Expression of Multiple Cytochrome P450 Enzymes and Multidrug Resistance- Associated Transport Proteins in Human Skin Keratinocytes Jens M. Baron, Daniela.

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Expression of Multiple Cytochrome P450 Enzymes and Multidrug Resistance- Associated Transport Proteins in Human Skin Keratinocytes Jens M. Baron, Daniela Höller Journal of Investigative Dermatology Volume 116, Issue 4, Pages 541-548 (April 2001) DOI: 10.1046/j.1523-1747.2001.01298.x Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Reverse transcription–PCR of proliferating keratino cytes derived from the human epidermis of different donors with primers specific for various P450 enzymes and β-actin. Reverse transcription–PCR analysis of (a) CYP 1A1, 1B1, 2B6, 2E1, and (b) CYP3A4, 3A5, and β-actin expression. Lane 1, donor A; lane 2, donor B; lane 3, donor C; lane 4, donor D; lane 5, donor E; lane 6, DNA marker pBR322 HaeIII digest. Induction of cells was performed with dexamethasone (10-7 M) or benzanthracene (10-6 M) for 24 h. Journal of Investigative Dermatology 2001 116, 541-548DOI: (10.1046/j.1523-1747.2001.01298.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Reverse transcription–PCR of keratinocytes at different levels of subconfluency with primers specific for MDR1, MRP1–6, and LRP. Reverse transcription–PCR analysis of (a) MDR1, MRP1, MRP3, MRP4, and (b) MRP5, MRP6, and LRP expression. Lanes 1–3, keratinocytes 60% subconfluency (donor A); lanes 4 and 5, keratinocytes 90% subconfluency (donor A); lane 6, DNA marker pBR322 HaeIII digest. Induction of cells was performed with dexamethasone (10-7 M) for 72 h. Journal of Investigative Dermatology 2001 116, 541-548DOI: (10.1046/j.1523-1747.2001.01298.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Immunoblots with antibodies against CYP enzymes and MDR-associated transport proteins. (a) Microsomal preparations from untreated proliferating human foreskin keratinocytes were separated on sodium dodecyl sulfate–polyacrylamide gel electrophoresis, blotted on to nitrocellulose sheets and subsequently treated with antibodies for cytochromes P450 1A1, 2B6, 2E1, and 3A5, respectively, and an alkaline phosphatase coupled secondary antibody. Arrows indicate the bands representing the particular constitutive cytochrome P450 enzyme. Lanes 1–3, donors A–C; lane 4, molecular weight marker: 106, 77, 50.8, 35.6, 28.1, 20.9 kDa. (b) The expression of MDR1, MRP1, MRP3, and MRP5 was detected via a 7.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis using specific monoclonal antibodies. Generally, 60 µg of protein are loaded per lane and only for the detection of MDR1 keratinocytes were induced with dexamethasone (10-7 M) for 72 h. The protein bands were detected on an autoradiography film using the enhanced chemiluminescence. Lanes 1–6, donors A–E (2×). Rainbow-colored protein molecular weight marker (myosin 220 kDa, phosphorylase b 97.4 kDa, bovine serum albumin 66 kDa). Journal of Investigative Dermatology 2001 116, 541-548DOI: (10.1046/j.1523-1747.2001.01298.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Expression of cytochrome P450 by intracellular staining in differentiated keratinocytes with an antibody against CYP 1A1. Intracellular immunohistochemical staining of differentiated normal epidermal keratinocytes after 48 h of Ca2+ preincubation and subsequent incubation with solvent (a) or (b) benzanthracene (2 × 10-5 M) for 72 h. After APAAP staining with an antibody against CYP1A1, (a) shows only a few positive cells, whereas in (b) an enhancement of CYP1A1 expression is shown. Journal of Investigative Dermatology 2001 116, 541-548DOI: (10.1046/j.1523-1747.2001.01298.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Expression of cytochrome P450 and MRP1 by intracellular staining in proliferating keratinocytes. Intracellular immunohistochemical staining of proliferating normal epidermal keratinocytes. Using antibodies against CYP 1A1 (a), 2B6 (b), 2E1 (c), 3A (d), MDR1 (e), and MRP1 (f) the expression of these proteins were shown by immunofluorescence. Journal of Investigative Dermatology 2001 116, 541-548DOI: (10.1046/j.1523-1747.2001.01298.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions