Low Levels Of Apoptosis Research Articles

Transcriptomic profiling and pathway analysis of cultured human lung microvascular endothelial cells following ionizing radiation exposure

The vascular system is sensitive to radiation injury, and vascular damage is believed to play a key role in delayed tissue injury such as pulmonary fibrosis. However, the response of endothelial cells to radiation is not completely understood. We examined the response of primary human lung microvascular endothelial cells (HLMVEC) to 10 Gy (1.15 Gy/min) X-irradiation. HLMVEC underwent senescence (80–85%) with no significant necrosis or apoptosis. Targeted RT-qPCR showed increased expression of genes CDKN1A and MDM2 (10–120 min). Western blotting showed upregulation of p2/waf1, MDM2, ATM, and Akt phosphorylation (15 min–72 h). Low levels of apoptosis at 24–72 h were identified using nuclear morphology. To identify novel pathway regulation, RNA-seq was performed on mRNA using time points from 2 to 24 h post-irradiation. Gene ontology and pathway analysis revealed increased cell cycle inhibition, DNA damage response, pro- and anti- apoptosis, and pro-senescence gene expression. Based on published literature on inflammation and endothelial-to-mesenchymal transition (EndMT) pathway genes, we identified increased expression of pro-inflammatory genes and EndMT-associated genes by 24 h. Together our data reveal a time course of integrated gene expression and protein activation leading from early DNA damage response and cell cycle arrest to senescence, pro-inflammatory gene expression, and endothelial-to-mesenchymal transition.

Hyperosmotic Stress Induces a Specific Pattern for Stress Granule Formation in Human-Induced Pluripotent Stem Cells.

Stress granules (SGs) are assemblies of selective messenger RNAs (mRNAs), translation factors, and RNA-binding proteins in small untranslated messenger ribonucleoprotein (mRNP) complexes in the cytoplasm. Evidence indicates that different types of cells have shown different mechanisms to respond to stress and the formation of SGs. In the present work, we investigated how human-induced pluripotent stem cells (hiPSCs/IMR90-1) overcome hyperosmotic stress compared to a cell line that does not harbor pluripotent characteristics (SH-SY5Y cell line). Gradient concentrations of NaCl showed a different pattern of SG formation between hiPSCs/IMR90-1 and the nonpluripotent cell line SH-SY5Y. Other pluripotent stem cell lines (hiPSCs/CRTD5 and hESCs/H9 (human embryonic stem cell line)) as well as nonpluripotent cell lines (BHK-21 and MCF-7) were used to confirm this phenomenon. Moreover, the formation of hyperosmotic SGs in hiPSCs/IMR90-1 was independent of eIF2α phosphorylation and was associated with low apoptosis levels. In addition, a comprehensive proteomics analysis was performed to identify proteins involved in regulating this specific pattern of hyperosmotic SG formation in hiPSCs/IMR90-1. We found possible implications of microtubule organization on the response to hyperosmotic stress in hiPSCs/IMR90-1. We have also unveiled a reduced expression of tubulin that may protect cells against hyperosmolarity stress while inhibiting SG formation without affecting stem cell self-renewal and pluripotency. Our observations may provide a possible cellular mechanism to better understand SG dynamics in pluripotent stem cells.

Expression of miR-34a and miR-15b during the progression of cervical cancer in a murine model expressing the HPV16 E7 oncoprotein.

The high-risk human papillomavirus (HR-HPV) E7 oncoprotein appears to be a major determinant for cell immortalization and transformation altering critical processes such as cell proliferation, apoptosis, and immune response. This oncoprotein plays an essential role in cervical carcinogenesis, but other cofactors such as long-term use of hormonal contraceptives are necessary to modulate the risk of cervical cancer (CC). The role of HR-HPVs in the alteration of microRNA (miRNA) levels in persistent viral infections currently remains unclear. The aim of this study was to evaluate the miR-34a and miR-15b expression levels in the murine HPV16K14E7 (K14E7) transgenic model after chronic estrogen (E2) treatment and their involvement in CC. Interestingly, results showed that, although miR-34a expression is elevated by the HPVE7 oncogene, this expression was downregulated in the presence of both the E7 oncoprotein and chronic E2 in cervical carcinoma. On the other hand, miR-15b expression was upregulated along cervical carcinogenesis mainly by the effect of E2. These different changes in the expression levels of miR-34a and miR-15b along cervical carcinogenesis conduced to low apoptosis levels, high cell proliferation and finally, to cancerous cervical tissue development. In this work, we also determined the relative mRNA expression of Cyclin E2 (Ccne2), Cyclin A2 (Ccna2), and B cell lymphoma 2 (Bcl-2) (target genes of miR-34a and miR-15b); Sirtuin 1 (Sirt1), Cmyc, and Bax (miR-34a target genes); and p21/WAF1 (mir15b target gene) and the H-ras oncogene. Given the modifications in the expression levels of miR-34a and miR-15b during the development of cervical cancer, it will be useful to carry out further investigation to confirm them as molecular biomarkers of cancer.

Heterogeneity of neutrophils in arterial hypertension

Cellular heterogeneity and diversity are recognized to contribute to the functions of neutrophils under homeostatic and pathological conditions. We previously suggested that the chronic inflammatory responses associated with hypertension (HTN) are related to the participation of different subpopulations of neutrophils. Two populations of neutrophils can be obtained by density gradient centrifugation: normal-density neutrophils (NDN) and low-density neutrophils (LDN). However, the lack of standardized functional protocols has limited phenotypic characterization and functional comparisons of LDN and NDN.Based on their capability to incorporate Na+, maturity and activation stage, we characterized NDN and LDN in blood samples from ten patients with HTN and ten healthy individuals (HI) using flow cytometry. We compared the levels of reactive oxygen species (ROS), generation of neutrophil extracellular traps (NETs) and levels of apoptosis in NDN and LDN. In general, the NDN and LDN subpopulations from patients with HTN exhibited higher levels of sodium influx and ROS, and lower levels of apoptosis than the corresponding NDN and LDN subsets from HI. Transmission electron microscopy revealed NDN and LDN from patients with HTN exhibited alterations to mitochondrial morphology and fewer cytoplasmic granules than the corresponding HI subpopulations.Our results indicate both the NDN and LDN subpopulations enhance the effects of inflammation that contribute to the pathophysiology of HTN. Further detailed studies are required to characterize the events during ontogeny of the myeloid lineage that result in the diverse phenotypic characteristics of each subpopulation of LDN and NDN.

Computational model of cardiomyocyte apoptosis identifies mechanisms of tyrosine kinase inhibitor-induced cardiotoxicity

Despite clinical observations of cardiotoxicity among cancer patients treated with tyrosine kinase inhibitors (TKIs), the molecular mechanisms by which these drugs affect the heart remain largely unknown. Mechanistic understanding of TKI-induced cardiotoxicity has been limited in part due to the complexity of tyrosine kinase signaling pathways and the multi-targeted nature of many of these drugs. TKI treatment has been associated with reactive oxygen species generation, mitochondrial dysfunction, and apoptosis in cardiomyocytes. To gain insight into the mechanisms mediating TKI-induced cardiotoxicity, this study constructs and validates a computational model of cardiomyocyte apoptosis, integrating intrinsic apoptotic and tyrosine kinase signaling pathways. The model predicts high levels of apoptosis in response to sorafenib, sunitinib, ponatinib, trastuzumab, and gefitinib, and lower levels of apoptosis in response to nilotinib and erlotinib, with the highest level of apoptosis induced by sorafenib. Knockdown simulations identified AP1, ASK1, JNK, MEK47, p53, and ROS as positive functional regulators of sorafenib-induced apoptosis of cardiomyocytes. Overexpression simulations identified Akt, IGF1, PDK1, and PI3K among the negative functional regulators of sorafenib-induced cardiomyocyte apoptosis. A combinatorial screen of the positive and negative regulators of sorafenib-induced apoptosis revealed ROS knockdown coupled with overexpression of FLT3, FGFR, PDGFR, VEGFR, or KIT as a particularly potent combination in reducing sorafenib-induced apoptosis. Network simulations of combinatorial treatment with sorafenib and the antioxidant N-acetyl cysteine (NAC) suggest that NAC may protect cardiomyocytes from sorafenib-induced apoptosis.

Genetically Based Physiological Responses to Overwinter Starvation in Gibel Carp (Carassius gibelio).

Normally, fish will decrease food intake or even stop feeding during the winter. In previous studies, two widely cultured gibel carp strains (strain A and strain F) showed differences in lipid and glucose metabolism. Therefore, we hypothesized that the physiological changes during the overwintering period would be different between the two strains. Thus, the two strains were starved for 77 days, after which the levels of glucose and lipid metabolism, ER stress, autophagy, and apoptosis were determined. The starvation increased hepatic glycogenolysis and fatty acid β-oxidation but suppressed lipogenesis in both strains overwintering. Considering the effects of genotype, strain F had higher levels of ER stress and autophagy but lower levels of apoptosis than strain A, suggesting that strain F might be more resistant to overwintering starvation. The interactions between strains and starvation periods were observed in plasma triglyceride contents and the mRNA levels of pyruvate kinase (pk), sterol regulatory element binding protein 1 (srebp1), activating transcription factor 4 (atf4), and autophagy protein 12 (atg12). In conclusion, long-term starvation during winter could induce hepatic glycogenolysis and fatty acid β-oxidation but suppress lipogenesis, ER stress, autophagy, and apoptosis in gibel carp, and strain F may be more resistant to starvation during winter. Taken together, these results discovered the responses to prolonged starvation stress during winter in two strains of gibel carp and could provide information for genotype selection, especially for selecting strains better adapted to winter.

AKT, ERK and NFKB Signaling Pathways in Neurons of Hypothalamic Supraoptic and Paraventricular Nuclei in Aging Transgenic Mice with HER2/neu Overexpression

Epidermal growth factor receptors are widely expressed in various tissues, including the brain, and play an important role in the regulation of such biological processes as proliferation, cell migration and death, etc. This study aimed to study the involvement of the cell survival-related signaling pathways (AKT, ERK, NF-kB cascades) in the regulation of hypothalamic neuronal apoptosis in experimental models of physiological and pathological (HER2/neu overexpression) aging, as well as the effect of FASL and TNF-alpha cytokines on these signaling cascades. Experiments were carried out on HER2/neu transgenic mice with accelerated senescence compared to the wild type mice (FVB/N strain). In the hypothalamic supraoptic and paraventricular nuclei, expression of the anti-apoptotic protein survivin was determined by immunohistochemistry, while expression of proteins of the cytokine-dependent cascades (FASL, TNF-alpha) and protein members of the cell survival-related signaling pathways (phosphorylated AKT, ERK, NF-kB) was assayed by Western blotting. It was found that in FVB/N mice the AKT and ERK cascades do not contribute significantly to neuronal senescence, whereas activation of FAS signaling and a decrease in the NF-kB pathway activity can lead to an increased proportion of dying neurons. In HER2/neu mice, neuronal resistance to apoptosis during aging is ensured by a combination of different cell survival-related signaling pathways: the ERK cascade in the supraoptic nucleus and the NF-kB and AKT cascades in the paraventricular nucleus. In addition, a suppression of FAS signaling was revealed, also leading to a low level of apoptosis.

Analysis of proliferation and apoptosis, MUC-1 and Cdx-2 expression in distal colonic polyps

Background. The universal molecular characteristics of malignancy are activation of cell proliferation and inactivation of apoptotic cell death. Besides genes-regulators of the processes other molecules are involved, including mucins and Cdx-2. Aim . To conduct comparative and correlation analysis of indexes of immunohistochemical expression of Ki-67, p53, caspase-3, as well as MUC-1 and Cdx-2 in distal colonic polyps. Methods. Pathohistological and immunohistochemical studies of biopsies of adenomas and hyperplastic polyps from 40 patients were carried out. Results. Distal colonic polyps are characterized by medium proliferation level: the median of Ki-67 expression for hyperplastic polyps is 42,20 (35,27; 52,38) %, while the median for adenomas is 65,39 (52,28; 76,23) % (р˂0,05). The polyps are characterized by low p53 expression levels: the median of the marker expression for hyperplastic polyps is 1,53 (1,17; 3,16) %, for adenomas – 9,15 (8,26; 12,39) % (р˂0,05). The studied polyps are characterized by low apoptosis levels: the median of caspase-3 expression for hyperplastic polyps is 33,78 (30,28; 42,34) CUOD, for adenomas – 22,19 (16,84; 40,51) CUOD (р˂0,05). Distal colonic polyps are characterized by medium MUC-1 and Cdx-2 expression levels: the medians of the markers expression for hyperplastic polyps are 62,13 (34,20; 71,85) CUOD and 71,81 (62,36; 77,77) CUOD, for adenomas – 80,25 (52,14; 95,21) CUOD and 95,14 (81,19; 112,17) CUOD, respectively (р˂0,05). The correlation analysis revealed several correlations: reverse medium strength correlation between p53 and caspase-3 expression levels (r = -0,57, р˂0,05), direct medium strength correlation between caspase-3 and MUC-1 expression levels (r = 0,52, p˂0,05) take place in hyperplastic polyps, whereas reverse strong correlation between caspase-3 and MUC-1 expression levels 1 (r = -0,88, p˂0,05) takes place in adenomas. Conclusions. Distal colonic adenomas differ by higher proliferation and p53 expression levels, comparing to hyperplastic polyps, and by lower apoptosis level that is associated with higher MUC-1 expression level as well. Hyperplastic polyps differ by higher apoptosis level that is associated with lower MUC-1 expression level. Cdx-2 expression increases on benign adenoma stage and does not correlate with proliferation and apoptosis indexes.

In ovo culturing of turkey (Meleagris gallopavo) ovarian tissue to assess graft viability and maturation of prefollicular germ cells and follicles

Biobanking of turkey ovarian tissue appears to be the most cost-effective method for the long-term preservation of female genetics. However, to ensure the successful transplantation of biobanked ovarian tissue for breed or line revival, the transplantation and development of fresh ovarian tissue must be evaluated. To assess transplantability, ovaries from poults 1 to 15 days posthatch (dph) were cultured in ovo in chicken eggs for 6 d and compared with the equivalent fresh tissue. The viability of cultured ovarian tissue was evaluated visually, whereas the level of late-stage apoptosis was measured via the TUNEL assay. In addition, the diameter and density of prefollicular germ cells and follicles (primordial and primary) were measured to assess maturation. Results showed that all cultured grafts (74/74), on surviving chicken chorioallantoic membrane, were viable with low levels (0.8 ± 0.1%) of late-stage apoptosis. The diameter of prefollicular germ cells in cultured ovaries from poults at 5 and 7 dph were larger (P < 0.002) than that of their preculture counterparts but were not able to reach their in vivo size. No significant follicular growth was observed in ovaries cultured in ovo; however, prefollicular germ cell density was over 4-fold greater in ovaries cultured from 7 dph poults (81,030 ± 17,611/mm3) than in their in vivo counterpart (16,463 ± 6,805/mm3). Interestingly, cultured ovaries from all other ages displayed equal or lower (P ≤ 0.05) prefollicular germ cell densities than their in vivo counterparts. Cultured ovaries from poults at 5 and 7 dph also exhibited an increase (P ≤ 0.05) in follicle density compared with their preculture counterparts; whereas, cultured ovaries from 15 dph poults had decreased densities (P < 0.001) compared with their preculture counterparts. This study demonstrated that, although age of ovarian tissue cultured in ovo did not affect the overall viability, 7 dph ovaries appeared to have a better cellular morphology after culturing in ovo than other ages. In addition, we also demonstrated for the first time that avian follicles can form during tissue culturing in ovo.

Abstract 414: A Canine Myocardial Slice Model of Doxorubicin Induced Cardiotoxicity to Study Autophagy Modulation and Its Effect on Extracellular Vesicle Associated Mirna

Dogs with cancer treated with chemotherapy agents such as doxorubicin (DOX) develop cardiovascular toxicity, providing an opportunity to evaluate cardioprotective strategies in the setting of cancer treatment translatable to human disease. However, due to the lack of a suitable approach to culture primary adult canine cardiomyocytes, mechanistic interrogation of cardiotoxicity after cancer therapy remains a challenge. Our study thus aims to validate a canine myocardial slice culture model to study autophagy modulation and the role of extracellular vesicle (EV) associated miRNA in the setting of DOX induced cardiotoxicity. We hypothesize that induction of autophagy in canine myocardial tissue will reduce apoptosis, exert early changes to EVs, and ameliorate DOX cardiotoxicity. Left ventricular tissue from client-owned donated adult dog hearts was sectioned with a vibratome and viability of the cultured myocardial slices was evaluated by histology and MTT assay. Apoptosis was quantified by TUNEL, and autophagy by fluorescent LC3 protein puncta. Secreted EVs were isolated from cultured tissue by size exclusion chromatography and characterized by nanoparticle tracking analysis, transmission electron microscopy and immunoblot. Canine myocardial slices are consistently viable for 7 days in culture - cardiomyocyte morphology is maintained with low levels of apoptosis, and baseline autophagy is observed. Induction of autophagy with rapamycin treatment results in reduced apoptosis. Cardiac tissue derived extracellular vesicles showed typical size, morphology and enrichment of proteins including tetraspanin CD9 and will be evaluated for changes to EV miRNA profile with autophagy modulation. The canine myocardial slice model allows, for the first-time, the elucidation of complex cross-talk among apoptosis, autophagy, and EVs with molecular and cellular resolutions. Detecting early changes in canine cardiomyocyte autophagy to halt cardiac damage rather than managing its consequences is a paradigm shift translatable towards preventing chemotherapy associated cardiotoxicity.

Gentamicin-Induced Acute Kidney Injury in an Animal Model Involves Programmed Necrosis of the Collecting Duct.

Gentamicin is a potent aminoglycoside antibiotic that targets gram-negative bacteria, but nephrotoxicity limits its clinical application. The cause of gentamicin-induced AKI has been attributed mainly to apoptosis of the proximal tubule cells. However, blocking apoptosis only partially attenuates gentamicin-induced AKI in animals. Mice treated with gentamicin for 7 days developed AKI, and programmed cell death pathways were examined using pharmacologic inhibitors and in RIPK3-deficient mice. Effects in porcine and murine kidney cell lines were also examined. Gentamicin caused a low level of apoptosis in the proximal tubules and significant ultrastructural alterations consistent with necroptosis, occurring predominantly in the collecting ducts (CDs), including cell and organelle swelling and rupture of the cell membrane. Upregulation of the key necroptotic signaling molecules, mixed lineage kinase domain-like pseudokinase (MLKL) and receptor-interacting serine/threonine-protein kinase 3 (RIPK3), was detected in gentamicin-treated mice and in cultured renal tubule cells. In addition, gentamicin induced apical accumulation of total and phosphorylated MLKL (pMLKL) in CDs in mouse kidney. Inhibiting a necroptotic protein, RIPK1, with necrostatin-1 (Nec-1), attenuated gentamicin-induced necrosis and upregulation of MLKL and RIPK3 in mice and cultured cells. Nec-1 also alleviated kidney inflammation and fibrosis, and significantly improved gentamicin-induced renal dysfunction in mice. Furthermore, deletion of RIPK3 in the Ripk3-/- mice significantly attenuated gentamicin-induced AKI. A previously unrecognized role of programmed necrosis in collecting ducts in gentamicin-induced kidney injury presents a potential new therapeutic strategy to alleviate gentamicin-induced AKI through inhibiting necroptosis.

Abstract A29: Anticancer effects of polo-like kinase-4 inhibitor (CFI-400945) in ovarian cancer

Abstract Background: Dysregulation of cell cycle and mitosis plays a pivotal role in carcinogenesis. Polo-like kinase (PLK) regulates centriole duplication and mitosis and is crucial in maintaining the genomic stability in cancer cells. Disruption of this process may affect cancer cell growth and so PLK represents a druggable target in cancer therapy. CFI-400945 is a small molecule against PLK4 activity, and this study aims to investigate its anticancer efficacy and mechanisms in ovarian cancer cells. Methods: Expression of PLK4 in ovarian cancer patients was determined by Taqman quantitative PCR (qPCR) at transcriptional level. To assess the in vitro growth inhibition effect of CFI-400945, XTT cell viability assay was employed to compare the IC50 of CFI-400945 in a panel of 25 ovarian cancer cell lines of different histologic origins, and the antiproliferative results were assessed by direct cell counting assay. The anticancer activity of CFI-400945 in ovarian cancer cells were further studied by (1) propidium iodide (PI) staining to assess its impact on cell cycle and cell division, (2) annexin V staining to determine the level of apoptosis, (3) gH2AX Western blotting and gH2AX/RAD51 co-immunofluorescence to reveal its effect on DNA damage and repair, and (4) beta-galactosidase activity staining to study the outcome of senescence induced by CFI-400945. To study the change of gene expression induced by CFI-400945 at molecular level, poly-A capture RNA sequencing (RNAseq) was performed in OCC1 and ES2. Results: The qPCR assay significantly demonstrated overexpression of PLK4 in ovarian cancer tumor tissue when compared to nontumor. The growth inhibition effect of CFI-400945 varied among different ovarian cancer cell lines, with IC50 ranging from 0.7 to 68.2nmol. PI-based cell cycle analysis clearly demonstrated the presence of aneuploidy up to 16N, indicating the failure of cell division after S phase DNA duplication. In addition to its impact on cell division, gH2AX immunofluorescent staining and Western blotting also revealed CFI-400945 induced DNA damage. However, induction of apoptosis was only rarely seen in the cell lines tested. The aneuploidy formation and low level of apoptosis was followed by cellular senescence, which was evidenced by 3-fold increase in the number of cells having positive beta-galactosidase staining after CFI-400945 treatment as compared to control. RNAseq of OCC1 and ES2 showed an induction of senescence-associated secretory phenotypes, like IL6 and P21, and suppression of DNA repair gene expression, like RAD51 and BRCA1, which were in agreement with our in vitro observation. Conclusion: CFI-400945 had antiproliferative effect in ovarian cancer cell lines of different histologic origins, and its anticancer activity was attributable to DNA damage, its suppressive effect on DNA repair, failure of cell division, and subsequent cell senescence. Further study would focus on enhancing the anticancer activity of CFI-400945 through drug combination against DNA damage repair. Citation Format: Ka Yu Tse, Kin Tak Chan, Vincent Yuen, Misty Shuo Zhang, Philip Pun Ching Ip, Hextan Yeung Sheung Ngan, Mark Bray, Carmen Chai Lui Wong, Tak Wah Mak. Anticancer effects of polo-like kinase-4 inhibitor (CFI-400945) in ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A29.

Silencing Signal Transducer and Activator of Transcription 3 (STAT3) and Use of Anti-Programmed Cell Death-Ligand 1 (PD-L1) Antibody Induces Immune Response and Anti-Tumor Activity.

BackgroundThe treatment of cancer is still unable to meet the needs of patients and remains a huge challenge. This study investigated the immune response and anti-cancer effect of silencing STAT3 combined with the use of anti-PD-L1 antibody.Material/MethodsTransfected CT26.WT cells were used to subcutaneously inoculate C57B/L6 mice, which were subsequently injected with anti-PD-L1 antibody. Treated mice were examined for tumor formation and inflammation using HE staining. Tumors were investigated for apoptosis using the TUNEL assay. The expression of STAT3, PD-L1, and C-met was studied immunohistochemistrially and by using PCR and Western blot analysis.ResultsFour weeks after inoculation, tumors were observed in the inoculated mice. HE staining showed obvious inflammation in mice injected with cells that were silenced for STAT3 and injected with PD-L1 antibody. TUNEL assay showed low level of apoptosis in mice injected with cells silenced for STAT3 or injected with PD-L1 antibody, and higher level of apoptosis following combined treatment of STAT3 silencing and PD-L1 antibody injection. Immunohistochemistry, PCR, and Western blot analyses revealed that the expression of C-met, PD-L1, and STAT3 was significantly reduced in tumors following the combined treatment. Compared with treatment of STAT3 silencing or PD-L1 antibody injection, the combined treatment enhanced apoptosis.ConclusionsSilencing STAT3 and PD-L1 antibody injection in combination increased apoptosis in tumor cells and thus offers better anti-cancer activity.

Expression of CD44v6-Containing Isoforms Influences Cisplatin Response in Gastric Cancer Cells.

CD44v6-containing isoforms are frequently de novo expressed in gastric cancer (GC). Whether CD44v6 has a central role in GC transformation and/or progression, whether it conditions response to therapy or whether it is only a bystander marker is still not known. Therefore, we aimed to clarify the role of CD44v6 in GC. We generated GC isogenic cell lines stably expressing CD44s or CD44v6 and tested them for different cancer hallmarks and response to cisplatin, and we further confirmed our findings in cells that endogenously express CD44v6. No correlation between overexpression of CD44v6 and the tested cancer hallmarks was observed, suggesting CD44v6 is not a driver of GC progression. Upon cisplatin treatment, CD44v6+ cells survive better and have lower apoptosis levels than CD44v6− cells, possibly due to concomitant activation of STAT3 and P38. In co-culture experiments, we discovered that CD44v6+ cells are involved in GC cell overgrowth after cisplatin treatment. In conclusion, we show that CD44v6 expression increases cell survival in response to cisplatin treatment in GC cells and that these cells override CD44v6-negative cells after cisplatin-treatment. This suggests that tumor expression of CD44v6-containing variants may condition the outcome of GC patients treated with chemotherapy.

Fiber Diameter Differentially Regulates Function of Retinal Pigment and Corneal Epithelial Cells on Nanofibrous Tissue Scaffolds.

Biomaterials have significant functions as tissue scaffolds to support cells for regeneration. Nanofibrous scaffolds which mimic the architecture of the extracellular matrix are well suited to support epithelial cells for ocular tissue engineering. This study aimed at investigating the role of scaffold architecture, if any, on the response of ocular epithelial cells. Thus, we have cultured two different types of ocular epithelial cells on nanofibrous scaffolds of two different diameters to evaluate their generic and cell-specific properties. Human adult retinal pigment epithelial (ARPE-19) and human corneal epithelial (HCE-T) cells were cultured on poly(ε-caprolactone) (PCL) nanofibers of different diameters, nominally 500 and 1300 nm. Moduli of the fiber mats were marginally different at 7.4 and 11.1 kPa for 500 and 1300 nm diameter, respectively. The molecular changes in the cells in response to the different fibers were analyzed by qRT-PCR, Western blot, immunofluorescence, ELISA, flow cytometry, MTT assay, and SEM to assess properties such as proliferation, apoptosis, membrane potential, epithelial-mesenchymal transition, stem cell population, VEGF-A secretion, differentiation, and metabolic status of the cells. HCE-T cells revealed characteristic morphology along with higher expression of proliferation, differentiation, and lower apoptotic markers when cultured on PCL nanofibers of 500 nm. However, on nanofibers of 1300 nm, the cells showed higher expression of the corneal stem/progenitor as well as pluripotent stem cell markers. ARPE-19 cells exhibited characteristic hexagonal morphology with elevated expression levels of proliferative markers, phagocytic activity, and lower apoptosis levels. However, on 500 nm nanofibers, they expressed higher levels of pluripotent markers and secretion of VEGF-A. These findings demonstrate that the response can differ markedly from scaffold architecture even if derived from the same tissue and originating from the same germ layer. Furthermore, it paves the way for a target specific outcome and, thereby, for personalized translational medicine.

Identification of a Native Novel Oncolytic Immunoglobulin on Exfoliated Colon Epithelial Cells: A Bispecific Heterodimeric Chimera of IgA/IgG*

Understanding the nature of cell surface markers on exfoliated colonic cells is a crucial step in establishing criteria for a normally functioning mucosa. We have found that colonic cells isolated from stool samples (SCSR-010 Fecal Cell Isolation Kit, NonInvasive Technologies, Elkridge, MD), preserved at room temperature for up to one week, with viability of >85% and low levels of apoptosis (8% - 10%) exhibit two distinct cell size subpopulations, in the 2.5 μM - 5.0 μM and 5.0 μM - 8.0 μM range. In addition to IgA, about 60% of the cells expressed a novel heterodimeric IgA/IgG immunoglobulin that conferred a broad-spectrum cell mediated cytotoxicity against tumor cells. In a cohort of 58 subjects the exclusive absence of this immunoglobulin in two African-Americans was suggestive of a germline deletion. Serial cultures in stem cell medium retained the expression of this heterodimer. Since a majority of the cystic cells expressed the stem cell markers Lgr5 and Musashi-1 we termed these cells as gastrointestinal progenitor stem cells (GIP-C**). CXCR-4, the cytokine co-receptor for HIV was markedly expressed. These cells also expressed CD20, IgA, IgG, CD45, and COX-2. We assume that they originated from mature columnar epithelium by dedifferentiation. Our observations indicate that we have a robust noninvasive method to study mucosal pathophysiology and a direct method to create a database for applications in regenerative medicine.

АKT-, ERK-, NF-KB-СИГНАЛЬНЫЕ КАСКАДЫ В НЕЙРОНАХ СУПРАОПТИЧЕСКОГО И ПАРАВЕНТРИКУЛЯРНОГО ЯДЕР ГИПОТАЛАМУСА ПРИ СТАРЕНИИ У МЫШЕЙ СО СВЕРХЭКСПРЕССИЕЙ HER2/NEU

Epidermal growth factor receptors are widely expressed in the various tissues, including the brain, and play an important role in the regulation of such biological processes as proliferation, cell migration, cell death, etc. This study aimed to study the involvement of signaling pathways related with cell survival (AKT, ERK, NF-kB cascades) in the regulation of hypothalamic neuronal apoptosis in experimental models of physiological and pathological (HER2/neu overexpression) aging, as well as the effect of FASL and TNF-alpha cytokines on these signaling cascades. Experiments were carried out on HER2/neu transgenic mice with accelerated cellular senescence compared to the wild type mice (FVB/N strain). In the hypothalamic supraoptic and paraventricular nuclei, expression of the antiapoptotic protein survivin was determined by immunohistochemistry, while expression of proteins of the cytokine-dependent cascades (FASL, TNF-alpha) and protein members of the survival signaling pathways (phosphorylated AKT, ERK, NF-kB) was assayed by Western blotting. It was found that in FVB/N mice the AKT, ERK cascades do not play a significant role in aging, whereas activation of FAS signaling and a decrease in the NF-kB pathway activity can lead to an increased proportion of dying neurons. In HER2/neu mice, neuronal tolerance to apoptosis during aging is provided by a combination of different survival signaling pathways: the ERK cascade is activated in the supraoptic nucleus and the NF-kB and AKT cascades in the paraventricular nucleus. In addition, a suppression of FAS signaling was revealed, also leading to a low level of apoptosis.

XLF/Cernunnos Loss Impairs Mouse Brain Development by Altering Symmetric Proliferative Divisions of Neural Progenitors

XLF/Cernunnos is a component of the ligation complex used in classical non-homologous end joining (cNHEJ), a major DNA double strand break (DSB) repair pathway.We evidenced neurodevelopmental delays and significant behavioral alterations associated to microcephaly in Xlf-/- mice. This phenotype, reminiscent of clinical and neuropathologic features in humans deficient in cNHEJ, is related to a low level of apoptosis of differentiating neural cells, but primarily to a premature neurogenesis during brain development. We show that premature neurogenesis, which consists in an early shift of neural progenitors from proliferative to neurogenic divisions, results from an increase in chromatid breaks affecting mitotic spindle orientation, highlighting a direct link between asymmetric chromosome segregation and asymmetric neurogenic divisions. This study reveals thus that XLF is required for maintaining symmetric proliferative divisions of neural progenitors during brain development and shows that premature neurogenesis may play a major role in neurodevelopmental pathologies caused by NHEJ deficiency and/or genotoxic stress.

English

Tea Tree oil (TTO), the essential oil from the Australian native Melaleuca alternifolia has demonstrated a variety of beneficial efficacies including antimicrobial, antifungal, antiviral and anti-inflammatory. This report discusses data obtained on the in vitro activity of TTO on human glioblastoma cells U87MG. Cell viability was examined by 2-(4,5-dimethyl-2-thiazolyl)-3,5-diphenyl-2H-tetrazolium bromide MTT assay. Growth was investigated by incubating cells with various concentrations of TTO (0.025 and 0.05 %) for 24, 48 or 72 h and daily cell count. Cell cycle and apoptosis assay were assessed by flow citometry. TTO decreased cell viability in a dose and time-dependent manner. . The cell cycle distribution showed that TTO enhanced the accumulation of the cells in G0/G1 phase. The analysis by Western blot of protein related to cell cycle (CDK2 and p27), cell apoptosis (caspase 6 and 8), necrosis (TNFR1 and RIP1) demonstrated that TTO induces U87MG growth inhibition by more synergic mechanisms: necrosis, low level apoptosis and cell cycle arrest. TTO induces also in vivo glioblastoma tumor growth inhibition in a murine subcutaneous model. Key words: Brain cancer, tea tree oil, terpinol, glioblastoma, natural drug, adjuvant chemotherapy, temozolomide, apoptosis, cell cycle. &nbsp; &nbsp;

P4488Organotypic culture of the epicardium: a new tool for cardiovascular research

Abstract Background The epicardium, the most external layer of the heart, is composed of a layer of epithelial cells and underlying connective tissue. Following myocardial infarction, epicardial cells are activated and provide a source of paracrine factors and progenitor cells. In the border zone of the ischaemic tissue, the activated epicardial cells support cardiac and vascular regeneration by releasing pro-angiogenic and pro-survival factors, and by differentiating towards multiple cell lineages. During this process, activated epicardial cells migrate to the site of injury where they contribute to both post-ischemic remodelling and fibrosis. There is limited knowledge of the cellular and molecular regulation of these processes in large animals and humans, in part due to the lack of robust and representative models. Purpose In this project, we developed an ex vivo 3D organotypic model derived from porcine hearts, amenable to culture, which enables structural, molecular and cellular studies of the epicardium. Methods Thin epicardial/cardiac tissue slices (EpCardio-TS) were obtained by using a vibratome to cut the first layer of tissue from the epicardial side of porcine heart cubes. Slices were cultured for up to 72h in a bioreactor that uses a 3D printed chamber connected to a control system that allows maintenance and adjustment of culture conditions, and ensures continuous media flow. Local intracellular delivery of fluorescent quantum-dots (Qdots) was performed using nanoneedle chips to track epicardial cells, whilst cell fate is visualised in 3D by performing immunofluorescence on decolourised slices. Results Intact EpCardio-TS obtained from porcine heart included a viable epicardium, expressing typical epicardial markers (wt-1, mesothelin, uroplakin), and an electrically active myocardium. Live/dead staining showed epicardial (67.8±16.2%, N=5) and myocardial (40.8±28.6%, N=3) viability, and TUNEL assay confirmed low levels of apoptosis (6.3±5.1% of wt-1+ epicardial cells N=1). Moreover, the presence of proliferating epicardial cells (PCNA+), the increase in wt-1+ cells, and the increase in epicardial gene expression (Tbx18 and TCF21) suggested that cells maintain their progenitor phenotype and undergo activation in culture. Nanoinjection of fluorescent Qdots to EpCardio-TS localized them to the wt-1+ cells on the slice surface, presenting a strategy to mark the epicardial layer. This, combined with the successful decolourisation of the slices, provides an in vitro platform to track the role of epicardial cells in cardiac remodelling and fibrosis. Conclusions EpCardio-TS represents a robust ex vivo model merging the complexity of a 3D organotypic culture with the simplicity of the in vitro culture. EpCardio-TS are amenable to culture and cell tracking, and can therefore find application in toxicology and gene therapy screening for the modulation of epicardial interactions with myocardial and non-myocardial cells of the heart.