Select de novo Gene and Protein Expression During Renal Epithelial Cell Culture in Rotating Wall Vessels is Shear Stress Dependent

Kaysen, James H.; Campbell, W. C.; Majewski, R. R.; Goda, F. O.; Navar, L. Gabriel; Lewis, Fawn C.; Goodwin, Thomas J.; Hammond, Timothy G.

doi:10.1007/s002329900499

Cited by 58 publications

(18 citation statements)

References 26 publications

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“…Further work by Kaysen et al . supports this hypothesis, showing a shear stress dependence of selected de novo g ene and protein expression during renal epithelial cell culture in RWV [42]. Therefore, different shear stress situations on both devices used in our study seem to be a cause of differences in the results obtained.…”

Section: Discussionsupporting

confidence: 83%

Spheroid formation of human thyroid cancer cells under simulated microgravity: a possible role of CTGF and CAV1

et al. 2014

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BackgroundMulticellular tumor spheroids (MCTS) formed scaffold-free under microgravity are of high interest for research and medicine. Their formation mechanism can be studied in space in real microgravity or on Earth using ground-based facilities (GBF), which simulate microgravity. On Earth, these experiments are more cost-efficient and easily performable. However, each GBF might exert device-specific and altered superimposingly gravity-dependent effects on the cells.ResultsFTC-133 human thyroid cancer cells were cultivated on a 2D clinostat (CN) and a random positioning machine (RPM) and compared with corresponding 1 g control cells. Harvested cell samples were investigated by microscopy, quantitative realtime-PCR and Multi-Analyte Profiling. Spheroid formation and growth occurred during 72 h of cultivation on both devices. Cytokine secretion and gene activation patterns frequently altered in different ways, when the cells were cultured either on the RPM or the CN. A decreased expression of CAV1 and CTGF in MCTS compared to adherent cells was observed after cultivation on both machines.ConclusionThe development of MCTS proceeds similarly on the RPM and the CN resembling the situation observed under real microgravity conditions, while no MCTS formation was observed at 1 g under identical experimental conditions. Simultaneously, changes in the regulation of CTGF and CAV1 appeared in a comparable manner on both machines. A relationship between these molecules and MCTS formation is discussed.

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Section: Discussionsupporting

confidence: 83%

Spheroid formation of human thyroid cancer cells under simulated microgravity: a possible role of CTGF and CAV1

et al. 2014

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“…The microarray analysis was performed as in Figure 2, but using genes identified in stress-response studies [30]–[33].…”

Section: Resultsmentioning

confidence: 99%

Identification of G1-Regulated Genes in Normally Cycling Human Cells

et al. 2008

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BackgroundObtaining synchronous cell populations is essential for cell-cycle studies. Methods such as serum withdrawal or use of drugs which block cells at specific points in the cell cycle alter cellular events upon re-entry into the cell cycle. Regulatory events occurring in early G1 phase of a new cell cycle could have been overlooked.Methodology and FindingsWe used a robotic mitotic shake-off apparatus to select cells in late mitosis for genome-wide gene expression studies. Two separate microarray experiments were conducted, one which involved isolation of RNA hourly for several hours from synchronous cell populations, and one experiment which examined gene activity every 15 minutes from late telophase of mitosis into G1 phase. To verify synchrony of the cell populations under study, we utilized methods including BrdU uptake, FACS, and microarray analyses of histone gene activity. We also examined stress response gene activity. Our analysis enabled identification of 200 early G1-regulated genes, many of which currently have unknown functions. We also confirmed the expression of a set of genes candidates (fos, atf3 and tceb) by qPCR to further validate the newly identified genes.Conclusion and SignificanceGenome-scale expression analyses of the first two hours of G1 in naturally cycling cells enabled the discovery of a unique set of G1-regulated genes, many of which currently have unknown functions, in cells progressing normally through the cell division cycle. This group of genes may contain future targets for drug development and treatment of human disease.

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“…Suspension cultures in rotating wall vessels reinstate many structural and function elements found in vivo [27, 31, 34, 35]. Human renal proximal tubular cells in suspension culture express the endocytic scavenger proteins cubilin and megalin and the associated highly differentiated vesicular transport system not seen in 2D cultures.…”

Section: The Role Of Shear Stress In Maintaining Cellular Differenmentioning

confidence: 99%

“…Human renal proximal tubular cells in suspension culture express the endocytic scavenger proteins cubilin and megalin and the associated highly differentiated vesicular transport system not seen in 2D cultures. They also regain the microvilli and tight junctions lost in 2-dimensional culture [34, 35]. …”

Section: The Role Of Shear Stress In Maintaining Cellular Differenmentioning

confidence: 99%

“…Initially expensive and limited to large culture volumes during development more than three decades ago, new materials, additive manufacturing, and injection molding manufacturing techniques should allow production of small volume vessels appropriate for high throughput hepatotoxicity testing that are four orders of magnitude cheaper than the original device [27, 31, 34, 35]. Given this dramatic change in feasibility, the rotating wall suspension culture of hepatic cells deserves more thorough exploration and definition so that it can be implemented for drug toxicity screening in high throughput assays.…”

Section: Practical Recommendations: Cyp2b6 Expression and Polymorpmentioning

confidence: 99%

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Hepatocyte CYP2B6 Can Be Expressed in Cell Culture Systems by Exerting Physiological Levels of Shear: Implications for ADME Testing

Hammond

Birdsall

2017

Journal of Toxicology

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Cytochrome 2B6 (CYP2B6) has substantial clinical effects on morbidity and mortality and its effects on drug metabolism should be part of hepatotoxicity screening. Examples of CYP2B6's impacts include its linkage to mortality during cyclophosphamide therapy and its role in determining hepatotoxicity and CNS toxicity during efavirenz therapy for HIV infection. CYP2B6 is key to metabolism of many common drugs from opioids to antidepressants, anesthetics, and anticonvulsants. But CYP2B6 has been extremely difficult to express in cell culture, and as a result, it has been largely deemphasized in preclinical toxicity studies. It has now been shown that CYP2B6 expression can be supported for extended periods of time using suspension culture techniques that exert physiological levels of shear. New understanding of CYP2B6 has identified five clinically significant genetic polymorphisms that have a high incidence in many populations and that convey a substantial dynamic range of activity. We propose that, with the use of culture devices exerting physiological shear levels, CYP2B6 dependent drug testing, including definition of polymorphisms and application of specific inhibitors, should be a standard part of preclinical absorption, distribution, metabolism, and excretion (ADME) testing.

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Select de novo Gene and Protein Expression During Renal Epithelial Cell Culture in Rotating Wall Vessels is Shear Stress Dependent

Cited by 58 publications

References 26 publications

Spheroid formation of human thyroid cancer cells under simulated microgravity: a possible role of CTGF and CAV1

Spheroid formation of human thyroid cancer cells under simulated microgravity: a possible role of CTGF and CAV1

Identification of G1-Regulated Genes in Normally Cycling Human Cells

Hepatocyte CYP2B6 Can Be Expressed in Cell Culture Systems by Exerting Physiological Levels of Shear: Implications for ADME Testing

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