Human Nasopharyngeal Epithelial Source Of Cytochrome P450 2e1 Cdna Clone Of In Vitro Metabolic Function

Objective To investigate the role of CYP2E1 gene in chemical carcinogen-induced nasopharyngeal carcinogenesis, and to provide new evidences about etiology and pathogenesis of nasopharyngeal carcinoma.Methods RT-PCR was used to clone the CYP2E1 gene in human embryonic nasopharyngeal epithelial (HENE) cell, the transformed nasopharyngeal epithelial cell line (7429) and the nasopharyngeal carcinoma cell line (HNEi). The cloned segments were inserted into pGEM-T Easy vector by DNA recombination technique and sequenced respectively. A tetracycline (Tc)-controlled expression system that quantitatively controls gene expression in eukaryotic cells was used to express CYP2E1 in NIH 3T3 cells in culture. First we constructed the Tet/3T3 cell lines that constitutively express the reverse Tc-controlled transactivator (rtTA). Then the cloned 7429 CYP2E1 cDNA was subcloned into the Tc-controlled expression vector (pRevTRE) and transfected into the Tet/3T3 cell lines. Here a line of NIH 3T3 cells expressing human CYP2E1 (Tet/3T3-2El) in a Tc-dependent manner was described. Dox was used to regulate the expression of CYP2E1, and the dose-dependent expression of CYP2E1 was determined by RT-PCR and western blot. CYP2E1 catalytic activity was assayed by the 6-hydroxylation of chlorzoxazone (CZ), and the 6-hydroxychlorzoxazone (6-OH-CZ) was determined by HPLC.Results ?In comparison with HENE-2E1 cDNA, there were two point mutations at positions 846(A to T), 901 (A to G) in 7429-2E1 cDNA and only one point mutation at position 901 (A to G) in HNE1-2E1 cDNA. In comparison with Human (adult, emanol-inducible) liver CYP2E1 gene (GenBank NO.J02843), HENE-2E1 cDNA had one point mutation at position 901(G to A); 7429-2E1 had one point mutation at 846(A to T). All these point mutations didn't change the amino acid sequence. But no base change was found in HNE1-2E1 cDNA. (2)A Tet/3T3-2el cell clone expressing high level of CYP2E1 were established. Varying theconcentration of Dox in the media could also modulate the level of CYP2E1 expression. (3)The increased formation of 6-OH-CZ was correlated with an increase in protein level of CYP2E1.Conclusion (1)There are a few of base substitutions among HENE-2E1 cDNA, 7429-2E1 cDNA and HNE1 cDNA sequences, all these point mutations are synonymous mutation. The study reconfirm that the human CYP2E1 gene is relatively well conserved. (2)The lines of NIH 3T3 cell expressing human CYP2E1 (Tet/3T3-2El) in a Tc-dependent manner was reconstructed. This cell line offers a useful system to investigate post-translational regulation of the enzyme and CYP2E1-dependent metabolic activation; and to study the role of CYP2E1 in related mechanism of carcinogenesis and the cytotoxicity of xenobiotics. (3)In the hydroxylation of Chlorzoxazone, the catalytic activity of the enzyme has an increase in the presence of Dox, which depended on the increase in the level of the expression of the protein.