Browsing by Subject "Cell lines"

Now showing 1 - 7 of 7
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    Beta cell extracellular vesicle miR-21-5p cargo is increased in response to inflammatory cytokines and serves as a biomarker of type 1 diabetes
    (Springer Nature, 2018-05) Lakhter, Alexander J.; Pratt, Rachel E.; Moore, Rachel E.; Doucette, Kaitlin K.; Maier, Bernhard F.; DiMeglio, Linda A.; Sims, Emily K.; Pediatrics, School of Medicine
    AIMS/HYPOTHESIS: Improved biomarkers are acutely needed for the detection of developing type 1 diabetes, prior to critical loss of beta cell mass. We previously demonstrated that elevated beta cell microRNA 21-5p (miR-21-5p) in rodent and human models of type 1 diabetes increased beta cell apoptosis. We hypothesised that the inflammatory milieu of developing diabetes may also increase miR-21-5p in beta cell extracellular vesicle (EV) cargo and that circulating EV miR-21-5p would be increased during type 1 diabetes development. METHODS: MIN6 and EndoC-βH1 beta cell lines and human islets were treated with IL-1β, IFN-γ and TNF-α to mimic the inflammatory milieu of early type 1 diabetes. Serum was collected weekly from 8-week-old female NOD mice until diabetes onset. Sera from a cross-section of 19 children at the time of type 1 diabetes diagnosis and 16 healthy children were also analysed. EVs were isolated from cell culture media or serum using sequential ultracentrifugation or ExoQuick precipitation and EV miRNAs were assayed. RESULTS: Cytokine treatment in beta cell lines and human islets resulted in a 1.5- to threefold increase in miR-21-5p. However, corresponding EVs were further enriched for this miRNA, with a three- to sixfold EV miR-21-5p increase in response to cytokine treatment. This difference was only partially reduced by pre-treatment of beta cells with Z-VAD-FMK to inhibit cytokine-induced caspase activity. Nanoparticle tracking analysis showed cytokines to have no effect on the number of EVs, implicating specific changes within EV cargo as being responsible for the increase in beta cell EV miR-21-5p. Sequential ultracentrifugation to separate EVs by size suggested that this effect was mostly due to cytokine-induced increases in exosome miR-21-5p. Longitudinal serum collections from NOD mice showed that EVs displayed progressive increases in miR-21-5p beginning 3 weeks prior to diabetes onset. To validate the relevance to human diabetes, we assayed serum from children with new-onset type 1 diabetes compared with healthy children. While total serum miR-21-5p and total serum EVs were reduced in diabetic participants, serum EV miR-21-5p was increased threefold compared with non-diabetic individuals. By contrast, both serum and EV miR-375-5p were increased in parallel among diabetic participants. CONCLUSIONS/INTERPRETATION: We propose that circulating EV miR-21-5p may be a promising marker of developing type 1 diabetes. Additionally, our findings highlight that, for certain miRNAs, total circulating miRNA levels are distinct from circulating EV miRNA content.
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    Classification of Breast Cancer Cell Lines into Subtypes Based on Genetic Profiles
    (2015-03-16) Pawar, Aniruddha Vikram; Li, Lang
    Today we know that there are several different types of breast cancer. Accurate identification breast cancer subtype is extremely important in treating this disease effectively. Consequently the process of invtro development of drugs to treat this disease should be naturally subtype specific. Until now several studies have identified multiple breast cancer cell lines and these cell lines have served as invaluable invitro tumor models. However very few of these cell lines are classified as per their subtypes. In this thesis an effort is made to classify 59 of such breast cancer cell lines using genetic profile comparison approach. This approach is based on comparing characteristic features such as copy number and gene expression of a given cell line to those observed from the tissue samples of different breast subtypes. The tissue data for this comparison comes from The Cancer Genome Atlas (TCGA) while cell line data is taken from Cancer Cell Line Encyclopedia (CCLE).
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    Comprehensive comparison of molecular portraits between cell lines and tumors in breast cancer
    (BioMed Central, 2016-08-22) Jiang, Guanglong; Zhang, Shijun; Yazdanparast, Aida; Li, Meng; Pawar, Aniruddha Vikram; Liu, Yunlong; Inavolu, Sai Mounika; Cheng, Lijun; Department of Medical and Molecular Genetics, IU School of Medicine
    Background: Proper cell models for breast cancer primary tumors have long been the focal point in the cancer’s research. The genomic comparison between cell lines and tumors can investigate the similarity and dissimilarity and help to select right cell model to mimic tumor tissues to properly evaluate the drug reaction in vitro. In this paper, a comprehensive comparison in copy number variation (CNV), mutation, mRNA expression and protein expression between 68 breast cancer cell lines and 1375 primary breast tumors is conducted and presented. Results: Using whole genome expression arrays, strong correlations were observed between cells and tumors. PAM50 gene expression differentiated them into four major breast cancer subtypes: Luminal A and B, HER2amp, and Basal-like in both cells and tumors partially. Genomic CNVs patterns were observed between tumors and cells across chromosomes in general. High C > T and C > G trans-version rates were observed in both cells and tumors, while the cells had slightly higher somatic mutation rates than tumors. Clustering analysis on protein expression data can reasonably recover the breast cancer subtypes in cell lines and tumors. Although the drug-targeted proteins ER/PR and interesting mTOR/GSK3/TS2/PDK1/ER_P118 cluster had shown the consistent patterns between cells and tumor, low protein-based correlations were observed between cells and tumors. The expression consistency of mRNA verse protein between cell line and tumors reaches 0.7076. These important drug targets in breast cancer, ESR1, PGR, HER2, EGFR and AR have a high similarity in mRNA and protein variation in both tumors and cell lines. GATA3 and RP56KB1 are two promising drug targets for breast cancer. A total score developed from the four correlations among four molecular profiles suggests that cell lines, BT483, T47D and MDAMB453 have the highest similarity with tumors. Conclusions: The integrated data from across these multiple platforms demonstrates the existence of the similarity and dissimilarity of molecular features between breast cancer tumors and cell lines. The cell lines only mirror some but not all of the molecular properties of primary tumors. The study results add more evidence in selecting cell line models for breast cancer research.
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    MicroRNA 21 targets BCL2 mRNA to increase apoptosis in rat and human beta cells
    (Springer, 2017-06) Sims, Emily K.; Lakhter, Alexander; Anderson-Baucum, Emily; Kono, Tatsuyoshi; Tong, Xin; Evans-Molina, Carmella; Pediatrics, School of Medicine
    AIMS/HYPOTHESIS: The role of beta cell microRNA (miR)-21 in the pathophysiology of type 1 diabetes has been controversial. Here, we sought to define the context of beta cell miR-21 upregulation in type 1 diabetes and the phenotype of beta cell miR-21 overexpression through target identification. METHODS: Islets were isolated from NOD mice and mice treated with multiple low doses of streptozotocin, as a mouse model of diabetes. INS-1 832/13 beta cells and human islets were treated with IL-1β, IFN-γ and TNF-α to mimic the milieu of early type 1 diabetes. Cells and islets were transfected with miR-21 mimics or inhibitors. Luciferase assays and polyribosomal profiling (PRP) were performed to define miR-21-target interactions. RESULTS: Beta cell miR-21 was increased in in vivo models of type 1 diabetes and cytokine-treated cells/islets. miR-21 overexpression decreased cell count and viability, and increased cleaved caspase 3 levels, suggesting increased cell death. In silico prediction tools identified the antiapoptotic mRNA BCL2 as a conserved miR-21 target. Consistent with this, miR-21 overexpression decreased BCL2 transcript and B cell lymphoma 2 (BCL2) protein production, while miR-21 inhibition increased BCL2 protein levels and reduced cleaved caspase 3 levels after cytokine treatment. miR-21-mediated cell death was abrogated in 828/33 cells, which constitutively overexpress Bcl2. Luciferase assays suggested a direct interaction between miR-21 and the BCL2 3' untranslated region. With miR-21 overexpression, PRP revealed a shift of the Bcl2 message towards monosome-associated fractions, indicating inhibition of Bcl2 translation. Finally, overexpression in dispersed human islets confirmed a reduction in BCL2 transcripts and increased cleaved caspase 3 production. CONCLUSIONS/INTERPRETATION: In contrast to the pro-survival role reported in other systems, our results demonstrate that miR-21 increases beta cell death via BCL2 transcript degradation and inhibition of BCL2 translation.
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    Monoallelically expressed noncoding RNAs form nucleolar territories on NOR-containing chromosomes and regulate rRNA expression
    (eLife Sciences, 2024-01-19) Hao, Qinyu; Liu, Minxue; Daulatabad, Swapna Vidhur; Gaffari, Saba; Song, You Jin; Srivastava, Rajneesh; Bhaskar, Shivang; Moitra, Anurupa; Mangan, Hazel; Tseng, Elizabeth; Gilmore, Rachel B.; Frier, Susan M.; Chen, Xin; Wang, Chengliang; Huang, Sui; Chamberlain, Stormy; Jin, Hong; Korlach, Jonas; McStay, Brian; Sinha, Saurabh; Janga, Sarath Chandra; Prasanth, Supriya G.; Prasanth, Kannanganattu V.; Biomedical Engineering and Informatics, Luddy School of Informatics, Computing, and Engineering
    Out of the several hundred copies of rRNA genes arranged in the nucleolar organizing regions (NOR) of the five human acrocentric chromosomes, ~50% remain transcriptionally inactive. NOR-associated sequences and epigenetic modifications contribute to the differential expression of rRNAs. However, the mechanism(s) controlling the dosage of active versus inactive rRNA genes within each NOR in mammals is yet to be determined. We have discovered a family of ncRNAs, SNULs (Single NUcleolus Localized RNA), which form constrained sub-nucleolar territories on individual NORs and influence rRNA expression. Individual members of the SNULs monoallelically associate with specific NOR-containing chromosomes. SNULs share sequence similarity to pre-rRNA and localize in the sub-nucleolar compartment with pre-rRNA. Finally, SNULs control rRNA expression by influencing pre-rRNA sorting to the DFC compartment and pre-rRNA processing. Our study discovered a novel class of ncRNAs influencing rRNA expression by forming constrained nucleolar territories on individual NORs.
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    A novel intraperitoneal metastatic xenograft mouse model for survival outcome assessment of esophageal adenocarcinoma
    (Plos, 2017-02-22) Hassan, Md Sazzad; Awasthi, Niranjan; Li, Jun; Schwarz, Margaret A.; Schwarz, Roderich E.; von Holzen, Urs; Department of Surgery, IU School of Medicine
    Esophageal adenocarcinoma (EAC) has become the dominant type of esophageal cancer in United States. The 5-year survival rate of EAC is below 20% and most patients present with locally advanced or widespread metastatic disease, where current treatment is largely ineffective. Therefore, new therapeutic approaches are urgently needed. Improvement of EAC patient outcome requires well-characterized animal models in which to evaluate novel therapeutics. In this study we aimed to establish a peritoneal dissemination xenograft mouse model of EAC that would support survival outcome analyses. To find the best candidate cell line from 7 human EAC cell lines of different origin named ESO26, OE33, ESO51, SK-GT-2, OE19, OACM5.1C and Flo-1 were injected intraperitoneally/subcutaneously into SCID mice. The peritoneal/xenograft tumor formation and mouse survival were compared among different groups. All cell lines injected subcutaneously formed tumors within 3 months at variable rates. All cell lines except OACM5.1C formed intraperitoneal tumors within 3 months at variable rates. Median animal survival with peritoneal dissemination was 108 days for ESO26 cells (5X106), 65 days for OE33 cells (5X106), 88 days for ESO51 cells (5X106), 76 days for SK-GT-2 cells (5X106), 55 days for OE19 cells (5X106), 45 days for OE19 cells (10X106) and 82 days for Flo-1 cells (5X106). Interestingly, only in the OE19 model all mice (7/7 for 5X106 and 5/5 for10X106) developed bloody ascites with liver metastasis after intraperitoneal injection. The median survival time of these animals was the shortest (45 days for 10X106 cells). In addition, median survival was significantly increased after paclitaxel treatment compared with the control group (57 days versus 45 days, p = 0.0034) along with a significant decrease of the relative subcutaneous tumor volume (p = 0.00011). Thus peritoneal dissemination mouse xenograft model for survival outcome assessment after intraperitoneal injection of OE19 cells will be very useful for the evaluation of cancer therapeutics.
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    α-COP binding to the survival motor neuron protein SMN is required for neuronal process outgrowth
    (Oxford University Press, 2015-12-20) Li, Hongxia; Custer, Sara K.; Gilson, Timra; Hao, Le Thi; Beattie, Christine E.; Androphy, Elliot J.; Department of Dermatology, IU School of Medicine
    Spinal muscular atrophy (SMA), a heritable neurodegenerative disease, results from insufficient levels of the survival motor neuron (SMN) protein. α-COP binds to SMN, linking the COPI vesicular transport pathway to SMA. Reduced levels of α-COP restricted development of neuronal processes in NSC-34 cells and primary cortical neurons. Remarkably, heterologous expression of human α-COP restored normal neurite length and morphology in SMN-depleted NSC-34 cells in vitro and zebrafish motor neurons in vivo. We identified single amino acid mutants of α-COP that selectively abrogate SMN binding, retain COPI-mediated Golgi-ER trafficking functionality, but were unable to support neurite outgrowth in cellular and zebrafish models of SMA. Taken together, these demonstrate the functional role of COPI association with the SMN protein in neuronal development.