Establishment of novel in vitro mouse chief cell and SPEM cultures identifies MAL2 as a marker of metaplasia in the stomach

Oxyntic atrophy in the stomach leads to chief cell transdifferentiation into spasmolytic polypeptide expressing metaplasia (SPEM). Investigations of preneoplastic metaplasias in the stomach are limited by the sole reliance on in vivo mouse models, owing to the lack of in vitro models for distinct normal mucosal lineages and metaplasias. Utilizing the Immortomouse, in vitro cell models of chief cells and SPEM were developed to study the characteristics of normal chief cells and metaplasia. Chief cells and SPEM cells isolated from Immortomice were cultured and characterized at both the permissive (33°C) and the nonpermissive temperature (39°C). Clones were selected on the basis of their transcriptional expression of specific stomach lineage markers (named ImChief and ImSPEM) and protein expression and growth were analyzed. The transcriptional expression profiles of ImChief and ImSPEM cells were compared further by using gene microarrays. ImChief cells transcriptionally express most chief cell markers and contain pepsinogen C and RAB3D-immunostaining vesicles. ImSPEM cells express the SPEM markers TFF2 and HE4 and constitutively secrete HE4. Whereas ImChief cells cease proliferation at the nonpermissive temperature, ImSPEM cells continue to proliferate at 39°C. Gene expression profiling of ImChief and ImSPEM revealed myelin and lymphocyte protein 2 (MAL2) as a novel marker of SPEM lineages. Our results indicate that the expression and proliferation profiles of the novel ImChief and ImSPEM cell lines resemble in vivo chief and SPEM cell lineages. These cell culture lines provide the first in vitro systems for studying the molecular mechanisms of the metaplastic transition in the stomach.

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Kaposi sarcoma-associated herpesvirus (KSHV) induces heme oxygenase-1 expression and activity in KSHV-infected endothelial cells

Blood ◽

10.1182/blood-2003-08-2781 ◽

2004 ◽

Vol 103 (9) ◽

pp. 3465-3473 ◽

Cited By ~ 63

Author(s):

Shane C. McAllister ◽

Scott G. Hansen ◽

Rebecca A. Ruhl ◽

Camilo M. Raggo ◽

Victor R. DeFilippis ◽

...

Keyword(s):

Endothelial Cells ◽

Heme Oxygenase ◽

Cell Biology ◽

Molecular Mechanisms ◽

Expression Profiles ◽

Kaposi Sarcoma ◽

Heme Oxygenase 1 ◽

Spindle Cell Proliferation

Abstract Kaposi sarcoma (KS) is the most common AIDS-associated malignancy and is characterized by angiogenesis and the presence of spindle cells. Kaposi sarcoma-associated herpesvirus (KSHV) is consistently associated with all clinical forms of KS, and in vitro infection of dermal microvascular endothelial cells (DMVECs) with KSHV recapitulates many of the features of KS, including transformation, spindle cell proliferation, and angiogenesis. To study the molecular mechanisms of KSHV pathogenesis, we compared the protein expression profiles of KSHV-infected and uninfected DMVECs. This comparison revealed that heme oxygenase-1 (HO-1), the inducible enzyme responsible for the rate-limiting step in heme catabolism, was up-regulated in infected endothelial cells. Recent evidence suggests that the products of heme catabolism have important roles in endothelial cell biology, including apoptosis and angiogenesis. Here we show that HO-1 mRNA and protein are up-regulated in KSHV-infected cultures. Comparison of oral and cutaneous AIDS-KS tissues with normal tissues revealed that HO-1 mRNA and protein were also up-regulated in vivo. Increased HO-1 enzymatic activity in vitro enhanced proliferation of KSHV-infected DMVECs in the presence of free heme. Treatment with the HO-1 inhibitor chromium mesoporphyrin IX abolished heme-induced proliferation. These data suggest that HO-1 is a potential therapeutic target for KS that warrants further study. (Blood. 2004;103: 3465-3473)

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Cortical Spheroid Model for Studying the Effects of Ischemic Brain Injury

10.1101/2021.10.16.464587 ◽

2021 ◽

Author(s):

Rachel M McLaughlin ◽

Amanda Laguna ◽

Ilayda Top ◽

Christien Hernadez ◽

Liane L Livi ◽

...

Keyword(s):

Molecular Mechanisms ◽

Three Dimensional ◽

Cost Effective ◽

Glucose Deprivation ◽

In Vitro Models ◽

3D Architecture ◽

A Cell ◽

Antioxidant Agent

Stroke is a devastating neurological disorder and a leading cause of death and long-term disability. Despite many decades of research, there are still very few therapeutic options for patients suffering from stroke or its consequences. This is partially due to the limitations of current research models, including traditional in vitro models which lack the three-dimensional (3D) architecture and cellular make-up of the in vivo brain. 3D spheroids derived from primary postnatal rat cortex provide an in vivo-relevant model containing a similar cellular composition to the native cortex and a cell-synthesized extracellular matrix. These spheroids are cost-effective, highly reproducible, and can be produced in a high-throughput manner, making this model an ideal candidate for screening potential therapeutics. To study the cellular and molecular mechanisms of stroke in this model, spheroids were deprived of glucose, oxygen, or both oxygen and glucose for 24 hours. Both oxygen and oxygen-glucose deprived spheroids demonstrated many of the hallmarks of stroke, including a decrease in metabolism, an increase in neural dysfunction, and an increase in reactive astrocytes. Pretreatment of spheroids with the antioxidant agent N-acetylcysteine (NAC) mitigated the decrease in ATP seen after 24 hours of oxygen-glucose deprivation. Together, these results show the utility of our 3D cortical spheroid model for studying ischemic injury and its potential for screening stroke therapeutics.

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Histone methyltransferase SET8 is regulated by miR-192/215 and induces oncogene-induced senescence via p53-dependent DNA damage in human gastric carcinoma cells

Cell Death and Disease ◽

10.1038/s41419-020-03130-4 ◽

2020 ◽

Vol 11 (10) ◽

Author(s):

Xiaojing Zhang ◽

Yin Peng ◽

Yuan Yuan ◽

Yuli Gao ◽

Fan Hu ◽

...

Keyword(s):

Dna Damage ◽

Molecular Mechanisms ◽

Biological Activities ◽

Expression Profiles ◽

Carcinoma Cells ◽

Negative Regulator ◽

Expression Change ◽

Human Gastric Carcinoma Cells

Abstract Gastric cancer (GC) is the most common cancer throughout the world. Despite advances of the treatments, detailed oncogenic mechanisms are largely unknown. In our previous study, we investigated microRNA (miR) expression profiles in human GC using miR microarrays. We found miR-192/215 were upregulated in GC tissues. Then gene microarray was implemented to discover the targets of miR-192/215. We compared the expression profile of BGC823 cells transfected with miR-192/215 inhibitors, and HFE145 cells transfected with miR-192/-215 mimics, respectively. SET8 was identified as a proposed target based on the expression change of more than twofold. SET8 belongs to the SET domain-containing methyltransferase family and specifically catalyzes monomethylation of H4K20me. It is involved in diverse functions in tumorigenesis and metastasis. Therefore, we focused on the contributions of miR-192/215/SET8 axis to the development of GC. In this study, we observe that functionally, SET8 regulated by miR-192/215 is involved in GC-related biological activities. SET8 is also found to trigger oncogene-induced senescence (OIS) in GC in vivo and in vitro, which is dependent on the DDR (DNA damage response) and p53. Our findings reveal that SET8 functions as a negative regulator of metastasis via the OIS-signaling pathway. Taken together, we investigated the functional significance, molecular mechanisms, and clinical impact of miR-192/215/SET8/p53 in GC.

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A Novel in Vitro Model to Investigate Behavior of Articular Chondrocytes in Osteoarthritis

The Journal of Rheumatology ◽

10.3899/jrheum.080080 ◽

2009 ◽

Vol 37 (2) ◽

pp. 426-431 ◽

Cited By ~ 2

Author(s):

KENNETH S. RANKIN ◽

RACHEL L. LAKEY ◽

CRAIG H. GERRAND ◽

ANDREW P. SPROWSON ◽

ANDREW W. McCASKIE ◽

...

Keyword(s):

Molecular Mechanisms ◽

Articular Chondrocytes ◽

Type Ii Collagen ◽

Type Ii ◽

Transcriptional Expression ◽

Matrix Metalloproteinase 1 ◽

Standard Culture ◽

Cells Cultured

Objective. To investigate in vivo simulation of the microenvironment in which osteoarthritis (OA) chondrocytes are cultured in vitro.Methods. Human articular chondrocytes were cultured under normoxic and hypoxic conditions. Cells were cultured on standard culture plastic or a porous polyHEMA surface that closely resembles the in vivo cartilage microarchitecture. Morphological changes to the cells were demonstrated by fluorescent staining with DAPI and vinculin. Proteoglycan and type II collagen protein levels were assessed using established techniques. Matrix metalloproteinase-1 (MMP-1) production was assessed by ELISA. The gene expression of type II collagen and SOX9 was measured using real-time polymerase chain reaction.Results. Cells grown on culture plastic were seen to be flat and hexagonal. Cells cultured on the porous polyHEMA surface exhibited morphology in keeping with the in vivo microenvironment. Glycosaminoglycan release in hypoxia was high from cells cultured on standard culture plastic. Transcriptional expression of type II collagen was upregulated in hypoxia and by culture on the polyHEMA surface. Transcriptional expression of SOX9 in hypoxia was upregulated compared to normoxia; no significant effect was seen by varying the culture surface. Translational expression of type II collagen was upregulated at 20% oxygen on the polyHEMA surface compared to culture plastic and this was related to MMP-1 expression.Conclusion. Culture of chondrocytes in hypoxia and on a porous surface simulates the in vivo microenvironment and illustrates the molecular mechanisms of OA.

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Molecular signature of anastasis for reversal of apoptosis

F1000Research ◽

10.12688/f1000research.10568.2 ◽

2017 ◽

Vol 6 ◽

pp. 43 ◽

Cited By ~ 5

Author(s):

Ho Man Tang ◽

C. Conover Talbot Jr ◽

Ming Chiu Fung ◽

Ho Lam Tang

Keyword(s):

Gene Expression ◽

Time Course ◽

Molecular Mechanisms ◽

Expression Profiles ◽

Cancer Cell Line ◽

Molecular Signature ◽

Cell Recovery ◽

Therapeutic Implications

Anastasis (Greek for "rising to life") is a cell recovery phenomenon that rescues dying cells from the brink of cell death. We recently discovered anastasis to occur after the execution-stage of apoptosis in vitro and in vivo. Promoting anastasis could in principle preserve injured cells that are difficult to replace, such as cardiomyocytes and neurons. Conversely, arresting anastasis in dying cancer cells after cancer therapies could improve treatment efficacy. To develop new therapies that promote or inhibit anastasis, it is essential to identify the key regulators and mediators of anastasis – the therapeutic targets. Therefore, we performed time-course microarray analysis to explore the molecular mechanisms of anastasis during reversal of ethanol-induced apoptosis in mouse primary liver cells. We found striking changes in transcription of genes involved in multiple pathways, including early activation of pro-cell survival, anti-oxidation, cell cycle arrest, histone modification, DNA-damage and stress-inducible responses, and at delayed times, angiogenesis and cell migration. Validation with RT-PCR confirmed similar changes in the human liver cancer cell line, HepG2, during anastasis. Here, we present the time-course whole-genome gene expression dataset revealing gene expression profiles during the reversal of apoptosis. This dataset provides important insights into the physiological, pathological, and therapeutic implications of anastasis.

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miR-205-5p inhibits human endometriosis progression by targeting ANGPT2 in endometrial stromal cells

Stem Cell Research & Therapy ◽

10.1186/s13287-019-1388-5 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 5

Author(s):

Chen-Fei Zhou ◽

Min-Juan Liu ◽

Wei Wang ◽

Sha Wu ◽

Yu-Xin Huang ◽

...

Keyword(s):

Molecular Mechanisms ◽

Expression Profiles ◽

Target Prediction ◽

Luciferase Reporter ◽

Endometrial Stromal Cells ◽

Candidate Target ◽

Candidate Target Gene ◽

Angiopoietin 2

Abstract Background miRNA expression profiles in ectopic endometrium (EC) serving as pathophysiologic genetic fingerprints contribute to determining endometriosis progression; however, the underlying molecular mechanisms remain unknown. Methods miRNA microarray analysis was used to determine the expression profiling of EC fresh tissues. qRT-PCR was performed to screen miR-205-5p expression in EC tissues. The roles of miR-205-5p and its candidate target gene, angiopoietin-2 (ANGPT2), in endometriosis progression were confirmed on the basis of both in vitro and in vivo systems. miR-205-5p and ANGPT2 expression were measured by in situ hybridization and immunochemistry, and their clinical significance was statistically analysed. Results miR-205-5p was screened as a novel suppressor of endometriosis through primary ectopic endometrial stromal cell migration, invasion, and apoptosis assay in vitro, along with endometrial-like xenograft growth and apoptosis in vivo. In addition, ANGPT2 was identified as a direct target of miR-205-5p through bioinformatic target prediction and luciferase reporter assay. Re-expression and knockdown of ANGPT2 could respectively rescue and simulate the effects induced by miR-205-5p. Importantly, the miR-205-5p-ANGPT2 axis was found to activate the ERK/AKT pathway in endometriosis. Finally, miR-205-5p and ANGPT2 expression were closely correlated with the endometriosis severity. Conclusion The newly identified miR-205-5p-ANGPT2-AKT/ERK axis illustrates the molecular mechanism of endometriosis progression and may represent a novel diagnostic biomarker and therapeutic target for disease treatment.

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In Vitro Models to Study Mechanisms of Lung Toxicity

Alternatives to Laboratory Animals ◽

10.1177/026119299001800127.1 ◽

1990 ◽

Vol 18 (1_part_1) ◽

pp. 267-281

Author(s):

Åke Ryrfeldt ◽

Ian A. Cotgreave ◽

Peter Moldéus

Keyword(s):

Molecular Mechanisms ◽

In Vitro Models ◽

Lung Toxicity ◽

Lung Mechanics ◽

Lung Cells ◽

Suitable Model ◽

Clara Cells ◽

Functional Studies

Several functioning in vitro systems of varying complexity are currently in use for the study of mechanisms of lung toxicity. The isolated perfused lung is the model closest to the in vivo situation. It is a suitable model for combining metabolic and functional studies. It is, for instance, possible to relate changes in lung mechanics and lung perfusion flow to the release of various mediators during exposure of the lung to various agents. A simpler model may be constructed from lung slices which are less viable but suitable for uptake as well as metabolism studies. Specific lung cells such as Clara cells and type II pneumocytes have been isolated and cultured and are valuable tools for studies of the molecular mechanisms of lung toxicity, particularly in cases of cell-specific toxicity. There is, however, a great need to develop techniques for the isolation and culture of other types of lung cells and also to improve the culturing techniques for those already isolated.

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Zearalenone Exposure Disrupts Blood–Testis Barrier Integrity through Excessive Ca2+-Mediated Autophagy

Toxins ◽

10.3390/toxins13120875 ◽

2021 ◽

Vol 13 (12) ◽

pp. 875

Author(s):

Jinjin She ◽

Nannan Feng ◽

Wanglong Zheng ◽

Hao Zheng ◽

Peirong Cai ◽

...

Keyword(s):

Molecular Mechanisms ◽

In Vitro Models ◽

Reproductive Disorders ◽

Junction Protein ◽

Testicular Morphology ◽

Related Proteins ◽

Junction Proteins ◽

Blood Testis Barrier

Zearalenone (ZEA), a common mycotoxin in grains and animal feeds, has been associated with male reproductive disorders. However, the potential toxicity mechanism of ZEA is not fully understood. In this study, in vivo and in vitro models were used to explore the effects of ZEA on the blood–testis barrier (BTB) and related molecular mechanisms. First, male BALB/C mice were administered ZEA orally (40 mg/kg·bw) for 5–7 d. Sperm motility, testicular morphology, and expressions of BTB junction proteins and autophagy-related proteins were evaluated. In addition, TM4 cells (mouse Sertoli cells line) were used to delineate the molecular mechanisms that mediate the effects of ZEA on BTB. Our results demonstrated that ZEA exposure induced severe testicular damage in histomorphology and an ultrastructural, time-dependent decrease in the expression of blood–testis barrier junction-related proteins, accompanied by an increase in the expression of autophagy-related proteins. Additionally, similar to the in vitro results, the dose-dependent treatment of ZEA increased the level of cytoplasmic Ca2+ and the levels of the autophagy markers LC3-II and p62, in conjunction with a decrease in the BTB junction proteins occludin, claudin-11, and Cx43, with the dislocation of the gap junction protein Cx43. Meanwhile, inhibition of autophagy by CQ and 3-MA or inhibition of cytoplasmic Ca2+ by BAPTA-AM was sufficient to reduce the effects of ZEA on the TM4 cell BTB. To summarize, this study emphasizes the role of Ca2+-mediated autophagy in ZEA-induced BTB destruction, which deepens our understanding of the molecular mechanism of ZEA-induced male reproductive disorders.

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Activation of the Anaphase Promoting Complex reverses multiple drug resistant cancer

10.1101/2020.05.26.115337 ◽

2020 ◽

Author(s):

T.G. Arnason ◽

V. MacDonald-Dickinson ◽

J.F. Davies ◽

L. Lobanova ◽

C. Gaunt ◽

...

Keyword(s):

Gene Expression ◽

Molecular Mechanisms ◽

Multiple Drug Resistance ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Multiple Drug ◽

Anaphase Promoting Complex ◽

Insulin Sensitizer

ABSTRACTLike humans, canines spontaneously develop lymphomas that are treated by chemotherapy cocktails and frequently develop multiple drug resistance (MDR). Their shortened clinical timelines and tumor accessibility make them excellent models to study MDR mechanisms. We previously demonstrated that adjunct treatment of in vitro MDR cell lines with insulin-sensitizers effectively restored MDR chemosensitivity and prevented MDR development. This study extends the use of an insulin-sensitizer to clinical and tumor responses in vivo in volunteer canines with MDR lymphoma, including assessing changes in MDR protein biomarkers and global gene expression. Longitudinal tumor sampling and analysis of MDR cases throughout treatment allowed a correlation between in vivo molecular mechanisms and clinical responsiveness. We found reduced MDR biomarkers within all tumors, yet only one canine entered clinical remission. Analysis of tumor samples during remission and relapse allowed comparison of gene expression profiles. This revealed the Anaphase Promoting Complex (APC), a ubiquitin-E3 ligase regulating cell cycle progression, was impaired during chemoresistance/MDR and restored during remission. Validating in vitro tests restored MDR chemosensitivity upon APC activation, supporting the idea that APC activity is an important underlying cellular mechanism associated with treatment resistance, and a novel potential therapeutic target.

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Mechanistic Basis of Arsenic Induced Carcinogenesis: Differential miRNA Expression

10.21203/rs.3.rs-1141158/v1 ◽

2022 ◽

Author(s):

Placheril J. John ◽

Navneet Kumar

Keyword(s):

Cancer Progression ◽

Mirna Expression ◽

Molecular Mechanisms ◽

Expression Profiles ◽

Inorganic Arsenic ◽

Ros Generation ◽

Mirna Expression Profiles ◽

Mechanistic Basis

Abstract Arsenic, a toxic metalloid, provokes many detrimental consequences to human health. It is prevalent in earth's crust and poses a major threat to humans globally. Inorganic arsenic exposure occurs mainly via drinking water or food and is metabolized in mammals to form organic metabolites/ end products. Chronic exposure to arsenic causes lung, skin and urinary bladder cancers and increases the risks of liver, kidney and prostate cancers. Arsenic-induced ROS generation, disturbances in several signaling pathways, DNA repair inhibition, chromosomal aberrations, and epigenetic changes including alterations in DNA methylation, histone modifications and differential miRNA expression profiles are involved in cancer progression, and malignant transformation. However, details of arsenic-induced carcinogenesis and molecular mechanisms involved are still remaining obscure. MicroRNAs are post-transcriptional gene expression regulators and themselves may act as oncogenes and tumor suppressor genes. Differential miRNA expression is implicated in several human cancers. This review covers general mechanistic basis of arsenic-induced carcinogenesis, explores recent in-vitro, in-vivo and cohort studies on differential miRNA expression profiles and shares associated molecular mechanistic data on miRNA dysregulation and their functional consequences leading to arsenic induced tumorigenesis, metastasis and cancer, also discusses the future directions.

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