Cytochrome cultured cell expression

In cell cultures, the expression of the reductase mRNA, like the tdc and stric-tosidine synthase sts) genes, was found to be induced by elicitors and down-regulated by auxins. GlOH was found to be localized in provacuolar membranes and not in the endoplasmic reticulum like many other cytochrome P450 enzymes. Interestingly, this enzyme is inhibited by the end product, alkaloid catharanthine, but not by vindoline and vinblastine. Therefore, feedback regulation may also operate in vivo, provided that the catharanthine and GlOH are within the same cellular compartment (Facchini and De Luca, 2008). 

For C. roseus suspension-cultured cells, elicitation with fungal elicitors results in the induction of TDC activity (99,186,202,203,284,329-331). This is due to the induction of expression of the Tdc gene. Similarly, SSS activity is induced (202,203,284,329,330). The induction by the Pythium aphanider-matum or yeast elicitor of the transcription of both genes is not affected by cycloheximide that is, the induction is independent of de novo protein biosynthesis, and thus follows an already available signal-transduction chain. The response is quite fast, for the enhanced transcription can already be measured 15 min after elicitation (202,203). Also, the NADPH cytochrome P-450 reductase mRNA level is induced by elicitation with fungal elicitors (113). Moreno et al (99,151) measured activities of a number of enzymes involved in secondary metabolism in C. roseus before and after elicitation with a P. aphanidermatum preparation. GlOH activity was found to be slightly decreased by elicitation and IPP-isomerase showed similar behavior. The pattern of terpenoids formed by the crude enzyme extracts from elicited and nonelicited cells was different. The total incorporation decreased, that is, the activities of the enzymes of the terpenoid pathway were lower. The relative incorporation decreased particularly for squalene. 

Oxidative metabolic prohling of pesticides using transgenic tobacco cell suspension cultures, which express human cytochrome P450 isozymes (B. Schmidt, RWTH Aachen Univ., Germany)... 

A study of the modulation of benzo(a-)pyrene metabolism and regulation of cytochrome CYPlAl gene expression by piperine in 5 L cells in culture revealed that piperine-mediated inhibition of AHH activity and consequent suppression of the procarcinogen activation results from direct interaction of piperine with... 

Bulun SE, Simpson ER, Word RA (1994) Expression of the CYP19 gene and its product aromatase cytochrome P450 in human uterine leiomyoma tissue and cells in culture. J Clin Endocrinol Metab 78 736-743... 

Messenger RNA molecules for both subunits of the cytochrome and the two cytosolic components are detectable in unstimulated bloodstream cells. Experiments involving incubation of neutrophil suspensions with the protein synthesis inhibitor cycloheximide indicate that constitutive expression of one or more components of the oxidase is required for the neutrophil to maintain its ability to generate reactive oxidants. For example, when neutrophils are incubated in vitro with cycloheximide, their ability to generate reactive oxidants declines more rapidly than in control cells, as they age in culture (Fig. 7.12). This decline in oxidase activity when protein biosynthesis is blocked is not due to cell death, because cells treated with cycloheximide for this time still exclude trypan blue. Furthermore, when protein biosynthesis is stimulated in neutrophils by the addition of GM-CSF for 24 h in vitro, the ability to generate reactive oxidants is enhanced considerably above the levels observed in untreated cells. 

The background cytochrome P450 complement of the cell line and the conditions under which these enzymes are expressed. CYPlAl is sometimes expressed in cultured mammalian cells upon treatment with appropriate inducers (Diamond et al., 1980 Crespi et al., 1985). 

CYP3A4 has been expressed in human lymphoblasts (Crespi et al., 1991a). The expression level in this report was quite low. Recent modifications to the promoter for cDNA expression and coexpression of OR have led to a 40-fold increase in catalytic activity. The mean testosterone 6j8-hydroxylase activity in microsomes from h3A4/OR cells (cultured in the presence of dexamethasone to induce cytochrome P450 reductase activity) [1000 pmol/(mg min)] is comparable to the mean values observed in human liver microsomes [1070 pmol/(mg min) Yamazaki et al., 1993]. The turnover number for testosterone and CYP3A4 in the human lymphoblasts was 15/min for endogenous OR levels and increased to 44/min with OR coexpression. 

A feature of some pterocarpan phytoalexins (e.g., pisatin and glyceollin of pea and soybean, respectively) is their hydroxylation at position 6a, a reaction catalyzed by a microsomal cytochrome P450 monooxygenase.49 50 A cDNA encoding this enzyme was recently characterized from elicited soybean cell cultures.51 The microsomal protein, expressed in yeast cells, catalyzed the stereoselective hydroxylation of (6a/ , lla/ )-3,9-dihydroxypterocarpan to its 6a-hydroxy derivative. It was also demonstrated that the enzyme expression is regulated at the transcriptional level.51... 

Peters WHM, Reolofs HMJ (1989) Time-dependent activity and expression of glutathion-S-transferases in the human adenocarcinoma cell line CACO-2. Biochem J 264 613-616 Rosenberg DW, Leff T (1993) Regulation of cytochrom P450 in cultured human colonic cells. Arch Biochem Biophys 300 186-192... 

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