Induction of two cytochrome P450 genes, Cyp6a2 and Cyp6a8, of Drosophila melanogaster by caffeine in adult flies and in cell culture.

Bhaskara S, Dean ED, Lam V, Ganguly R
Gene. 2006 377: 56-64

PMID: 16713132 · DOI:10.1016/j.gene.2006.02.032

To examine whether caffeine, the most widely used xenobiotic compound, would induce insect cytochrome P450 or CYP gene expression, upstream DNA fragments of Cyp6a2 (0.12, 0.26, 0.52 and 0.98-kb) and Cyp6a8 (0.06, 0.1, 0.2, 0.5 and 0.8-kb) genes of Drosophila melanogaster were individually fused to the firefly luciferase (luc) reporter gene. Promoter activities of these constructs were examined in Drosophila SL-2 cells using luciferase assays. Activity of 0.2- and 0.8-kb upstream DNA of Cyp6a8 was also measured in transgenic female flies. When these flies were treated with 2 mM pure caffeine or Vivarin caffeine, both DNA fragments showed a 4-5-fold induction of promoter activity. Endogenous Cyp6a8 and Cyp6a2 genes in these flies also showed caffeine-induced expression. In addition, both 0.2- and 0.8-kb DNAs showed differential basal and caffeine-induced activity in head, ovaries, gut, cuticle plus fat body and malpighian tubules. However, in all tissues 0.8-kb DNA always showed higher basal and caffeine-induced activities compared to the 0.2-kb DNA, suggesting that the additional DNA present in the 0.8-kb fragment has sequences that enhance both activities. In SL-2 cells, all reporter constructs of each Cyp6 gene showed significantly higher basal activity than the empty vector. Sequences that boost basal activity are located in -265/-129 and -983/-522 DNA of Cyp6a2, and -199/-109 and -491/-199 DNA of Cyp6a8 genes. While the 0.12- and 0.1-kb upstream DNAs of Cyp6a2 and Cyp6a8 genes respectively did not show caffeine-inducibility in SL-2 cells, the longest upstream DNA of each gene gave the highest level of induction. Caffeine-responsive sequences are not clustered at one place; they appear to be dispersed in -983/-126 and -761/-109 regions of Cyp6a2 and Cyp6a8 genes which also contain many binding sites for activator protein 1 (AP1) and cyclic AMP response element binding protein (CRE-BP). Significance of these binding sites in caffeine-inducibility has been discussed.

MeSH Terms (17)

Animals Animals, Genetically Modified Base Sequence Caffeine Cell Line Cytochrome P-450 Enzyme System Cytochrome P450 Family 6 DNA Primers Drosophila melanogaster Drosophila Proteins Female Gene Expression Genes, Insect Genes, Reporter Mixed Function Oxygenases Plasmids Promoter Regions, Genetic

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