Bcl-2 Gene Family Research Articles

Role of Bcl-2 Family Proteins in Photodynamic Therapy Mediated Cell Survival and Regulation.

Photodynamic therapy (PDT) is a treatment modality that involves three components: combination of a photosensitizer, light and molecular oxygen that leads to localized formation of reactive oxygen species (ROS). The ROS generated from this promising therapeutic modality can be lethal to the cell and leads to consequential destruction of tumor cells. However, sometimes the ROS trigger a stress response survival mechanism that helps the cells to cope with PDT-induced damage, resulting in resistance to the treatment. One preferred mechanism of cell death induced by PDT is apoptosis, and B-cell lymphoma 2 (Bcl-2) family proteins have been described as a major determinant of life or death decision of the death pathways. Apoptosis is a cellular self-destruction mechanism to remove old cells through the biological event of tissue homeostasis. The Bcl-2 family proteins act as a critical mediator of a life–death decision of cells in maintaining tissue homeostasis. There are several reports that show cancer cells developing resistance due to the increased interaction of the pro-survival Bcl-2 family proteins. However, the key mechanisms leading to apoptosis evasion and drug resistance have not been adequately understood. Therefore, it is critical to understand the mechanisms of PDT resistance, as well as the Bcl-2 family proteins, to give more insight into the treatment outcomes. In this review, we describe the role of Bcl-2 gene family proteins’ interaction in response to disease progression and PDT-induced resistance mechanisms.

Status of kinases in Epstein-Barr virus and Helicobacter pylori Coinfection in gastric Cancer cells

BackgroundHelicobacter pylori (H. pylori) and Epstein - Barr virus (EBV) plays a significant role in aggressive gastric cancer (GC). The investigation of genes associated with these pathogens and host kinases may be essential to understand the early and dynamic progression of GC.AimThe study aimed to demonstrate the coinfection of EBV and H. pylori in the AGS cells through morphological changes, expression of the kinase and the probable apoptotic pathways.MethodsGenomic DNA isolation of H. pylori and its characterization from clinical samples were performed. RT-qPCR of kinases was applied to scrutinize the gene expression of kinases in co-infected GC in a direct and indirect (separated through insert size 0.45 μm) H. pylori infection set up. Morphological changes in co-infected GC were quantified by measuring the tapering ends of gastric epithelial cells. Gene expression profiling of apoptotic genes was assessed through RT-qPCR.ResultsAn interleukin-2-inducible T-cell kinase (ITK) showed significant upregulation with indirect H. pylori infection. Moreover, Ephrin type-B receptor six precursors (EPHB6) and Tyrosine-protein kinase Fyn (FYN) showed significant upregulation with direct coinfection. The tapering ends in AGS cells were found to be extended after 12 h. A total of 24 kinase genes were selected, out of which EPHB6, ITK, FYN, and TYK2 showed high expression as early as 12 h. These kinases may lead to rapid morphological changes in co-infected gastric cells. Likewise, apoptotic gene expression such as APAF-1 and Bcl2 family genes such as BAD, BID, BIK, BIM, BAX, AND BAK were significantly down-regulated in co-infected AGS cells.ConclusionAll the experiments were performed with novel isolates of H. pylori isolated from central India, for the functional assessment of GC. The effect of coinfection with EBV was more profoundly observed on morphological changes in AGS cells at 12 h as quantified by measuring the tapering of ends. This study also identifies the kinase and apoptotic genes modulated in co-infected cells, through direct and indirect approaches. We report that ITK, EPHB6, TYK2, FYN kinase are enhanced, whereas apoptotic genes such as APAF-1, BIK, FASL, BAX are significantly down-regulated in AGS cells coinfected with EBV and H. pylori.

Genome-wide identification and expression analysis of Bcl-2 gene family under low-temperature stress in tilapia (Oreochromis niloticus)

Low temperature stress can lead to variety of changes, including apoptosis in tilapia (Oreochromis niloticus). The B cell lymphoma-2 (Bcl-2) gene family plays an important role in the process of apoptosis. The present study conducted genome-wide characterization of the Bcl-2 family genes in tilapia and their mRNA expression profiles were analysed in different tissues of tilapia under the low temperature stress (10°C). Twenty-four Bcl-2 family genes were identified, containing 2~8 exons. These genes were classified into two subfamilies (Bcl-2 homologs and BH3-only) based on their conserved domains. Besides, these BCL-2 proteins in tilapia possess at least one of the four conserved BH domains. The phylogenetic analysis showed that the Bcl-2 family genes did not aggregate by species, demonstrating sequence conservation of different types of Bcl-2 family members. Real-time quantitative PCR (RT-qPCR) analysis showed that Bcl-2 family genes were broadly expressed in different tissues of tilapia. When reared at 10 °C, the transcriptional expression levels of most of anti-apoptotic Bcl-2 homologs subgroup members and other BH3-only subgroup members in most tissues of tilapia were higher than those at 30°C. However, most of other Bcl-2 family members revealed a lower expression. The results suggested that hypothermia had significantly induced apoptotic in tilapia.

Integrated analysis of patient samples identifies biomarkers for venetoclax efficacy and combination strategies in acute myeloid leukemia.

Deregulation of the BCL2 gene family plays an important role in the pathogenesis of acute myeloid leukemia (AML). The BCL2 inhibitor, venetoclax, has received FDA approval for the treatment of AML. However, upfront and acquired drug resistance ensues due, in part, to the clinical and genetic heterogeneity of AML, highlighting the importance of identifying biomarkers to stratify patients onto the most effective therapies. By integrating clinical characteristics, exome and RNA sequencing, and inhibitor data from primary AML patient samples, we determined that myelomonocytic leukemia, upregulation of BCL2A1 and CLEC7A, as well as mutations of PTPN11 and KRAS conferred resistance to venetoclax and multiple venetoclax combinations. Venetoclax in combination with an MCL1 inhibitor AZD5991 induced synthetic lethality and circumvented venetoclax resistance.

Local anesthetic articaine ameliorates LPS-induced acute kidney injury via inhibition of NF-ĸB activation and the NLRP3 inflammasome pathway.

The present study was conducted to determine the protective effect of articaine (ART) in anlipopolysaccharide (LPS)-induced acute kidney injury (AKI) animal model. The results suggest ART causes a significant decrease in serum blood urea nitrogen, creatinine, and serum cystatin C level, showing a protective effect against LPS-induced AKI. This has been further supported by histopathological findings of kidney tissues. The level of tumor necrosis factor-α, interleukin (IL)-6, and IL-1β in serum and kidney tissues was remarkably inhibited by ART in a dose-dependent manner. ART causes a significant reduction of malondialdehyde and increases the activities of glutathione and superoxide dismutase with an increase in dose as compared to the LPS-treated group. Moreover, the ART-treated group showed dose-dependent inhibition of LPS-induced nuclear factor-κB activation and TLR4 expression as confirmed by Western blot analysis. The level of Bcl-2 family genes (Bcl-2 and Bax) was restored near to normal by ART. Collectively, all the above results indicated that ART had protective effects against LPS-induced AKI by blocking inflammatory and oxidative responses.

Zinc oxide nanoparticles enhance expression of maspin in human breast cancer cells.

Toxic and apoptotic impacts of zinc oxide nanoparticle (ZNP) on different cancer cells have been reported. Maspin (a mammary serine protease inhibitor) as a tumor suppressor gene can inhibit tumor growth and metastasis. The expression of maspin is modulated by p53, Bcl-2 family genes, and estrogen receptor α (ER-α). This study aimed to assess the ZNP effects on maspin expression in MCF-7 cells (a breast cancer cell). Experimental groups (ZNP5, ZNP10, and ZNP20) received 5, 10, and 20μM/mL ZNP for 48h, respectively. 17-β-estradiol (E2) was used to evaluate the role of ER-α in the anticancer impact of ZNP. Cell viability, Annexin V, migration assay, gene expression, and western blotting methods were applied to evaluate ZNP effects on the MCF-7 cells. ZNP at the concentrations of 10 and 20μM/mL could significantly decrease the viability and migration rate, and significantly increase apoptosis percentage in the MCF-7 cells. ZNP significantly enhanced mRNA expression and protein level of maspin in MCF-7 cells in a concentration-dependent way. ZNP concentration-dependently elevated mRNA expression and protein level of p53 and Bax while reduced the expression of Bcl-2 and ER-α. E2 promoted cancer cell growth by enhancing survival and migration rates. E2 treatment reduced mRNA expression and protein level of maspin and p53, and elevated Bcl-2 expression. ZNP considerably changed these events induced by E2 in the MCF-7 cells. It is concluded that the maspin overexpression is one of the toxic mechanisms of the ZNP on the ER-α-positive breast cancer cells, and can suppress the migration of these cells.

Tiao Geng decoction for treating menopausal syndrome exhibits anti-aging effects likely via suppressing ASK1/MKK7/JNK mediated apoptosis in ovariectomized rats

Ethnopharmacological relevanceTG-decoction (Tiao Geng decoction) is the extract of a Chinese herb mixture that has been used for treating menopausal symptoms for over 30 years. We have previously reported anti-aging and anti-oxidative effects of the TG-decoction on hypothalamic neurons in ovariectomized (OVX) rats.Aim of the study: The present study further investigates the effects of TG-decoction on the prevention of aging-related ultrastructural changes in menopausal hypothalamic neurons and the likely molecular mechanism. Materials and methodsA total of 120 four-month-old female SPF Sprague Dawley rats were divided into six groups. Five groups were ovariectomized (OVX) and one group served as a sham control. Three OVX groups received TG-decoction at three different doses. The remaining two OVX groups served as positive and negative controls by receiving estradiol valerate and saline solution. The sham group received saline. After one month, aging-related ultrastructural alterations in hypothalamic neurons were evaluated using transmission electron microscopy. Nissl staining was used to assess the pathomorphological changes of the hypothalamic neurons. Cell apoptosis was evaluated by TUNEL. Expression of Bcl-2 family genes was studied using qRT-PCR. Expression of the apoptosis-related proteins ASK1, MKK7, JNK, c-Jun, Bax, Casp3 and Bcl-2 was studied using western blotting. ResultsOvariectomy of female rats led to visible damage and aging-like alterations in the mitochondria and endoplasmic reticulum as well as large deposits of lipofuscin in hypothalamic tissue. TG-decoction treatment prevented this visible damage and lipofuscin deposition, increased the number of nerve cells and normally-shaped Nissl bodies, and reduced the number of TUNEL-positive cells. Expression of Bcl-2 gene was increased, while Bax gene reduced. Expression of the proteins ASK1, MKK7, JNK, c-Jun, Bax and Casp3 was reduced, while that of Bcl-2 was increased. ConclusionTG-decoction reduces aging-related ultrastructural changes in hypothalamic neurons, likely by suppressing ASK1/MKK7/JNK-mediated apoptosis in neuronal mitochondria or nuclei.

B-cell lymphoma 2 family genes show a molecular pattern of spatiotemporal heterogeneity in gynaecologic and breast cancer.

ObjectivesBCL2 family proteins have been widely studied over the past decade due to their essential roles in apoptosis, oncogenesis and anti‐cancer therapy. However, the similarities and differences in the spatial pattern of the BCL2 gene family within the context of chromatin have not been well characterized. We sought to fill this knowledge gap by assessing correlations between gene alteration, gene expression, chromatin accessibility, and clinical outcomes in gynaecologic and breast cancer.Materials and methodsIn this study, the molecular characteristics of the BCL2 gene family in gynaecologic cancer were systematically analysed by integrating multi‐omics datasets, including transcriptomics, chromatin accessibility, copy number variation, methylomics and clinical outcome.ResultsWe evaluated spatiotemporal associations between long‐range regulation peaks and tumour heterogeneity. Differential expression of the BCL2 family was coupled with widespread chromatin accessibility changes in gynaecologic cancer, accompanied by highly heterogeneous distal non‐coding accessibility surrounding the BCL2L1 gene loci. A relationship was also identified between gene expression, gene amplification, enhancer signatures, DNA methylation and overall patient survival. Prognostic analysis implied clinical correlations with BAD, BIK and BAK1. A shared protein regulatory network was established in which the co‐mutation signature of TP53 and PIK3CA was linked to the BCL2L1 gene.ConclusionsOur results provide the first systematic identification of the molecular features of the BCL2 family under the spatial pattern of chromatin in gynaecologic and breast cancer. These findings broaden the therapeutic scope of the BCL2 family to the non‐coding region by including a significantly conserved distal region overlaying an enhancer.

Synthetic antiprotozoal thiazolide drug induced apoptosis in colorectal cancer cells: implications of IL-6/JAK2/STAT3 and p53/caspases-dependent signaling pathways based on molecular docking and in vitro study.

Colorectal cancer (CRC) is a global pressing healthcare priority. Dysregulation of the IL6/JAK2/STAT3 and p53/caspase downstreaming pathways are significantly involved in the progression of CRC, and mainly affecting apoptosis. Discovery of new anti-cancer agents is laborious, time consuming, and costly with obvious socioeconomic burden. In the present study, we are proposing new molecular insights on the anti-proliferative and apoptotic therapeutic effects of nitazoxanide (NTZ) on CRC. NTZ is FDA-approved thiazolide antiparasitic agent, which has excellent safety and pharmacokinetic profiles. The molecular docking study revealed that NTZ has better binding affinity and docking score against JAK2 and BCL2 proteins compared to 5-Fluorouracil, which is the standard drug for treatment of CRC. The current in vitro work on a human HCT116 cell line displayed that NTZ had lower IC50 value (11.20µM) than 5-flurouracil (23.78µM), and NTZ induced a statistically significant down-regulation of IL6/JAK2/STAT3. NTZ also modulated significantly the p53/caspases-dependent signaling pathways, leading to enhancement of apoptosis and an increase of DNA fragmentation. Moreover, NTZ regulated the Bcl-2 gene family and promoted the loss of mitochondrial function which was depicted by release of cytochrome c (Cyt c), and caspase activation in apoptotic HCT116 cells. Additionally, NTZ was able to reduce the expression of VEGF in CRC cell line, which needs future thorough molecular investigations. In conclusion, our findings provided a novel evidence that NTZ could be a dual potential IL6/JAK2/STAT3 signaling inhibitor and p53/caspases-dependent pathway activator in CRC cell line. These potentials support further exploratory molecular researches targeting the therapeutic roles of NTZ in CRC; individually and simultaneously with current approved chemotherapeutic regimens.

Id1 and Id3 Maintain Steady-State Hematopoiesis by Promoting Sinusoidal Endothelial Cell Survival and Regeneration

Investigating mechanisms that regulate endothelial cell (EC) growth and survival is important for understanding EC homeostasis and how ECs maintain stem cell niches. We report here that targeted loss of Id genes in adult ECs results in dilated, leaky sinusoids and a pro-inflammatory state that increases in severity over time. Disruption in sinusoidal integrity leads to increased hematopoietic stem cell (HSC) proliferation, differentiation, migration, and exhaustion. Mechanistically, sinusoidal ECs (SECs) show increased apoptosis because of reduced Bcl2-family gene expression following Id gene ablation. Furthermore, Id1-/-Id3-/- SECs and upstream type H vessels show increased expression of cyclin-dependent kinase inhibitors p21 and p27 and impaired ability to proliferate, which is rescued by reducing E2-2 expression. Id1-/-Id3-/- mice do not survive sublethal irradiation because of impaired vessel regeneration and hematopoietic failure. Thus, Id genes are required for the survival and regeneration of BM SECs during homeostasis and stress to maintain HSC development.

Co-inhibition of BET proteins and PI3Kα triggers mitochondrial apoptosis in rhabdomyosarcoma cells.

Remodeling transcription by targeting bromodomain and extraterminal (BET) proteins has emerged as promising anticancer strategy. Here, we identify a novel synergistic interaction of the BET inhibitor JQ1 with the PI3Kα-specific inhibitor BYL719 to trigger mitochondrial apoptosis and to suppress tumor growth in models of rhabdomyosarcoma (RMS). RNA-Seq revealed that JQ1/BYL719 co-treatment shifts the overall balance of BCL-2 family gene expression towards apoptosis and upregulates expression of BMF, BCL2L11 (BIM), and PMAIP1 (NOXA) while downregulating BCL2L1 (BCL-xL). These changes were confirmed by qRT-PCR and western blot analysis. Ingenuity pathway analysis (IPA) of RNA-Seq data followed by validation qRT-PCR and western blot identified MYC and FOXO3a as potential transcription factors (TFs) upstream of the observed gene expression pattern. Immunoprecipitation (IP) studies showed that JQ1/BYL719-stimulated increase in BIM expression enhances the neutralization of antiapoptotic BCL-2, BCL-xL, and MCL-1. This promotes the activation of BAK and BAX and caspase-dependent apoptosis, as (1) individual silencing of BMF, BIM, NOXA, BAK, or BAX, (2) overexpression of BCL-2 or MCL-1 or (3) the caspase inhibitor N-Benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethylketone (zVAD.fmk) all rescue JQ1/BYL719-induced cell death. In conclusion, co-inhibition of BET proteins and PI3Kα cooperatively induces mitochondrial apoptosis by proapoptotic re-balancing of BCL-2 family proteins. This discovery opens exciting perspectives for therapeutic exploitation of BET inhibitors in RMS.

Comparative analysis of TTF-1 binding DNA regions in small-cell lung cancer and non-small-cell lung cancer.

Thyroid transcription factor‐1 (TTF‐1, encoded by the NKX2‐1 gene) is highly expressed in small‐cell lung carcinoma (SCLC) and lung adenocarcinoma (LADC), but how its functional roles differ between SCLC and LADC remains to be elucidated. Here, we compared the genome‐wide distributions of TTF‐1 binding regions and the transcriptional programs regulated by TTF‐1 between NCI‐H209 (H209), a human SCLC cell line, and NCI‐H441 (H441), a human LADC cell line, using chromatin immunoprecipitation‐sequencing (ChIP‐seq) and RNA‐sequencing (RNA‐seq). TTF‐1 binding regions in H209 and H441 cells differed by 75.0% and E‐box motifs were highly enriched exclusively in the TTF‐1 binding regions of H209 cells. Transcriptome profiling revealed that TTF‐1 is involved in neuroendocrine differentiation in H209 cells. We report that TTF‐1 and achaete‐scute homolog 1 (ASCL1, also known as ASH1, an E‐box binding basic helix–loop–helix transcription factor, and a lineage‐survival oncogene of SCLC) are coexpressed and bound to adjacent sites on target genes expressed in SCLC, and cooperatively regulate transcription. Furthermore, TTF‐1 regulated expression of the Bcl‐2 gene family and showed antiapoptotic function in SCLC. Our findings suggest that TTF‐1 promotes SCLC growth and contributes to neuroendocrine and antiapoptotic gene expression by partly coordinating with ASCL1.

Effects of Me2SO and Trehalose on the Cell Viability, Proliferation, and Bcl-2 Family Gene (BCL-2, BAX, and BAD) Expression in Cryopreserved Human Breast Cancer Cells.

Long-term cryopreservation of the viability and metabolic state of cells in cancer cell/tissue specimens has significant implications for diagnostic verification of disease progression in cancer patients and selection of effective treatment options via development of the patient-derived xenograft (PDX) models for drug screening. The purpose of this study is to investigate the effects of cryoprotectant agents (CPAs) on the expression of BCL-2 family genes (BCL-2, BAX, and BAD) that are involved in the growth and development of breast cancers. MCF-7 cells were cryopreserved in Dulbecco's modified Eagle's medium (DMEM) with 20% (v/v) fetal bovine serum, using 10% (v/v) Me2SO (dimethyl sulfoxide, DMSO) or 7.5% (v/v) Me2SO with 100is-300 mM trehalose as cryoprotectant solutions. After storage at -80°C for 7 days, the cells were thawed for evaluation. The use of Me2SO and trehalose has affected cell survival, proliferation, apoptotic state, as well as BCL-2 family gene expression. The conventional 10% (v/v) Me2SO method yields ∼80% post-thaw cell survival and good cell proliferation, but it drastically alters the pattern of the BCL-2 family gene expression. The antiapoptotic gene BCL-2 is downregulated, whereas two proapoptotic genes BAX and BAD are upregulated. The partial substitution of Me2SO with 200 or 300 mM trehalose enhances cell proliferation of survived cells after cryopreservation. The presence of trehalose upregulates the expression of both the antiapoptotic gene BCL-2 and proapoptotic genes BAX and BAD. Cryopreservation could tip off the checkpoint of the apoptotic pathway regulated by the BCL-2 family members, and the effect may be protectant dependent. The findings of this study demonstrate the importance of paying attention to the potential change of gene expression and metabolic state of cancer cells after cryopreservation in an attempt to development of the PDX models from cryopreserved cancer cells or tissue specimens.

Induction of apoptosis in hematological cancer cells by dorsomorphin correlates with BAD upregulation

AMPK is generally a tumor suppressor. However, once cancer arises, AMPK becomes a tumor promoter instead, driving cancer development. For such AMPK-driven cancers, AMPK blockade may be a valuable therapeutic strategy. Here we show that AMPK is upregulated in a variety of hematological cancers and plays key roles in maintaining viability of tumor cells. Blockade of AMPK signaling by dorsomorphin markedly induces apoptosis in Jurkat, K562 cell lines as well as primary cancerous B cells. Mechanistically, dorsomorphin significantly upregulates the expression of BAD, a pro-apoptotic member of the Bcl-2 gene family involved in initiating apoptosis. Reduction of BAD expression by RNA interference prevents apoptosis in response to AMPK inhibition. Thus, our data found BAD integrates the pro-apoptotic effects of dorsomorphin and provided novel insights into the mechanisms by which AMPK facilitates survival signaling in hematologic tumor cells.

The RAS-PI3K-AKT-NF-κB pathway transcriptionally regulates the expression of BCL2 family and IAP family genes and inhibits apoptosis in fibrous epulis.

BackgroundEpulis has a tumor‐like appearance but is considered to be a massive reactive lesion rather than a true neoplasia. Limited information about the pathogenesis of epulis is available. The purpose of our study was to identify potential signaling pathways in fibrous epulis through transcriptome profiling.MethodsDifferentially expressed genes (DEGs) between fibrous epulis lesions and normal gingival tissues were detected using RNA sequencing (RNAseq). The expression levels of eighteen genes were validated using quantitative real‐time PCR (qRT‐PCR).ResultsRNAseq identified 533 upregulated genes and 732 downregulated genes. The top 10 upregulated genes were IL11, OSM, MMP3, KRT75, MMP1, IL6, IL1B, IL24, SP7, and ADGRG3. The top 10 downregulated genes were BCHE, TYR, DCT, KRT222, RP11‐507K12.1, COL6A5, PMP2, GFRA1, SCN7A, and CDH19. KEGG pathway analysis further indicated that the DEGs were enriched in “Pathways in cancer” and the “Ras signaling pathway”. quantitative real‐time PCR verified that the expression levels of SOS1, HRAS, PIK3CA, AKT3, IKBKA, IKBKB, NFKB1, BCL2, BCL2L1, XIAP, BIRC2, and BIRC3 were increased significantly.ConclusionsThe current transcriptomic profiling study reveals that in fibrous epulis, RAS‐PI3K‐AKT‐NF‐κB pathway transcriptionally regulates the expression of BCL2 family and IAP family genes, leading to increased proliferation and apoptosis inhibition.

Genetically Engineered Hematopoietic Progenitors Derived from Human Induced Pluripotent Stem Cells Achieve the Feeder-Free and Robust Production of Neutrophils with the Functional Capacity In Vivo

Chemotherapies for cancers induce granulocytopenia and additively increase the risk of infection. Infections in patients with granulocytopenia, especially neutropenia, have remained as a major problem. Though granulocyte transfusion therapy (GTX) is a therapeutic option against neutropenic infections refractory to supportive therapies, GTX has not been in wide use mainly due to its physical burden on donors. We have previously reported the establishment of genetically engineered hematopoietic progenitors priming neutrophils (NeuP-HPCs) from human induced pluripotent stem cells (iPSCs) by inducible expression of c-MYC and BMI1, which cultured on OP9 feeder cells, kept expanding until 12 weeks after the induction and were differentiated into functional neutrophils four days after silencing c-MYC and BMI1. In the current study, we sophisticated the previous production system and established a robust and feeder-free production system of neutrophils, for the purpose of donor-free GTX. To enhance the expandability of NeuP-HPCs to be preferably applied in clinical settings, we investigated genes enhancing the expandability of NeuP-HPCs. we performed single-cell analysis of focused 83 transcriptional factors in HPCs and identified the population committing neutrophils in early phase, which enriched the expression of BCL6, CEBPB, CEBPD, and LITAF. As CEBPB provided the two-weeks extension of the expandability among four genes and had a lot of target genes, we conducted the search for target genes of CEBPB which purely enhanced the expandability. Motif analysis of CEBPB revealed that BCL2A1 was the gene associated with the cell proliferation in top-ranked ten target genes, suggesting that BCL2 family genes enhanced the expandability of NeuP-HPCs. Consistent with the result, the expression of BCL2A1, BCL-XL or MCL1, which were the major BCL2 family genes in the development of myeloid cells, enhanced the expandability and kept expanding for at least 16 weeks, indicating that one NeuP-HPC was estimated to generate thousands aliquot of 5.0 x 1010 cells, clinically meaningful scale, in 10 weeks. Notably, NeuP-HPCs expressing BCL-XL (NeuP-HPCs-XL) achieved the feeder-free expansion, suggesting that NeuP-HPCs-XL were preferable to clinical application. Whereas the constitutive expression of BCL2 family genes inhibited the neutrophilic differentiation, NeuP-HPCs-XL with the inducible expression of c-MYC, BMI1, and BCL-XL generated the functional neutrophils, which showed expression of neutrophil-specific markers, CD16b and CD66b, formed toxic granules with lipopolysaccharides (LPS) and produced reactive oxygen species (ROS) with phorbol-12-myristate-13 acetates (PMA). Gene expression profiling by RNA sequence comparing the neutrophils with or without LPS revealed the activation of NF-kB pathway, which was the critical pathway through LPS stimulation. Furthermore, the neutrophils with LPS stimulation recapitulated the expression profile of peripheral blood in patients with gram-negative sepsis. Gene ontology enrichment analysis showed the enrichment of inflammatory response, innate immune response, cytokine activity (including higher expression of IL1A, IL1B, IL6 and TNFa), cell adhesion (including higher expression of ICAM1, also a functional marker of phagocytosis) and chemotaxis (including higher expression of CXCL2, CXCL8, CCL19 and CCL20) with LPS stimulation. These findings indicated that the neutrophils from NeuP-HPCs-XL harbored the principal neutrophil functions including adhesion, chemotaxis, phagocytosis, ROS generation and cytokine production. To assess the function of neutrophils from NeuP-HPCs-XL in vivo, we performed in vivo imaging of the neutrophils expressing luciferase in the mice model of LPS-induced inflammation. In NSG mice harboring the local inflammation induced by intraperitoneal injection of LPS, intraperitoneally administrated neutrophils aggregated specifically at the inflammation site and showed the persistent survival for 72 hours, in marked contrast to the diffuse distribution and the shorter survival for 24 hours in NSG mice without LPS injection. These findings suggested that neutrophils from NeuP-HPCs-XL were functional in vivo. In summary, we achieved the feeder-free and robust production of neutrophils with the functional capacity in vivo. NeuP-HPCs-XL are potential sources of neutrophils for donor-free GTX. Figure Disclosures Miyauchi: Kyowa Hakko Kirin Co. Ltd.: Research Funding. Iwasaki:Kyowa Hakko Kirin Co. Ltd.: Employment. Kawagoshi:Kyowa Hakko Kirin Co. Ltd.: Employment. Kagoya:Nihon Shinyaku: Research Funding; Kyowa Kirin: Speakers Bureau. Kurokawa:Nippon Shinyaku Co., Ltd.: Research Funding; Teijin Limited: Research Funding; Shire Japan K.K.: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Chugai Pharmaceutical Company: Consultancy, Research Funding, Speakers Bureau; ONO PHARMACEUTICAL CO., LTD.: Speakers Bureau; Bioverativ Japan ltd.: Consultancy; MSD K.K.: Consultancy, Research Funding, Speakers Bureau; Celgene K.K.: Consultancy, Speakers Bureau; Boehringer Ingelheim: Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Daiichi Sankyo Conpany: Speakers Bureau; Sumitomo Dainippon Pharma Co.,Ltd.: Research Funding, Speakers Bureau; Janssen Pharmaceutical K.K.: Speakers Bureau; Shionogi & Co., Ltd: Consultancy, Honoraria; Otsuka Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; Astellas Pharma Inc.: Research Funding, Speakers Bureau; Takeda Pharmaceutical Company Limited.: Research Funding, Speakers Bureau; Yakult Honsha Company: Speakers Bureau; Pfizer Japan Inc.: Research Funding; Eisai Co., Ltd.: Research Funding, Speakers Bureau; Novartis Pharma K.K.: Research Funding.

Cage Transcriptome Analysis Reveals BCL2A1 Upregulation in FLT3-ITD/D835 Dual Mutated AML Cells Harboring Complex Co-Mutations

Cage Transcriptome Analysis Reveals BCL2A1 Upregulation in FLT3-ITD/D835 Dual Mutated AML Cells Harboring Complex Co-Mutations

Completion of BAX recruitment correlates with mitochondrial fission during apoptosis

BAX, a member of the BCL2 gene family, controls the committed step of the intrinsic apoptotic program. Mitochondrial fragmentation is a commonly observed feature of apoptosis, which occurs through the process of mitochondrial fission. BAX has consistently been associated with mitochondrial fission, yet how BAX participates in the process of mitochondrial fragmentation during apoptosis remains to be tested. Time-lapse imaging of BAX recruitment and mitochondrial fragmentation demonstrates that rapid mitochondrial fragmentation during apoptosis occurs after the complete recruitment of BAX to the mitochondrial outer membrane (MOM). The requirement of a fully functioning BAX protein for the fission process was demonstrated further in BAX/BAK-deficient HCT116 cells expressing a P168A mutant of BAX. The mutant performed fusion to restore the mitochondrial network. but was not demonstrably recruited to the MOM after apoptosis induction. Under these conditions, mitochondrial fragmentation was blocked. Additionally, we show that loss of the fission protein, dynamin-like protein 1 (DRP1), does not temporally affect the initiation time or rate of BAX recruitment, but does reduce the final level of BAX recruited to the MOM during the late phase of BAX recruitment. These correlative observations suggest a model where late-stage BAX oligomers play a functional part of the mitochondrial fragmentation machinery in apoptotic cells.

Deregulation of BCL2 family genes in glioblastoma cells consequent to poly(butyl cyanoacrylate) nanoparticles treatment

Brain tumours develop resistance to chemotherapy mainly due to the presence of the blood brain barrier which partially blocks the penetration of drugs into the brain. Poly(butyl cyanoacrylate) nanoparticles have been extensively proposed for delivering drugs into the brain and have shown their ability to cross the blood brain barrier in vitro and in vivo. However, the application of nanoparticles could be limited by their potential side effects. In this study, two glioblastoma cell lines with the poly(butyl cyanoacrylate) nanoparticles were treated at different concentrations. Through MTT assay, the viability of treated cells was quantified. Induction of apoptosis was analysed by flowcytometry as well as quantitative PCR assay for the expression of BCL2 family genes. The results of this study showed that the treatment with poly(butyl cyanoacrylate) nanoparticles induced neither apoptosis nor necrosis in these cells. The quantitative real-time PCR results showed that poly(butyl cyanoacrylate) nanoparticles treatment caused significant changes in the expression of some BCL2 family members. In conclusion, it seems that poly(butyl cyanoacrylate) nanoparticles treatment at non-toxic dose can change the expression of some apoptosis-related genes in glioblastoma cells while has no significant effect on their viability.

Temperature-induced testicular germ cell loss and recovery in Nile tilapia Oreochromis niloticus

Water temperature is a critical external factor influencing gonadal development in fish. This research aimed to study the impact of elevated temperature on testicular germ cell survival and reproductive capacity of Nile tilapia. Male Nile tilapia were exposed to high temperatures of either 36 (HT1) or 37 °C (HT2) for 3000 degree-days (DD) and thereafter returned to the control temperature of 27 °C (CT) for 2200 DD. The deleterious effects on testicular germ and somatic cells were observed histologically, characterised by vacuolisation, atrophy and the loss of spermatogenic cells in testes with a more severe impact of HT2 compared to HT1. Interestingly, serum 11-ketotestosterone (11-KT) and testosterone (T) levels tended to be higher during the heat treatments than CT. Expression levels of germline-specific genes piwil1, piwil2 and nanos2 and Bcl-2 family genes, bcl-xLb and baxa were significantly reduced during the heat treatment compared to CT, more so in the HT2, while the levels of nanos3 and gfra1 transcripts were only significantly reduced in HT2, implying a significant loss of spermatogonial stem cell (SSC) and spermatogonia in HT2. The effect of HT2 is further evidenced by the significantly reduced sperm density and fertilisation rate compared to CT and HT1 at the end of the recovery period but complete sterility was not induced by HT2. Overall, the present study showed significant effects of HT2 on germ cell survival with histological changes in testes, reduced milt quality, increased 11-KT, and decreased expression of germline-specific genes, SSC marker genes and Bcl-2 family genes in testes which could therefore be potential target genes for sterilisation by genome editing.