Polarized cell cultures for integrated studies of drug metabolism and transport

Brimer-Cline, Cynthia; Schuetz, Erin G.

doi:10.1016/s0076-6879(02)57690-4

Cited by 3 publications

(6 citation statements)

References 9 publications

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“…These CYP3A4/CYP3A5 cDNAs were then recombined into an ϳ38.2-kb AdV-BGFP-Gateway destination vector that also encoded ␤-galactosidase and green fluorescent protein. The CYP3A4/CYP3A5 AdV and the AdV-BGFP (vector control) were grown, propagated, and purified, and their titer was determined essentially as detailed (33,34). CYP3A4/CYP3A5 AdV and the AdV-BGFP (100 multiplicity of infection) were transduced into 80 -90% confluent HepG2 cells (Ϸ5 ϫ 10 6 ) in 25-cm 2 flasks with 10 g/ml polybrene.…”

Section: Methodsmentioning

confidence: 99%

Multisite Phosphorylation of Human Liver Cytochrome P450 3A4 Enhances Its gp78- and CHIP-mediated Ubiquitination

Wang

Guan²,

Acharya

et al. 2012

Molecular & Cellular Proteomics

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CYP3A4, an integral endoplasmic reticulum (ER)-anchored protein, is the major human liver cytochrome P450 enzyme responsible for the disposition of over 50% of clinically relevant drugs. Alterations of its protein turnover can influence drug metabolism, drug-drug interactions, and the bioavailability of chemotherapeutic drugs. Such CYP3A4 turnover occurs via a classical ER-associated degradation (ERAD) process involving ubiquitination by both UBC7/gp78 and UbcH5a/CHIP E2-E3 complexes for 26 S proteasomal targeting. These E3 ligases act sequentially and cooperatively in CYP3A4 ERAD because RNA interference knockdown of each in cultured hepatocytes results in the stabilization of a functionally active enzyme. We have documented that UBC7/gp78-mediated CYP3A4 ubiquitination requires protein phosphorylation by protein kinase (PK) A and PKC and identified three residues (Ser-478, Thr-264, and Ser-420) whose phosphorylation is required for intracellular CYP3A4 ERAD. We document herein that of these, Ser-478 plays a pivotal role in UBC7/ gp78-mediated CYP3A4 ubiquitination, which is accelerated and enhanced on its mutation to the phosphomimetic Asp residue but attenuated on its Ala mutation. Intriguingly, CYP3A5, a polymorphically expressed human liver CYP3A4 isoform (containing Asp-478) is ubiquitinated but not degraded to a greater extent than CYP3A4 in HepG2 cells. This suggests that although Ser-478 phosphorylation is essential for UBC7/gp78-mediated CYP3A4 ubiquitination, it is not sufficient for its ERAD. Additionally, we now report that CYP3A4 protein phosphorylation by PKA and/or PKC at sites other than Ser-478, Thr-264, and Ser-420 also enhances UbcH5a/CHIP-mediated ubiquitination. Through proteomic analyses, we identify (i) 12 additional phosphorylation sites that may be involved in CHIP-CYP3A4 interactions and (ii) 8 previously unidentified CYP3A4 ubiquitination sites within spatially associated clusters of Asp/Glu and phosphorylatable Ser/Thr residues that may serve to engage each E2-E3 complex. Collectively, our findings underscore the interplay between protein phosphorylation and ubiquitination in ERAD and, to our knowledge, provide the very first example of gp78 substrate recognition via protein phosphorylation. Molecular & Cellular Proteomics 11: 10.1074/mcp.M111.010132, 1-17, 2012. Hepatic cytochromes P450 (P450s)1 are endoplasmic reticulum (ER)-anchored hemoproteins involved in the metabolism of numerous endo-and xenobiotics. Of these, CYP3A4 is particularly noteworthy because it comprises 30% of the human liver microsomal P450 complement and is responsible for the metabolism of Ͼ50% clinically relevant drugs, as well as hepatotoxins such as aflatoxin B1 (1). In common with other ER-integral P450s, it is a monotopic protein, N-terminally anchored to the ER membrane, with the bulk of its catalytic domain in the cytosol. We have documented that CYP3A4, in common with its CYP3A orthologs, incurs ubiquitin (Ub)-dependent proteasomal degradation (UPD) in a classical ER-associated degradation (ERAD) process...

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Section: Methodsmentioning

confidence: 99%

Multisite Phosphorylation of Human Liver Cytochrome P450 3A4 Enhances Its gp78- and CHIP-mediated Ubiquitination

Wang

Guan²,

Acharya

et al. 2012

Molecular & Cellular Proteomics

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“…Those most important for the transport studies include very low expression of cytochrome P450,(1726) especially that of the 3A4 isoenzyme that is important in drug metabolism,(1651) a lower content of efflux pumps and transporters for active absorption of vitamins, bile acids, amino acids, nucleosides and peptides,(1727) and tighter cell junctions reminiscent of those in the colon, as opposed to those in the small intestine. 1667,1728 …”

Section: Disposition Of Chemicalsmentioning

confidence: 99%

“…1760–1762 Other cell lines forming the polarized monolayer and having a high expression of both transporters and enzymes can be used to study their cooperation. (1726)…”

Section: Disposition Of Chemicalsmentioning

confidence: 99%

“…Although the Caco-2 cells in monolayer attain several morphological and functional characteristics of mature enterocytes, some differences do exist, primarily because of the colonic origin of the cell line. Those most important for the transport studies include very low expression of cytochrome P450, 1726 especially that of the 3A4 isoenzyme that is important in drug metabolism, 1651 a lower content of efflux pumps and transporters for active absorption of vitamins, bile acids, amino acids, nucleosides and peptides, 1727 and tighter cell junctions reminiscent of those in the colon, as opposed to those in the small intestine. 1667,1728 Variability of the Caco-2 monolayers, caused by cellular factors and culture conditions, complicates the interlaboratory comparisons.…”

Section: Cell Monolayersmentioning

confidence: 99%

“…[1760][1761][1762] Other cell lines forming the polarized monolayer and having a high expression of both transporters and enzymes can be used to study their cooperation. 1726 Among other than Caco-2 cellular monolayers, the Madin-Darby canine kidney (MDCK) cell line 1763 has been deployed most frequently. The initial focus on tissue cultures that were suitable for the monitoring of viral propagation, 1764,1765 oncogenesis, 1766 and physiological processes 1647,1767 was extended to the monolayers grown on a porous support, which were used to study the transport of drugs 1768,1769 and ions, imitating tubular reabsorption.…”

Section: Cell Monolayersmentioning

confidence: 99%

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Modeling Kinetics of Subcellular Disposition of Chemicals

Baláž

2009

Chem. Rev.

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Generation of tetracycline-controllable CYP3A4-expressing Caco-2 cells by the piggyBac transposon system

Ichikawa

Akamine

Murata

et al. 2021

Sci Rep

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Caco-2 cells are widely used as an in vitro intestinal epithelial cell model because they can form a monolayer and predict drug absorption with high accuracy. However, Caco-2 cells hardly express cytochrome P450 (CYP), a drug-metabolizing enzyme. It is known that CYP3A4 is the dominant drug-metabolizing enzyme in human small intestine. In this study, we generated CYP3A4-expressing Caco-2 (CYP3A4-Caco-2) cells and attempted to establish a model that can simultaneously evaluate drug absorption and metabolism. CYP3A4-Caco-2 cells were generated by piggyBac transposon vectors. A tetracycline-controllable CYP3A4 expression cassette (tet-on system) was stably transduced into Caco-2 cells, thus regulating the levels of CYP3A4 expression depending on the doxycycline concentration. The CYP3A4 expression levels in CYP3A4-Caco-2 cells cultured in the presence of doxycycline were similar to or higher than those of adult small intestine. The CYP3A4-Caco-2 cells had enough ability to metabolize midazolam, a substrate of CYP3A4. CYP3A4 overexpression had no negative effects on cell proliferation, barrier function, and P-glycoprotein activity in Caco-2 cells. Thus, we succeeded in establishing Caco-2 cells with CYP3A4 metabolizing activity comparable to in vivo human intestinal tissue. This cell line would be useful in pharmaceutical studies as a model that can simultaneously evaluate drug absorption and metabolism.

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Polarized cell cultures for integrated studies of drug metabolism and transport

Cited by 3 publications

References 9 publications

Multisite Phosphorylation of Human Liver Cytochrome P450 3A4 Enhances Its gp78- and CHIP-mediated Ubiquitination

Multisite Phosphorylation of Human Liver Cytochrome P450 3A4 Enhances Its gp78- and CHIP-mediated Ubiquitination

Modeling Kinetics of Subcellular Disposition of Chemicals

Generation of tetracycline-controllable CYP3A4-expressing Caco-2 cells by the piggyBac transposon system

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