Epigenic regulation and posttranslational modification of human cytochrome P450s : focus on CYP2W1, CYP1A2 and CYP2C18
Author: Gomez, Alvin V
Date: 2008-12-12
Location: Föreläsningssalen Rockefeller, Nobels väg 11, Karolinska Institutet
Time: 13.00
Department: Institutionen för fysiologi och farmakologi / Department of Physiology and Pharmacology
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Thesis (598.0Kb)
Abstract
The cytochrome P450 superfamily represents a very important group of
enzymes that are responsible for the metabolism of drugs as well as of
endogenous compounds. Many P450s are genetically polymorphic causing
important interindividual variability in P450 expression and activity
whereas in some cases the basis for interindividual variation has still
not been resolved. In this thesis, epigenetic and posttranslational
aspects of the regulation of P450s are discussed, with special emphasis
on CYP2W1, CYP1A2 and CYP2C18.
CYP2W1 was identified through search in the dbEST and Celera sequence databases and cloned. CYP2W1 is a 5.5 kb long gene located on chromosome 7p22.3, was found to be expressed in HepG2 cells, and later in Caco2TC7 cells. Transient cDNA-based expression in HEK293 cells yielded a protein that is properly folded, as evidenced by its typical P450 spectrum and was active in the catalysis of arachidonic acid metabolism. Gene analysis revealed a high density of CG dinucleotides and the presence of CpG islands in promoter and exon 1-intron 1 region. Experiments using 5-Aza-2′-deoxycytidine treatment of HepG2 and B16A2 cells suggested involvement of DNA methylation in the regulation of CYP2W1 expression. Interestingly, the CYP2W1 enzyme was found to be expressed almost entirely in fetal colon (rat) and in human colorectal cancers. In these tumors there was a reverse correlation of DNA methylation and CYP2W1 expression, which further proved the involvement of DNA methylation in CYP2W1 regulation. The colorectal cancer-specific expression of CYP2W1 offers an attractive means of developing an anticancer chemotherapeutic strategy by prodrug activation. We found that the enzyme is localized at the cell surface, thus raising the possibility for cancer antibody therapy. It is posttranslationally modified by N-linked glycosylation on Asn177, which is the first time glycosylation is described for P450s in families 1-3. The functional role of glycosylation in CYP2W1 is yet to be determined.
Although the extensive variability in CYP1A2 expression and activity is well documented, the main genetic basis for this is poorly understood. We studied DNA methylation in the CYP1A2 gene in relation to interindividual differences in hepatic expression as revealed from a human liver bank. The DNA methylation level of the CpG island was shown to be inversely correlated with the CYP1A2 mRNA levels. When evaluated against allele-specific expression, however, no correlation was found with DNA methylation. Interestingly, site-specific changes in DNA methylation correlated with allele-specific expression, suggesting the possible role of transcription regulators that bind to specific sites and are influenced by methylation changes. In addition, evidence is provided for the microRNA silencing of CYP2C18 explaining its lack of expression at the protein level.
In conclusion a new colorectal cancer specific form of P450 (CYP2W1) has been cloned and characterized and suggested to constitute a possible drug target. Epigenetic mechanisms that control certain P450 expression have been identified and could potentially provide additional understanding to the important interindividual differences in expression of these pharmacologically and physiologically important genes.
CYP2W1 was identified through search in the dbEST and Celera sequence databases and cloned. CYP2W1 is a 5.5 kb long gene located on chromosome 7p22.3, was found to be expressed in HepG2 cells, and later in Caco2TC7 cells. Transient cDNA-based expression in HEK293 cells yielded a protein that is properly folded, as evidenced by its typical P450 spectrum and was active in the catalysis of arachidonic acid metabolism. Gene analysis revealed a high density of CG dinucleotides and the presence of CpG islands in promoter and exon 1-intron 1 region. Experiments using 5-Aza-2′-deoxycytidine treatment of HepG2 and B16A2 cells suggested involvement of DNA methylation in the regulation of CYP2W1 expression. Interestingly, the CYP2W1 enzyme was found to be expressed almost entirely in fetal colon (rat) and in human colorectal cancers. In these tumors there was a reverse correlation of DNA methylation and CYP2W1 expression, which further proved the involvement of DNA methylation in CYP2W1 regulation. The colorectal cancer-specific expression of CYP2W1 offers an attractive means of developing an anticancer chemotherapeutic strategy by prodrug activation. We found that the enzyme is localized at the cell surface, thus raising the possibility for cancer antibody therapy. It is posttranslationally modified by N-linked glycosylation on Asn177, which is the first time glycosylation is described for P450s in families 1-3. The functional role of glycosylation in CYP2W1 is yet to be determined.
Although the extensive variability in CYP1A2 expression and activity is well documented, the main genetic basis for this is poorly understood. We studied DNA methylation in the CYP1A2 gene in relation to interindividual differences in hepatic expression as revealed from a human liver bank. The DNA methylation level of the CpG island was shown to be inversely correlated with the CYP1A2 mRNA levels. When evaluated against allele-specific expression, however, no correlation was found with DNA methylation. Interestingly, site-specific changes in DNA methylation correlated with allele-specific expression, suggesting the possible role of transcription regulators that bind to specific sites and are influenced by methylation changes. In addition, evidence is provided for the microRNA silencing of CYP2C18 explaining its lack of expression at the protein level.
In conclusion a new colorectal cancer specific form of P450 (CYP2W1) has been cloned and characterized and suggested to constitute a possible drug target. Epigenetic mechanisms that control certain P450 expression have been identified and could potentially provide additional understanding to the important interindividual differences in expression of these pharmacologically and physiologically important genes.
List of papers:
I. Karlgren M, Gomez A, Stark K, Svärd J, Rodriguez-Antona C, Oliw E, Bernal ML, Ramón y Cajal S, Johansson I, Ingelman-Sundberg M (2006). "Tumor-specific expression of the novel cytochrome P450 enzyme, CYP2W1." Biochem Biophys Res Commun 341(2): 451-8.
Fulltext (DOI)
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II. Gomez A, Karlgren M, Edler D, Bernal ML, Mkrtchian S, Ingelman-Sundberg M (2007). "Expression of CYP2W1 in colon tumors: regulation by gene methylation." Pharmacogenomics 8(10): 1315-25.
Fulltext (DOI)
Pubmed
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III. Gomez A, Edler D, Wigzell H, Mkrtchian S, Ingelman-Sundberg M (2008). "Glycosylation and cell surface localization of the colon cancer cell-specific cytochrome P450 2W1 (CYP2W1)." [Manuscript]
IV. Ghotbi R, Gomez A, Milani L, Syvänen A-C, Bertilsson L, Ingelman-Sundberg M, Aklillu E (2008). "Allele-specific expression and gene methylation in the control of CYP1A2 mRNA level in human livers." Pharmacogenomics J. Provisionally. [Accepted]
Fulltext (DOI)
Pubmed
View record in Web of Science®
I. Karlgren M, Gomez A, Stark K, Svärd J, Rodriguez-Antona C, Oliw E, Bernal ML, Ramón y Cajal S, Johansson I, Ingelman-Sundberg M (2006). "Tumor-specific expression of the novel cytochrome P450 enzyme, CYP2W1." Biochem Biophys Res Commun 341(2): 451-8.
Fulltext (DOI)
Pubmed
View record in Web of Science®
II. Gomez A, Karlgren M, Edler D, Bernal ML, Mkrtchian S, Ingelman-Sundberg M (2007). "Expression of CYP2W1 in colon tumors: regulation by gene methylation." Pharmacogenomics 8(10): 1315-25.
Fulltext (DOI)
Pubmed
View record in Web of Science®
III. Gomez A, Edler D, Wigzell H, Mkrtchian S, Ingelman-Sundberg M (2008). "Glycosylation and cell surface localization of the colon cancer cell-specific cytochrome P450 2W1 (CYP2W1)." [Manuscript]
IV. Ghotbi R, Gomez A, Milani L, Syvänen A-C, Bertilsson L, Ingelman-Sundberg M, Aklillu E (2008). "Allele-specific expression and gene methylation in the control of CYP1A2 mRNA level in human livers." Pharmacogenomics J. Provisionally. [Accepted]
Fulltext (DOI)
Pubmed
View record in Web of Science®
Issue date: 2008-11-21
Rights:
Publication year: 2008
ISBN: 978-91-7409-274-5
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