Date of Award

8-2005

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Ranjan Ganguly

Committee Members

Bruce McKee, Albrecht von Arnim, Jae Park, Sundar Venkatachalam

Abstract

Cytochrome P450 monooxygenases or CYPs comprise a large family of enzymes that are found in all classes of living organisms, from bacteria to man. These enzymes are involved in the metabolism of many endogenous and xenobiotic (foreign) compounds. In insects, CYPs confer resistance to various insecticides, and resistance-associated overexpression of multiple CYP genes in resistant insects is a common phenomenon. In Drosophila, multiple Cyp genes including Cyp6a2 and Cyp6a8 show higher level of expression in resistant strains than in the susceptible ones. To date, molecular basis of CYP gene overexpression has not been examined in detail. Barbiturate compounds such as phenobarbital and barbital induce both these genes. An unpublished observation from our laboratory showed that Cyp6a2 as well as Cyp6a8 are induced by over-the-counter caffeine tablet, Vivarin. In the present study, I used pure caffeine as a tool to better understand the mechanism of Cyp6a2 and Cyp6a8 gene regulation in Drosophila.

The specific objectives of this project has been to (1) map the upstream DNA of both genes for the sequences responsible for caffeine-induction; (2) examine whether adenosine receptors and/or cAMP-specific phosphodiesterase (cPDE) are involved in transduction of caffeine signal to induce the two Cyp6 genes; and (3) investigate whether Drosophila AP-l transcription factors (D-JUN and D-FOS) playa role in caffeine induction of Cyp6a8 gene. For these objectives, several Cyp-luc reporter plasmids carrying firefly lueiferase (luc) reporter gene under the control of different regions of Cyp6a8 and Cyp6a2 upstream DNAs were constructed to transfect Drosophila embryonic cells, Schneider line 2 (SL-2). Two transgenic reporter strains carrying firefly lueiferase (luc) reporter gene under the control of O.8-kb or 0.2-kb upstream DNA of Cyp6a8 gene were also used to study the mechanism of caffeine induction in vivo.

Results of Northern blot analysis showed that caffeine induces endogenous Cyp6a2 and Cyp6a8 genes at the steady-state mRNA levels both in reporter transgenic and wild-type flies. Transfection experiments with SL-2 cells showed that -983/-1 and 7611-11 upstream DNAs of Cyp6a2 and Cyp6a8, respectively, have sequences for caffeine-induced expression. Further transfection experiments with reporter plasmids carrying luc reporter gene attached to truncated upstream DNAs of Cyp6a2 and Cyp6a8 genes showed that the regions between -265/-129 of Cyp6a2 and -199/-109 of Cyp6a8 have sequences that confer caffeine-induced expression. However, the level of both constitutive and induced expression was highest with -981/-1 DNA of Cyp6a2and -761111 DNA of Cyp6a8genes. Sequence analysis identified several putative binding sites for Activator Protein -1 (AP-l) and cyclic AMP response element CRE binding protein. (CREB) motifs in the upstream DNA of both genes. Moreover, when the four core bases of the single AP-l site present in the -109/-11 DNA of Cyp6a8 were mutated, constitutive expression decreased by 8-fold, suggesting the positive role of AP-l in Cyp6a8 gene expression.

To examine whether caffeine signaling is mediated via adenosine receptor (AdoR) and/or via cPDE inhibition, SL-2 cells transfected with Cyp6a2 and Cyp6a8 reporter constructs were treated with AdoR agonists or with antagonists or with cPDE inhibitors. The Cyp6a8-luc reporter transgenic lines were also treated with these chemicals. The results showed that caffeine signaling is mediated by PDE inhibition and via increase in the intracellular cAMP level. Indeed, treatment with dibutyryl cAMP induces Cyp6a2 as well as Cyp6a8 promoters. Since induction of cAMP pathway is known to upregulate AP-I transcription factors, effect of overexpression of Drosophila D-FOS and (or) DJUN (components of AP-I) on Cyp6a8 promoter activity in SL-2 cells was examined. Surprisingly, activity of Cyp6a8-1uc reporter construct was inhibited when D-FOS or DJUN proteins were overexpressed, suggesting that AP-I proteins are inhibitory for Cyp6 gene expression. In contrast, the Cyp6a8 promoter activity was upregulated, when cells were transfected with anti-D-JUN plasmid or when cells transfected with D-JUN or DFOS sense construct were treated with caffeine. When relation between caffeine and the two AP-I proteins was examined, it was found that caffeine treatment significantly lowers the D-JUN protein level both in SL-2 cells as well as in adult flies. Reporter gene assays and Northern blot analysis showed that caffeine treatment has no effect on the transcriptional activity of the D-jun and D-fos genes. Taken together, it might be concluded that caffeine induction of Cyp6a2 and Cyp6a8 genes is mediated via degradation of D-JUN that acts as a repressor for the promoter of Cyp6a8. Induction of the cAMP pathway and subsequent phosphorylation of the AP-l proteins may relieve the AP-l mediated-repression by promoting the degradation of these proteins. Further investigation is required to resolve these possibilities.

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