Prolonged Cell Survival Research Articles

Eukaryotic response to hypothermia in relation to integrated stress responses.

Eukaryotic cells respond to hypothermic stress through a series of regulatory mechanisms that preserve energy resources and prolong cell survival. These mechanisms include alterations in gene expression, attenuated global protein synthesis and changes in the lipid composition of the phospholipid bilayer. Cellular responses to hyperthermia, hypoxia, nutrient deprivation and oxidative stress have been comprehensively investigated, but studies of the cellular response to cold stress are more limited. Responses to cold stress are however of great importance both in the wild, where exposure to low and fluctuating environmental temperatures is common, and in medical and biotechnology settings where cells and tissues are frequently exposed to hypothermic stress and cryopreservation. This means that it is vitally important to understand how cells are impacted by low temperatures and by the decreases and subsequent increases in temperature associated with cold stress. Here, we review the ways in which eukaryotic cells respond to hypothermic stress and how these compare to the well-described and highly integrated stress response systems that govern the cellular response to other types of stress.

Intraperitonial/ Subcutaneous delivery of fit autologous Mesenchymal Stromal cells effectively cure DSS induced colitis

Background & Aim Background Bone marrow derived Mesenchymal stromal cells (BM-MSCs) with their excellent regenerative and immunomodulatory function, offer great potential for diverse clinical application. However, the success rate of advanced clinical trials has not been proven to be as promising as pre-clinical animal data in a wide array of disease models. Proper knowledge of the biological and pharmacological aspects of MSC based therapy is required to meet the medical demand of human clinical trials. AIM To execute a controlled, side-by-side comparison of the effects of delivery route, metabolic fitness and immune compatibility on dwell time and therapeutic efficacy of BM-MSC in mouse model of colitis. Methods, Results & Conclusion Methods/procedures Here we have compared the efficacy of 1) allogenic vs. autologous 2) metabolically active vs. inactive and 3) IP/IV/SC route of BM-MSC transplantation in a model of DSS-induced colitis mice, Finally, using in vivo optical imaging, we identified the most effective mode of BM-MSC application by tracking the bio-distribution and survival of bioluminescent BM-MSC. Main findings BM-MSC delivered by intraperitoneal or subcutaneous route is equivalently efficacious and superior than intravenous route of administration in reduction of colitis severity. Metabolically active BM-MSC supports prolonged cell survival as well as shows best biological activity. Immunocompetent colitis mice reject allogenic MHC-mismatched MSC. Conclusion Culture-rescued, metabolically active autologous BM-MSC achieves maximal potency if administered via IP or SC route into mouse model of colitis.

Stem Cell/Oxygen-Releasing Microparticle Enhances Erectile Function in a Cavernous Nerve Injury Model.

Erectile dysfunction caused by damage to the cavernous nerve is a common complication of radical prostatectomy for patients with localized prostate cancer. Various studies have investigated repair of damaged tissue and prevention of fibrosis in the corpus cavernosum using stem cell therapy. However, stem cell therapy has limitations, including insufficient nutrient and oxygen supply to transplanted stem cells. This study investigated whether stem cell/oxygen-releasing hollow microparticles (HPs) had therapeutic effect on erectile dysfunction in a rat model of bilateral cavernous nerve injury (BCNI). Therapeutic effects were observed in the BCNI model at 1, 2, and 4 weeks postcavernous nerve injury. Erectile function further improved after treatment with stem cell/oxygen-releasing HP system compared with treatment with only stem cells at 4 weeks. Stem cell/oxygen-releasing HP system increased cyclic guanosine monophosphate (cGMP) level and neuronal nitric oxide synthase (nNOS), endothelial nitric oxide synthase (eNOS), α-smooth muscle actin (α-SMA), and muscarinic acetylcholine receptor 3 (M3) expression while decreasing fibrosis and apoptosis in the corpus cavernosum. Our results clearly show that stem cell survival increases around transplanted stem cell/oxygen-releasing hybrid system site. Taken together, an oxygen-releasing HP system supported prolonged stem cell survival, sustaining the paracrine effect of the stem cells, and consequently enhancing erectile function. These findings show promise with regard to prolonged stem cell survival in stem cell applications for various diseases and types of tissue damage. Impact statement In this study, we used an oxygen-releasing hollow microparticles (HPs) system with stem cells to attempt to overcome certain limitations of stem cell therapy, including insufficient nutrient and oxygen supplies for transplanted stem cells. Our results demonstrated that a stem cell/oxygen-releasing HP hybrid system could further improve erectile function, cyclic guanosine monophosphate (cGMP) level, and NOS level in a bilateral cavernous nerve injury rat model through prolonged stem cell survival. Our data suggest that a stem cell/oxygen-releasing HP system is a promising clinical treatment option for postprostatectomy erectile dysfunction. Furthermore, this system may be relevant in different disease therapies and regenerative medicine.

The X-linked trichothiodystrophy-causing gene RNF113A links the spliceosome to cell survival upon DNA damage

Prolonged cell survival occurs through the expression of specific protein isoforms generated by alternate splicing of mRNA precursors in cancer cells. How alternate splicing regulates tumor development and resistance to targeted therapies in cancer remain poorly understood. Here we show that RNF113A, whose loss-of-function causes the X-linked trichothiodystrophy, is overexpressed in lung cancer and protects from Cisplatin-dependent cell death. RNF113A is a RNA-binding protein which regulates the splicing of multiple candidates involved in cell survival. RNF113A deficiency triggers cell death upon DNA damage through multiple mechanisms, including apoptosis via the destabilization of the prosurvival protein MCL-1, ferroptosis due to enhanced SAT1 expression, and increased production of ROS due to altered Noxa1 expression. RNF113A deficiency circumvents the resistance to Cisplatin and to BCL-2 inhibitors through the destabilization of MCL-1, which thus defines spliceosome inhibitors as a therapeutic approach to treat tumors showing acquired resistance to specific drugs due to MCL-1 stabilization.

Autophagy Machinery as a Promising Therapeutic Target in Endometrial Cancer.

Endometrial cancer is the fourth most frequent neoplasia for women worldwide, and over the past two decades it incidence has increased. The most common histological type of endometrial cancer is endometrioid adenocarcinoma, also known as type 1 endometrial cancer. Endometrioid endometrial cancer is associated with diverse epidemiological risk factors including estrogen use, obesity, diabetes, cigarette smoking, null parity, early menarche, and late menopause. Clinical effectiveness of chemotherapy is variable, indicating that novel molecular therapies against specific cellular processes associated to cell survival and resistance to therapy, such as autophagy, urged to ameliorate the rates of success in endometrial cancer treatment. Autophagy (also known as macroautophagy) is a specialized mechanism that maintains cell homeostasis which is activated in response to cellular stressors including nutrients deprivation, amino acids starvation, hypoxia, and metabolic stress to prolong cell survival via lysosomal degradation of cytoplasmic macromolecules and organelles. However, in human cancer cells, autophagy has a controversial function due to its dual role as self-protective or apoptotic. Conventional antitumor therapies including hormones, chemotherapy and ionizing radiation, may activate autophagy as a pro-survival tumor response contributing to treatment resistance. Intriguingly, if autophagy continues above reversibility of cell viability, autophagy can result in apoptosis of tumor cells. Here, we have reviewed the mechanisms of autophagy described in endometrial cancers, including the role of PI3K/AKT/mTOR, AMPK-mTOR, and p53 signaling pathways that trigger or inhibit the process and thus representing potential molecular targets in therapeutic clinical approaches. In addition, we discussed the recent findings indicating that autophagy can be modulated using repurposing drugs which may leads to faster experimentation and validation, as well as more easy access of the medications to patients. Finally, the promising role of dietary compounds and microRNAs in autophagy modulation is also discussed. In conclusion, although the research about autophagy is scarce but ongoing in endometrial cancer, the actual findings highlight the promising usefulness of novel molecules for directing targeted therapies.

Enhancing the Effect of CLL-1 CAR T Cells with Interleukin-15 for Treatment of Acute Myeloid Leukemia

Introduction: C-type lectin 1 (CLL-1, CD371) is highly expressed on the malignant cells from many patients with AML, and CAR T cells directed to this antigen can selectively target both leukemic progenitor cells (LSC) as well as AML blasts whilst sparing normal tissues. We previously showed (1) that such CAR-Ts can recognize and eliminate both AML blasts and primitive AML colony-forming cells in a low tumor-burden model. We have now modified the structure of the CLL-1 CAR and added transgenic expression of IL15 to enhance performance sufficiently for activity even against more extensive disease. Material and Methods: We assessed the phenotype and cytolytic ability of T cells transduced with 5 CLL-1 CAR constructs, varying in their spacer, transmembrane and costimulatory sequences (CD28z-CD8, CD28z-sh, CD28z-CH3, 4-1BBz-sh, 4-1BBz-CH3), and compared these with the effects of our published construct (4-1BBz-CD8)(1). We used flow cytometry to determine the effects of each construct on T cell phenotype and differentiation, and sequential (recursive) co-culture assays with tumor-cell targets to determine the durability of the anti-tumor activity. The most active constructs (CD28z-CD8 and 4-1BBz-CD8) were then evaluated in NOD.SCID IL-2Rg-/- (NSGS) mice engrafted with 1.5x10ˆ6 FFLuc-modified HL 60 AML cells, which received 2x10ˆ6 CLL-1 CAR T cells on day 3. To determine if we could further potentiate the in vivo expansion, persistence and anti-tumor activity of the CLL-1 CAR-T cells, we used a second retroviral vector to co-express transgenic IL15, measuring the effects in vitro and in vivo. Mice engrafted with 1.5x10ˆ6 tumor cells and received 2.5x10ˆ6 CLL-1 CAR T cells on week 3 in patient derived xenograft (PDX) model. We determined antitumor activity by bioluminescence imaging and weekly bleeding and measured serum cytokines by multiplex analysis (Luminex, TX). After euthanasia, we examined formalin-fixed/paraffin embedded sections. Results: Modified CLL-1 CAR constructs were expressed by 70-80% of cells irrespective of CAR sequence, but CD28z-CD8 CAR T cell expansion was significantly higher than CAR T cells with 4-1BBz endodomains (p<0.001), in part because of a higher death rate/lower viability in 4-1BBz cells (p<0.001). Consistent with these differences, both CD4 and CD8 T cell populations had more terminally differentiated cells (CCR7-CD45RA+) in CD28z versus 41BBz CAR T cells. In sequential co-culture assays against HL 60 (E:T=1:4) and THP-1 (E:T=1:4), CD28z-CD8 CAR T cells continued to expand well producing the greatest antitumor effect. In vivo models showed reduction in tumor signal in mice receiving either CD28z-CD8 CAR T or 4-1BBz-CD8 CAR T cells, but that only CD28z-CD8 CAR T cells prolonged survival (p<0.01). Nonetheless, all mice ultimately relapsed, usually with extramedullary disease, in association with limited CAR T persistence. We therefore incorporated transgenic IL15 as a "signal 3" for CD28z-CD8 CAR T cells, and determined the effects of forced IL15 expression on T cell phenotype, expansion, and antitumor activity in vitro and in vivo. In vitro, CD28z-CD8 CAR T cells with IL15 were less terminally differentiated and had superior expansion compared to CD28z-CD8 CAR T cells without IL15 (p<0.001). In both AML PDX and AML cell line animal models, CD28z-CD8 CAR T co-expressing transgenic IL15 initially (week 1) expanded better than CD28z-CD8 CAR T without IL15 (p<0.001) (Fig 1a), but produced severe acute toxicity associated with high level production of human IL15, TNF alpha and IFN gamma (Fig 1b). Histopathology showed marked inflammatory changes with tissue damage in lung and liver. This acute toxicity could be managed by 2 strategies, individually or in combination. The excessive TNF alpha secretion could be blocked with anti-TNF alpha antibody (1mg/kg/mouse) (BioLegend, CA USA) weekly, while excessive T cell expansion could be arrested by activation of an inducible caspase 9 safety switch by administration of dimerizing drug (2). Both strategies successfully prolonged tumor free survival (Fig 2,b). Conclusion: Addition of transgenic IL15 to CLL-1-CD28z-CD8 CAR augmented activity against AML in a range of cell line and PDX models, and toxicity associated with exuberant CART expansion could be prevented by cytokine blockade and/or an inducible safety switch.

Mesenchymal Stromal Cells from Patients with Cyanotic Congenital Heart Disease are Optimal Candidate for Cardiac Tissue Engineering

Engineered heart tissues (EHTs) are regarded as being the most promising alternative to synthetic materials, and autologous mesenchymal stem cells (MSCs) are widely used as seeding cells. However, few studies have evaluated the feasibility of using MSCs from patients with cyanotic congenital heart disease (C-CHD) as seeding cells for EHTs, in comparison with cells from patients of acyanotic congenital heart disease (A-CHD). In the present study, we cultured MSCs from A-CHD and C-CHD patients in normoxia or hypoxia conditions, and compared their pro-angiogenic, anti-apoptotic and inflammation-modulatory potentials. In vivo, we seeded the cells into collagen patches conjugated with, or without, proangiogenic cytokines, which were used to repair the right ventricular outflow tract (RVOT) of rats. The in vitro results showed that C-CHD MSCs expressed higher levels of VEGFA and VEGFR2, and secreted more pro-angiogenic and anti-inflammatory cytokines under hypoxic conditions. On the other hand, apoptosis-related genes from C-CHD MSCs were modulated adaptably, converting these cells into an anti-apoptotic phenotype. In vivo studies demonstrated that in 4 weeks after RVOT reconstruction, cytokine-immobilized patches seeded with C-CHD MSCs exhibited preserved morphology, prolonged cell survival and enhanced angiogenesis compared to A-CHD MSCs. C-CHD MSCs that undergo “naturally hypoxic precondition” present a better cell source for EHTs, which would provide a promising individualized biomaterial for C-CHD patients.

Melatonin Enhances Mitophagy by Upregulating Expression of Heat Shock 70 kDa Protein 1L in Human Mesenchymal Stem Cells under Oxidative Stress.

Human mesenchymal stem cells (hMSCs) are a potent source of cell-based regenerative therapeutics used to treat patients with ischemic disease. However, disease-induced oxidative stress disrupts mitochondrial homeostasis in transplanted hMSCs, resulting in hMSC apoptosis and reducing their efficacy post-transplantation. To address this issue, we evaluated the effects of melatonin on cellular defense mechanisms and mitophagy in hMSCs subjected to oxidative stress. H2O2-induced oxidative stress increases the levels of reactive oxygen species and reduces membrane potential in hMSCs, leading to mitochondrial dysfunction and cell death. Oxidative stress also decreases the expression of 70-kDa heat shock protein 1L (HSPA1L), a molecular chaperone that assists in the recruitment of parkin to the autophagosomal mitochondrial membrane. Decreased expression of HSPA1L destabilizes parkin, thereby impairing mitophagy. Our results indicate that treating hMSCs with melatonin significantly inhibited mitochondrial dysfunction induced by oxidative stress, which decreased hMSCs apoptosis. In damaged hMSCs, treatment with melatonin increased the levels of HSPA1L, which bound to parkin. The interaction between HSPA1L and parkin increased membrane potential and levels of oxidative phosphorylation, resulting in enhanced mitophagy. Our results indicate that melatonin increased the expression of HSPA1L, thereby upregulating mitophagy and prolonging cell survival under conditions of oxidative stress. In this study, we have shown that melatonin, a readily available compound, can be used to improve hMSC-based therapies for patients with pathologic conditions involving oxidative stress.

Inhibiting Autophagy in Renal Cell Cancer and the Associated Tumor Endothelium.

The clear cell subtype of kidney cancer encompasses most renal cell carcinoma cases and is associated with the loss of von Hippel-Lindau gene function or expression. Subsequent loss or mutation of the other allele influences cellular stress responses involving nutrient and hypoxia sensing. Autophagy is an important regulatory process promoting the disposal of unnecessary or degraded cellular components, tightly linked to almost all cellular processes. Organelles and proteins that become damaged or that are no longer needed in the cell are sequestered and digested in autophagosomes upon fusing with lysosomes, or alternatively, released via vesicular exocytosis. Tumor development tends to disrupt the regulation of the balance between this process and apoptosis, permitting prolonged cell survival and increased replication. Completed trials of autophagic inhibitors using hydroxychloroquine in combination with other anticancer agents including rapalogues and high-dose interleukin 2 have now been reported. The complex nature of autophagy and the unique biology of clear cell renal cell carcinoma warrant further understanding to better develop the next generation of relevant anticancer agents.

124 Characterization of the cooperative role of co-amplified PIK3Cα and ΔNp63α in squamous tumor initiation and progression

The chromosome region between 3q26 and 3q28, which includes proto-oncogenes PIK3CA and TP63, is frequently amplified in a spectrum of squamous cell carcinomas. Using primary murine epidermal keratinocytes and an orthotopic nude mouse grafting model, our lab has previously established that elevated ΔNp63α cooperates with the oncogenic H-Ras pathway to drive malignant progression of H-Ras-initiated tumors. The PI3K pathway operates downstream of Ras and is often activated in cancers by amplification of the wild-type PIK3Cα gene (wt) or by gain-of-function mutation (H1047R or E545K, mt). We asked whether activation of PI3K pathway by overexpressing wt or mt PIK3Cα is sufficient to initiate tumorigenesis and/or cooperate with ΔNp63α to drive malignant progression. H-Ras and wt or mt PIK3Cα were overexpressed individually and in combination with ΔNp63α or vector control in primary mouse keratinocytes by lentiviral gene transduction. The expression of wt or mt PIK3Cα activated downstream target AKT, indicated by the phosphorylation of Ser473 and Thr308 residues. Transduction of PIK3Cα (wt or mt), did not activate Ras/MEK/ERK pathway, and did not induce the typical senescent phenotype observed with H-Ras expression in primary keratinocytes, suggesting that PI3K pathway is not responsible for oncogene induced senescence in this context. Lentiviral driven PIK3C α expression in primary keratinocytes enhanced BrdU incorporation and prolonged cell survival but was not sufficient to support cell passaging. In preliminary in vivo grafting studies in athymic nude mice, keratinocytes transduced with PIK3Cα (wt or mt) did not form tumors or cooperate with ΔNp63α in tumor progression, as is seen with Ras. This multistep model allows for the assessment of additional genes, both co-amplified (e.g. SOX2 and ACTL6A) and on other chromosomes, for their contribution to squamous tumor initiation and progression.

MON-466 Transcriptome Dynamics of in vitro Human Pituitary Tumor Primary Culture

Lack of an immortalized or long-term hormone-secreting human pituitary tumor in vitro cell line has significantly hampered studies of etiopathogenesis and development of targeted therapies for pituitary tumors. Various media, vessels, substrates and additives have been explored to optimize primary cultures. Some methods have prolonged cell survival time but maintaining hormone secretion is still a major challenge. We have recently developed methodology to maintain robust hormone secretion in human prolactinoma, somatotroph and corticotroph tumors in vitro for up to 9 months. To gain insight into the changes in gene expression profiles that occurred prior to/coincident with loss of hormone secretion during in vitro human pituitary tumor cultures, we compared the global transcriptiome landscape in serial passages from two human prolactinoma primary cultures. We obtained 27.1 million quality filtered reads per sample of which ~90% were exonic reads detecting an average of 16,000 genes. 3D principal component analysis identified 3 distinct clusters, with close similarity between cell passages 1-2, those beyond passage 4-5 and a stand-alone passage 3. Analysis of differential gene expression (DEG) over time using DAVID knowledgebase and hierarchical clustering demonstrated that most changes in gene expression occurred in the first 1-2 weeks in in vitro culture. During the first week of in vitro culture, although the cells maintained robust PRL secretion, reduced activation of angiogenesis and growth signal pathways (PI-3K, MAPK pathways, cell adhesion, GTPase activity, cell proliferation and cell-cell junction assembly) was observed. In the following 1-2 weeks, the cell population maintained PRL secretion but exhibited continued loss of survival signals, with increased expression of extracellular matrix (ECM) and surface adhesion molecules consistent with development of a fibroblastic phenotype that dominated later stage cultures. Based on our findings, we propose that the molecular events underpinning loss of hormone production in primary pituitary tumor cultures represent transition from endocrine cells to pituitary specific fibroblastic folliculo-stellate cell likely selected by the in vitro culture conditions. Our data further reveal that underexpression of several GPCRs and resultant downregulation of the PKA pathway may lead to loss of hormone production in parallel with loss of cell survival signals. In summary, our study defines a temporal progressive transcriptome transition signature to explain loss of hormone secretion and morphological alterations observed during human pituitary tumor in vitro culture and may provide molecular targets to further optimize our culture methodology to attain “immortalized” hormone-producing human pituitary tumor cell-lines.

Short Variant of Mitochondrial Dynamin OPA1 Renders Improved Cell Survival under Stress Conditions

The protein optic atrophy 1 (OPA1) is a dynamin‐related membrane remodeling protein associated with the inner mitochondrial membrane. OPA1 is a critical factor for mitochondrial structure and function by mediating mitochondrial inner membrane fusion and maintaining cristae structure. Under cellular stress associated with mitochondrial dysfunction, the inner membrane‐anchored long OPA1 (L‐OPA1) undergoes proteolytic cleavage to form short soluble OPA1 (S‐OPA1) lacking the transmembrane domain. The prevalent thought has been that S‐OPA1 is a functionally insignificant proteolytic product of L‐OPA1. Hence, the loss of L‐OPA1 and accumulation of S‐OPA1 by OPA1 cleavage under stress conditions is thought to be detrimental to cell survival. However, we recently demonstrated that S‐OPA1 is fully competent for maintaining energetics and cristae structure. Therefore, in this study, we used cells exclusively expressing L‐, S‐OPA1, or both, and tested the capacity of L‐ and S‐OPA1 in supporting cell survival under stress conditions. Under hydrogen peroxide‐mediated oxidant stress, we found that S‐OPA1‐expressing cells have better survival than L‐OPA1 cells. Interestingly, this difference in cell survival under oxidant stress was only observed in glucose‐free OXPHOS media but not in the presence of glucose, suggesting that S‐OPA1 may confer higher mitochondrial energetic activity compared with L‐OPA1. We also observed better survival of S‐OPA1‐expressing cells under nutrient starvation. Under apoptotic stress by treating cells with the transcription inhibitor actinomycin D, S‐OPA1 cells showed greatly decreased cytochrome c release compared to L‐OPA1 cells. These observations suggest that S‐OPA1 is more protective during apoptosis than L‐OPA1. Mechanistically, we found that S‐OPA1 cells have higher mitochondrial electron transport activity in nutrient depletion. Furthermore, by testing mitochondrial calcium retention capacity, we found that S‐OPA1 cells show increased resistance to mitochondrial permeability transition induced by mitochondrial calcium overload, compared with L‐OPA1 only cells. These data indicate that S‐OPA1 confers more efficient energy metabolism and more resistance to mitochondrial stress, allowing prolonged cell survival under stress.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Natural Killer Cells Integrate Signals Received from Tumour Interactions and IL2 to Induce Robust and Prolonged Anti-Tumour and Metabolic Responses.

Natural Killer (NK) cells are lymphocytes with an important role in anti-tumour responses. NK cells bridge the innate and adaptive arms of the immune system; they are primed for immediate anti-tumour function but can also have prolonged actions alongside the adaptive T cell response. However, the key signals and cellular processes that are required for extended NK cell responses are not fully known. Herein we show that murine NK cell interaction with tumour cells induces the expression of CD25, the high affinity IL2 receptor, rendering these NK cells highly sensitive to the T cell-derived cytokine IL2. In response to IL2, CD25high NK cells show robust increases in metabolic signalling pathways (mTORC1, cMyc), nutrient transporter expression (CD71, CD98), cellular growth and in NK cell effector functions (IFNγ, granzyme B). Specific ligation of an individual activating NK cell receptor, NK1.1, showed similar increases in CD25 expression and IL2-induced responses. NK cell receptor ligation and IL2 collaborate to induce mTORC1/cMyc signalling leading to high rates of glycolysis and oxidative phosphorylation (OXPHOS) and prolonged NK cell survival. Disrupting mTORC1 and cMyc signalling in CD25high tumour interacting NK cells prevents IL2-induced cell growth and function and compromises NK cell viability. This study reveals that tumour cell interactions and T cell-derived IL2 cooperate to promote robust and prolonged NK cell anti-tumour metabolic responses.

Bcl-2 expression in reactive oral lesions with atypical epithelium and in oral epithelial dysplasia associated with carcinogen exposure.

Background:Reactive lesions of the oral cavity are nonneoplastic proliferations with very similar appearance to benign neoplastic lesions and are associated with chronic local irritation or trauma. Although these lesions are uncommonly associated with carcinogen exposure, at times, they present histopathologically with dysplastic epithelium, thus making it difficult to differentiate it from true potentially malignant disorders. Hence, the present study was conducted to evaluate the expression of Bcl-2 protein, an antiapoptotic marker, in reactive lesions with and without atypical epithelium and in true epithelial dysplasia, which clinically presents as premalignant disorders.Materials and Methods:The samples included 15 cases each of normal oral mucosa (NOM), reactive lesions with and without dysplasia and oral epithelial dysplasia (OED) associated with carcinogen exposure. All the samples were subjected to immunohistochemical staining using Bcl-2 antibody. The total number of cells in the basal and parabasal layers in each field and total number of cells expressing Bcl-2 among them and the staining intensity were assessed.Statistical Analysis:Kruskal–Wallis ANOVA test was used to compare the number of positive cells among the four groups. The comparison of average percentage of positive cells between the study groups was done using Mann–Whitney U-test.Results:The immunohistochemical staining for Bcl-2 protein was identified in few cells in the basal layers of NOM, reactive lesions without atypical epithelium and in the basal and parabasal layers in reactive lesions with atypical epithelium and OED, as a granular cytoplasmic staining and as an accentuation around the nuclear membrane. There was a gradual increase in the expression and intensity of staining from Group I to IV.Interpretation and Conclusion:The altered or increased expression of Bcl-2 oncoprotein in reactive lesions with atypical epithelium and in OED with carcinogen exposure may lead to prolonged cell survival and can be considered as an early molecular event in carcinogenesis, helping us in understanding the nature of dysplasia in reactive lesions, which was not considered during the histopathology reporting.

STAT6 degradation and ubiquitylated TRIML2 are essential for activation of human oncogenic herpesvirus.

Aberrations in STAT6-mediated signaling are linked to the development of multiple cancer types. Increasing evidence has shown that activation of human oncogenic herpesvirus lytic replication is crucial for viral tumorigenesis. However, the role of STAT6 in herpesvirus lytic replication remains elusive. Here, by using Kaposi’s sarcoma-associated herpesvirus (KSHV) as a model, we revealed that RTA, the master regulator of lytic replication, interacts with STAT6 and promotes lysine 48 (K48) and K63-linked ubiquitylation of STAT6 for degradation via the proteasome and lysosome systems. Moreover, degradation of STAT6 is dramatically associated with the increased ubiquitylated form of tripartite motif family like 2 (TRIML2, a tumor suppressor) for prolonged cell survival and virion production, which is also commonly observed in lytic activation of Epstein-Barr virus, herpes simplex virus 1 and cytomegalovirus. These results suggest that degradation of STAT6 is important for the lytic activation of KSHV and as such, may be an attractive therapeutic target.

Engineering Protective Polymer Coatings for Liver Microtissues.

Three-dimensional (3D) hepatocyte microtissues (MT), also known as spheroids, have proven to be advantageous in providing more accurate information and physiologically relevant and predictive data for liver-related in vivo tests; therefore, spheroids have increasingly been used to study hepatotoxicity, drug delivery to the liver, and tissue engineering. However, variabilities in the generation of 3D MT remain a major challenge. Methods that encapsulate and protect hepatocytes offer a promising pathway in prolonging cell survival, as well as maintaining its liver cell functions. Herein, we studied the encapsulation and resultant protective effects of hydrogen bonded, biocompatible polymer coatings for hepatocyte MT in 3D cell culture. We exposed the MT to hepatotoxic nanomaterials (NMs), such as graphene oxide (GO) and cobalt oxide (Co3O4), to assess the protective effects of poly(vinylpyrrolidone) (PVPON) and tannic acid (TA) coatings. The polymer coating allowed the MT to maintain its morphology. More significantly, it increased the viability of hepatocyte-composed MT by hampering the cellular interaction between hostile NMs and hepatocytes. Based on alanine transaminase (ALT) and aspartate aminotransferase (AST) levels, the liver cell function was maintained throughout the coating process, including after NM treatment. The study provides a straightforward and safe methodology for maintaining the morphology as well as cellular function of hepatocyte MT in vitro.

Calcium phosphate buffer formed in the mitochondrial matrix during preconditioning supports ΔpH formation and ischemic ATP production and prolongs cell survival –A hypothesis

Ischemic preconditioning makes cells less sensitive to oxygen deprivation. A similar effect can be achieved by increasing the calcium concentration and applying potassium channel openers. A hypothetical mechanism of preconditioning is presented. In the mitochondrial matrix, there is a calcium hydroxide buffer consisting of a few insoluble calcium phosphate minerals. During ischemia, calcium ions stored in the matrix buffer start to leak out, forming an electric potential difference, while hydroxyl ions remain in the matrix, maintaining its pH and the matrix volume. Preconditioning factors increase the matrix buffer capacity. Production of ATP during ischemia might be the relic of a pre-endosymbiotic past.

Adipose-derived stem cells-conditioned medium improved osteogenic differentiation of induced pluripotent stem cells when grown on polycaprolactone nanofibers.

Considering that the common osteogenic growth factors cannot be transplanted with stem cells to the patients, many studies are underway to find a replacement for these factors. Recently, it has been determined that mesenchymal stem cell (MSC)-derived conditioned medium (CM) contains effective factors in the bone formation process. In the current study, the synergistic effect of adipose-derived MSC's CM, and polycaprolactone (PCL) scaffold was investigated on the osteogenic differentiation potential of human induced pluripotent stem cells (iPSCs). After scaffold fabrication by electrospinning and characterization by scanning electron microscopy, iPSCs proliferation in the presence of CM, PCL, and both was evaluated using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide. Then, iPSCs osteogenic differentiation was investigated while cultured on tissue culture plate and PCL under CM compared with the osteogenic medium using alizarin red staining, calcium content, alkaline phosphatase activity and gene and protein expression analysis. Proliferation rate of the iPSCs was increased while cultured under CM and its effect was synergistically enhanced by culture on PCL. Evaluation of the osteogenic markers was showed CM alone could induce osteogenic differentiation into the iPSCs and this potential was significantly increased while combined with PCL nanofibrous scaffold. According to the results, it was demonstrated that CM has an osteogenic induction property almost the same of the common osteogenic medium and it can also be used potentially with stem cells when transplant to the patients. CM can also help by prolonging cell survival at the site of the defect as well as accelerating healing process.

Advancement in Stem Cell Therapy for Ischemic Myocardial Cell: A Systematic Review

Background: Cardiac muscle possesses a limited capacity to regenerate its tissue on its own. It is less likely to reverse the altered cardiac functioning to its normal physiological state after a major myocardial infarction. Stem cell transplantation provided a unique therapeutic approach in managing such injuries. There has been a substantial debate about the complexity, scope and medical application of stem cell transplantation in past few years. Materials and Methods: An extensive review of medical literature was conducted to establish the consensus about the possible mechanism of cell renewal, associated complications and risks of failure of this technique. Twenty cases of mammalian animals and twenty-four cases of stem cell transplantation in human subjects were reviewed. Results: Most common associated complication was re-stenosis of coronary artery. Few clinical trials reported the failure in improving cardiac functioning. The success rate of stem cell transplantation was remarkable in the literature related to experimental animal subjects. Conclusion: It was concluded that renewal of the cardiac cell is a result of induction of angiogenesis and prolonged cell survival. This topic still requires an immense amount of research to fill the gap in adequate knowledge.

Cell survival regulation during receptor-mediated endocytosis of chemically-modified lipoproteins associated to the formation of an Amphiphysin 2 (Bin1)/c-Myc complex

Amphiphysin 2 and members of the BAR-domain family of proteins participate in a wide array of cellular processes including cell cycle and endocytosis. Given that amphiphysin 2 is related to diverse cell responses as a result of metabolic stress, we investigated in macrophages whether oxidative stress originated by the internalization of oxidized low density lipoproteins (oxLDL) affect both, the expression of amphiphysin 2 and its binding partner c-Myc. Here we report that under oxidative stress, a complex formation between amphiphysin 2(Bin1) and c-Myc allows the cell to develop a novel survival equilibrium state established between cell proliferation and cell death. We propose that under conditions of oxidative stress given by the internalization of oxLDL, macrophages employ the formation of the amphiphysin 2(Bin1)/c-Myc complex as a control mechanism to initially avoid the process of cell death in an attempt to prolong cell survival.