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Cell structure, function and adaptation
Published in C. Simon Herrington, Muir's Textbook of Pathology, 2020
Not all cells retain the ability to enter the cell cycle. Permanent cells are terminally differentiated and confined to G0, e.g. neurons in the central nervous system (CNS) in adults. Stable cell populations, such as hepatocytes, normally have a very low rate of proliferation but are able to proliferate under certain conditions in the presence of growth factors. Labile populations are constantly cycling, even though a relatively small proportion of their cells is actively cycling at any given time. As cell proliferation is such a key event in the life of an organism, and as inappropriate cycling activity could have such devastating effects, there are many tight controls over proliferation.
SBA Answers and Explanations
Published in Vivian A. Elwell, Jonathan M. Fishman, Rajat Chowdhury, SBAs for the MRCS Part A, 2018
Vivian A. Elwell, Jonathan M. Fishman, Rajat Chowdhury
Apoptosis may be physiological (as a normal part of growth and development), as well as pathological, where balancing the production of new cells enables a stable cell population. A good example of physiological apoptosis is the shaping of the hands in embryogenesis. Under normal conditions, apoptosis is precisely regulated by pro-apoptotic (p53, c-myc, Bax, Bad) and anti-apoptotic (Bcl-2, Bcl-XL) factors. Alteration in the fine balance of pro-apoptotic and anti-apoptotic factors may result in neoplasia (e.g., loss of p53 is found in many tumours).
Role of Histones in Cell Differentiation
Published in Gerald M. Kolodny, Eukaryotic Gene Regulation, 2018
It is suggestive, however, that in the lower eukaryotes, primitive forms of stable cell differentiation have also started to appear. They are expressed as an intracellular differentiation of the nucleus into macro- and micronucleus in protozoa, as an intraco-lonial differentiation into two major cell types — somatic and reproductive — in the aggregates of colonial organisms, and as more complex developmental changes in some other lower eukaryotes. Thus, the appearance of histones in lower eukaryotes coincides with the emergence in these organisms of some primitive forms of stable cellular differentiation.
A high-throughput cell-based gaussia luciferase reporter assay for measurement of CYP1A1, CYP2B6, and CYP3A4 induction
Published in Xenobiotica, 2021
Han Li, Yu-Guang Wang, Zeng-Chun Ma, Gao Yun-Hang, Song Ling, Chen Teng-Fei, Zhang Guang-Ping, Yue Gao
In recent years, the CYP promoter is linked to the luciferase reporter gene, and then transferred into cells. This cell model has been shown to be a suitable hepatic model for testing the hepatotoxicity of bioactive drugs and constitutes a valuable alternative for hepatotoxicity testing (Tolosa 2012, Liao et al. 2020). There are many reports on the study of CYP induction by this method, but most of the transfection methods are transient transfection (Kumagai et al. 2016, Chen et al. 2020, Yu et al. 2021). Compared with the transient transfection process, it takes a lot of time and energy to establish a stable cell line. It is inefficient to insert the target gene expression vector into the host cell genome. Moreover, only a few cells can integrate the vector into a highly transcriptional region and produce enough recombinant proteina (Satoh et al. 2017). Even so, transgene expression is often silenced upon long-term cell culture. Therefore, a large number of clones need to be isolated and characterised in the process of transfection and screening, which may take several months (Büssow 2015). The over-expression of CYP1A1 or CYP1A2 in stable cell lines has been used to identify inhibitors of the activities of these P450s and determine inhibitory kinetic parameters (Yueh et al.2005). Co-transfection of a hPXR expression vector and a reporter plasmid (p3A4-hPXRE-Luc) into HepG2 has been demonstrated to be a useful tool in screening for inducers of CYP3A, which can activate hPXR and/or hVDR (Sekimoto et al. 2012).
Modulation of ABCG2 surface expression by Rab5 and Rab21 to overcome multidrug resistance in cancer cells
Published in Xenobiotica, 2020
The expression and localisation of EGFP-ABCG2 fusion protein was analysed after the transient transfection of MCF-7 cells with the construct. Both fluorescence microscopy and western blot analysis showed that the expression of EGFP tagged ABCG2 protein was higher after 48 h of transfection (data not shown). After transient expression analysis, we stably expressed EGFP-ABCG2 in the MCF-7 cell line. Single-cell clones were obtained using selection antibiotic G418. Two ABCG2 over-expressing clones were selected for ABCG2 expressional analysis in comparison to parental MCF-7 wild type cells. Both qRT-PCR and western immunoblotting showed that the expression of ABCG2 in clone 2 was higher than in clone 1 (Figure 1(A,B)). Clone 2 was used for the functional analysis of ABCG2, labelled as MCF-7/G2 in text. To assess whether the ABCG2 was localised on cell surface or intracellular compartments, immunocytochemistry was performed using anti-ABCG2 primary antibody and Alexa 568 conjugated secondary antibody. The fluorescence of ABCG2 was mainly confined to the cell surface in MCF-7/G2 as compared to the parental MCF-7 wild type cells (Figure 1(C)). These results demonstrate a similar expression and localisation of ABCG2 in transiently and stably transfected MCF-7 cells with ABCG2. All subsequent experiments were performed using the stable cell line. We have also found numerous extracellular vesicle-like structures where vesicular membrane showed high amount of ABCG2. In contrast, MCF-7/wt cells did not show any detectable extracellular vesicle labelled with ABCG2 (Figure 1(C)).
Hepatitis A virus structural protein pX interacts with ALIX and promotes the secretion of virions and foreign proteins through exosome-like vesicles
Published in Journal of Extracellular Vesicles, 2020
Wang Jiang, Pengjuan Ma, Libin Deng, Zhi Liu, Xu Wang, Xiyu Liu, Gang Long
Huh-7 and Huh-7.5.1-GA cells [36] were cultured in DMEM medium supplemented with 2 mM L-glutamine, nonessential amino acids, 100 U penicillin per ml, 100 μg streptomycin per ml and 10% FBS (complete DMEM). HM175/18f, HM175/18f-pX-N-Δ32-64 and HM175/18f-pX-mut4-9 RNAs were in vitro transcribed by using MEGAscript® Kits (Life Technologies) according to the manufacturer’s protocol. Then, 10 μg RNA was electroporated into 2 × 10⁶ Huh-7.5.1-GA cells suspended in 400 μl cytomix (containing 2 mM ATP and 5 mM glutathione) at 960 mF and 270 V using a GenePulser system (Bio-Rad). Immediately after electroporation, the cells were resuspended in complete DMEM and seeded as required. After Huh-7.5.1-GA cells showed 99% HAV infection, the supernatant was harvested for titration and stored at −80°C. Stable cell lines were constructed as previously described [36]. Briefly, psPAX2 (a packaging plasmid), pMD2.G (a G protein expressing plasmid) and lentiviral vectors were cotransfected into 293T cells; 48 hours later, the supernatants containing lentivirus were harvested and filtered through 0.45-μm filters. Cell lines were selected with puromycin (10 μg/ml).