Converting cell death into senescence by PARP1 inhibition improves recovery from acute oxidative injury.

Excessive amounts of reactive oxygen species (ROS) lead to macromolecular damage and high levels of cell death with consequent pathological sequelae. We hypothesized that switching cell death to a tissue regenerative state could potentially improve the short-term and long-term detrimental effects of ROS-associated acute tissue injury, although the mechanisms regulating oxidative stress-induced cell fate decisions and their manipulation for improving repair are poorly understood. Here, we show that cells exposed to high oxidative stress enter a poly (ADP-ribose) polymerase 1 (PARP1)-mediated regulated cell death, and that blocking PARP1 activation promotes conversion of cell death into senescence (CODIS). We demonstrate that this conversion depends on reducing mitochondrial Ca2+ overload as a consequence of retaining the hexokinase II on mitochondria. In a mouse model of kidney ischemia-reperfusion damage, PARP inhibition reduces necrosis and increases transient senescence at the injury site, alongside improved recovery from damage. Together, these data provide evidence that converting cell death into transient senescence can therapeutically benefit tissue regeneration.

Detection of SARS-CoV-2 B.1.1.529 (Omicron) variant by SYBR Green-based RT-qPCR.

The coronavirus disease 2019 (COVID-19) pandemic is unceasingly spreading across the globe, and recently a highly transmissible Omicron SARS-CoV-2 variant (B.1.1.529) has been discovered in South Africa and Botswana. Rapid identification of this variant is essential for pandemic assessment and containment. However, variant identification is mainly being performed using expensive and time-consuming genomic sequencing. In this study, we propose an alternative RT-qPCR approach for the detection of the Omicron BA.1 variant using a low-cost and rapid SYBR Green method. We have designed specific primers to confirm the deletion mutations in the spike (S Δ143-145) and the nucleocapsid (N Δ31-33) which are characteristics of this variant. For the evaluation, we used 120 clinical samples from patients with PCR-confirmed SARS-CoV-2 infections, and displaying an S-gene target failure (SGTF) when using TaqPath COVID-19 kit (Thermo Fisher Scientific, Waltham, USA) that included the ORF1ab, S, and N gene targets. Our results showed that all the 120 samples harbored S Δ143-145 and N Δ31-33, which was further confirmed by whole-genome sequencing of 10 samples, thereby validating our SYBR Green-based protocol. This protocol can be easily implemented to rapidly confirm the diagnosis of the Omicron BA.1 variant in COVID-19 patients and prevent its spread among populations, especially in countries with high prevalence of SGTF profile.

Observational cohort study of the clinical outcomes associated with B.1.1.7/SGTF among hospitalized COVID-19 patients in Lebanon.

Background: The B.1.1.7 SARS-CoV-2 variant results in spike gene target failure (SGTF) in reverse transcription-quantitative polymerase chain reaction (RT-PCR) assays. Few studies have been published on the clinical impact of B.1.1.7/SGTF. Aims: To assess the incidence of B.1.1.7/SGTF and its associated clinical characteristics among hospitalized COVID-19 patients. Methods: This observational, single-centre, cohort study was conducted between December 2020 and February 2021 and included 387 hospitalized COVID-19 patients. The Kaplan-Meier method was used for survival analysis, and logistic regression to identify risk factors associated with B.1.1.7/SGTF. Results: By February 2021, B.1.1.7/SGTF (88%) dominated the SARS-CoV-2 PCR results in a Lebanese hospital. Of the 387 eligible COVID-19 patients confirmed by SARS-CoV-2 RT-PCR, 154 (40%) were non-SGTF and 233 (60%) were B.1.1.1.7/SGTF; this was associated with a higher mortality rate among female patients [22/51 (43%) vs 7/37 (19%); P = 0.0170]. Among patients in the B.1.1.7/SGTF group, most were aged ≥ 65 years [162/233 (70%) vs 74/154 (48%); P < 0.0001]. Independent predictors of B.1.1.7/SGTF infection were hypertension (OR = 0.415; CI: 0.242-0.711; P = 0.0010), age ≥ 65 years (OR = 0.379; CI: 0.231-0.622; P < 0.0001), smoking (OR = 1.698; CI: 1.023-2.819; P = 0.0410), and cardiovascular disease (OR = 3.812; CI: 2.215-6.389; P < 0.0001). Only non-SGTF patients experienced multi-organ failure [5/154 (4%) vs 0/233 (0%); P = 0.0096]. Conclusion: There was a clear difference between the clinical features associated with B.1.1.7/SGTF and non-SGTF lineages. Tracking viral evolution and its clinical impact is crucial for proper understanding and management of the COVID-19 pandemic.

Impact of pre-graft serology on risk of BKPyV infection post-renal transplantation.

OBJECTIVES: BK polyomavirus-associated nephropathy is a troublesome disease caused by BK polyomavirus (BKPyV) infection in immunocompromised renal graft recipients. There are no effective treatments available, making immunosuppression reduction the only management option. Thus, pre-graft predictive BKPyV replication markers are needed for identification of patients at high risk of viraemia. METHODS: We conducted a retrospective study to assess the correlation between pre-transplantation BKPyV serostatus and post-transplantation incidence of BKPyV infection. Sera from 329 recipients and 222 matched donors were tested for anti-BKPyV antibodies against BKPyV serotypes I and IV by using a virus-like particle-based immunoglobulin G enzyme-linked immunosorbent assay, and BKPyV DNA load was monitored for at least 1 year post-transplantation. RESULTS: Eighty recipients were viruric and 59 recipients were viraemic post-transplantation. In the post-transplantation period, the probability of developing viraemia for serotype I increased from 4.3% for the D-/R+ group to 12.1% for the D+/R+ group, climbing to 37.5% for the D+/R- group (P < 0.05). When calculating recipient mean titres for serotypes I and IV, we observed a clear difference in the proportions of viraemia, decreasing from 50% for mean titres <400 to 13.5% for titres ≥400 (P < 0.001), as well as a higher proportion of presumptive nephropathy (50% versus 23.1%, respectively; P < 0.05). In univariate analysis, this parameter had an odds ratio of 6.41 for the risk of developing post-transplantation BKPyV viraemia (95% confidence interval 3.16-13.07; P < 0.0001). CONCLUSIONS: Determination of both donor and recipient BKPyV seropositivity before transplantation and antibody titre measurements may serve as a predictive tool to manage clinical BKPyV infection by identification of patients at high risk.

Apoptotic and Non-Apoptotic Modalities of Thymoquinone-Induced Lymphoma Cell Death: Highlight of the Role of Cytosolic Calcium and Necroptosis.

Targeting non-apoptotic modalities might be therapeutically promising in diffuse large B cell lymphoma (DLBCL) patients with compromised apoptotic pathways. Thymoquinone (TQ) has been reported to promote apoptosis in cancer cells, but little is known about its effect on non-apoptotic pathways. This work investigates TQ selectivity against DLBCL cell lines and the cell death mechanisms. TQ reduces cell viability and kills cell lines with minimal toxicity on normal hematological cells. Mechanistically, TQ promotes the mitochondrial caspase pathway and increases genotoxicity. However, insensitivity of most cell lines to caspase inhibition by z-VAD-fmk (benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone) pointed to a critical role of non-apoptotic signaling. In cells dying through non-apoptotic death, TQ increases endoplasmic reticulum (ER) stress markers and substantially increases cytosolic calcium ([Ca2+]c) through ER calcium depletion and activation of store-operated calcium entry (SOCE). Chelation of [Ca2+]c, but not SOCE inhibitors, reduces TQ-induced non-apoptotic cell death, highlighting the critical role of calcium in a non-apoptotic effect of TQ. Investigations showed that TQ-induced [Ca2+]c signaling is primarily initiated by necroptosis upstream to SOCE, and inhibition necroptosis by necrostatin-1 alone or with z-VAD-fmk blocks the cell death. Finally, TQ exhibits an improved selectivity profile over standard chemotherapy agents, suggesting a therapeutic relevance of the pro-necroptotic effect of TQ as a fail-safe mechanism for DLBCL therapies targeting apoptosis.

A retrospective analysis of 902 hospitalized COVID-19 patients in Lebanon: clinical epidemiology and risk factors.

BACKGROUND: The clinical epidemiology of hospitalized COVID-19 patients has never been described before in Lebanon. Moreover, the hospital admission and PCR positivity rates have not been assessed and compared yet. OBJECTIVES: To describe the characteristics and outcomes of hospitalized patients with coronavirus induced disease 2019 (COVID-19) in Lebanon and identify risk factors for severe disease or death. STUDY DESIGN: This is a retrospective mono-center cohort study in which we used patients' files to extract and analyse data on demographic and clinical characteristics, as well as mortality. Moreover, we tracked the pandemic by recording the daily total and ICU inpatient census and the PCR positivity rate for admitted and outpatients. RESULTS: Although the total admission rate increased from September to April, the ICU census switched this trend in December to stabilize at an average of around 10 patients/day until April. The case fatality rate was 19% for the 902 hospitalized patients, of which the majority (80%) had severe COVID-19. The severity odds ratio is significantly decreased in immunosuppressed cases (OR, 0.18; CI, 0.05-0.67; p=0.011). Additionally, the odds of COVID-19 related death are significantly greater if consolidations are found in the chest computed tomography (CT) scan (OR, 12; CI, 2.63-55.08; p=0.0013). CONCLUSION: Consolidations in the lungs significantly increase the COVID-19 death risk. Risk factors identification is important to improve patients' management and vaccination strategies. In addition, hospital statistics are good indicators of a pandemic's track.

Variant analysis of the first Lebanese SARS-CoV-2 isolates.

Recently the first genome sequences for 11 SARS-CoV-2 isolates from Lebanon became available. Here, we report the detection of variants within the genome of these strains. Pairwise alignment analysis using blastx was performed between these sequences and the UniProtKB data for the SARS-CoV-2 coronavirus to identify amino acid variations. Variants analysis was performed using multiple Bioinformatics tools. We noticed for the first time 18 mutations that have never been reported before. Among those, a frame shift (8651A>) in NSP4, a stop codon 6887A > T in NSP3 and two missense mutations in spike S2 were found. In addition, we found 28 variants in ORF1ab alone. A previously reported variant, 23403A > G, in the spike protein S2 was mostly seen. Two other known mutations 25563G > T in ORF3a and 14408C > T in ORF1ab were detected respectively in 6 and 8 out of the 11 isolates. Our results may help to prognose forthcoming infections in this region.

Disruption of gap junctions attenuates acute myeloid leukemia chemoresistance induced by bone marrow mesenchymal stromal cells.

The bone marrow (BM) niche impacts the progression of acute myeloid leukemia (AML) by favoring the chemoresistance of AML cells. Intimate interactions between leukemic cells and BM mesenchymal stromal cells (BM-MSCs) play key roles in this process. Direct intercellular communications between hematopoietic cells and BM-MSCs involve connexins, components of gap junctions. We postulated that blocking gap junction assembly could modify cell-cell interactions in the leukemic niche and consequently the chemoresistance. The comparison of BM-MSCs from AML patients and healthy donors revealed a specific profile of connexins in BM-MSCs of the leukemic niche and the effects of carbenoxolone (CBX), a gap junction disruptor, were evaluated on AML cells. CBX presents an antileukemic effect without affecting normal BM-CD34+ progenitor cells. The proapoptotic effect of CBX on AML cells is in line with the extinction of energy metabolism. CBX acts synergistically with cytarabine (Ara-C) in vitro and in vivo. Coculture experiments of AML cells with BM-MSCs revealed that CBX neutralizes the protective effect of the niche against the Ara-C-induced apoptosis of leukemic cells. Altogether, these results suggest that CBX could be of therapeutic interest to reduce the chemoresistance favored by the leukemic niche, by targeting gap junctions, without affecting normal hematopoiesis.

Correction: Disruption of gap junctions attenuates acute myeloid leukemia chemoresistance induced by bone marrow mesenchymal stromal cells.

The original version of this Article omitted the following from the Acknowledgements: This research was also supported by grants to KZ (UL and L-CNRS). This has now been corrected in both the PDF and HTML versions of the Article.

Pre-Transplantation Assessment of BK Virus Serostatus: Significance, Current Methods, and Obstacles.

The immunosuppression required for graft tolerance in kidney transplant patients can trigger latent BK polyomavirus (BKPyV) reactivation, and the infection can progress to nephropathy and graft rejection. It has been suggested that pre-transplantation BKPyV serostatus in donors and recipients is a predictive marker for post-transplantation BKPyV replication. The fact that research laboratories have used many different assay techniques to determine BKPyV serostatus complicates these data analysis. Even studies based on the same technique differed in their standard controls choice, the antigenic structure type used for detection, and the cut-off for seropositivity. Here, we review the different BKPyV VP1 antigens types used for detection and consider the various BKPyV serostatus assay techniques' advantages and disadvantages. Lastly, we highlight the obstacles in the implementation of a consensual BKPyV serologic assay in clinics (e.g., the guidelines absence in this field).

Circulating miR-29c, miR-30c, miR-193a-5p and miR-885-5p: Novel potential biomarkers for HTLV-1 infection diagnosis.

Human T-cell leukemia virus type 1 (HTLV-1) is an oncoretrovirus that infects 5-10 million people worldwide. Currently, different methods are used to test HTLV-1 infection. However, a biomarker that could enable an early and accurate diagnosis of HTLV-1 infection is still lacking. Here, we compared the serum miRNA expression profile in HTLV-1 infected patients versus healthy individuals to identify a potential biomarker for diagnosis of HTLV-1 infection.TaqMan miRNA microarray (TLDA) was carried out to compare the miRNA expression profile in infected versus healthy individuals. Quantitative real-time RT-PCR (qRT-PCR) was applied to validate TLDA results. Receiver-operator characteristic (ROC) curve analysis was performed to determine the diagnostic accuracy of the most highly and significantly identified deregulated miRNA(s) as potential biomarker(s). We identified deregulated expression for ten miRNAs with miR-127, miR-136, miR-142-3p, miR-221, and miR-423-5p being down-regulated whilst let-7b, miR-29c, miR-30c, miR-193a-5p, and miR-885-5p being up-regulated in infected individuals. ROC curve analyses showed an AUC (Areas Under the ROC Curve) of 0.875 (95% CI: 0.7819-0.9581; P = .0021), 0.861 (95% CI: 0.7596-0.9754; P = .003), 0.856 (95% CI: 0.689-0.895; P = .011), and 0.849 (95% CI: 0.678-0.855; P = .017) for miR-29c, miR-30c, miR-193a-5p, and miR-885-5p respectively. Combined ROC analyses using these 4 miRNAs showed a greater AUC of 0.907 (95% CI: 0.809-1; P = .000001) indicating a robust diagnostic value of these 4 miRNAs. Our findings highlight serum miR-29c, miR-30c, miR-193a-5p and miR-885-5p as novel potential biomarkers important for HTLV-1 diagnosis.

Constitutive IP3 signaling underlies the sensitivity of B-cell cancers to the Bcl-2/IP3 receptor disruptor BIRD-2.

Anti-apoptotic Bcl-2 proteins are upregulated in different cancers, including diffuse large B-cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL), enabling survival by inhibiting pro-apoptotic Bcl-2-family members and inositol 1,4,5-trisphosphate (IP3) receptor (IP3R)-mediated Ca2+-signaling. A peptide tool (Bcl-2/IP3R Disruptor-2; BIRD-2) was developed to abrogate the interaction of Bcl-2 with IP3Rs by targeting Bcl-2's BH4 domain. BIRD-2 triggers cell death in primary CLL cells and in DLBCL cell lines. Particularly, DLBCL cells with high levels of IP3R2 were sensitive to BIRD-2. Here, we report that BIRD-2-induced cell death in DLBCL cells does not only depend on high IP3R2-expression levels, but also on constitutive IP3 signaling, downstream of the tonically active B-cell receptor. The basal Ca2+ level in SU-DHL-4 DLBCL cells was significantly elevated due to the constitutive IP3 production. This constitutive IP3 signaling fulfilled a pro-survival role, since inhibition of phospholipase C (PLC) using U73122 (2.5 µM) caused cell death in SU-DHL-4 cells. Milder inhibition of IP3 signaling using a lower U73122 concentration (1 µM) or expression of an IP3 sponge suppressed both BIRD-2-induced Ca2+ elevation and apoptosis in SU-DHL-4 cells. Basal PLC/IP3 signaling also fulfilled a pro-survival role in other DLBCL cell lines, including Karpas 422, RI-1 and SU-DHL-6 cells, whereas PLC inhibition protected these cells against BIRD-2-evoked apoptosis. Finally, U73122 treatment also suppressed BIRD-2-induced cell death in primary CLL, both in unsupported systems and in co-cultures with CD40L-expressing fibroblasts. Thus, constitutive IP3 signaling in lymphoma and leukemia cells is not only important for cancer cell survival, but also represents a vulnerability, rendering cancer cells dependent on Bcl-2 to limit IP3R activity. BIRD-2 seems to switch constitutive IP3 signaling from pro-survival into pro-death, presenting a plausible therapeutic strategy.

Reciprocal sensitivity of diffuse large B-cell lymphoma cells to Bcl-2 inhibitors BIRD-2 versus venetoclax.

Bcl-2 is often upregulated in cancers to neutralize the BH3-only protein Bim at the mitochondria. BH3 mimetics (e.g. ABT-199 (venetoclax)) kill cancer cells by targeting Bcl-2's hydrophobic cleft and disrupting Bcl-2/Bim complexes. Some cancers with elevated Bcl-2 display poor responses towards BH3 mimetics, suggesting an additional function for anti-apoptotic Bcl-2 in these cancers. Indeed, Bcl-2 via its BH4 domain prevents cytotoxic Ca2+ release from the endoplasmic reticulum (ER) by directly inhibiting the inositol 1,4,5-trisphosphate receptor (IP3R). The cell-permeable Bcl-2/IP3R disruptor-2 (BIRD-2) peptide can kill these Bcl-2-dependent cancers by targeting Bcl-2's BH4 domain, unleashing pro-apoptotic Ca2+-release events. We compared eight "primed to death" diffuse large B-cell lymphoma cell lines (DLBCL) for their apoptotic sensitivity towards BIRD-2 and venetoclax. By determining their IC50 using cytometric cell-death analysis, we discovered a reciprocal sensitivity towards venetoclax versus BIRD-2. Using immunoblotting, we quantified the expression levels of IP3R2 and Bim in DLBCL cell lysates, revealing that BIRD-2 sensitivity correlated with IP3R2 levels but not with Bim levels. Moreover, the requirement of intracellular Ca2+ for BIRD-2- versus venetoclax-induced cell death was different. Indeed, BAPTA-AM suppressed BIRD-2-induced cell death, but promoted venetoclax-induced cell death in DLBCL cells. Finally, compared to single-agent treatments, combining BIRD-2 with venetoclax synergistically enhanced cell-death induction, correlating with a Ca2+-dependent upregulation of Bim after BIRD-2 treatment. Our findings suggest that some cancer cells require Bcl-2 proteins at the mitochondria, preventing Bax activation via its hydrophobic cleft, while others require Bcl-2 proteins at the ER, preventing cytotoxic Ca2+-signaling events via its BH4 domain.

HA14-1 potentiates apoptosis in B-cell cancer cells sensitive to a peptide disrupting IP 3 receptor / Bcl-2 complexes.

Anti-apoptotic B-cell lymphoma 2 (Bcl-2) is commonly upregulated in hematological cancers, including B-cell chronic lymphocytic leukemia (B-CLL) and diffuse large B-cell lymphoma (DLBCL), thereby protecting neoplastic cells from oncogenic-stress-induced apoptosis. Bcl-2 executes its anti-apoptotic function at two different sites in the cell. At the mitochondria, Bcl-2 via its hydrophobic cleft interacts with pro-apoptotic Bcl-2 family members to inhibit apoptosis. At the endoplasmic reticulum (ER), Bcl-2 via its Bcl-2 homology (BH)4 domain, prevents excessive Ca(2+) signals by interacting with the inositol 1,4,5-trisphosphate receptor (IP3R), an intracellular Ca(2+)-release channel. A peptide tool (BIRD-2) that targets the BH4 domain of Bcl-2 reverses Bcl-2's inhibitory action on IP3Rs and can trigger pro-apoptotic Ca(2+)signals in B-cell cancer cells. Here, we explored whether HA14-1, a Bcl-2 inhibitor that also inhibits sarco/endoplasmic reticulum Ca(2+)-ATPases (SERCA), could potentiate BIRD-2-induced cell death. We measured apoptosis in Annexin V/7-AAD stained cells using flow cytometry and intracellular Ca(2+) signals in Fura2-AM-loaded cells using an automated fluorescent plate reader. HA14-1 potentiated BIRD-2-induced Ca(2+) release from the ER and apoptosis in both BIRD-2-sensitive DLBCL cell lines (SU-DHL-4) and in primary B-CLL cells. BIRD-2-resistant DLBCL cells (OCI-LY-1) were already very sensitive to HA14-1. Yet, although BIRD-2 moderately increased Ca(2+) levels in HA14-1-treated cells, apoptosis was not potentiated by BIRD-2 in these cells. These results further underpin the relevance of IP3R-mediated Ca(2+) signaling as a therapeutic target in the treatment of Bcl-2-dependent B-cell malignancies and the advantage of combination regimens with HA14-1 to enhance BIRD-2-induced cell death.

Leakage evaluation of original and compatible implant-abutment connections: In vitro study using Rhodamine B.

Leakage has been addressed as a major contributing factor to inflammatory reactions at the implant-abutment connection, leading to problems such as oral malodor, inflammation, and marginal bone loss. The aim of this study was to investigate in vitro the leakage at implant-abutment interface of OsseoSpeed™ implants connected to original and compatible abutments. A total of 28 OsseoSpeed implants were divided into four groups (n = 7). Each group was connected to four different abutments according to manufacturers' recommendations: group A (TiDesign™); group B (Natea™); group C (Dual™); and group D (Implanet™) abutments. The inner volume of each implant-abutment combination was calculated and leakage was detected for each group with spectrophotometric analysis at 1 h (D0) and 48 h (D1) of incubation time using Rhodamine B. At 1 h, leakage volume was significantly lower in TiDesign and Dual than in Natea and Implanet (P < 0.001). At 48 h, however, leakage was significantly lower between TiDesign and all other systems (P < 0.005). Compatible abutments do not fit internal connection of OsseoSpeed implants perfectly, which increases the leakage of the final assembly.

In vitro evaluation of leakage at implant-abutment connection of three implant systems having the same prosthetic interface using rhodamine B.

Objectives. Hollow space between implant and abutment may act as reservoir for commensal and/or pathogenic bacteria representing a potential source of tissue inflammation. Microbial colonization of the interfacial gap may ultimately lead to infection and bone resorption. Using Rhodamine B, a sensitive fluorescent tracer dye, we aim in this study to investigate leakage at implant-abutment connection of three implant systems having the same prosthetic interface. Materials and Methods. Twenty-one implants (seven Astra Tech, seven Euroteknika, and seven Dentium) with the same prosthetic interface were connected to their original abutments, according to the manufacturers' recommendation. After determination of the inner volume of each implant systems, the kinetic quantification of leakage was evaluated for each group using Rhodamine B (10(-2) M). For each group, spectrophotometric analysis was performed to detect leakage with a fluorescence spectrophotometer at 1 h (T0) and 48 h (T1) of incubation time at room temperature. Results. Astra Tech had the highest inner volume (6.8 µ L), compared to Dentium (4 µ L) and Euroteknika (2.9 µ L). At T0 and T1, respectively, the leakage volume and percentage of each system were as follows: Astra Tech 0.043 µ L or 1.48% (SD 0.0022), 0.08 µ L or 5.56% (SD 0.0074), Euroteknika 0.09 µ L or 6.93% (SD 0.0913), 0.21 µ L or 20.55% (SD 0.0035), and Dentium 0.07 µ L or 4.6% (SD 0.0029), 0.12 µ L or 10.47% (SD 0.0072). Conclusion. The tested internal conical implant-abutment connections appear to be unable to prevent leakage. In average, Astra Tech implants showed the highest inner volume and the least leakage.

A dual role for the anti-apoptotic Bcl-2 protein in cancer: mitochondria versus endoplasmic reticulum.

Anti-apoptotic Bcl-2 contributes to cancer formation and progression by promoting the survival of altered cells. Hence, it is a prime target for novel specific anti-cancer therapeutics. In addition to its canonical anti-apoptotic role, Bcl-2 has an inhibitory effect on cell-cycle progression. Bcl-2 acts at two different intracellular compartments, the mitochondria and the endoplasmic reticulum (ER). At the mitochondria, Bcl-2 via its hydrophobic cleft scaffolds the Bcl-2-homology (BH) domain 3 (BH3) of pro-apoptotic Bcl-2-family members. Small molecules (like BH3 mimetics) can disrupt this interaction, resulting in apoptotic cell death in cancer cells. At the ER, Bcl-2 modulates Ca(2+) signaling, thereby promoting proliferation while increasing resistance to apoptosis. Bcl-2 at the ER acts via its N-terminal BH4 domain, which directly binds and inhibits the inositol 1,4,5-trisphosphate receptor (IP3R), the main intracellular Ca(2+)-release channel. Tools targeting the BH4 domain of Bcl-2 reverse Bcl-2's inhibitory action on IP3Rs and trigger pro-apoptotic Ca(2+) signaling in cancer B-cells, including chronic lymphocytic leukemia (CLL) cells and diffuse large B-cell lymphoma (DLBCL) cells. The sensitivity of DLBCL cells to BH4-domain targeting tools strongly correlated with the expression levels of the IP3R2 channel, the IP3R isoform with the highest affinity for IP3. Interestingly, bio-informatic analysis of a database of primary CLL patient cells also revealed a transcriptional upregulation of IP3R2. Finally, this review proposes a model, in which cancer cell survival depends on Bcl-2 at the mitochondria and/or the ER. This dependence likely will have an impact on their responses to BH3-mimetic drugs and BH4-domain targeting tools. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.

The selective BH4-domain biology of Bcl-2-family members: IP3Rs and beyond.

Anti-apoptotic Bcl-2-family members not only neutralize pro-apoptotic proteins but also directly regulate intracellular Ca(2+) signaling from the endoplasmic reticulum (ER), critically controlling cellular health, survival, and death initiation. Furthermore, distinct Bcl-2-family members may selectively regulate inositol 1,4,5-trisphosphate receptor (IP3R): Bcl-2 likely acts as an endogenous inhibitor of the IP3R, preventing pro-apoptotic Ca(2+) transients, while Bcl-XL likely acts as an endogenous IP3R-sensitizing protein promoting pro-survival Ca(2+) oscillations. Furthermore, distinct functional domains in Bcl-2 and Bcl-XL may underlie the divergence in IP3R regulation. The Bcl-2 homology (BH) 4 domain, which targets the central modulatory domain of the IP3R, is likely to be Bcl-2's determining factor. In contrast, the hydrophobic cleft targets the C-terminal Ca(2+)-channel tail and might be more crucial for Bcl-XL's function. Furthermore, one amino acid critically different in the sequence of Bcl-2's and Bcl-XL's BH4 domains underpins their selective effect on Ca(2+) signaling and distinct biological properties of Bcl-2 versus Bcl-XL. This difference is evolutionary conserved across five classes of vertebrates and may represent a fundamental divergence in their biological function. Moreover, these insights open novel avenues to selectively suppress malignant Bcl-2 function in cancer cells by targeting its BH4 domain, while maintaining essential Bcl-XL functions in normal cells. Thus, IP3R-derived molecules that mimic the BH4 domain's binding site on the IP3R may function synergistically with BH3-mimetic molecules selectivity suppressing Bcl-2's proto-oncogenic activity. Finally, a more general role for the BH4 domain on IP3Rs, rather than solely anti-apoptotic, may not be excluded as part of a complex network of molecular interactions.

Altered Ca(2+) signaling in cancer cells: proto-oncogenes and tumor suppressors targeting IP3 receptors.

Proto-oncogenes and tumor suppressors critically control cell-fate decisions like cell survival, adaptation and death. These processes are regulated by Ca(2+) signals arising from the endoplasmic reticulum, which at distinct sites is in close proximity to the mitochondria. These organelles are linked by different mechanisms, including Ca(2+)-transport mechanisms involving the inositol 1,4,5-trisphosphate receptor (IP3R) and the voltage-dependent anion channel (VDAC). The amount of Ca(2+) transfer from the endoplasmic reticulum to mitochondria determines the susceptibility of cells to apoptotic stimuli. Suppressing the transfer of Ca(2+) from the endoplasmic reticulum to the mitochondria increases the apoptotic resistance of cells and may decrease the cellular responsiveness to apoptotic signaling in response to cellular damage or alterations. This can result in the survival, growth and proliferation of cells with oncogenic features. Clearly, proper maintenance of endoplasmic reticulum Ca(2+) homeostasis and dynamics including its links with the mitochondrial network is essential to detect and eliminate altered cells with oncogenic features through the apoptotic pathway. Proto-oncogenes and tumor suppressors exploit the central role of Ca(2+) signaling by targeting the IP3R. There are an increasing number of reports showing that activation of proto-oncogenes or inactivation of tumor suppressors directly affects IP3R function and endoplasmic reticulum Ca(2+) homeostasis, thereby decreasing mitochondrial Ca(2+) uptake and mitochondrial outer membrane permeabilization. In this review, we provide an overview of the current knowledge on the proto-oncogenes and tumor suppressors identified as IP3R-regulatory proteins and how they affect endoplasmic reticulum Ca(2+) homeostasis and dynamics.