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The effects of newborn calf serum and foetal bovine serum on the Na+ + K+-activated adenosine triphosphatase activity in 3T3 and SV3T3 cells grown to confluency

Tenang EM, McCaldin B

Biochem. Int., 1988 Feb;16(2):193-8.
PMID: 2835046

The effects of cell density and growth in 10% foetal bovine serum and 10% newborn calf serum on the activity of the enzyme (Na+ + K+)-ATPase were studied in 3T3 and SV3T3 cells. The enzyme activity decreases in 3T3 cells grown in foetal bovine serum as the cells approach confluency while in those grown in newborn calf serum the enzyme activity increases. The (Na+ + K+)-ATPase activity does not change with increase in cell density in SV3T3 cells grown in foetal bovine serum while the enzyme activity in those grown in newborn calf serum increases with increase in cells density up to about 1.35 x 10(5) cells/sq. cm. and then decreases with further increase in cell number. At confluency it was found that the enzyme activity is higher in the SV3T3 as compared to the 3T3 cells when the cells were grown in 10% foetal bovine serum, whereas in those grown in 10% newborn calf serum the enzyme activity is higher in the 3T3 as compared to the SV3T3 cells.

Matched MeSH terms: Cell Line, Transformed/drug effects; Cell Line, Transformed/enzymology
Golden SusPtrit: a genetically well transformable barley line for studies on the resistance to rust fungi

Yeo FK, Hensel G, Vozábová T, Martin-Sanz A, Marcel TC, Kumlehn J, et al.

Theor Appl Genet, 2014 Feb;127(2):325-37.
PMID: 24247233 DOI: 10.1007/s00122-013-2221-7

KEY MESSAGE: We developed 'Golden SusPtrit', i.e., a barley line combining SusPtrit's high susceptibility to non-adapted rust fungi with the high amenability of Golden Promise for transformation. Nonhost and partial resistance to Puccinia rust fungi in barley are polygenically inherited. These types of resistance are principally prehaustorial, show high diversity between accessions of the plant species and are genetically associated. To study nonhost and partial resistance, as well as their association, candidate gene(s) for resistance must be cloned and tested in susceptible material where SusPtrit would be the line of choice. Unfortunately, SusPtrit is not amenable to Agrobacterium-mediated transformation. Therefore, a doubled haploid (DH) mapping population (n = 122) was created by crossing SusPtrit with Golden Promise to develop a 'Golden SusPtrit', i.e., a barley line combining SusPtrit's high susceptibility to non-adapted rust fungi with the high amenability of Golden Promise for transformation. We identified nine genomic regions occupied by resistance quantitative trait loci (QTLs) against four non-adapted rust fungi and P. hordei isolate 1.2.1 (Ph.1.2.1). Four DHs were selected for an Agrobacterium-mediated transformation efficiency test. They were among the 12 DH lines most susceptible to the tested non-adapted rust fungi. The most efficiently transformed DH line was SG062N (11-17 transformants per 100 immature embryos). The level of non-adapted rust infection on SG062N is either similar to or higher than the level of infection on SusPtrit. Against Ph.1.2.1, the latency period conferred by SG062N is as short as that conferred by SusPtrit. SG062N, designated 'Golden SusPtrit', will be a valuable experimental line that could replace SusPtrit in nonhost and partial resistance studies, especially for stable transformation using candidate genes that may be involved in rust-resistance mechanisms.

Matched MeSH terms: Cell Line, Transformed
Abnormality in DNA-protein binding in ataxia-telangiectasia nuclear extracts

Mohamed R, Lavin MF

Biochem Biophys Res Commun, 1989 Feb 15;158(3):749-54.
PMID: 2537634

Anomalies in DNA replication, repair and recombination in ataxia-telangiectasia (A-T) point to a defect in structure or function of chromatin. In this study we have compared DNA-protein binding in nuclear extracts from control and A-T cells using two assay systems, filter-binding and DNA-accessibility. Interestingly, the extent of DNA protein binding over a range of protein concentration was significantly lower in A-T extracts. In addition the accessibility of the restriction enzyme Eco R1 to protein-bound plasmid was greater when A-T extracts were used. This is in keeping with the reduced binding observed in the filter-binding assay.

Matched MeSH terms: Cell Line, Transformed
Global and Targeted Proteomics of Prostate Cancer Cell Secretome: Combination of 2-Dimensional Image-Converted Analysis of Liquid Chromatography and Mass Spectrometry and In Silico Selection Selected Reaction Monitoring Analysis

Nurdin A, Hoshi Y, Yoneyama T, Miyauchi E, Tachikawa M, Watanabe M, et al.

J Pharm Sci, 2016 Nov;105(11):3440-3452.
PMID: 27665127 DOI: 10.1016/j.xphs.2016.08.013

Prostate-specific antigen is currently the only protein biomarker routinely used as a diagnostic tool for early detection and treatment monitoring of prostate cancer. However, it remains questionable whether prostate-specific antigen-based screening can sensitively and selectively identify the presence and progression status of primary and metastatic prostate cancers. Hence, the purpose of this study was to identify potential biomarker candidates in the secretome of primary and metastatic prostate cancer cells by using a combination of global and targeted proteomics. Quantitative comparisons among secretome proteins derived from androgen-responsive primary cancer cells (P-22Rv1), androgen-irresponsive bone metastatic cancer cells (M-PC-3), and noncancerous prostate cells (N-PNT2) were performed using 2-dimensional image-converted analysis of liquid chromatography and mass spectrometry followed by in silico selection selected reaction monitoring analysis. Mediator of RNA polymerase II transcription subunit 13-like, insulin-like growth factor-binding protein 2, and hepatocyte growth factor were identified as highly secreted proteins from P-22Rv1 cells compared with N-PNT2 cells. Prostate-associated microseminoprotein, proactivator polypeptide, collagen-α-1 (VI) chain, and neuropilin-1 were identified as predominantly secreted proteins in M-PC-3 cells compared with N-PNT2 cells. These proteins in biological fluids are considered to be candidate biomarkers of primary and/or metastatic prostate cancer.

Matched MeSH terms: Cell Line, Transformed
Fulltext A three-dimensional collagen construct to model lipopolysaccharide-induced activation of BV2 microglia

Haw RT, Tong CK, Yew A, Lee HC, Phillips JB, Vidyadaran S

J Neuroinflammation, 2014 Jul 30;11:134.
PMID: 25074682 DOI: 10.1186/1742-2094-11-134

BACKGROUND: We report a novel method of culturing microglia in three dimension (3D) using collagen as a substrate. By culturing microglia within a matrix, we aim to emulate the physical state of microglia embedded within parenchyma.
METHODS: BV2 microglia cell suspensions were prepared with type I collagen and cast into culture plates. To characterise the BV2 microglia cultured in 3D, the cultures were evaluated for their viability, cell morphology and response to lipopolysaccharide (LPS) activation. Conventional monolayer cultures (grown on uncoated and collagen-coated polystyrene) were set up concurrently for comparison.
RESULTS: BV2 microglia in 3D collagen matrices were viable at 48 hrs of culture and exhibit a ramified morphology with multiplanar cytoplasmic projections. Following stimulation with 1 μg/ml LPS, microglia cultured in 3D collagen gels increase their expression of nitric oxide (NO) and CD40, indicating their capacity to become activated within the matrix. Up to 97.8% of BV2 microglia grown in 3D cultures gained CD40 positivity in response to LPS, compared to approximately 60% of cells grown in a monolayer (P

Matched MeSH terms: Cell Line, Transformed
Inhibition of lysosomal vacuolar proton pump down-regulates cellular acidification and enhances E. coli bactofection efficiency

Akinsola RO, Lee CW, Sim EUH, Narayanan K

Anal Biochem, 2021 03 01;616:114088.
PMID: 33358938 DOI: 10.1016/j.ab.2020.114088

Endosomal escape is considered a crucial barrier that needs to be overcome by integrin-mediated E. coli for gene delivery into mammalian cells. Bafilomycin, a potent inhibitor of the H+ proton pump commonly employed to lower endosomal pH, was evaluated as part of the E. coli protocol during delivery. We found an increase in green fluorescent protein expression up 6.9, 3.2, 5.0, 2.8, and 4.5 fold in HeLa, HEK-293, A549, HT1080, and MCF-7 respectively, compared to untreated cells. Our result showed for the first time that Inhibition of lysosomal V-ATPase enhances E. coli efficiency.

Matched MeSH terms: Cell Line, Transformed
Fulltext A Human Corneal Epithelial Cell Line Model for Limbal Stem Cell Biology and Limbal Immunobiology

Shaharuddin B, Ahmad S, Md Latar N, Ali S, Meeson A

Stem Cells Transl Med, 2017 03;6(3):761-766.
PMID: 28297580 DOI: 10.5966/sctm.2016-0175

Limbal stem cell (LSC) deficiency is a visually debilitating condition caused by abnormal maintenance of LSCs. It is treated by transplantation of donor-derived limbal epithelial cells (LECs), the success of which depends on the presence and quality of LSCs within the transplant. Understanding the immunobiological responses of these cells within the transplants could improve cell engraftment and survival. However, human corneal rings used as a source of LSCs are not always readily available for research purposes. As an alternative, we hypothesized that a human telomerase-immortalized corneal epithelial cell (HTCEC) line could be used as a model for studying LSC immunobiology. HTCEC constitutively expressed human leukocyte antigen (HLA) class I but not class II molecules. However, when stimulated by interferon-γ, HTCECs then expressed HLA class II antigens. Some HTCECs were also migratory in response to CXCL12 and expressed stem cell markers, Nanog, Oct4, and Sox2. In addition because both HTCECs and LECs contain side population (SP) cells, which are an enriched LSC population, we used these SP cells to show that some HTCEC SP cells coexpressed ABCG2 and ABCB5. HTCEC SP and non-side population (NSP) cells also expressed CXCR4, but the SP cells expressed higher levels. Both were capable of colony formation, but the NSP colonies were smaller and contained fewer cells. In addition, HTCECs expressed ΔNp63α. These results suggest the HTCEC line is a useful model for further understanding LSC biology by using an in vitro approach without reliance on a supply of human tissue. Stem Cells Translational Medicine 2017;6:761-766.

Matched MeSH terms: Cell Line, Transformed
Genotoxicity assessment of biphasic calcium phosphate of modified porosity on human dental pulp cells using Ames and Comet assays

Wahab NFAC, Kannan TP, Mahmood Z, Rahman IA, Ismail H

Toxicol In Vitro, 2018 Mar;47:207-212.
PMID: 29247761 DOI: 10.1016/j.tiv.2017.12.002

Biphasic Calcium Phosphate (BCP) with a ratio of 20/80 Hydroxyapatite (HA)/Beta-tricalcium phosphate (β-TCP) promotes the differentiation of human dental pulp cells (HDPCs). In the current study, the genotoxicity of locally produced BCP of modified porosity (65%) with a mean pore size of 300micrometer (μm) was assessed using Comet and Ames assays. HDPCs were treated with BCP extract at three different inhibitory concentrations which were obtained based on cytotoxicity test conducted with concurrent negative and positive controls. The tail moment of HDPCs treated with BCP extract at all three concentrations showed no significant difference compared to negative control (p>0.05), indicating that BCP did not induce DNA damage to HDPCs. The BCP was evaluated using five tester strains of Salmonella typhimurium TA98, TA100, TA102, TA1537 and TA1538. Each strain was incubated with BCP extract with five different concentrations in the presence and absence of metabolic activation system (S9) mix. Concurrently, negative and positive controls were included. The average number of revertant colonies per plate treated with the BCP extract was less than double as compared to the number of revertant colonies in negative control plate and no dose-related increase was observed. Results from both assays suggested that the BCP of modified porosity did not exhibit any genotoxic effect under the present test conditions.

Matched MeSH terms: Cell Line, Transformed
De novo hotspot variants in CYFIP2 cause early-onset epileptic encephalopathy

Nakashima M, Kato M, Aoto K, Shiina M, Belal H, Mukaida S, et al.

Ann Neurol, 2018 04;83(4):794-806.
PMID: 29534297 DOI: 10.1002/ana.25208

OBJECTIVE: The cytoplasmic fragile X mental retardation 1 interacting proteins 2 (CYFIP2) is a component of the WASP-family verprolin-homologous protein (WAVE) regulatory complex, which is involved in actin dynamics. An obvious association of CYFIP2 variants with human neurological disorders has never been reported. Here, we identified de novo hotspot CYFIP2 variants in neurodevelopmental disorders and explore the possible involvement of the CYFIP2 mutants in the WAVE signaling pathway.
METHODS: We performed trio-based whole-exome sequencing (WES) in 210 families and case-only WES in 489 individuals with epileptic encephalopathies. The functional effect of CYFIP2 variants on WAVE signaling was evaluated by computational structural analysis and in vitro transfection experiments.
RESULTS: We identified three de novo CYFIP2 variants at the Arg87 residue in 4 unrelated individuals with early-onset epileptic encephalopathy. Structural analysis indicated that the Arg87 residue is buried at an interface between CYFIP2 and WAVE1, and the Arg87 variant may disrupt hydrogen bonding, leading to structural instability and aberrant activation of the WAVE regulatory complex. All mutant CYFIP2 showed comparatively weaker interactions to the VCA domain than wild-type CYFIP2. Immunofluorescence revealed that ectopic speckled accumulation of actin and CYFIP2 was significantly increased in cells transfected with mutant CYFIP2.
INTERPRETATION: Our findings suggest that de novo Arg87 variants in CYFIP2 have gain-of-function effects on the WAVE signaling pathway and are associated with severe neurological disorders. Ann Neurol 2018;83:794-806.

Matched MeSH terms: Cell Line, Transformed
Fulltext Mesenchymal stem cells exert anti-proliferative effect on lipopolysaccharide-stimulated BV2 microglia by reducing tumour necrosis factor-α levels

Jose S, Tan SW, Ooi YY, Ramasamy R, Vidyadaran S

J Neuroinflammation, 2014;11:149.
PMID: 25182840 DOI: 10.1186/s12974-014-0149-8

Progression of neurodegenerative diseases occurs when microglia, upon persistent activation, perpetuate a cycle of damage in the central nervous system. Use of mesenchymal stem cells (MSC) has been suggested as an approach to manage microglia activation based on their immunomodulatory functions. In the present study, we describe the mechanism through which bone marrow-derived MSC modulate the proliferative responses of lipopolysaccharide-stimulated BV2 microglia.

Matched MeSH terms: Cell Line, Transformed
Oral cancer secretome: identification of cancer-associated proteins

Chang HY, Hor SY, Lim KP, Zain RB, Cheong SC, Rahman MA, et al.

Electrophoresis, 2013 Aug;34(15):2199-208.
PMID: 23712713 DOI: 10.1002/elps.201300126

This study aims to identify cancer-associated proteins in the secretome of oral cancer cell lines. We have successfully established four primary cell cultures of normal cells with a limited lifespan without human telomerase reverse transcriptase (hTERT) immortalization. The secretome of these primary cell cultures were compared with that of oral cancer cell lines using 2DE. Thirty five protein spots were found to have changed in abundance. Unambiguous identification of these proteins was achieved by MALDI TOF/TOF. In silico analysis predicted that 24 of these proteins were secreted via classical or nonclassical mechanisms. The mRNA expression of six genes was found to correlate with the corresponding protein abundance. Ingenuity Pathway Analysis (IPA) core analysis revealed that the identified proteins were relevant in, and related to, cancer development with likely involvements in tumor growth, metastasis, hyperproliferation, tumorigenesis, neoplasia, hyperplasia, and cell transformation. In conclusion, we have demonstrated that a comparative study of the secretome of cancer versus normal cell lines can be used to identify cancer-associated proteins.

Matched MeSH terms: Cell Line, Transformed
Fulltext Suppression of cell division-associated genes by Helicobacter pylori attenuates proliferation of RAW264.7 monocytic macrophage cells

Tan GM, Looi CY, Fernandez KC, Vadivelu J, Loke MF, Wong WF

Sci Rep, 2015;5:11046.
PMID: 26078204 DOI: 10.1038/srep11046

Helicobacter pylori at multiplicity of infection (MOI ≥ 50) have been shown to cause apoptosis in RAW264.7 monocytic macrophage cells. Because chronic gastric infection by H. pylori results in the persistence of macrophages in the host's gut, it is likely that H. pylori is present at low to moderate, rather than high numbers in the infected host. At present, the effect of low-MOI H. pylori infection on macrophage has not been fully elucidated. In this study, we investigated the genome-wide transcriptional regulation of H. pylori-infected RAW264.7 cells at MOI 1, 5 and 10 in the absence of cellular apoptosis. Microarray data revealed up- and down-regulation of 1341 and 1591 genes, respectively. The expression of genes encoding for DNA replication and cell cycle-associated molecules, including Aurora-B kinase (AurkB) were down-regulated. Immunoblot analysis verified the decreased expression of AurkB and downstream phosphorylation of Cdk1 caused by H. pylori infection. Consistently, we observed that H. pylori infection inhibited cell proliferation and progression through the G1/S and G2/M checkpoints. In summary, we suggest that H. pylori disrupts expression of cell cycle-associated genes, thereby impeding proliferation of RAW264.7 cells, and such disruption may be an immunoevasive strategy utilized by H. pylori.

Matched MeSH terms: Cell Line, Transformed
Elevated secretory IgA antibodies to Epstein-Barr virus (EBV) and presence of EBV DNA and EBV receptors in patients with cervical carcinoma

Se Thoe SY, Wong KK, Pathmanathan R, Sam CK, Cheng HM, Prasad U

Gynecol Oncol, 1993 Aug;50(2):168-72.
PMID: 8397152

Epstein-Barr virus (EBV) receptors (EBV/C3d receptors) were detected, using the monoclonal antibody HB5, on 23 ectocervical and 5 endocervical biopsies of the uterine cervix. Elevated IgA titers against the viral capsid antigen and early antigen of EBV were also found in the cervical secretions from cervical carcinoma patients (83%), compared with samples from patients with cervical intraepithelial neoplasia (75%), herpes simplex virus-infected patients (0%), and gynecologic patients with nonmalignant conditions (0%). EBV DNA was present in 63% of cervical carcinoma biopsies detected by in situ hybridization. These observations suggest a positive association between EBV and carcinoma of the cervix.

Matched MeSH terms: Cell Line, Transformed

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