, migration, invasion, metastasis, and apoptosis, thus tumor cells often exhibit increased expression of Ca2+ regulatory networks [2+ signaling, it is not surprising that cells tightly and precisely regulate proteins handling Ca2+ signals, including receptors, channels, and transporters [2+ homeostasis includes transient Ca2+ release from intracellular stores  as well as more sustained influx of extracellular Ca2+ [2+ entry pathway (SOCE) is a major Ca2+ entry pathway in non-excitable cells and SOCE activity is known to modulate insensitivity to antigrowth signals via multiple routes, as reviewed by Prevarskaya et al. [2+ levels, and deciphering the role and interplay of individual members in facilitating tumor progression remains challenging. There is a great deal of controversy surrounding the role of TRPC1; while some reports suggest it is an ion channel, other reports suggest that TRPC1 alone is not sufficient to form an ion channel and functions as a modulator of other TRPC channels. Additionally, the expression of TRPC1 as a prognostic marker in cancer appears to be context specific as TRPC1 expression has been reported to be associated with poor clinical outcomes for certain types of cancers, while in other indications it is reported to be associated with improved outcomes. This review will focus on the role of TRPC1 expression as a determinant of SOCE and cancer progression.Calcium is a ubiquitous second messenger that is known to regulate a myriad of physiological cellular functions in normal cells . Levels ll death . Ion chanetworks ,4. Moreosporters . Regulatlar Ca2+ . The stoDrosophila melanogaster by studying photo-transduction [Drosophila TRP and were initially classified into three subfamilies TRP-Canonical, TRP-Vanilloid, and TRP-Melastatin  [2+-activated monovalent-selective cation channels [2+ to varying degrees [The transient receptor potential (TRP) ion channels were first discovered in sduction . The TRPctively) . Later, ctively) . Except channels ,11, TRP  degrees . TRP cha degrees .2+ stores through ryanodine receptors (RyRs) or inositol 1,4,5-trisphosphate receptors (IP3R) [2) into IP3 and diacylglycerol (DAG). The depletion of Ca2+ stores from the ER is sensed by the transmembrane protein stromal interaction molecules (STIM1 and STIM2), as Ca2+ dissociates from the EF domain of STIM1 and/or STIM2 [2+-selective CRAC channels that allow for both cytosolic Ca2+ signaling and replenishing of ER stores [In addition to TRP channels, the SOCE mechanism of action is dependent on the depletion of the endoplasmic reticulum (ER) Cas (IP3R) ,14. SOCEs (IP3R) . This reor STIM2 . STIM moR stores . AdditioR stores ,17,18,192+, Na+, and K+ [2+ selective Icrac currents [2+ entry  [2+-permeable channel by itself, but is essential in forming heteromers with all other members of the TRPC subfamily [2+ permeability in heteromeric complexes containing TRPC1, but the effect of TRPC1 in Ca2+ entry and as a member of the SOCE pathway is tissue dependent.The TRPC subfamily consists of seven members (TRPC1-7), and they are known to function as non-selective cation channels, with permeability to Ca, and K+ . The rol, and K+ . Howevercurrents ,23,24,25currents . This ex2+ entry B [27,28,5-TRPC5) . Howeverubfamily . Further2+ signaling through the SOCE pathway involving STIM1 and ORAI1 has been reported previously to affect the prognosis of cervical, colorectal, breast, esophageal, multiple myeloma, and lung cancers by affecting tumor growth, proliferation, metastasis, and survival [The role of Casurvival ,32,33,342+ signaling modulates, including kinases, phosphatases, proteases, and metabolic enzymes, the role of Ca2+ signaling in the progression of cancer is likely multi-factorial. However, one attractive Ca2+-dependent candidate pathway is the activation of NFAT , Bucher et al. reported that NFAT activity is chronically elevated in tumor cells; a finding that correlated with elevated Ca2+ levels. In addition, inhibition of calcineurin with cyclosporin A or FK506 treatment reduced NFAT target genes including EGR2, IL10, NFKB1A, and Jun, and induced cell death in DLCBCL cell lines [Considering the numerous pathways that Cay Putney ). Increaressions . Ca2+ bill lines . Similarll lines . The autll lines   and its translocation to the plasma membrane. Further, TGFβ activated PKCα-dependent cellular motility and migration, by inhibiting the tumor suppressor PTEN [2+ entry [3+ abrogated the TGFβ-dependent increase in cytosolic Ca2+ levels. Blocking PKCα by the selective PKCα inhibitor Gö-6976 also inhibited TGFβ-induced Ca2+ entry [Epithelial-mesenchymal transition (EMT) is a known modulator and a key step into tumor invasion and metastasis. One of the key modulators of EMT is TGF-β, which has been reported previously to induce EMT in mammary epithelial cells . The rolignaling ,56. In S2+ entry . Pharmac2+ entry . Further2+ entry .2+ flux was associated with ORAI1 downregulation, while TRPC1 expression was not altered [2+ levels, but increased SOCE activity was noted by depleting intracellular stores using sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) inhibitor cyclopiazonic acid [It has previously been reported that epithelial–mesenchymal transition (EMT) is associated with upregulation of SOCE via increased levels of STIM1 and ORAI1 in MCF7 and MDA-MB-231 cell lines, promoting invasion and proliferation of breast cancer cells . However altered . Interesnic acid . Reducinnic acid .2+-sensing receptor (CaR) by extracellular Ca2+ or its agonist spermine [2+-mediated CaR activation in MCF7 cells results in activation of PLC and PKC [2+-activated K+ channels (KCa3.1) [MCF7 breast cancer cells proliferate in response to activation of the Caspermine . El Hian and PKC . They fu and PKC . Cell cy(KCa3.1) ,55. Inte(KCa3.1) . In huma(KCa3.1) . Taken t2+ influx and proliferation as well as the formation of multinucleated cells, a characteristic of GBM [2+ channel involved in SOCE, its function was blocked using a polyclonal-TRPC1 antibody, which reduced the Ca2+ entry by 25%, whereas no effect was seen with TRPC5 inhibition [2+ entry was significantly decreased following the reduction of TRPC1 expression using shRNA [In D54MG cells, a model for malignant gliomas or glioblastoma multiforme (GBM), the pharmacological inhibition of SOCE with 2-APB, SKF96365, and MRS1845 significantly reduced both Cac of GBM . To defihibition . Similarng shRNA . The knong shRNA .2+ channel, as TRPC channels contain a caveolin-1 binding domain [2+ signaling, the use of SOCE inhibitors MRS1845 and SKF96365 disrupted glioma migration [It has been reported that lipid rafts microdomains (LRDs) and plasma membrane caveolin-1 are critical for TRPC1 insertion into the plasma membrane and its activity as a Cag domain ,70,71. Ig domain . Consistigration . Evidencigration .3 receptor inhibitor and SOCE modulator 2-APB, or the specific TRPC1 antibody T1E3, inhibited proliferation of A549 cells [Lung cancer is the leading cause of death from cancer in men and women and thus clinical data continue to indicate the need for new treatment strategies to improve patient outcomes . Levels 2+ levels in A549 cells related to constitutive SOCE activity. Downregulating HIF-1α was associated with low expression of ORAI1, TRPC1, and TRPC6, and resulted in decreased proliferation of A549 cells. Silencing TRPC1 decreased hypoxia-induced autophagy [2+ influx through the SOCE pathway [As mentioned earlier, TRPC1 levels in breast cancer were dependent on HIF-1α following hypoxia-induced EMT. Wang et al. reported similar results in lung cancer by exposing A549 cells to high nicotine levels, which in turn resulted in increased HIF-1α levels , leadinutophagy . Autophautophagy , albeit  pathway .2+ levels have been shown to be higher in HT29 colon carcinoma cell lines with higher increase in cytosolic Ca2+ in response to agonists like ATP and carbachol, compared to the normal human mucosa cell line NCM460 [2+-activated K+ channels (SK3), ORAI1, and TRPC1 in the lipid rafts [In colon cancer, the role of SOCE members, mainly TRPC1 and ORAI1, has been discussed thoroughly by Villalobos et al. . Basal Ce NCM460 . Althougid rafts . Further2+ signaling to support proliferation, invasion, and metastasis, whereas excessive Ca2+ levels lead to apoptosis and cell death; both the suppression and enhancement of the SOCE pathways may be exploited for treatment of cancer. In triple-negative breast cancer (TNBC), Grant et al. reported that TRPC1 and TRPC4 were overexpressed in some TNBC cell lines. For example, the Hs578T TNBC cell line showed high expression levels of TRPC1 and TRPC4 compared to the MD-MB-231 TNBC cell line, and the BT-549 TNBC cell line had high levels of TRPC4 when compared to MD-MB-231 cell line [2+ overload and cell death through channel agonists, rather than pharmacological inhibition to reverse hallmarks of cancer.Cancer requires robust Caell line . The hig2+ overload being a vulnerability for cancer is that TNBC cell lines with increased TRPC4 and TRPC5 showed increased sensitivity to the TRPC4 and TRPC5 activator englerin A (EA) compared to cell lines with reduced expression [2+-dependent cell death, indicating the importance of the heteromeric formation of TRPC members. Although TRPC1 levels in BT-549 cells were comparable to the EA-resistant cell line HCC1806, BT-549 cells showed increased sensitivity to EA-induced Ca2+ entry and cell death when compared to Hs578T, when TRPC4 alone is predominantly and highly expressed. A possible explanation for this is the shift of the heteromeric TRPC1/TRPC4 channel towards more Na+ flux [2+ levels and followed by cell death The Ca2+ entry was due to activation of TRPC4 channels, as was demonstrated by patch-clamp studies. Further, while the expression of only TRPC1 did not contribute as a Ca2+ channel to the englerin A-induced Ca2+ overload and cell death, the co-expression of TRPC1 and TRPC4 and their heteromeric formation reproduced the same Ca2+ entry currents in HEK293 cells [Support of Capression . InteresNa+ flux . In rena93 cells .2+ levels in multiple myeloma cells lines [3 receptor, SERCA pump, PLCβ, TRPC1, and TRPM7. Treatment with the pharmacological inhibitor 2-ABP attenuated MTI-101-induced Ca2+ influx, a finding that correlated with decreased cell death. U266 and MM.1S myeloma cells with reduced expression of TRPC1 using shRNA strategies showed a reduction in MTI-101-induced cell death [2+ entry and whether MTI-101-induced activity is dependent on activation of TRPC1 heteromers and regulation of the SOCE pathway, thereby allowing for sustained Ca2+ influx and leading to caspase-independent cell death [2+ overload may be a unique vulnerability to cancer cells, as experimental evidence indicates that cancer cells remodel Ca2+ handling pathways to favor the Ca2+ influx required to facilitate the hallmarks of cancer.These findings corroborate the importance of TRPC1 expression for the overall functionality of the SOCE pathway. Our laboratory has shown that the novel cyclic peptide referred to as MTI-101 induces a robust and sustained increase in intracellular Cals lines ,79. Emerll death . Further2+ signals generated by STIM1-ORAI1 activation [As outlined above, several TRPC members have been studied and implicated in the promotion of cancer through tumor cell proliferation, migration, invasion, and survival, as observed in lung cancer, malignant glioma, neuroblastoma, renal cell carcinoma, hepatoma, thyroid cancer, colon cancer, and breast cancer ,57,80,81tivation . Thus, mExperimental evidence indicates that although TRPC1 may not represent a driver of cancer, the expression of TRPC1 contributes to the hallmarks of cancer. To further the understanding of TRPC1 functionality in vivo, various TRPC knockout mice were created and used in different settings. A Hepta-KO cell line was created by combining cell lines with 5 TRPC knockout alleles, with TRPC2 KO, along with TRPC4 knockout, formulating a null cell line devoid of all seven TRPC members. Surprisingly, the Hepta-KO model has a functional SOCE system regardless of TRPC expression . These d


Our results showed that TET2, EZH2, MAD2 and CDC20 were aberrantly expressed in AML patients. The expression level of MAD2 or CDC20 was positively correlated with that of TET2 or EZH2. Hypermethylation of the TET2 gene down-regulated its transcription. Down-regulation of EZH2 or TET2 expression inhibited apoptosis, affected MAD2 and CDC20 expression, and promoted CIN in AML cells. Decitabine treatment restored TET2 methylation and EZH2 transcription and ameliorated CIN in AML. Therefore, TET2 and EZH2 play a tumor-inhibiting role in AML that affects CIN via MAD2 and CDC20.TET2 and EZH2 play important roles in the epigenetic regulation in many cancers. However, their specific roles in acute myeloid leukemia (AML) pathogenesis remain unknown. Here, the expression, methylation or mutation of EZH2 and TET2 was determined and further correlated with the levels of the chromosome instability (CIN) genes MAD2 and CDC20. We down-regulated EZH2 and TET2 in AML cell lines and assessed the effect on CIN using fluorescence  Methylation at carbon atom 5 of the nucleotide cytosine (5-methylcytosine or 5-mC) usually induces inhibition of gene expression. Ten-eleven translocation methylcytosine dioxygenase 2 (TET2) is an evolutionarily conserved dioxygenase that can regulate gene transcription through DNA demethylation by catalyzing the conversion of 5-mC to 5-hydroxymethyl-cytosine (5-hmC)5. TET2 mutations are frequently acquired during the progression of myeloproliferative neoplasms (MPN) or myelodysplastic syndromes (MDS) to AML6. It was reported that TET2 down-regulation, independent of its mutational status, was correlated with poor survival in MDS or AML patients7. Moreover, a lower level of TET2 methylation can define a subgroup of AML that is highly curable and cannot be identified solely by genetic and cytogenetic analyses8. However, few studies regarding the correlation between the expression and the methylation status of TET2 in AML are reported.AML comprises a heterogeneous group of malignancies in which immature and dysfunctional hematopoietic progenitors proliferate and accumulate in the bone marrow. Epigenetic modifications, including DNA methylation and histone methylation, have been shown to play a key role in the pathophysiology of AML9. Somatic EZH2 mutations or amplifications are observed in many cancers, including prostate cancer, colon carcinoma and non-Hodgkin lymphoma. Gain-of-function somatic mutations of EZH2 (Tyr641) are frequently found in patients with follicular lymphoma and germinal center diffuse large B-cell lymphoma10. Overexpression of EZH2 has also been shown to play a critical role in various B cell tumors11. On the other hand, EZH2 inactivation in myeloid malignancies such as MDS or MDS/MPN is associated with poor prognosis and contributes to disease pathogenesis12. A recent study also showed that loss of EZH2 function induced multidrug resistance in AML13. However, the specific role of EZH2 in AML remains unclear.Enhancer of zeste homolog 2 (EZH2), another epigenetic factor, is the catalytic component of polycomb repressive complex 2 (PRC2). EZH2 plays a controversial role in cancer development, acting as either an oncogene or a tumor suppressor depending on the type of cancer. Frequent mutations in the region encoding the catalytic SET domain of EZH2, such as the EZH2 (Tyr646) somatic mutation in humans or EZH2 (Tyr641) somatic mutation in mice, have been reported13. Either elevated or reduced levels of MAD2 can cause abnormal mitotic checkpoint function in mice, resulting in CIN and tumor progression. Moreover, cell division cycle protein 20 homologue (CDC20), another mitotic checkpoint protein, is very important for chromosome segregation and spindle assembly. CDC20 regulates the anaphase-promoting complex or cyclosome (APC/C) ubiquitinase activity on its substrates and subsequent degradation of these proteins by proteasomes14. CIN promotes tumor development primarily through the aberrant expression of the mitotic checkpoint proteins MAD2 and CDC20. However, the specific roles of the CIN genes MAD2 and CDC20 in the pathogenesis of AML have not yet been identified.The epigenetic factors TET2 and EZH2 can catalyze histone modification to alter chromatin structure and modulate gene expression. Therefore, deregulation of histone modification and acquisition of chromosome instability (CIN) due to abnormal expression, methylation or mutation of TET2 and EZH2 are crucial for AML development. CIN refers to the inability of cells to maintain correct chromosome complements after mitosis, resulting in aneuploidy. CIN usually arises at the mitotic checkpoint, and a key mediator of this pathway is the mitotic checkpoint protein MAD2. The expression level of MAD2 has been found to be significantly altered in many cancersTo investigate the roles of the epigenetic/CIN/mitosis axis in AML, we determined the expression, methylation or mutation of TET2, EZH2, MAD2 and CDC20 and explored their clinical relevance in AML patients. Moreover, we down-regulated the epigenetic factors TET2 and EZH2 to further explore their therapeutic potential in AML.A total of 115 subjects were recruited in this study, comprising 58 AML patients who were newly diagnosed (ND) and 57 patients who were complete remission (CR), at Qilu Hospital of Shandong University . The individual characteristics of these AML patients are shown in Supplementary Table 2O to make a final volume of 10 μL. The PCR primer sequences for the relevant genes are shown in Table 15.Total RNA was abstracted from all samples using TRIzol reagent  according to the producer’s protocol. Then, cDNA was synthesized from approximately 1 µg of total RNA using a PrimeScript RT Reagent Kit . Reverse transcription was operated at 37 °C for 15 min followed by 85 °C for 5 s. Quantitative real-time PCR (qPCR) was performed using a LightCycler 480 II Real-time PCR system . The PCR mixture contained 5 μL of 2× SYBR Green Real-time PCR Master Mix, 1 μL of cDNA, 0.8 μL each of the forward and reverse primers, and 3.2 μL of ddH16.Genomic DNA was isolated using a DNA Extraction Kit  and was then chemically modified using an EZ DNA Methylation-Gold Kit  according to the manufacturer’s instructions. The methylation status of the TET2 promoter was determined by methylation-specific PCR (MSP-PCR). As shown in Table 2O, 1 µL of purified PCR product (10 ng/µL), 4 µL of BigDye (2.5×), 2 µL of BigDye Seq Buffer (5×) and 1 µL each of the forward and reverse primers to make up a final volume of 20 μL. The primer sequences are shown in Table 17.Genomic DNA isolated from AML samples was used to amplify the mutated hotspots in EZH2 (E641 and Y646 on exon 16). The PCR mixture contained 12 µL of ddH2. Demethylation was performed using decitabine . AML cells were seeded at a density of 1 × 106 cells/mL in 6-well culture plates and treated with decitabine for 48 h. Then, cells were harvested for subsequent analysis.The AML cell lines HL-60, THP1 and U937 were cultured in RPMI 1640 medium  with 10% fetal bovine serum. The cells were incubated at 37 °C in humidified air containing 5% COTET2-siRNA, EZH2-siRNA and the respective controls were purchased from GenePharma . The siRNA was transfected into AML cells with Lipofectamine 2000 reagent  or Micropoly transfecter TM cell reagent  according to the manufacturer’s recommendation. The inhibition efficiency of the targeted genes was evaluated using qRT-PCR 24 h after transfection. The siRNA which has the highest knockdown efficiency was used for subsequent functional studies.5 cells were harvested, resuspended in 100 µl of flow cytometry binding buffer, and stained with 5 µL of Annexin V/FITC and 1 µL of PI. Then, the cells were incubated in the dark for 15 min at 4 °C, and 400 µL of binding buffer was added. The cells were immediately analyzed on a Galios flow cytometer 18.Apoptosis was detected using an Annexin V/fluorescein isothiocyanate (FITC) and propidium iodide (PI) apoptosis detection kit . Briefly, after treatment with decitabine or transfection with TET2-siRNA or EZH2-siRNA for 48 h, 2 × 1018. In brief, cells were hypotonically treated, fixed with methanol and acetic acid, and placed dropwise on slides. The fixed cells on the slides were hybridized with fluorescent probes against 5q, 7q, 20q12, +8, −Y, RB1, 1q21, D13S319 and IgH for 16 h. After being stained with DAPI, the cells were observed under a fluorescence microscope for measurement of the fluorescence signals. A total of 400 cells were counted for each sample, and the results were shown as the ratio of positive-signal cells to all counted cells.Hybridization and FISH analysis were performed using standard procedures according to the published recommendationsData are presented as the means ± SDs or the medians (ranges). The statistical significance of differences between two groups was determined using two-sided t tests when the data were normally distributed or using the Mann-Whitney test when the data were non-normally distributed. Statistical analyses were performed using SPSS version 17.0 . Correlations between the epigenetic genes (TET2 or EZH2) and mitotic genes (MAD2 or CDC20) were analyzed using the Spearman correlation test. The value of the correlation coefficient indicates the presence or absence of correlation. P value less than 0.05 indicated statistical significance.All procedures performed in studies involving human participants were in accordance with the ethical standards of the Medical Ethics Committee of Qilu Hospital of Shandong University and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.Informed consent was obtained from all individual participants included in the study.P < 0.0001), EZH2  and CDC20 , and marginally significantly decrease of MAD2  , EZH2 , MAD2  and CDC20  at the CR stage compared with the ND stage  Fig. . In addiage Fig. .Figure 1P = 0.0305) and of MAD2 and CDC20   or between the levels of TET2 and either MAD2 or CDC20 .Moreover, correlations between the relative expression levels of the epigenetic genes and the mitotic genes were investigated. In ND AML patients, we observed a significantly positive correlation between the levels of EZH2 and CDC20  Fig. . HoweverP = 0.068)  in U937 cells  with decitabine, then performed the TET2 methylation analysis. Our data showed that the demethylation effect of decitabine is more apparent in U937 cells compared with HL60 and THP1 Fig. . We alsolls Fig. . These flls Fig. .Figure 3P < 0.001), THP1  and HL-60  cells were observed after transfection  and THP1  was decreased significantly by siRNA. Our results obtained via the cell counting method that proliferation was inhibited in U937 cells more apparently than in THP1 or HL-60 cells . Significant decreases in the EZH2 level in U937 . Moreover, markedly decreased expression of MAD2  was found. However, no significant decrease in CDC20  was found in EZH2-siRNA cells compared to control cells . However, no significant increase was found in the expression level of MAD2  or CDC20  in TET2-knockdown cells  or EZH2  in TET2- or EZH2-siRNA U937 cells compared with decitabine-untreated control cells. However, decitabine reduced MAD2 expression in TET2-siRNA U937 cells and did not significantly affect other genes  AML patients but in none of the controls. Moreover, there was a marginal decrease in TET2 mRNA levels in AML patients with TET2 hypermethylation compared to those without TET2 hypermethylation. Our data showed that the hypermethylation status of TET2 may contribute to the decrease in TET2 expression in AML patients. Furthermore, there is rare study about the role of TET2 methylation in tumor prognosis. In our manuscript, because of the short-term follow-up of the AML patients, we have no survival data of these patients and cannot further analyze the correlation between methylation status and prognosis. If possible, we will follow up these patients and do this correlation analysis in the future.21. However, it was also proved that EZH2 as an oncogenic mutation induced lymphoma and melanoma through a vast reorganization of chromatin structure22. EZH2 plays dual roles in malignant diseases. To investigate the specific role of EZH2 in AML, we examined samples from 68 newly diagnosed patients with AML. However, neither of the mutated hotspots in EZH2 exon 16 was found in any of these patients. A significant increase was found in the expression of EZH2 at CR stage compared with that at ND stage, suggesting the mitigation of dys-regulated gene expression after chemotherapy.Suppression of EZH2 expression was reported to induce chemoresistance in AML cell lines and primary cells. Decreased EZH2 levels can result in derepression of the HOX gene, and inhibiting expression of HOXB7 and HOXA9 in resistant cells could enhance cellular sensitivity to tyrosine kinase inhibitors and other chemotherapeutic drugs. These suggested that restoration of EZH2 was a potential method to overcome chemoresistance in AML24, and studies in animals indicated that either high or low MAD2 levels could induce aberrant mitotic checkpoint functions, resulting in CIN and tumor progression26. MAD2 primarily senses the connection between microtubules and the centromere. During mitosis, MAD2 recruits CDC20 to form the MAD2-CDC20 complex, which accumulates and thus inhibits APC/C activity to ensure normal mitosis. Thus, either low or high expression of MAD2 or CDC20 can lead to aneuploidy and even tumor formation. In the present study, we observed a positive relationship between the EZH2 and MAD2/CDC20 levels. In U937 cells, interestingly, TET2 expression increased in response to EZH2 knockdown, while the EZH2 level decreased with knockdown of TET2. This interesting phenomenon may be due to the dual function of EZH2. On the other hand, we considered that TET2 may be upstream of EZH2 and that negative feedback from EZH2 expression leads to the increased TET2 mRNA expression. This observation indicates that the balance of TET2 and EZH2 mRNA expression is important for the proliferation and apoptosis of AML cells. Further research is needed to explore the mechanism.In the process of mitosis, the duplicated chromosomes are split and evenly scattered to progeny cells leading by the spindle. Many researches indicated a mitotic checkpoint which inhibits chromosome segregation until all of the chromosomes are appropriately affixed to microtubules by kinetochores. Many kinetochore-localized proteins, including MAD2 and CDC20, have been involved in this process. Abnormal expression of MAD2 and CDC20 was reported to induce tumor cell aneuploidy in various malignancies. However, MAD2 levels vary significantly in human tumorsMany ongoing studies focused on the CIN inhibitors. The role of CIN in hematological malignancies is currently an attractive topic. Though it is widely accepted that aberrant CIN promotes the proliferation and survival of AML cells, the interactions between epigenetic regulation and CIN are not fully understood in AML. Our results showed that knockdown of either EZH2 or TET2 induced an increase in the level of CIN compared with that in controls in U937 cells. Decitabine treatment of EZH2- or TET2-siRNA-transfected U937 cells decreased the number of abnormally marked positions. These results suggest that EZH2 and TET2 can act antagonistically to regulate cell growth by modulating CIN by influencing MAD2 or CDC20 expression in AML.27.In summary, down-regulation of EZH2 or TET2 affected cell proliferation and influenced MAD2 or CDC20 expression, ultimately ameliorating CIN. Decitabine may partly ameliorate these abnormal changes. Further studies to clarify the specific roles of these mediators in AML are awaited and will ultimately provide a perspective for clinical treatmentDataset 1.


Myeloid disorders are conditions being characterized by abnormal proliferation and development of myeloid lineage including granulocytes , monocytes, erythroids, and megakaryocytes precursor cells. Myeloid leukemia, based on clinical presentation and proliferative rate of neoplastic cells, is divided into acute (AML) and myeloproliferative neoplasms (MPN). The most commonly myeloid leukemia reported in horses are AML-M4 (myelomonocytic) and AML-M5 (monocytic). Isolated cases of AML-M6B (acute erythroid leukemia), and chronic granulocytic leukemia have also been reported. Additionally, bone marrow disorders with dysplastic alterations and ineffective hematopoiesis affecting single or multiple cell lineages or myelodysplastic diseases (MDS), have also been reported in horses. MDSs have increased myeloblasts numbers in blood or bone marrow, although less than 20%, which is the minimum level required for diagnosis of AML. This review performed a detailed description of the current state of knowlegde of the myeloproliferative disorders in horses following the criteria established by the World Health Organization. Leukemia is defined as a progressive malignant disease of blood-forming organs, characterized by abnormal proliferation and development of leukocytes and their precursors in blood and bone marrow. Depending on the proportion of blast cells, cell morphology, and expression of specific antigens in the neoplastic cells, leukemia is divided into lymphocytic and myeloid types, and both of them could be acute and chronic –4. MyeloAML is a clonal expansion of myeloid stem cells, which either minimally, partially, or fully differentiation into a particular myeloid lineage . Often, AML is an aggressive rapidly progressive condition with excessive numbers of neoplastic undifferentiated or blast cells which exceed 20% (formerly 30%) of bone marrow or blood cells. These immature cells are increased in bone marrow and in some cases, they might circulate in blood. Aspirate biopsies or necropsy studies of bone marrow can provide cytologic details of the cell population, which might help to identify cell lineage and dysplastic changes .Myeloproliferative neoplasms  occurs when the neoplastic transformation occurs at a later stage of differentiation, when cells are mature. In MPN, the cells resemble the mature cells of the affected lineage, but they appear increased in numbers in blood or/and bone marrow. Typically, MPN is less aggressive than AML, with slower progression, since that the neoplastic transformation occurs at a later stage of differentiation. However, the division between AML and MPN is not absolute. Some cases share characteristics of both and also some chronic type leukemias could progress to acute disease or "blast cell crisis , 8. In aMDS is a disorder characterized by dysplastic changes  in one or more cell lineages  with a hypercellular marrow, blasts count lower than 20% and concurrent peripheral cytopenias due to ineffective hematopoiesis. Some authors consider MDS as a predecessor for the development of leukemia –12.Identification of different subtypes of AML, MPN, and MDS cases can be performed by cytopathology, blood, and bone marrow cells morphologic findings, cytochemical staining used to identify myeloid cell lineage, histopathology, immunologic expression of cell antigens identified with immunoassays such as immunohistochemistry and flow cytometry . These lThis review reviews the current state-of-art of clinical cases of myelopoietic neoplasias in the horse, classified following the guidelines established by the World Health Organization (WHO) for myeloid leukemias.http://www.ncbi.nlm.nih.gov/pubmed) using the words "myeloid leukemia, myeloproliferative neoplasm, myeloproliferative neoplasia, equine, horse". The titles and abstracts were evaluated, expanding in detail the articles referring to these disorders. As additional sources, own studies were used. The experience of the authors in this area has made it possible to adapt the cases described in the literature to the current categorization criteria.The search for information was initially carried out in the PubMed database  described a classification of myeloid leukemia in veterinary medicine. This classification adopted the criteria and definitions of the French-American-British Cooperative Group (FAB) and the National Cancer Institute workshop used for human medicine.The common terminology for AML classification includes 8 categories (M0 to M8) and was based on the morphological and cytochemical characteristics of blast cells. Lately, cytochemical staining was replaced by immunophenotypic characterization of the cell lineage. Subsequently, the WHO revised this classification and added two new criteria such as chromosomal translocations and evidence of dysplasia . AML is AML-M0 (acute myeloid leukemia) is diagnosed when blast cell number is greater than or equal to 90% of myeloid cells. Less than or equal to 5% of blast cells in general circulation are stained with myeloperoxidase (MPO) and no Auer blast cells are observed. Auer blast cells are groups of azurophilic granular material forming elongated needles (Auer rods) in the cytoplasm of myeloid leukemic blast cells. To the authors’ knowledge, this subtype has not been documented in the horse yet . AML-M0 A separate entity in the context of AML es the acute undifferentiated leukemia (AUL). Based on FAB and WHO, AUL is characterized by the presence of blastic hematopoietic cell within the bone marrow and/or blood at proportions greater than 20–30%, even up to 100% of the total nucleated cells. Because of the immaturity and atypia of these blasts, differentiation of the cells on morphological, cytochemical and phenotypic characteristics into lymphoid or myeloid is difficult. In human medicine, AUL which is positive for myeloid markers is classified as AML-M0, whereas cases negative for lymphoid and myeloid markers are classified as ‘undifferentiated’ or better still, ‘unclassifiable’ leukemia. In veterinary medicine, AUL is extremely uncommon, although it has been reported in dogs .9/L. In bone marrow aspirate, the differential cell count was composed of 42% lymphoid cells, 5% differentiated myeloid cells, 24% erythroid cells (predominantly rubricytes and metarubricytes), and 29% blast cells with occasional plasma cells [Acute myeloblastic leukemia (AML-M1 and M2) may be granulocytic or neutrophil and is subdivided into M1 where > 90% of non-erythroid cells are myeloblasts, and M2 when 30–90% of non-erythroid cells are myeloblasts . AML-M2 ma cells . Becausema cells .In AML-M3 or promyelocytic, there is a predominance of promyelocytes in both circulating blood and bone marrow . AML-M3 Both granulocyte and monocyte differentiation occur in AML-M4 or myelomonocytic. At least 20% of both tumor cell lines stain for neutrophil or the monocyte series; at least 20% of blast cells in blood or bone marrow and at least 20% of bone marrow cells must be of the monocyte lineage to distinguish AML-M2 from AML-M4 . The perAML-M5 or monocytic is characterized by peripheral monocytosis with neoplastic monocytes comprising more than 80% of nonerythroid cells in the bone marrow . Two subAML-M6 or acute erythroid leukemia includes two subtypes of erythroid/myeloid leukemia (AML-M6A) and pure erythroid leukemia (AML-M6B). In AML-M6A there is a dual lineage with a co-production of myeloblasts and erythroblasts. More than 50% of the total nucleated cells in the bone marrow are erythroids and at least 30% are myeloblast precursors  identifiAML-M7 or megakaryocytic is diagnosed when at least 30% of the nucleated cell population within the bone marrow are megakaryocytes , but notAcute basophilic leukemia , although it a rare form of AML, has been reported in dogs , cats 3, in a caMyeloproliferative neoplasms or CML have primarily mature cells in blood and/or bone marrow, without a prominent increase in blast cells. CML is commonly found in adult animals. Although these proceses are chronic in nature, they can progress to AML. Subgroups of this neoplasm are quite rare in horses and include .Chronic granulocytic leukemia (CGL) is characterized by a proliferation of cell lineages of relatively mature neutrophils, eosinophils, or basophils. Within the bone marrow, relatively mature granulocytes predominate, with an M:E ratio from 5:1 to 20:1 . An earlMore recently, Johansson et al.  reportedNeutrophilic leukemia, a subtype of MPN, has been described in horses with progressive loss of physical condition, anorexia, limb edema, fever, clotting disorders, icterus from hemolytic anemia, and recurrent infections , 40. ChaChronic eosinophilic leukemia (CEL) is a clonal proliferation of eosinophilic precursors that might result in a persistently high number of eosinophils in blood, bone marrow, or tissues . AlthougChronic basophilic leukemia (CBL) is an extremely rare disease, described in humans , in one Polycythemia vera or primary erythrocytosis is rare in horses. It is the result of the malignant transformation of bone marrow precursors of all bone marrow cell lines and manifests itself with erythrocytosis. It may be accompanied by thrombocytosis and leukocytosis, but serum erythropoietin (EPO) is typically normal . In horsJAK2). JAK2 is a nonreceptor tyrosine kinase that downstream of the EPO receptor that directs the signaling cascade triggered by the binding of EPO to its receptor in red blood cell precursors in the bone marrow. In this way, signaling promotes cell division and erythropoiesis. Normally, JAK2 is only activated, by phosphorylation of tyrosine residues, when the EPO binds to its receptor. However, up to 90% of patients with polycythemia vera have a homozygous mutation of a single amino acid in JAK2. In these cases, the valine is replaced by phenylalanine in the amino acid 617, which makes the enzyme constitutively active. This leads to erythropoiesis, either independently of EPO or because the precursors become hypersensitive to normal EPO concentrations [JAK2 has been identified in dogs [In humans, polycythemia vera is due to the mutation (V1617F) in the pseudokinase domain of Janus-activated kinase 2 (trations . Polycyt in dogs .JAK2 gene in blood cells has been described [9/L) with a positive Coombs test. Peripheral blood smear revealed the presence of basophilia and a large number of abnormally shaped megakaryocytes was found in bone marrow. The diagnosis of ET should only be considered in animals with sustained and unexplained thrombocytosis longer than 1–2 months and without evidence of MPN . The presence of sensitive markers of inflammation (e.g. acute reactive proteins) might help to distinguish between reactive thrombocytosis and ET [Essential thrombocythemia (ET) represents an overproduction of megakaryocytes in bone marrow that might result in the release of abundant platelets into blood. These high amounts of platelets may not be completely functional, and might cause vascular thrombosis and bleeding disorders. In humans with ET, a mutation of the escribed . In catsescribed –55. In ts and ET , 55.Chronic monocytic leukemia (CMoL) is a clonal stem cell disorder characterized by an excessive proliferation of granulocytic and monocytic cells. A case of CMoL was reported in a 6-year-old Hessian gray gelding with a history of impaired performance, cough, colic, edema of the ventral abdomen, prepuce and limbs, reduced skin turgor, pale mucous membranes, forced costoabdominal breathing, reduced venous return, enlarged lymph nodes, and splenomegaly. The hematological analysis revealed anemia, leukocytosis, and a high percentage of monocytoid leukemic cells. Generalized lymphadenopathy, splenomegaly, ascites, hydrothorax, and a diffusely thickened gut wall were found at necropsy. Massive infiltration with monocytoid leukemic cells was detected in lymph nodes, spleen, bone marrow, liver, gut wall, kidneys, and choroid plexus. Incubation of living cells obtained from a leukocyte concentrate with latex particles revealed phagocytosis in the leukemic cells on light and electron microscopy. The leukemic cells were marked mainly with ANBE and CAE, showing their monocytic origin Table . On scan9/L), with neutrophilia and monocytosis and a pronounced leftward shift in both cell series. Circulating myeloid blast cells can represent between 5 and 10% of leukocytes. Hypersegmented granulocytes and monocytes are frequently found, as well as mild to severe normochromic macrocytic anemia and thrombocytopenia. As with CMoL, repeated blood and bone marrow tests with special staining or immunophenotyping are needed to confirm the diagnosis of CMMoL [Chronic myelomonocytic leukemia (CMMoL) is often included among MDS because of its multilinear dyshematopoiesis and its tendency to progress to AML. In cats and dogs, CMMoL usually has extreme leukocytosis  represents a group of disorders characterized by ineffective hematopoiesis and dysplastic alterations in single to multiple cell lineages with a hypercellular marrow, blasts counts lower than 20% and concurrent peripheral cytopenias due to ineffective hematopoiesis . HoweverTable Although uncommon, myeloid leukemias (both acute and chronic) and myelodysplasic syndromes have been reported in horses. The most commonly described myeloid leukemia reported in horses has been AML-M4 (myelomonocytic) and AML-M5 (monocytic). Isolated cases of AML-M5 (monocytic), AML-M6B (acute erythroid leukemia) and chronic granulocytic leukemia have also been reported. Unreported myeloid leukemias are AML-M0 (acute myeloid leukemia), AML-M1 (acute myeloblastic leukemia), AML-M3 (promyelocytic), AML-6A (acute erythroid leukemia), AML-M7 (megakaryocytic), chronic basophilic leukemia, essential thombocythemia and, chronic myelomonocytic leukemia. Until now, the classification of leukemic processes in the horse has combined hematological and bone marrow cytological studies, some stains for the cytochemical study of progenitor and/or mature cells, and scarce markers for the immunophenotypic study of the myeloid series. Unlike what happens in humans and other animal species like dogs and cats, the limited available methodology for the immunohistochemical or cytogenetic study currently makes it difficult to differentiate the different types of myeloid leukemias in the horse. Therefore, future research based on these areas would be needed to correctly classify these malignancies in the equine species.


PEITC is a naturally occurring isothiocyanate that has been shown to have efficacy against various types of cancer in preclinical studies. We first incorporated PEITC into a carbomer gel. A uniform formulation was prepared, and its viscosity was appropriate for topical application. We then demonstrated the release of PEITC from the gel into and through a Strat-M skin-like membrane. Finally, the effects of the PEITC-containing gel were tested against SCC and normal keratinocytes skin cells in culture, and these results were compared to those obtained for free 5-fluoruracil (5-FU), a commonly used skin-cancer drug. Our results show that a homogeneous PEITC-containing topical gel can be prepared and used to kill SCC cells. Thus, our formulation may be useful for treating SCC in the clinic.It is currently estimated that one in every five Americans will develop skin cancer during their lifetime. Squamous cell carcinoma (SCC) is a common type of skin cancer that can develop due to the skin’s exposure to the sun. Herein, we prepared a topical gel containing 0.5%  Isothiocyanates (ITCs) are organic compounds with a –N=C=S functional group that can react with cellular nucleophiles. Naturally occurring ITCs are contained in cruciferous vegetables  as glucosinolates and are released via hydrolysis by the enzyme myrosinase. Myrosinase is also contained in these vegetables and in the human gut. ITCs have been widely investigated as potential cancer chemopreventive and therapeutic agents; they may prevent tumor initiation, promotion, and progression . StudiesPEITC is a naturally occurring ITC, and gluconasturtiin, the precursor of PEITC, is found in watercress. PEITC has been shown to have efficacy against various cancers in preclinical animal models . MicromoIt is estimated that approximately one in five Americans will develop skin cancer at some point in their lives by the age of seven . The two50′s of 8 µM and 21.5 µM, respectively. Although this SCC cancer cell line was derived from tongue epithelial cells, they have also been used to develop a 3D in vitro skin cancer model [Recently, Lam-ubol et al. demonstrated that PEITC is toxic toward the human SCC-25 cell line . After 7er model . Thus, wer model ,21.PEITC was purchased from Sigma Aldrich ; 5-Fluorouracil was purchased from TCI America ; and 1,2-benzenedithiol (BDT) was purchased from VWR . Carbopol 940 and triethanolamine were purchased from Fisher Scientific , and dimethyl sulfoxide (DMSO) was from VWR . The MTS assay kit was purchased from Promega , and human SCC-25 and keratinocytes were purchased from the American Type Culture Collection (ATCC) .The human SCC-25 cells were cultured in a DMEM/Ham’s F12 medium purchased from Sigma Aldrich  and were supplemented with 10% fetal bovine serum (FBS), 0.4 µg/mL hydrocortisone, 15 mM HEPES, sodium bicarbonate, and L-glutamine, all of which were also purchased from Sigma Aldrich. The culture medium was changed every 2–3 days, and the cells were subcultured at 70–80% confluency. The human primary epidermal keratinocytes were cultured in a dermal cell basal medium from the ATCC and were supplemented with penicillin-streptomycin-amphotericin B, phenol red, and a supplemental kit, as recommended by the ATCC. The culture medium was again changed every 2–3 days and subcultured at 70–80% confluency.w/v carbomer gel. PEITC (0.05 mL) was dissolved in 0.025 mL of DMSO, as PEITC is more soluble in DMSO than in water, and DMSO is miscible with the carbomer gel. This solution was then added to 5 mL of the 0.5% w/v carbomer gel, reducing the total carbomer concentration to 0.49% w/v. Finally, triethanolamine (10 µL) was added to the gel mixture, and the gel was mixed via probe sonication at an amplitude of 20 MHz for 10 s and was then vortexed for 1 min and gently stirred. A blank carbomer gel was prepared in the same manner but without adding PEITC. The pH of both gels was measured using a Mettler Toledo F20 pH-meter. Carbomer concentrations (0.5–2%) were tested to determine the best base. The 0.5% stock was chosen due to its viscosity. Carbopol 940 (0.05 g) was dispersed in 10 mL of deionized water. The mixture was kept at room temperature for 24 h to form a 0.5% R2 = 0.98) was used to determine the concentrations. For the standard curve, PEITC was first solubilized in DMSO and then mixed with potassium phosphate buffer. This stock was further diluted to 1:100 in potassium phosphate buffer. The final concentrations were then made in the phosphate buffer, and solubilized BDT in methanol was added to determine the PEITC concentrations. PETIC should be stored at 2–8 °C [The concentration of PEITC in each PEITC carbomer gel was determined using the 1,2-benzenedithiol-based cyclocondensation assay . First, t 2–8 °C ; thus, w−1 at 25, 32 and 37 °C. Samples were run in triplicate.The viscosities of the formulations were determined using a microVISC from RheoSense  at room temperature (20–25 °C) and at skin temperature (32 and 37 °C). After formulating a PEITC carbomer gel, the viscosity measurements were performed. The same study was performed using the blank carbomer gel and a commercially available gel, Alocane. Alocane  contains 4% Lidocaine HCl and the following inactive ingredients: 1,3-propanediol, aloe barbadensis leaf juice, dimethyl isosorbide, hydroxyethyl cellulose, phenoxyethanol, chlorphenesin, capryly glycol, tocopheryl acetate, and water, and has a pH of 5.3. Using the provided pipette for the instrument, 10 μL of sample were injected into the viscometer for each viscosity measurement, and the viscosity was measured at shear rates of 10 to 70 sR2 = 0.98).Transdermal diffusion tests were conducted using a Franz cell with a 11.28 mm orifice diameter and Millipore Strat-M membranes, a type of membrane commonly used in predictive studies for diffusion in human skin ,25. Fran4 cells/mL in 1.5 mL complete culture medium, per well. After 24 h, the cells, at approximately 50% confluency, determined visually at 10× using an Olympus inverted microscope, were treated with PEITC carbomer gel or blank carbomer gel at the corresponding weight .Human SCC-25 cells and keratinocytes were seeded in 12-well plates at approximately 5 × 10n = 3 for each dose  were calculated using the following equation in Solver:a and b were parameters determined by the program [Excel’s Solver program was used to generate curves for the diffusion and cell studies. All the concentrations leading to 50% cell proliferation inhibition (IC program . When usv/w PEITC (t = 30 and 55 days. At day 30, we measured 29.8 ± 4.1 mg, and at day 55, we measured 26.6 ± 1.9 mg. These data are similar to those collected at t = 0, meaning the formulation is relatively stable at 4 °C, which is the temperature that PEITC should be stored [As is shown in /w PEITC . To detee stored . FurtherViscosities of the blank carbomer gel, PEITC carbomer gel, and commercial Alocane gel were measured at temperatures of 25, 32, and 37 °C at various shear rates. The skin surface has been reported to range from 32 to 37 °C ,30; thusThe ability of the PEITC in the gel to permeate into and through the skin was then determined using a Strat-M synthetic membrane, which serves as a model for human skin . We perf50 values of 1.2 μM and 3.8 μM were calculated for SCC-25 cells and keratinocytes, respectively. When FBS was present in a culture medium during the 30-min treatment of SCC cells with PEITC, reduced toxicity was observed (The efficacy of the PEITC-containing carbomer gel was tested in a cell culture. Using 12-well plates, the gel was placed on inserts, and PEITC passed through the insert membranes to the cells below. We first determined that 31.5 µg of PEITC was contained in the reservoir after a 30-min exposure to 0.1 g gel. On the basis of this value, the amount of gel required for concentrations of 1–8 µM PEITC were calculated. The 30-min time point was chosen because it represented a reasonable therapy window for a patient, and the amount of gel used was reasonable for real-life applications. The weight equivalents of the blank carbomer gel were also studied in a cell culture. The blank carbomer gel was only slightly toxic toward both cell lines, although it appears that keratinocytes were more sensitive to it than the SCC-25 cells . As was observed . The Sol50 values of 5-FU against SCC-25 cells and keratinocytes were both approximately 0.4 mM after a 48-h exposure. A decrease in viability over 48 h is most likely due to 5-FU interfering with the S phase of the cell cycle [We then tested the toxicity of 5-FU toward these cell lines. Levy et al. determined that a total of 2.2% of 5-FU in a 5% 5-FU formulation is absorbed . From thll cycle .We herein incorporated PEITC into a topical carbomer gel for use against SCC lesions. We chose to prepare a gel with a low percentage of PEITC, as only low concentrations of PEITC are needed to elicit an effect in vitro and in vivo . We prepRecently, Lam-ubol et al. demonstrated that PEITC is toxic toward SCC-25 cells . The toxThe drug 5-FU is currently used against SCC of the skin and is administered orally or, more commonly, via a topical cream . It is kIn this work, our goal was to formulate a gel that could be used to treat SCC using naturally occurring isothiocyanates. We previously used PEITC to treat lung cancer cells and showed a reasonable therapeutic window . The bla


Reactive oxygen species (ROS) generated by the NADPH oxidase are crucial for antimicrobial host defense and also play a role in the regulation of inflammatory processes. Signals generated by formyl peptide receptor 2 (FPR2) activate the neutrophil ROS generating NADPH oxidase; such signals are mediated when the receptors bind an activating agonist, as well as when agonist desensitized FPR2 are reactivated by the receptor for platelet-activating factor (PAF). We present data on the effects of Idelalisib, a specific inhibitor for the PI3Kδ isoform, on ROS production during FPR2 activation and reactivation by PAF, respectively. Neutrophils were isolated from peripheral blood of healthy adults obtained from the blood bank at Sahlgrenska University Hospital and ROS release was measured using isoluminol-amplified chemiluminescence. Value of the Data•These data are the first to show the involvement of PI3K-δ in FPR2 mediated activation of the neutrophils NADPH-oxidase, an electron transport system that is essential for microbial killing and the regulation of inflammatory processes.•These results expand the current knowledge about GPCR signaling which may benefit research of several inflammatory diseases including the activating PI3K-delta syndrome (APDS) as well as research focused on the role of FPR2 as a regulator of inflammation.•The findings can be used to further characterize the role of PI3K-δ in inflammatory settings, by additional studies of the functions of PI3K-δ in regulating GPCR signaling and oxidative burst activity in inflammatory diseases.11.110 (agonist for FPR2), PAF (agonist for PAFR) and PMA (receptor-independent PKC activator). Neutrophil activation and ROS release induced by F2Pal10, PAF and PMA in the presence or absence of the PI3K-δ inhibitor are shown  were incubated for five min at 37 °C in the absence  or presence of the δ-isoform specific inhibitor Idelalisib  before stimulation with PMA, F2Pal10 or PAF and measurement of O2−production with an isoluminol-enhanced chemiluminescence system. (A-B) Neutrophils incubated with or without Idelalisib (1 µM) were stimulated with PMA (50 nM). Data are presented as A) one representative chemiluminescence trace out of three and B) peak O2− production compared with or without Idelalisib (1 µM) . Statistical analysis was performed using paired Student's t-test to compare the inhibitor treated cells to control cells. C-D) Neutrophils incubated with or without different concentrations of Idelalisib  were stimulated with F2Pal10 (500 nM). E-F) Neutrophils incubated with or without different concentrations of Idelalisib  were stimulated with PAF (100 nM). C, E) Data are presented as one representative trace with or without Idelalisib (1 µM) n ≥ 4. D and F) Idelalisib dose-dependent inhibition , as the normalized response in the absence of Idelalisib, and the fitted curve (mean ± SD n ≥ 4). The IC50 value and 95% of confidence interval (CI) are given.Neutrophils (10A) Neutrophils (105) were stimulated with the FPR2 agonist F2Pal10  or buffer (dashed line). When the response had declined, the FPR2 desensitized cells were subsequently activated with a second stimulation with PAF . B) F2Pal10 (500 nM) desensitized neutrophils incubated one min with Idelalisib (100 nM), FPR2 antagonist  or buffer before PAF stimulation . A-B) One representative O2− trace out of six is shown. C) Quantification of inhibitory effects of Idelalisib , FPR2 antagonist , and PAFR antagonist  on PAF-induced FPR2 reactivation. The inhibitors were added one min before stimulation of F2Pal10 (500 nM)-desensitized neutrophils with PAF (100 nM) and the peak O2−values for each condition were determined. The results are presented as the percent remaining PAF-induced NADPH-oxidase activity in FPR2-desensitized cells after treatment with the inhibitors as compared to FPR2-desensitized cells in the absence of inhibitors . Statistical analysis was performed using one-way ANOVA followed by Dunnet's multiple comparison to control.22.16/mL in Krebs-Ringer phosphate buffer (KRG) containing glucose (10 mM), Mg2+ (1.5 mM), and Ca2+ (1 mM). The cells were kept on ice until use.Human neutrophils were isolated from buffy coats as described 2.22− production was measured by an isoluminol-enhanced chemiluminescence system, described previously 2− production was measured in 6-channel Biolumat LB 9505 , using disposable 4-mL polypropylene tubes with a 0.9 mL reaction mixture. Neutrophils (1 × 105), HRP (4 U/mL), isoluminol (2 × 10−5 M) and optimal antagonist/inhibitor (KRG for control) were incubated (five min 37 °C) before stimulation with 0.1 mL agonist and the release of O2−was measured as light emission over time. FPR2 desensitized neutrophils were obtained by pre-activating neutrophils with the FPR2 specific agonist F2Pal10GPCR agonist-induced extracellular O2.310 , was from Caslo Laboratory . The PI3K-δ specific inhibitor Idelalisib was purchased from Selleckchem . PAF was obtained from Avanti Polar Lipids . The FPR2 peptidomimetic antagonist CN6 2+ (1 mM) and Mg2+ (1.5 mM)  was made in house. Dextran and Ficoll-Paque were obtained from GE Healthcare Life Sciences . Horseradish peroxidase (HRP) was obtained from Boehringer Mannheim (Germany).The FPR2 pepducin agonist F2Pal2.4t-test or a one-way ANOVA followed by Dunnet's multiple comparison test; details are stated in the respective figure legends. Statistical significant differences are indicated by *p ≤ 0.05. **p ≤ 0.01. Each independent experiment was performed as biological replicates with neutrophils isolated from individual blood donors.Data analysis was performed with Graph Pad Prism version 8.0a . Data were analyzed with a paired Student´s Buffy coat blood samples were obtained from healthy adults from the blood bank at Sahlgrenska University Hospital. Ethics approval was not needed since the buffy coats were provided anonymously and could not be traced back to a specific individual. This is in line with Swedish legislation section code 4§ 3p SFS 2003:460 (Lag om etikprövning av forskning som avser människor).The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article.


Dear Editor,1. We and others have shown that these patients are characterized by high genomic instability, along with defects of DNA damage response (DDR) genes3.Acute myeloid leukemia (AML) patients carrying complex karyotype or aneuploidies have a very poor prognosis, with a 5-year overall survival (OS) <20%PALB2) is a key player in the homology recombination (HR) pathway, since it functions as a scaffold for the HR complex4. PALB2 monoallelic germline mutations are associated with an increased risk of developing breast, pancreatic, and ovarian cancers, whereas biallelic mutations lead to Fanconi anemia5. Genomic lesions, including sequence mutations and copy-number alterations (CNAs) of HR and DDR genes, are rare events in sporadic cancers and no recurrent genomic alterations in these genes have been reported so far in AML.Partner and localizer of BRCA2  and acute erythroid leukemia (AEL) cohort (n = 159) were retrieved from https://www.cbioportal.org/ and https://pecan.stjude.cloud/, respectively. The GSE23452 SNP array (n = 144), the GSE14468 (n = 486), and The Cancer Genome Atlas  AML transcriptomic datasets were obtained from Gene Expression Omnibus collection (https://www.ncbi.nlm.nih.gov/gds) and https://cancergenome.nih.gov/, respectively. SNP array data files for the NGS-PTL cohort (n = 119) are available through the Gene Expression Omnibus public database (accession no. GSE160982). Detailed dataset information and analysis are described in the Single-nucleotide polymorphism (SNP) arrays of 119 adult AML cases and whole-exome sequencing (WES) data of 68 adult AML cases were downloaded from the NGS-PTL project repository. Mutational data from the Beat-AML study : one somatic frameshift mutation was identified in one AML patient  and two missense mutations  were identified in two AEL cases. We then asked whether PALB2 was affected by CNAs. PALB2 CN loss was detected in 12/233 patients (5.2%), with a minimal common deleted region of 6.6 kb at the C terminus. This region included exon 12, which encodes for the domain responsible for the interaction with the HR proteins RAD51 and BRCA2 and the DNA repair-related polymerase POLH . WNT and ERBB2 signaling pathways, cell adhesion, cytokines, and inflammatory response, metabolism, necroptotic process, senescence, and autophagy in cancer were the most relevant pathways enriched of genes affected by CN loss , in comparison to 19.6% of PALB2 wild-type cases , indicating that TP53 functionality was potentially lost in these cases. To identify additional mutations that cooperated with PALB2 loss, we integrated CN results with WES data (n = 68) of available AML cases from the NGS-PTL cohort3 that was enriched for patients having complex karyotype , PALB2 loss patients had mutations in DNMT3A , BCOR (n = 1), CDKN2A (n = 1), FLT3 (n = 1), NPM1 (n = 1), NRAS (n = 1), and WT1 . Specifically, patients with favorable prognosis  or inv(16) AML) expressed higher levels of PALB2, while intermediate and adverse risk cases had lower expression : 1.2–not reached; PALB2 wild-type: 16.2 months, 95% CIs: 12.9–19.1; p < 0.0001; Fig. PALB2 loss negatively affected the prognosis of AML patients with a hazard ratio of 2.74 . Therefore, PALB2 might be a novel biomarker of poor prognosis in AML.We then investigated the prognostic role of genetic alterations of PALB2 is a tumor suppressor affected by mutations and CNAs in patients with either a familiar history of breast, pancreatic, and ovarian cancers5, or, rarely, in sporadic solid and hematological tumors . Notably, PALB2 participates also in the Fanconi anemia pathway and biallelic germline mutations were associated with the development of AML, among other childhood cancers4.PALB2 is rarely mutated at the somatic level in adult AML. However, in a cohort of 233 patients, PALB2 CN loss was detected in 5.2% of cases and it was associated with TP53 genomic alterations. Large PALB2 genomic deletions were also reported in one AML, one myelodysplastic syndrome8, and four AEL cases9. Remarkably, the co-association of PALB2 and TP53 alterations has been previously described in PALB2-related breast cancer10. Moreover, in our cohort, we detected seven patients with a focal deletion involving exons 11 and 12, which encode for the WD40 domain. Mutations in this domain were reported to disrupt DNA repair activity6.In this study, we report that PALB2 loss pointed to the ERBB2 (HER2) signaling pathway . These data suggest that the phenotype of PALB2 loss patients might resemble that of HER2-negative breast cancers. Of note, breast cancer patients carrying PALB2 germline mutations were associated with the HER2-negative phenotype10. No familial history of cancer was reported in patients from our AML cohort, suggesting that the detected PALB2 CN loss might be somatic alterations acquired by the leukemic cells. However, we cannot exclude the germline origin of the deletion, since paired-germline samples were not available.Enrichment analysis of genes within chromosomal regions affected by CN loss and significantly associated with PALB2 genomic lesions and patients’ outcome so far11. We here show a negative impact of PALB2 genomic alterations on AML patients’ survival with cases carrying PALB2 loss having shorter OS. This predictive role was independent of other negative prognostic factors . Nevertheless, a limitation of our study is the heterogeneity of treatments and patients’ age, pointing out the need for a larger and homogeneous AML cohort to validate the potential of PALB2 as a biomarker for poor-prognosis AML.Few studies described an association between PALB2 mRNA levels predicted response to cisplatin–docetaxel in non-small cell lung cancer12, while PALB2-mutated pancreatic cancers were described to benefit from mitomycin C or PARP inhibition (PARPi) treatment13. Despite the potential relevance, these studies were characterized by a limited number of patients and the role of PALB2 alterations in therapy response deserves further investigation. This is particularly important in AML, where functional studies elucidating PALB2 role in AML cells are needed. PARPi is currently being explored as therapeutic options alone or in combination with chemotherapy, alkylating, or demethylating agents in AML2. Different studies reported a deregulated expression of HR genes in AML and preclinical studies showed that t and t AML are extremely sensitive to PARPi15. Taken together, our data indicate that therapies targeting the HR pathway may become a valuable therapeutic option for the subgroup of AML patients characterized by PALB2 CN loss and dismal outcome.At the therapeutic level, high Supplemental materialSupplemental Figure 1Supplemental Table 1Supplemental Table 2Supplemental Table 3Supplemental Table 4Supplemental Table 5


What is mentoring? Mentoring is defined as a professional relationship in which an experienced person assists another in developing specific skills and knowledge enhancing the mentee’s professional and personal growth. Mentoring benefits include: Knowledge transfer, creating a mentoring culture, and challenging the mentee to move beyond their comfort zone. Carmel and Paul (2015) describe self-selected mentoring as a process in which a mentee identifies a potential mentor based on similarities in interests and need. Findings indicated mentees experienced opportunities in career advancement, expanded thinking, scholarly confidence, facilitation of a collaborative culture, and understanding the importance of goal setting. Mentoring as a health promotion or intervention strategy has become widespread in communities. Two mentoring approaches will be discussed: a traditional format with students and faculty in academia, another utilizing an intergenerational approach with Latinx and Native American families. Recommendations will be discussed in terms of underlying core similarities across the venues.


Ca2+ is a ubiquitous second messenger whose fluctuations of its intracellular concentrations are handled by channels, pumps, exchangers, and Ca2+ binding proteins. The concerted action of the Ca2+ toolkit components encodes specific Ca2+ signals with defined spatio-temporal characteristics that determine the cellular responses. In this review, after a general overview of the adult brain NSCs and GSCs, we focus on the multiple roles of the Ca2+ toolkit in NSCs and discuss how GSCs hijack these mechanisms to promote tumor growth. Extensive knowledge of the role of the Ca2+ toolkit in the management of essential functions in healthy and pathological stem cells of the adult brain should help to identify promising targets for clinical applications.The brain of adult mammals, including humans, contains neural stem cells (NSCs) located within specific niches of which the ventricular-subventricular zone (V-SVZ) is the largest one. Under physiological conditions, NSCs proliferate, self-renew and produce new neurons and glial cells. Several recent studies established that oncogenic mutations in adult NSCs of the V-SVZ are responsible for the emergence of malignant primary brain tumors called glioblastoma. These aggressive tumors contain a small subpopulation of cells, the glioblastoma stem cells (GSCs), that are endowed with proliferative and self-renewal abilities like NSCs from which they may arise. GSCs are thus considered as the cells that initiate and sustain tumor growth and, because of their resistance to current treatments, provoke tumor relapse. A growing body of studies supports that Ca In the brain of adult mammals, including humans, neural stem cells (NSCs) reside within two major regions: the ventricular-subventricular zone  lining the lateral brain ventricles and the subgranular zone of the dentate gyrus in the hippocampus. Within these niches, NSCs sustain lifelong neurogenesis and gliogenesis . These Ca2+ toolkit components encode specific Ca2+ signals that are defined by their spatio-temporal profile and their magnitude  and mitochondria, Ca2+ pumps and exchangers that replenish Ca2+ stores or that extrude Ca2+ from the cell, and CaBP that buffer cytosolic free Ca2+ or act as effectors. The coordinated action of the Ca2+ toolkit components maintains low resting levels of cytosolic free Ca2+ and encodes specific Ca2+ signals that trigger selective cellular activities in response to extracellular signals , which is about 10,000-fold less than those of the extracellular medium. Thus, the opening of any Ca2+ channel located in the plasma membrane leads to a pronounced Ca2+ influx that sharply raises the intracellular Ca2+ concentration, that in turn, affects the activity of nearby proteins and elicits biological responses  mediate Cavia ligand-mediated activation of receptors coupled to the phospholipase C (PLC) that breaks down PIP2 to produce diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). DAG activates plasma membrane channels from the TRPC  subfamily, leading to a cationic current (Na+ and Ca2+), a process referred to as receptor-operated Ca2+ entry  represents a widespread type of Ca2+ entry that is triggered upon IP3-dependent depletion of ER Ca2+ stores  and elicit cellular responses  in T cells, Orai1 homopolymers form the core component of SOCs. Although Orai1 is the ubiquitous SOC protein, Orai2 and Orai3 mediate SOCE in certain specific tissues  can also be supported by TRPC tetrameric channels  or arachidonic acid metabolite Leukotriene C4 (LTC4)-regulated Ca2+ channels  and inositol 1,4,5-trisphosphate receptors (IP3Rs) in response to multiple factors that include free Ca2+ ion. Three different isoforms of RYRs , each encoded by a different gene, have been identified , Nuclear factor of activated T-cells (NFAT) or Ca2+/calmodulin-dependent protein kinase (CAMK) pathways  was shown to be preferentially activated by low frequency repeated Ca2+ transients, whereas NFAT activation needs higher frequencies and a long duration Ca2+ signals , a-amino-3-hydroxy-5-méthylisoazol-4-propionate (AMPA), and kainate subtypes  and CamKinase (CamK4), conducted to a decrease in the numbers of neuroblasts that reached the olfactory bulb, indicating that calmodulin and its effector are essential players for neuroblast migration  and TRPV  channels as compared to normal tissue  whose overexpression has been associated with a worse prognosis, have been found enriched in GSCs of glioblastoma as compared to either non-GSC-tumor cells or normal tissue  whose activity is mandatory for the maintenance of cell polarity. This induces cell depolarization, thereby precluding the Na+-dependent transport of nutrients and ultimately leading to starvation  that with NFAT1, controlled the maintenance of a naive stem cell state in GSCs (Jiang et al., Ca2+ in both NSCs and GSCs, which is correlated to prominence of Ca2+-related transcripts in both cell types. Indeed, NSCs and GSCs possess, each, a specific Ca2+ toolkit that allows to maintain Ca2+ homeostasis and to encode specific Ca2+ signals in response to the numerous signals from their microenvironment. Several functions of specific Ca2+ components in NSCs and GSCs have been unveiled. Current available data underpins that stemness is controlled by VGCCs, SOCs and IP3Rs in NSCs while it is regulated by VGCCs, nicotinic receptors, TRPVs and NFAT in GSCs, indicating that there might be cell type specific effects of Ca2+ toolkit components. Such differences may rely on either a differential expression of Ca2+ toolkit elements that, in turn, generates differential responses, or on yet unexplored functions of specific Ca2+ components in GSCs and NSCs, or both. It should also be kept in mind that the Ca2+ signals produced in a cell not only depend on Ca2+ release in the cytoplasm but also on its extrusion, a process that influences the spatial and temporal extent of the Ca2+ signal. Concerning the pumps, whose activity shapes the Ca2+ responses, little is known on their expression and functions in either NSCs or GSCs.Our review highlights a central role of CaIn vivo, cells evolve in microenvironments with multiple extracellular cues, many of which elicit Ca2+ signals and control cellular activities. Thus, it will be of importance to understand how the different signals are integrated and conduct to a specific Ca2+ signal within each cell type. Along with this latter question, the downstream effectors recruited by the different Ca2+ signals will require further investigation in order to understand how the specific spatio-temporal and magnitude profiles of Ca2+ signals select a cellular response. Reciprocally, it will be essential to understand the impact of the microenvironment and of pathological states on the remodeling of the Ca2+ toolkit of NSCs and GSCs, as it has been observed for endothelial cells (Lodola et al., 2+ toolkit in both NSCs and GSCs will contribute to understand the impact of Ca2+ alterations or dysregulation during aging and disease and will help to develop pharmacological strategies to combat brain diseases. Indeed, the remodeling of intracellular Ca2+ homeostasis is considered as a cellular and molecular hallmark of brain aging (Kumar et al., 2+ toolkit have not been explored in the V-SVZ so far. Yet, some molecules targeting the Ca2+ toolkit are already being tested in clinical trials for the treatment of glioblastoma. Among them, carboxyamidotriazole that inhibits non-VGCCs and disrupts Ca2+-mediated signal transduction, showed some promising results in a recent multicenter phase IB trial when used in combination with temozolomide, prompting further deciphering of the roles of Ca2+ channels in GSCs and NSCs (Omuro et al., An in-depth knowledge of the CaVC wrote the initial manuscript draft. VC, ET, ND, PA and BC revised and approved the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.