Protein Expression Levels Of Markers Research Articles

Ipatasertib exhibits anti‑tumorigenic effects and enhances sensitivity to paclitaxel in endometrial cancer invitro and invivo.

Endometrial cancer is the most common gynecologic cancer and one of the only cancers for which incidence and mortality is steadily increasing. Although curable with surgery in the early stages, endometrial cancer presents a significant clinical challenge in the metastatic and recurrent setting with few novel treatment strategies emerging in the past fifty years. Ipatasertib (IPAT) is an orally bioavailable pan‑AKT inhibitor, which targets all three AKT isoforms and has demonstrated anti‑tumor activity in pre‑clinical models, with clinical trials emerging for many cancer types. In the present study, the MTT assay was employed to evaluate the therapeutic efficacy of IPAT or IPAT in combination with paclitaxel (PTX) in endometrial cancer cell lines and primary cultures of endometrial cancer. The effect of IPAT and PTX on the growth of endometrial tumors was evaluated in a transgenic mouse model of endometrial cancer. Apoptosis was assessed using cleaved caspase assays and cellular stress was assessed using ROS, JC1 and tetramethylrhodamine ethyl ester assays. The protein expression levels of markers of apoptosis and cellular stress, and DNA damage were evaluated using western blotting and immunohistochemistry. IPAT significantly inhibited cell proliferation, caused cell cycle G1 phase arrest, and induced cellular stress and mitochondrial apoptosis in a dose dependent manner in human endometrial cancer cell lines. Combined treatment with low doses of IPAT and PTX led to synergistic inhibition of cell proliferation and induction of cleaved caspase 3 activity in the human endometrial cancer cell lines and the primary cultures. Furthermore, IPAT effectively reduced tumor growth, accompanied by decreased protein expression levels of Ki67 and phosphorylation of S6 in the Lkb1fl/flp53fl/fl mouse model of endometrioid endometrial cancer. The combination of IPAT and PTX resulted in increased expression of phosphorylated‑H2AX and KIF14, markers of DNA damage and microtubule dysfunction respectively, as compared with IPAT alone, PTX alone or placebo‑treated mice. The results of the present study provide a biological rationale to evaluate IPAT and the combination of IPAT and PTX in future clinical trials for endometrial cancer.

Hesperetin Ameliorates Inhibition of Neuronal and Oligodendroglial Cell Differentiation Phenotypes Induced by Knockdown of Rab2b, an Autism Spectrum Disorder-Associated Gene Product.

Autism spectrum disorder (ASD) is a central nervous system (CNS) neurodevelopmental disorder that includes autism, pervasive developmental disorder, and Asperger's syndrome. ASD is characterized by repetitive behaviors and social communication deficits. ASD is thought to be a multifactorial disorder with a range of genetic and environmental factors/candidates. Among such factors is the rab2b gene, although it remains unclear how Rab2b itself is related to the CNS neuronal and glial developmental disorganization observed in ASD patients. Rab2 subfamily members regulate intracellular vesicle transport between the endoplasmic reticulum and the Golgi body. To the best of our knowledge, we are the first to report that Rab2b positively regulates neuronal and glial cell morphological differentiation. Knockdown of Rab2b inhibited morphological changes in N1E-115 cells, which are often used as the neuronal cell differentiation model. These changes were accomplished with decreased expression levels of marker proteins in neuronal cells. Similar results were obtained for FBD-102b cells, which are used as the model of oligodendroglial cell morphological differentiation. In contrast, knockdown of Rab2a, which is another Rab2 family member not known to be associated with ASD, affected only oligodendroglial and not neuronal morphological changes. In contrast, treatment with hesperetin, a citrus flavonoid with various cellular protective effects, in cells recovered the defective morphological changes induced by Rab2b knockdown. These results suggest that knockdown of Rab2b inhibits differentiation in neuronal and glial cells and may be associated with pathological cellular phenotypes in ASD and that hesperetin can recover their phenotypes at the in vitro level at least.

IL-17A Promotes the Migration, Invasion and the EMT Process of Lung Cancer Accompanied by NLRP3 Activation.

Lung cancer is a deadly cancer worldwide, and its pathogenesis and treatment methods require continuous research and exploration. As a representative factor of adaptive immunity, the role of interleukin-17A (IL-17A) in lung cancer is still unclear. The purpose of the present study was to investigate the effect of IL-17A on the biological behaviour of lung cancer cells and the relative mechanism. The human lung adenocarcinoma A549 and H1299 cell lines were used for in vitro study. The effects of IL-17A on cell proliferation, migration and invasion were assessed by CCK-8 assay, wound-healing assay, transwell invasion assay and real-time cell analysis (RTCA). The expression levels of marker proteins in the process of epithelial-mesenchymal transition (EMT) were detected by western blot analysis. Caspase-1 activity and the concentration of IL-1β after NLRP3 inflammasome activation were measured by a Caspase-1 Activity Assay Kit and an IL-1β ELISA kit, respectively. Compared to the control group, IL-17A treatment did not affect the proliferation of A549 and H1299 cells in vitro, but it promoted cell migration, invasion and the EMT process. IL-17A treatment increased NLRP3 expression, caspase-1 activity and IL-1β level. Blockade of NLRP3 alleviated the cell migration, invasion and the EMT process induced by IL-17A. In conclusion, these findings indicated that NLRP3 participates in the migration, invasion and the EMT process of IL-17A-stimulated lung cells in vitro.

Effects of prometryn on cardiac and liver cell lines and mouse tissues

BackgroundPesticides are important biological agents that deter or kill pests. Globally, about 2 million tons of pesticides are being utilized each year which highlights their importance. Previous studies have suggested that high levels of pesticide exposure are associated with increased risk of chronic diseases, including cancers, cardiotoxicity, Parkinson’s disease, diabetes mellitus, neurological deficits, birth defects, and reproductive disorders. Prometryn is a commonly used selective triazine herbicide, which is currently used as an alternative to atrazine‐based products. It is widely applied to agriculture as it has lower acute toxicity than atrazine herbicides, stable chemical properties, and a long period of effectiveness. Although past studies have shown that triazine herbicides have carcinogenic potential in humans, the cytotoxic effects of prometryn on cardiac and hepatic systems are not well investigated or understood. The aim of this study was to determine the effects of prometryn on the heart and liver.MethodsH9c2 cardiac and HepG2 liver carcinoma cells were used to study the in‐vitro effects of prometryn. Cells were treated with varying concentrations of prometryn for 24 h. Cell viability, mitochondrial membrane potential, and reactive oxygen species (ROS) levels were assessed. For animal studies, male mice (10 weeks old) (n=24) were randomized to two treatment groups (control and prometryn). The control group received corn oil while the prometryn treated group were exposed to 185mg/kg b.w. prometryn dissolved in corn oil every 48 hours for 7 days. After a total of 4 doses, mice were euthanized, and the heart and liver tissues were collected. Proteasome and immunoproteasome activity were measured in the heart and liver. Protein expression levels of markers for oxidative stress, apoptosis, and proteasomal degradation such as GSTT2, GSTA1/2, Hsp70, Hsp27, NADPH oxidase, PARP‐1, p53, PSMD12, LMP2, DNP, VU‐1 were detected using immunoblotting. Protein oxidation and polyubiquitination were detected as an indicator of oxidative stress. A paired t‐test was used to assess group differences for protein levels and p values <0.05 were deemed statistically significant.ResultWe found that prometryn had a concentration‐dependent effects on H9c2 and HepG2 cell lines when we assessed cell viability, mitochondria function, and total intracellular reactive oxygen species formation. Increasing the concentration of prometryn increased the ROS formation, decreased cell viability and decreased mitochondrial membrane potential in these cell lines. Furthermore, in animal studies we observed changes in the proteasome and immunoproteasome activities in the liver but not in the heart. Changes in protein expression levels of oxidative stress and proteasomal degradation markers were only detected in the liver.ConclusionThese results indicate that H9c2 and HepG2 cells are sensitive to prometryn in‐vitro. However, prometryn effects on the heart and liver tissue in male mice is significantly different. Overall, our data supports the deduction that prometryn affected mitochondrial function, induced oxidative stress in cells, but also alters the ubiquitin‐proteasome system and increases oxidative stress in mice liver. These results help towards elucidation of the mechanism by which prometryn could cause diseases.

A Neonatal Mouse Model for Pressure Overload: Myocardial Response Corresponds to Severity.

The heart regeneration after apical resection and myocardial infarction in neonatal mice has been studied for years. However, the response of neonatal mouse heart under pressure overload is seldom explored. This study aimed to induce pressure overload in neonatal mice through a transverse aortic constriction (TAC) with different-gauge needles so as to investigate the effect of pressure overload on cardiomyocyte proliferation and hypertrophy in these mice. Myocardial hypertrophy was evaluated by echocardiographic, pathological, and molecular analyses. Cardiomyocyte proliferation was detected by immune-staining of phospho-histone H3, Ki67, and 5-bromo-2-deoxyuridine. Mild pressure overload induced with a 30-gauge needle stimulated cardiomyocyte proliferation, adaptive hypertrophy, and angiogenesis. The heart function was not hampered even 21 days after the surgery. Moderate pressure overload induced with a 32-gauge needle led to pathological myocardial hypertrophy, fibrosis, and heart failure 7 days after the surgery. The gene and protein expression levels of markers of hypertrophy and fibrosis increased in 32-gauge TAC group compared with that in sham and 30-gauge TAC groups. The mice barely survived after severe pressure overload induced with a 34-gauge needle. The findings of this study might provide new insights into cardiomyocyte proliferation and hypertrophy in neonatal mice under pressure overload.

The Effect of Zinc on the Marker Expression in Cell Transitional Activities

Zinc is a trace element that participates in protein structure, enzyme activity, and gene regulation. Zinc homeostasis helps to maintain strong immunity, bone health, and the levels of various hormones, and it is also involved in the regulation of oxidative stress, cell death, and aging processes. We are interested in studying the effect of zinc homeostasis on the marker expression in cell transitional activities, including markers for epithelial‐to‐mesenchymal transition (EMT) and stem cells. In my preliminary experiments, the dissociation of zinc from its binding sites has been achieved by acidic treatment because the binding between zinc and protein is tight at basic pH and loose at acidic pH. We hypothesize that the acid‐induced dissociation of zinc from its binding sites contributes to the cell transitional activities, from one type of cell to a different kind of cells during EMT and stem cell development. In my preliminary results, acid treatment resulted in the accumulation of spindle‐shaped cells, similar to the morphology of EMT cells suggested by the literature. It increased the expression of vimentin (a marker labeling EMT), CDK2 (a marker labeling G1 and S phases), and SSEA‐4 (a marker labeling stem cells). This acidic treatment also decreased the doubling time of mouse gastric cells during long‐term cell culture, suggesting that the acid‐induced zinc dissociation promoted cellular growth perhaps through the activation of EMT, the facilitation of cell cycle, or the development of stem cells. Moreover, the chelation of zinc alleviated the spindle cell accumulation, vimentin expression, and SSEA‐4 expression induced by the acid exposure, confirming the importance of zinc dissociation from its binding sites in the cell transitional activities. In conclusion, zinc functions in cell transitional activities through regulating the expression levels of marker proteins involved in EMT, cell cycle, and stem cell development.Support or Funding InformationFunding is provided partially by NIH (# NS081629) to YVL. Thanks to the Osteopathic Heritage Foundations, Graduate Assistantship Program at Ohio University Heritage College of Osteopathic Medicine for supporting graduate student YH.

Amentoflavone suppresses amyloid β1–42 neurotoxicity in Alzheimer's disease through the inhibition of pyroptosis

BackgroundThe accumulation of Amyloid β (Aβ) plays key roles in Alzheimer's disease (AD) by inducing intracellular reactive oxygen species (ROS) and neuronal cell death. In this study, we aimed to identify the neuroprotective mechanisms of amentoflavone (AF) in Aβ-induce neuronal cell injury. Materials and methodsThe animal model was established by injecting Aβ1-42 into the bilateral hippocampus. The effect of AF on Aβ1-42-induced neurological dysfunction was examined using the Y-maze and radical maze tests. The hippocampal neuron viability was examined using Nissl staining and TUNEL assay. On the other hand, in vitro studies were conducted using SH-SY5Y cells. The expression level of marker proteins was measured using western blot. The activity of caspase-1 and the levels of pro-inflammatory cytokines were determined using ELISA assay. AMPKα knock down was carried out by transfecting SH-SY5Y cells with siRNA against AMPK transcript. ResultsNeurological tests showed that AF significantly attenuated Aβ1-42-induced neurological dysfunction. AF suppressed Aβ1-42-induced pyroptosis in the hippocampal region of the rat model, which was associated with the modulation of AMPK/GSK3β signaling. Similar results were obtained in vitro in SH-SY5Y cells exposed to Aβ1-42, showing that the neuroprotective activity of AF is mediated by suppressing pyroptosis through AMPK/GSK3β signaling. ConclusionAF inhibits Aβ1-42-induced neurotoxicity in animal and cellular models through AMPK/GSK3β-mediated pyroptosis suppression. Our results highlight AF as a clinical compound for the prevention and treatment of AD.

Inhibition of miR-34a-5p protected myocardial ischemia reperfusion injury-induced apoptosis and reactive oxygen species accumulation through regulation of Notch Receptor 1 signaling.

Myocardial ischemia-reperfusion (I/R) injury is leading cause of death worldwide. miR-34a-5p was up-regulated in myocardial ischemia-reperfusion injury rats. We aim to explore how miR-34a-5p inhibition protected myocardium against I/R injury in both cell and animal models. In vivo rat and in vitro cell model were firstly constructed. quantitative real-time polymerase chain reaction was employed to investigate expression of miR-34a-5p and its target genes. Functional assays were conducted to detect the impact of miR-34a-5p on myocardial I/R injury. Enzyme-linked immunosorbent assay was performed to validate the expression levels of marker proteins of ischemia-reperfusion I/R-induced myocardial injury. MTT was performed to assess the cell viability and flow cytometry was utilized to detect cell apoptosis and reactive oxygen species accumulation. The interaction between miR-34a-5p and Notch Receptor 1 were also examined through luciferase reporter assay. miR-34a-5p was up-regulated post-reperfusion at rat myocardium. miR-34a-5p inhibitor attenuated myocardial ischemia-reperfusion injury, as shown by decreasing apoptosis rate, reducing infarct size and reactive oxygen species accumulation. In in vitro cell model, miR-34a-5p inhibitor also promoted cell proliferation, inhibited cell apoptosis and reactive oxygen species accumulation through targeting Notch Receptor 1 signaling. Our results revealed that miR-34a-5p knocking down attenuated myocardial I/R injury by promoting Notch Receptor 1 signaling-mediated inhibition of reactive oxygen species accumulation and cell apoptosis. Hence, miR-34a-5p might be a potential target for treatment of myocardial ischemia-reperfusion injury.

Knockdown of ferroportin accelerates erastin-induced ferroptosis in neuroblastoma cells.

Ferroptosis is a new-found iron-dependent form of non-apoptotic regulated cell death (RCD), which is activated on therapy with several antitumor agents, but the potential mechanism remains unclear. Erastin, exhibiting selectivity for RAS-mutated cancer cells, induces ferroptosis by increasing iron and lipid reactive oxygen species (ROS) levels in cell. Ferroportin (Fpn), the sole iron export protein, participates in the regulation of intracellular iron concentration. In this study, we investigated the role of Fpn on ferroptosis induced by erastin in SH-SY5Y cells. The cell viability was determined by CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay kit. The activity of caspase-3 was measured by ELISA kit. qRT-PCR was performed to examine the mRNA expression of Fpn. Western blot assay was conducted to examine the expression level of marker proteins. Specific commercial kits were used to examine the levels of MDA, ROS and iron in cells, respectively. Ferroptosis was evaluated by intracellular lipid ROS level and iron concentration. Hepcidin could prevent erastin-induced ferroptosis by degrading Fpn. Erastin (5 μg/mL) was observed to induce ferroptosis in neuroblastoma cells at 6 hours, which was promoted by knockdown of Fpn. The expression of Fpn gene and protein was decreased in SH-SY5Y cells treated with erastin. After treatment with erastin, Fpn siRNA transfection in SH-SY5Y cells was able to accelerate ferroptosis-associated phenotypic changes. Fpn acted as a negative regulator of ferroptosis by reducing intracellular iron concentration. Knockdown of Fpn enhanced anticancer activity of erastin. These results suggested that knockdown of Fpn accelerated erastin-induced ferroptosis by increasing iron-dependent lipid ROS accumulation, highlighting Fpn as a potential therapeutic target site for neuroblastoma. Thus, Fpn inhibitors may provide new access for chemosensitization of neuroblastoma.

Retinoic Acid Protects and Rescues the Development of Zebrafish Embryonic Retinal Photoreceptor Cells from Exposure to Paclobutrazol.

Paclobutrazol (PBZ) is a widely used fungicide that shows toxicity to aquatic embryos, probably through rain-wash. Here, we specifically focus on its toxic effect on eye development in zebrafish, as well as the role of retinoic acid (RA), a metabolite of vitamin A that controls proliferation and differentiation of retinal photoreceptor cells, in this toxicity. Embryos were exposed to PBZ with or without RA from 2 to 72 h post-fertilization (hpf), and PBZ-treated embryos (2–72 hpf) were exposed to RA for additional hours until 120 hpf. Eye size and histology were examined. Expression levels of gnat1 (rod photoreceptor marker), gnat2 (cone photoreceptor marker), aldehyde dehydrogenases (encoding key enzymes for RA synthesis), and phospho-histone H3 (an M-phase marker) in the eyes of control and treated embryos were examined. PBZ exposure dramatically reduces photoreceptor proliferation, thus resulting in a thinning of the photoreceptor cell layer and leading to a small eye. Co-treatment of PBZ with RA, or post-treatment of PBZ-treated embryos with RA, partially rescues photoreceptor cells, revealed by expression levels of marker proteins and by retinal cell proliferation. PBZ has strong embryonic toxicity to retinal photoreceptors, probably via suppressing the production of RA, with effects including impaired retinal cell division.

Vitamin D induces autophagy of pancreatic β-cells and enhances insulin secretion.

Epidemiological evidence indicates that vitamin D is involved in defense against diabetes; however, the precise underlying mechanism remains to be elucidated. In the present study, the effect of vitamin D on the pathogenesis of diabetes was investigated, with an emphasis on its direct effect on pancreatic β‑cells. A streptozotocin (STZ)‑induced type 1 diabetes mellitus (T1DM) mouse model and MIN6 mouse insulinoma β‑cells were subjected to vitamin D treatment. Histopathological analysis of pancreatic islets was performed to investigate insulitis, and reverse transcription-quantitative polymerase chain reaction and western blotting were used to determine the mRNA and protein expression levels of markers of autophagy [microtubule-associated protein 1A/1B‑light chain 3 (LC3) and Beclin 1] and regulation of apoptosis [B-cell lymphoma 2 (Bcl-2)]. Apoptosis of MIN6 cells was examined by flow cytometry following annexin V/propidium iodide labeling. The secretion of insulin was measured by ELISA. The results revealed that vitamin D reduced the incidence of T1DM, enhanced insulin secretion and relieved pancreatic inflammation in STZ‑treated mice. Furthermore, vitamin D increased the mRNA expression levels of LC3 and Beclin 1, and increased Bcl‑2 protein expression levels in STZ‑treated MIN6 cells, while decreasing the apoptosis rate. The results of the present study demonstrated, for the first time to the best of our knowledge, that vitamin D induces autophagy and suppresses apoptosis of pancreatic β‑cells, as well as preventing insulitis. These findings regarding vitamin D provide insights into its involvement in diabetes, and suggest a potential novel strategy for the treatment of diabetes via agents enhancing autophagy in pancreatic β-cells.

Beneficial effects of intravenous dexmedetomidine on cognitive function and cerebral injury following a carotid endarterectomy.

The present study aimed to investigate the effects of dexmedetomidine (DEX) on cognition following a carotid endarterectomy (CEA). In addition, the neuroprotective effects of DEX against ischemia-reperfusion injury during CEA were analyzed. Patients due to undergo elective CEA under general anesthesia were randomly assigned to either the DEX-treated group (group D; n=25) or the control group (group C; n=25). Patients in group D were treated with 0.3 µg/kg DEX pre-CEA, followed by 0.3 µg/kg/h DEX intraoperatively up to 30 min prior to the completion of surgery, and the patients in group C received an equal volume of normal saline. Cognitive function was assessed prior to CEA (T0), and at 24, 48, and 72 h, 7 days and 1 month post-surgery (T1-5, respectively), using the Mini-Mental State Examination (MMSE). Blood samples were drawn from the ipsilateral jugular bulb of all patients at 20 min prior to anesthesia (t0), and at 10 min following tracheal intubation, 15 min following clamping and unclamping of the carotid artery, and at 6 and 24 h postoperatively (t1-5, respectively). The protein expression levels of markers of cerebral ischemia and injury, namely S100 calcium-binding protein B (S100B) and neuron-specific enolase (NSE), and the concentration of the oxidative stress marker malondialdehyde (MDA), were analyzed. Patients in group D exhibited elevated MMSE scores at T2 and T3 post-CEA, as compared with group C. Furthermore, the protein expression level of S100B and the concentration of MDA in the jugular bulb of group D patients were markedly decreased compared with those in group C at t3-5 and t3, respectively. The results of the present study suggested that DEX was able to enhance the recovery of cognition following CEA, and this was associated with decreased cerebral damage and antioxidative effects.

SOX2 promotes tumorigenicity and inhibits the differentiation of I-type neuroblastoma cells.

SOX2 is a transcription factor associated with the pluripotency, proliferative potential, and self-renewing properties observed with embryonic stem cells and germ cells. SOX2 expression has been reported in several cancers and is implicated in tumorigenesis. We previously found that SOX2 expression was correlated to the clinical stage of neuroblastoma. Recently, we found that SOX2 overexpression occurs in I-type neuroblastoma cells (BE(2)-C cells). To elucidate the tumorigenic function of SOX2, we established a SOX2 overexpressed BE(2)-C cell line. SOX2 overexpressed cells showed higher tumorigenicity than control cells and exhibited decreased expression levels of marker proteins of N- or S-type cells after agent-induced differetiation. By contrast, in cells where SOX2 mRNA expression was knocked down by gene-specific siRNA, tumorigenicty was significantly decreased and the expression levels of marker proteins of N- or S-type cells were upregulated. In conclusion, our findings indicate an important function for SOX2 in promoting tumorigenicity of I-type neuroblastoma cells and in inhibiting their differentiation, suggesting that SOX2 might be a potential therapeutic target in neuroblastoma.

CCN4 regulates vascular smooth muscle cell migration and proliferation.

The migration and proliferation of vascular smooth muscle cells (VSMCs) are essential elements during the development of atherosclerosis and restenosis. An increasing number of studies have reported that extracellular matrix (ECM) proteins, including the CCN protein family, play a significant role in VSMC migration and proliferation. CCN4 is a member of the CCN protein family, which controls cell development and survival in multiple systems of the body. Here, we sought to determine whether CCN4 is involved in VSMC migration and proliferation. We examined the effect of CCN4 using rat cultured VSMCs. In cultured VSMCs, CCN4 stimulated the adhesion and migration of VSMCs in a dose-dependent manner, and this effect was blocked by an antibody for integrin α5β1. CCN4 expression was enhanced by the pro-inflammatory cytokine tumor necrosis factor α (TNF-α). Furthermore, knockdown of CCN4 by siRNA significantly inhibited the VSMC proliferation. CCN4 also could up-regulate the expression level of marker proteins of the VSMCs phenotype. Taken together, these results suggest that CCN4 is involved in the migration and proliferation of VSMCs. Inhibition of CCN4 may provide a promising strategy for the prevention of restenosis after vascular interventions.

Genetic backgrounds and redox conditions influence morphological characteristics and cell differentiation of osteoclasts in mice

Osteoclasts (OCLs) are multinucleated giant cells and are formed by the fusion of mononuclear progenitors of monocyte/macrophage lineage. It is known that macrophages derived from different genetic backgrounds exhibit quite distinct characteristics of immune responses. However, it is unknown whether OCLs from different genetic backgrounds show distinct characteristics. In this study, we showed that bone-marrow macrophages (BMMs) derived from C57BL/6, BALB/c and ddY mice exhibited considerably distinct morphological characteristics and cell differentiation into OCLs. The differentiation of BMMs into OCLs was comparatively quicker in the C57BL/6 and ddY mice, while that of BALB/c mice was rather slow. Morphologically, ddY OCLs showed a giant cell with a round shape, C57BL/6 OCLs were of a moderate size with many protrusions and BALB/c OCLs had the smallest size with fewer nuclei. The intracellular signaling of differentiation and expression levels of marker proteins of OCLs were different in the respective strains. Treatment of BMMs from the three different strains with the reducing agent N-acetylcysteine (NAC) or with the oxidation agent hydrogen peroxide (H(2)O(2)) induced changes in the shape and sizes of the cells and caused distinct patterns of cell differentiation and survival. Thus, genetic backgrounds and redox conditions regulate the morphological characteristics and cell differentiation of OCLs.

Abstract 1044: Role of autophagic mediators in the transition from endometriosis to endometriosis-associated ovarian cancers

Abstract Endometriosis, a common painful and inflammatory gynecological disease, has been proposed to contribute to the development of endometriosis-associated ovarian cancer, specifically the endometrioid and clear cell subtypes. The mechanism underlying this transition is unclear; however, many characteristics of endometriosis and endometrioid-ovarian cancer are similar with the exception of loss of cell cycle control. Autophagy, a survival mechanism which is activated in response to multiple stresses promotes tumorigenic development and may be an ideal target for therapy. To date, the role of autophagy in the development of endometrioid-ovarian carcinomas is unknown with the exception of attenuated mRNA levels of the autophagic marker, beclin-1, in an endometriosis-related condition. Since we have shown that SnoN/SkiL, a TGFβ co-transcriptional regulator, modulates autophagy in ovarian cancer cell lines, we propose that SnoN and/or autophagic mediators may be involved in the transition from endometriosis to ovarian cancer. Thus, we determined the basal expression levels of markers of TGFβ and autophagic pathways in primary and SV40 LTg immortalized endometriotic cells isolated from patients with endometriosis and compared their levels to endometrial/ovarian carcinoma cell lines. Cell lysates were prepared from confluent cultures of (1) five endometrial carcinoma cell lines (MFE296, MFE319, AN3-CA, KLE, and HEC-1A), (2) endometrioid-ovarian carcinoma cell line (TOV112D), (3) a clear cell carcinoma cell line (TOV21G), (4) three serous epithelial ovarian carcinoma cell lines (OVCAR8, HEY, and SKOV3), and (5) two primary (PNEs) as well as four SV40 LTg immortalized (INEs) normal endometriotic cells (derived from patients with endometriotic lesions). These were then assessed for protein expression levels of markers in various signaling pathways (autophagy, TGFβ, cell survival, and epithelial-mesenchymal transition) via western analysis and densitometric analysis. Relative to endometrial/serous epithelial ovarian carcinomas, PNEs and INEs expressed (1) low levels of oncogenes (EVI1, SnoN, and EGFR), (2) elevated levels of tumor suppressors (PTEN), (3) elevated levels of the EMT marker N-Cadherin, and (4) low levels of autophagic markers (i.e. ATG5, ATG7, and hVps34). Since we have observed marked differences in expression of autophagy and TGFβ signaling mediators, studies are ongoing to investigate the effects of knockdown and overexpression of ATGs and SnoN/SkiL in TOV112D and INEs, respectively, to determine their role in modulating this transition event. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1044. doi:10.1158/1538-7445.AM2011-1044

Phenotypic switching of Escherichia coli cells containing cyclic digenic systems with negative feedback upon changes in cultivation conditions

One of the mechanisms for the epigenetic control of cell phenotypes is based on switching the functioning regimes of bistable gene networks, which can maintain the two alternative levels of gene expression under the same conditions. Cyclic digenic systems with negative feedback represent an example of a simple bistable gene network. Cells carrying artificial cyclic digenic systems on plasmids inherit each alternative phenotype upon exponential growth on rich medium during several cell generations. The action of specific inducers is necessary for switching. In this work, the impact of changes in cell cultivation conditions on the phenotypic composition of the clonal Escherichia coli cell population containing artificial cyclic digenic systems with negative feedback was studied. Phenotypes differ with respect to the expression level of marker proteins: beta-galactosidase and GFP. Slow growth on a medium containing little-available carbon sources was shown to cause the transition from the phenotype Lac- to Lac+ in the absence of inducers. Phenotypic switching cannot be explained by transcriptional activation of the lactose operon, because 80 +/- 15% of cells inherit the acquired phenotype after replating bacteria on rich medium. Inheritance of the phenotype Lac- in batch culture depends on the medium and duration of cultivation. Dynamics of changes in the activity of beta-galactosidase and culture fluorescence suggests that a decrease in the level of metabolism resulted in the switch of these cyclic systems from bistable to monostable functioning regime, which corresponds to the Lac+ phenotype with respect to the ratio of regulatory proteins. Thus, the instability of growth conditions may cause phenotypic heterogeneity in the clonal population of cells containing bistable gene networks.

In vitro cultures of human pancreatic stem cells: Gene and protein expression of designated markers varies with passage

Adult stem cells may possess great plasticity, but the cellular mechanisms regulating their fate are not fully understood. Prior to application of stem cell populations in regenerative medicine, major challenges remain to be overcome. Fundamental questions about in vitro growth and spontaneous differentiation of adult stem cell populations must be resolved. In this study, we comprehensively characterized a stem cell population derived from human pancreatic tissue by analyzing mRNA and protein expression in consecutive passages. We examined transcription and protein expression levels of markers related to stem cells or differentiated cells, respectively, as well as the growth rate of a primary human pancreatic stem cell population. In particular, the course of spontaneous mRNA and protein expression of the genes for alpha-smooth muscle actin (alpha-SMA), neurofilaments (NF), cytokeratin 18 (CK18) and nestin was examined during 11 passages by means of RT-PCR and immunocytochemistry. The cell population showed exponential growth over 10 of the 11 examined passages. Both the spontaneous expression of stem cell-related mRNA and protein as well as the characteristics of spontaneous differentiation were variable. Changes in mRNA and protein expression showed no direct correlation. These results demonstrate the unpredictable behaviour of spontaneously differentiating stem cells, being influenced by numerous, barely traceable extrinsic factors. Characterization studies of stem cell populations therefore require improved analysis techniques together with strictly controlled cell cultivation conditions to improve global gene and protein expression analyses.