Gene 377 (2006) 56  64
www.elsevier.com/locate/gene
Induction of two cytochrome P450 genes, Cyp6a2 and Cyp6a8,
of Drosophila melanogaster by caffeine
in adult flies and in cell culture
N
Srividya Bhaskara, Erika Danielle Dean, Vita Lam, Ranjan Ganguly
Department of Biochemistry, Cellular and Molecular Biology, University of Tennessee, Knoxville, TN 37996, United States
Received 8 December 2005; received in revised form 7 February 2006; accepted 28 February 2006
Available online 5 April 2006
Received by Igor B. Rogozin
Abstract
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.
© 2006 Elsevier B.V. All rights reserved.
Keywords: Vivarin; Reporter gene assay; Transgenic flies; Cell transfection; Luciferase assay; Promoter assay; AP1 binding sites
1. Introduction perspectives. One such perspective is the induction and
regulation of mammalian and human CYP genes, especially in
Cytochrome P450 monooxygenases or CYPs comprise a relation to the metabolism of different toxic chemicals,
superfamily of enzymes that are involved in the biosynthesis of carcinogenic compounds and drugs including barbiturate com-
many biologically important compounds and metabolism of a pounds such as phenobarbital and barbital (Wang and Negishi,
variety of xenobiotic (foreign) chemicals to which living organ- 2003; Mandal, 2005). Interestingly, many of these xenobiotic
isms are exposed to on a daily basis (Guengerich, 2004). CYPs compounds have been used as tools to identify the cis-regulatory
have been studied in various organisms from different biological elements, receptors and other factors that regulate CYP gene
expression in mammals (Sueyoshi and Negishi, 2001; Hankin-
son, 2005).
Abbreviations: bp, base pair(s); CYP, cytochrome P450 monooxygenase;
Besides drugs and environmental toxicants, caffeine is
Cyp (or CYP), CYP-encoding gene; kb, kilobase(s) or 1000 bp; LUC,
probably another xenobiotic compound to which most people
Luciferase; luc, LUC-encoding gene; nt, nucleotide(s).
N are exposed everyday. Caffeine is naturally found in berries,
Corresponding author. Tel.: [phone]; fax: [phone].
E-mail address: [e-mail] (R. Ganguly). seeds and leaves of many plants including coffee, cocoa and tea.
0378-1119/$ - see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.gene.2006.02.032
S. Bhaskara et al. / Gene 377 (2006) 56 64 57
A variety of commercial food and drinks also contain a high ry506 host strain were used: 0.2-luc30-4 (also referred as 0.2-luc4),
quantity of caffeine. Because caffeine is consumed heavily and 0.8-luc110, 0.8-luc14, 0.8-luc121 and 3.1-luc2. Each of these
it is chemically very similar to other purines, extensive studies transgenic lines has a single reporter transgene with a firefly
have been done on the effect of caffeine on various biological luciferase (luc) reporter gene under the control of 0.2- (-11/
processes in humans and other mammals such as brain motor -199), 0.8- (-11/-761) or 3.1-kb (-11/-3100) upstream DNA of
activity, cognitive functions, sleep and hypertension (Fredholm the Cyp6a8 gene. The chromosomal locations of the transgene in
et al., 1999; Lorist and Tops, 2003). However, not much work different lines have been described earlier (Maitra et al., 2002). The
has been done on the effect of caffeine on gene expression 0.2-luc4 and 0.8-luc110 strains were made homozygous for their
except for three studies. One study showed that caffeine 2nd chromosome-linked transgene by using appropriate balancer
treatment increases CYP1A1 and CYP1A2 mRNA levels in rat stocks (Maitra et al., 2000) and two genetic crossing schemes. One
liver and kidney (Goasduff et al., 1996). In the other two studies scheme gave 0.2-luc4H and 0.8-luc110H stocks. Genotypes of
caffeine was found to increase the levels of c-fos, c-jun, and these flies are +/+; 0.2-luc/0.2-luc; +/+ and +/+; 0.8-luc/0.8-luc; +/
junB mRNAs in rat striatum (Svenningsson et al., 1995), and +, respectively. The transgene carrying 2nd chromosomes of these
sonic hedgehog mRNA in primary murine neuronal and stocks are from the ry506 strain that was used for transformation.
astroglial cells in culture (Sahir et al., 2004). The other scheme produced 0.2-luc4H-ry and 0.8-luc110H-ry
In insects, CYPs are known to be involved in conferring stocks. Besides the transgene carrying 2nd chromosomes, the X
insecticide resistance, and insecticide resistance-associated and 3rd chromosomes in these stocks are also from the ry506 strain
r r
overexpression of one or more CYP genes has been observed (indicated with r). Genotypes of these stocks are X /X ; 0.2-luc/
r r
in many insect species (see Scott, 1999 for review). In 0.2-luc; 3r/3r and X /X ; 0.8-luc/0.8-luc; 3r/3r, respectively. The
Drosophila also, at least five Cyp genes, including Cyp6a2 source of the 4th chromosome in any of these stocks is not known.
and Cyp6a8, show overexpression in DDT resistant strains Other reporter transgenic lines, 0.8-luc14, 0.8-luc121 and 3.1-
(Maitra et al., 1996; Dombrowski et al., 1998; Daborn et al., luc2, carrying 3rd chromosome-linked reporter transgenes are the
2001; Brandt et al., 2002). However, the mechanism of original transgenic lines made in the ry506 host strain. These strains
overexpression or the regulation of insect CYP genes in general have been maintained by crossing ry+ transgenic males and
is not well-understood. Since xenobiotic compounds have been females in every generation. Flies were reared at 25 °C on standard
successfully used to understand mammalian CYP gene regula- cornmeal-agar-molasses Drosophila medium.
tion, we surmised whether caffeine could also be used to study Schneider line 2 or SL-2 cells of D. melanogaster (Schneider,
insect CYP gene regulation. Therefore, in the present study we 1972), obtained from Invitrogen (Carlsbad, CA), were used for
used Drosophila as a model insect and examined the effect of promoter assays. Cells were maintained at 25 °C in Schneider s
caffeine on the Cyp6a2 and Cyp6a8 gene expression in adult fly Drosophila medium (Gibco-BRL) supplemented with 10% heat-
and also in Drosophila cells in culture. The results showed that inactivated bovine calf serum and 0.01% penicillin streptomy-
both genes are induced by caffeine, and Cyp6a8 shows cin (Sigma). Every fourth day the cells were transferred to fresh
differential constitutive and caffeine-induced expression in media.
different tissues of the adult flies.
2.2. Construction of reporter plasmids for Cyp6a2 and Cyp6a8
2. Materials and methods promoter assay in SL-2 cells
2.1. Fly strains and cell culture Recombinant plasmids carrying 0.9-kb (-983/-1) and 0.8-
kb (-761/-11) upstream DNA of Cyp6a2-91R and Cyp6a8-91R
The 91-C strain and the following five reporter transgenic lines alleles of 91-R strain respectively (Dombrowski et al., 1998;
of Drosophila melanogaster created by Maitra et al. (2002) in the Maitra et al., 2002) were used as templates to amplify different
Table 1
Sequences of the distal primers and PCR conditions used to amplify different regions of the Cyp6a2 and Cyp6a8 genes
a
Genes Primer names Regions amplified Primer sequences and regions they are identical to Tm (°C)
b
Cyp6a2 A2-C -983/-152 -ctcacgcgtTTCATTCGTTTTATCGCCG-32 (-983/965) 61
A2R-1M -522/-152 -ccacgcgtCAAGTGGGATCGTCCTGTAC-32 (-522/-503) 53
A2R-2M -265/-152 -ccacgcgtGCAGTGAAGCGTATGAGTATC-32 (-265/-245) 52
A2R-3M -129/-152 -ccacgcgtGCTAGCTAGCTCACATGCTGTC-32 (-129/-112) 56
c
Cyp6a8 A8-5F -491/-11 52 -ggcctcgagGTTGTGGTAGGTTAGTAGC-32 (-491/-473) 72.1
A8-1F -109/-11 52 -ggcctcgagCTAGCGACTGAGAATGCATC-32 (-109/-90) 78
A8-0.06F -67/-11 52 -ggcctcgagCAGCGTTTAAAAGCAGTTTGC-32 (-67/-47) 78.6
a
Extra bases added to each primer are shown in lower case. Bases of the upstream DNA are in upper case. For each gene ATG is at +1. The MluI and XhoI sites
engineered respectively for Cyp6a2 and Cyp6a8 are underlined.
b
The same A2A proximal primer (52 -ctcgtcgacTTTGCGTAGCTGCTCCC-32 ), complimentary to -1/-17 bases of the Cyp6a2 gene, was used with all distal
primers. Engineered bases are in lower case. Added SalI site is underlined. ATG is at +1. Tm = 63 °C.
c
G10 proximal primer (52 -cgttcgaaATGATCTTCGAATACG-32 ), complimentary to -11/-26 bases of the Cyp6a8 gene, was used with all distal primers.
Engineered bases are in lower case. The added HindIII site is underlined. ATG is at +1. Tm = 70 °C.
58 S. Bhaskara et al. / Gene 377 (2006) 56 64
regions of the upstream DNA of Cyp6a2 and Cyp6a8 genes via DNA mix. Cells to be transfected were resuspended in fresh
PCR. Table 1 shows the sequences of the distal and proximal SL-2 medium at 1 × 106 cells/ml density. To each well of a
primers used for each Cyp6 gene. Appropriate restriction 48-well microtiter plate, 500 źl of cells were dispensed and to
enzyme sites that were added to these primers for cloning each well, 84 źl of calcium phosphate-DNA mix was added
purpose are detailed in Table 1. PCR amplifications and in a drop-wise manner. After 18 h incubation at 25 °C, the
purification of the amplified DNAs were done by using Easy- medium was replaced with 500 źl of fresh medium and
Start PCR kit (Molecular Bioscience, Boulder) and kits from incubated at 25 °C for 24 h. The cells were then spun down
Qiagen (CA), respectively. Four amplified DNA fragments and resuspended in 500 źl of fresh SL-2 medium containing
representing the -983/-1, -522/-1, -265/-1 and -129/-1 8 mM caffeine (Sigma, St. Louis). Following 24 h incubation
regions (ATG at +1) of the Cyp6a2 gene were digested with MluI at 25 °C, cells were pelleted and 250 źl 1× passive lysis
and SalI, and cloned into MluI XhoI cut pGL3-basic vector buffer (Promega, WI) was added to each well. The plates
(Promega, WI). The resulting recombinant promoter-reporter were agitated on a plate shaker for 15 min at room
plasmids were named 0.98luc-A2, 0.52luc-A2, 0.26luc-A2 and temperature to lyse the cells and then centrifuged at
0.12luc-A2, respectively. In case of Cyp6a8 gene, three 1500 rpm for 5 min at 4 °C. The clear supernatant was
amplified upstream DNAs, representing -491/-11, -109/-11 collected from each well and assayed immediately for F-luc
and -67/-11 regions (ATG at +1) of Cyp6a8 were digested with and R-luc activities by using a dual luciferase assay kit
XhoI and HindIII and cloned into XhoI HindIII cut pGL2(N) (Promega, WI). Briefly, to 20 źl cell lysate, 100 źl luciferase
vector which is the pGL2-basic vector (Promega, WI) with SalI assay reagent II was mixed, and placed in a luminometer
site modified to NotI (Maitra et al., 2002). The recombinant within 10 s to measure the firefly luciferase (F-luc) activity.
promoter-reporter plasmids were named 0.5luc-A8, 0.1luc-A8 Immediately after recording the F-luc activity, 100 źl Stop
and 0.06luc-A8, respectively. In addition to these plasmids, and Glo reagent (Promega, WI) was added, vortexed gently
0.8luc-A8 and 0.2luc-A8 plasmids carrying -766/-11 and and the Renilla luciferase (R-luc) activity was measured
-199/-11 upstream DNA of Cyp6a8 (Maitra et al., 2002) were within 10 s. R-luc activity was used to normalize the data,
also used in the present study. All PCR products were sequenced which were expressed as the ratio of F-luc to R-luc activity.
and found to match with the reported sequence of Cyp6a2 and In each experiment, all reporter constructs of both Cyp6 genes
Cyp6a8 upstream DNAs (Dombrowski et al., 1998; Maitra et al., were used to transfect the same batch of cells. For each
2002). reporter construct, triplicate transfection was done. Two such
experiments were done with two different isolates of each
2.3. Treatment of flies, fly extract preparation, and luciferase assay reporter plasmid DNA to obtain the final mean F-luc/R-luc
ratio.
Transgenic female flies, 2 4 days old, were allowed to
feed for 24 h on instant Drosophila food made with aqueous 2.5. Northern blot hybridization
solution of 2.0 mM Vivarin caffeine (obtained from local
store) or 16 mM pure caffeine (Sigma, St. Louis), a dose The details of RNA blotting, probe synthesis and
found to be optimum for induction. Food made with water hybridization have been described previously (Maitra et al.,
was used as a control. After treatments, fly extracts were 2000). Briefly, total RNA was isolated from caffeine-treated
prepared from groups of ten females for luciferase and and untreated adult female flies by using Tri-reagent (Sigma,
protein assays. Details of fly handling, extract preparation, MO). For analysis, 20 źg of each RNA sample was dried,
firefly luciferase and protein assays have been described dissolved in 20 źl formaldehyde loading dye solution
previously (Maitra et al., 2002). Luciferase activity and total (Ambion, Austin, TX) and denatured by incubating at
protein in the fly extract were determined by using a 65 °C for 15 min. Samples were electrophoresed on a
luciferase assay reagent kit (Promega, Madison, WI) and 2.2 M formaldehyde -1.2% agarose gel and blotted onto
BCA protein assay kit (Pierce), respectively. Luciferase nylon membrane. The blots were cut into two pieces to probe
activity in fly extracts was expressed as average Registered CYP RNA in the upper half and the control RP49 mRNA in
Light Units per źg of total protein (RLU/źg). For each the lower half as described (Maitra et al., 2000). Upper
treatment, extracts prepared from three independent groups of halves of three blots made from three independent RNA
flies were assayed. samples and lower halves of these blots were prehybridized
in separate bottles for 2 h at 37 °C in Northern Max
2.4. SL-2 cell transfection, extract preparation and dual Prehybridization/Hybridization buffer (Ambion, TX). After
luciferase assay prehybridization,32P-labeled CYP (5 × 109 cpm/źg) and RP49
(1.2 × 108 cpm/źg) cDNA probes were added to the buffer in
For transient transfection of SL-2 cells, 1.0 źg of Cyp6a2 respective bottles and hybridization was done at 37 °C for
or Cyp6a8 firefly luciferase (F-luc) reporter plasmid and 22 24 h. The blots were washed under high stringent
0.5 źg of internal control Renilla luciferase (R-luc) plasmid conditions and scanned with a Packard Radioanalytical
(Promega, Madison) were dissolved in sterile 0.1 M CaCl2 Imager as described (Dombrowski et al., 1998). Data were
(Invitrogen, CA) and mixed with equal volume of 2× HEPES normalized using RP49 hybridization signal as the internal
buffer (Invitrogen, CA) at pH 7.1 to make calcium phosphate- control.
S. Bhaskara et al. / Gene 377 (2006) 56 64 59
Fig. 1. Expression of luciferase reporter gene in 0.2-luc4H and 0.8-luc110H reporter transgenic lines. Adult females of each line were allowed to feed on
medium containing water or 2.0 mM caffeine (Vivarin) for 24 h. Extracts and RNA prepared from these flies were assayed for luciferase activity (A) and LUC
mRNA level (B) as described in the Materials and methods. The results represent the means Ä… S.D. of three independent experiments. ANOVA p < 0.0001 for A
and B.
2.6. Statistics from four- to six-fold (Fig 2). In another transgenic line (0.2-
luc7), the 0.2-luc transgene located at different chromosomal
Data were analyzed using ANOVA program. location (Maitra et al., 2002) also showed about 5-fold induction
when treated with caffeine (data not shown). Since different
3. Results transgenic lines carrying reporter transgene at different chro-
mosomal position showed similar levels of induction, it may be
3.1. Vivarin and caffeine induce Cyp6a8 promoter activity concluded that caffeine induction is not a result of chromosomal
position effect; it is the function of the Cyp6a8 upstream DNA
To examine whether caffeine would induce Drosophila driving the luciferase reporter gene.
Cyp6a8 gene, adult females of 0.2-luc4H and 0.8-luc110H
transgenic lines were exposed to instant Drosophila medium 3.2. Caffeine induces the endogenous Cyp6a8 and Cyp6a2
made in aqueous solution of over-the-counter caffeine tablet genes in transgenic and wild type strains
called Vivarin. The final concentration of caffeine in the me-
dium was 2.0 mM. The results (Fig. 1A) showed that in both To rule out the possibility that induction of the luc reporter
transgenic lines, luciferase activity was significantly higher in gene by caffeine is not due to some peculiarity of the reporter
Vivarin-treated flies compared to the water-treated controls.
Expression of the luc reporter gene was also examined by
Northern blot analysis. A 3-fold induction of LUC mRNA was
observed in both transgenic lines following caffeine treatment
(Fig. 1B), suggesting that Cyp6a8 promoter is induced by
caffeine. Results also showed that LUC enzyme activity and
LUC mRNA levels were significantly higher in the 0.8-luc line
compared to the 0.2-luc line. These data agree with our previous
observations that 0.8-kb upstream DNA of Cyp6a8 has higher
promoter activity than the 0.2-kb DNA (Maitra et al., 2002).
Vivarin tablet contains many other ingredients besides
caffeine. Therefore, to examine whether the effect of Vivarin
could be mimicked by caffeine, 0.2-luc4H, 0.8-luc110H, 0.8-
luc14 and 0.8-luc121 transgenic lines were treated with pure
caffeine. Since the latter two strains were not made homozygous
for the reporter transgene, all transgenic lines were crossed to the
ry506 host strain used for germ line transformation, and the F1
Fig. 2. Effect of pure caffeine on the expression of luciferase reporter transgene
ry+ (transgene marker) females, heterozygous for the transgene,
in different transgenic lines. Each transgenic line was crossed to the ry506 host
were selected and treated with 16 mM caffeine, which was found
strain, and the ry+ (red-eyed) F1 females carrying the reporter transgene were
to be an optimum concentration in dose response experiments
allowed to feed on water or 16 mM caffeine containing medium for 24 h.
(data not shown). While a six-fold induction was observed in
Extracts prepared from these flies were assayed for luciferase activity. Data
0.2-luc4 line, in three 0.8-luc lines the level of induction ranged represent mean Ä… S.D. of three independent determinations. ANOVA p < 0.0001.
60 S. Bhaskara et al. / Gene 377 (2006) 56 64
Fig. 3. Northern blot analysis of endogenous Cyp6a8 and Cyp6a2 gene expression in 0.8-luc110H-ry reporter transgenic line and 91-C wild type strains of Drosophila.
Total RNA was extracted from adult females of each strain, which were treated with water or 16 mM caffeine as described in Fig. 2. RNA samples were fractionated on
agarose gel, blotted and hybridized with Cyp6a8 (A) and Cyp6a2 (B) gene probes. The levels of RP-49 RNA in the RNA samples were used as internal control to
correct RNA loading discrepancy between lanes. The results represent the means Ä… S.D. of three independent experiments done with three different RNA samples.
ANOVA p < 0.0001 for A and B.
transgenes or the host ry506 strain, RNA isolated from caffeine- line about a 12-fold induction of the reporter gene was observed
treated and untreated 0.8-luc110 (H-ry) transgenic flies in caffeine-treated flies (Fig. 5A). In the gut (Fig. 5B), con-
homozygous for the reporter transgene and a wild type strain stitutive expression of the reporter transgenes in the untreated
named 91-C were examined for endogenous Cyp6a8 gene transgenic flies was very low; it was more than one order of
expression by Northern blot analysis. We also examined magnitude lower than that found in the ovaries. In fact the
Cyp6a2, another Cyp6 gene of Drosophila that appears to be specific activities of luciferase enzyme in treated and untreated
under the same gene regulatory mechanism that controls the flies in the 0.2-luc4 (H-ry) line were too low to be considered as
Cyp6a8 gene (Maitra et al., 2000). Results showed that in both an expression of the transgene. In the 0.8-luc110 (H-ry) line,
0.8-luc110 (H-ry) transgenic line and 91-C strain endogenous constitutive expression was also very low but, approximately a
Cyp6a8 and Cyp6a2 genes were induced by caffeine at the 10-fold induction was observed in the caffeine-treated flies
steady-state mRNA level, although the level of induction of (Fig. 5B). In the cuticle plus fat body (Fig. 6A) and in the
these two genes varied between the two strains (Fig. 3). malpighian tubules (Fig. 6B) of the untreated flies of both trans-
genic lines, luciferase activities were found to be several orders
3.3. The 0.2- and 0.8-kb upstream DNAs of Cyp6a8 show magnitude higher than the activities observed in the ovary and
differential promoter activity in different tissues gut (Fig. 5). Both transgenes also showed a significant induction in
To investigate the promoter activities of 0.2- and 0.8-kb
upstream DNAs of Cyp6a8 in different parts of the body,
luciferase activity in head, body (whole abdomen plus thorax)
and whole female transgenic flies were measured. Differential
expression of both transgenes was observed in the head and body
of the transgenic flies. Firstly, activities of both 0.2luc-A8 and
0.8luc-A8 transgenes were much higher in the heads than in the
bodies of the untreated control flies (Fig. 4). Secondly, in the
heads and bodies of the control flies, activity of the 0.8luc-A8
transgene was much higher than the activity of the 0.2luc-A8
transgene. Thirdly, both transgenes were induced by caffeine, but
the level of induced LUC activity was consistently higher in the
0.8-luc110 (H-ry) transgenic line than in the 0.2-luc4 (H-ry) line.
Expression of the 0.2luc-A8 and 0.8luc-A8 transgenes was
also examined in the ovary (Fig. 5A), gut (Fig. 5B), abdominal
cuticle plus fat body (Fig. 6A) and malpighian tubules (Fig. 6B)
Fig. 4. Expression of the luciferase reporter transgene in different body parts of
of caffeine-treated and untreated adult female flies. In both
caffeine-treated and untreated female flies of 0.2-luc4H-ry and 0.8-luc110H-ry
transgenic lines, expression of the reporter gene was very low in
transgenic lines. Female flies of each transgenic line were fed with 16 mM
the ovaries of the untreated flies (Fig. 5A) and caffeine treatment
caffeine or water for 24 h and luciferase activity was measured as described. The
did not induce the reporter gene in the 0.2-luc4 (H-ry) line
results shown are the means Ä… S.D. of three independent experiments. ANOVA
(p = 0.34, Student s t-test). However, in the 0.8-luc110 (H-ry) p <0.0001.
S. Bhaskara et al. / Gene 377 (2006) 56 64 61
Fig. 5. Activities of the luciferase reporter gene in the ovary and gut tissues. Female flies of each transgenic line were fed with 16 mM caffeine or water for 24 h.
Ovaries (A) and guts (B) were dissected out from these females and luciferase activity was measured. Means Ä… S.D. of three independent experiments are shown.
ANOVA p < 0.0001.
the cuticle plus fat body of the caffeine-treated flies (Fig. 6A). upstream DNA of Cyp6a2 and Cyp6a8 genes were about 12.6
However, 0.8-luc110 (H-ry) line showed much higher induction and 3.3 times greater than the basal activity of the empty pGL3
than the 0.2-luc4 (H-ry) line. Two transgenic lines showed the vector, respectively (Fig. 7). This high basal activity may be due
most striking difference when the reporter gene activities in the to the presence of a putative TATA element in these DNA
malpighian tubules of the untreated flies were compared (Fig. 6B). fragments (Maitra et al., 2002). When 0.1luc-A8 reporter plas-
In the untreated flies, constitutive expression of the reporter gene mid was used, the basal activity increased about 36% relative to
in the 0.8luc-A8 transgenic line was about 350-fold greater than the activity of the 0.06luc-A8 plasmid and its final activity was
the expression found in the 0.2luc-A8 line (Fig. 6B). Although about 4.5-fold higher than the activity of the empty vector
caffeine treatment induced the reporter gene in both transgenic (Fig. 7B). The basal promoter activity increased another 4- and
lines, the induced activity was still about 160-fold higher in the 5-fold relative to the 0.12luc-A2 and 0.1luc-A8 reporter
0.8-luc110 (H-ry) than that observed in the 0.2-luc4 (H-ry) line. plasmids respectively, when 0.26luc-A2 and 0.2luc-A8 plasmids
were used. Compared to the empty vector, these plasmids
3.4. Analysis of the Cyp6a2 and Cyp6a8 upstream DNAs for showed about a 46- and 21-fold higher basal activity, re-
constitutive and caffeine-induced expression in SL-2 cells spectively (Fig. 7). These data suggest that -265/-129 Cyp6a2
DNA and -199/-109 Cyp6a8 have a strong positive effect on
To map the cis-regulatory DNA for constitutive and caffeine- the basal transcription.
induced expression, luc reporter plasmids carrying different In the case of Cyp6a2 gene, increasing the length of the
lengths of Cyp6a2 or Cyp6a8 upstream DNA were used to upstream DNA from -265 to -522 did not elevate the basal
transfect SL-2 cells. The results showed that the basal activity of promoter activity further; instead the promoter activity
0.12luc-A2 and 0.06luc-A8 plasmids carrying the shortest decreased by about 42% (Fig. 7A). Nevertheless, the activity
Fig. 6. Expression of luciferase reporter gene in fat body and malpighian tubules of adult flies. Female flies of two transgenic lines were treated with 16 mM caffeine or
water, and luciferase activities in the fly extracts were determined as described in Materials and methods. Data shown are the means Ä… S.D. of three independent
experiments. ANOVA p < 0.0001.
62 S. Bhaskara et al. / Gene 377 (2006) 56 64
Fig. 7. Promoter activities of different upstream DNAs of Cyp6a2 and Cyp6a8 genes in SL-2 cells. SL-2 cells were cotransfected with Renilla luciferase (R-luc) control
plasmid and firefly luciferase (F-luc) chimeric reporter constructs carrying different lengths of upstream DNA of Cyp6a2 or Cyp6a8 gene (ATG at +1). R-luc and F-luc
activities in the extracts of 8 mM caffeine-treated and untreated transfected cells were determined and compared. Each bar represents mean of two independent
transfection experiments done with two different batches of SL-2 cells and two different isolates of each chimeric reporter plasmid. In each experiment, three aliquots
of cells were transfected with each chimeric reporter plasmid. Relative basal activities were determined by comparing the F-luc/R-luc values of luciferase reporter
plasmid without any promoter and chimeric reporter plasmids. Putative TATA boxes (¾%) of Cyp6a2 and Cyp6a8 are located at -85 and -64 positions, respectively.
ANOVA p < 0.0003 for fold-induction and <0.0001 for basal activity.
of the 0.52luc-A2 plasmid was still about 26-fold greater than different. While the 0.12luc-A2 plasmid did not show much
the activity of the empty vector. Another 4-fold rise in the basal caffeine induction, a 4.5-fold caffeine induction was observed
promoter activity was observed when the upstream DNA length with the 0.26luc-A2 plasmid. Similar to the basal activity,
was increased from -522 to -983 (Fig. 7A). Thus, the region caffeine-induced activity dropped when the 0.52luc-A2 plasmid
between -522 and -983 of the Cyp6a2 gene also contains was used, but it increased again when the 0.98luc-A2 reporter
sequences that have a strong positive effect on the basal plasmid with longer upstream DNA was used. Cells transfected
transcription. In the case of Cyp6a8, such sequences appear to with this plasmid showed about a 6-fold induction in
be located between regions -199 and -491 because the basal transcriptional activity following caffeine treatment, suggesting
activity of the 0.5luc-A8 reporter plasmid carrying -491/-11 that the upstream DNA sequence located between -522 and
upstream DNA was about 5.5-fold greater than the activity of -983 regions plays a strong positive role in caffeine-induced
the 0.2luc-A8 reporter plasmid with the shorter (-199/-11) expression of the Cyp6a2 gene.
upstream DNA.
When transfected SL-2 cells were treated with 8 mM 4. Discussion
caffeine, all reporter plasmids, except 0.12luc-A2, 0.1-luc-A8
and 0.06luc-A8, showed significant induction (Fig. 7). For these The present investigation clearly demonstrated that the 0.2-
three plasmids, induction level ranged between 1.07- and 1.38- and 0.8-kb upstream DNA of Cyp6a8 show differential
fold, suggesting that about first 100 bp upstream DNA of both promoter activity in different body parts of adult female D.
Cyp6 genes probably lacks caffeine-responsive element. melanogaster. The basal promoter activity of both DNA
However, fold-induction increased steadily as the length of fragments was found to be highest in the head than in the
the upstream DNA was increased. The fold-inductions with body (thorax + abdomen) or the whole fly. When individual
0.2luc-A8, 0.5luc-A8 and 0.8luc-A8 reporter plasmids were tissues were examined, both DNA fragments showed three and
1.94, 2.56 and 3.82, respectively (Fig. 7B). In the case of four orders of magnitude higher promoter activities in the
Cyp6a2 upstream DNA, the fold-induction profile was a little cuticle plus fat body and malpighian tubules than in the ovaries
S. Bhaskara et al. / Gene 377 (2006) 56 64 63
and gut, respectively. However, in all tissues, body parts or activator protein or AP1, which binds with the AP1 element and
whole flies, the 0.8-kb upstream DNA always showed signi- enhances promoter activity. The AP1 proteins are products of
ficantly higher basal promoter activity than the 0.2-kb upstream immediate early genes, which are induced by various extracel-
DNA. In the malpighian tubules, the activity of the 0.8-kb DNA lular stimuli via cAMP pathway (Sng et al., 2004). Many cAMP
was unusually higher (<"350-fold) than the activity of the 0.2-kb responsive gene promoters are known to have AP1 and cAMP
DNA. When caffeine-inducibility was compared, both DNA response element or CRE, which binds with CRE binding
fragments showed similar fold-induction in head, body and protein called CREB or CRE-BP (Sng et al., 2004 and references
whole fly. However, in the ovaries, gut and cuticle plus fat body, therein). Analysis of the Cyp6a2 and Cyp6a8 upstream DNAs
0.8-kb DNA showed much higher caffeine-inducibility than the with MATCH"! program (Kel et al., 2003) at 0.8 core and matrix
0.2-kb DNA. Thus, the additional DNA (-761/-199) present in similarities identified 598 transcriptionally important sequence
the 0.8-kb DNA not only gives a higher basal promoter activity, motifs including many potential AP-1 and CREB sites (Table 2).
but it also gives a higher caffeine-inducibility in ovaries, gut and Since 0.12luc-A2 and 0.1luc-A8 DNA did not show any
cuticle plus fat body. Differential tissue and developmental
expression have also been observed for other Drosophila P450
genes (Bassett et al., 1997; Maibeche-Coisne et al., 2000).
Table 2
Locations of putative sequence motifs in the upstream DNA of Cyp6a2 and
In SL-2 cells, the longest upstream DNA of both Cyp6 genes
Cyp6a8 that participate in cAMP-mediated transcriptional regulation
also showed the highest basal and caffeine-induced promoter
a
Gene Position Core Matrix Sequence Factor
activity. However, basal promoter activity increased in two
(strand) match match (+ strand) name
steps when the length of the upstream DNA of Cyp6 gene was
increased. In the case of Cyp6a2 gene, sequences giving two- Cyp6a2 -865 (-) 0.850 0.834 aattaTGTCAtt CREB
-864 (-) 0.967 0.940 attaTGTCAtt AP-1
step increase of promoter activity are located in -265/-129 and
-863 (-) 0.848 0.865 ttATGTCa CRE-BP1
-983/-522 regions of the upstream DNA. These sequences
-741 (-) 0.800 0.804 gCTCCTta STRE
also increased caffeine-inducibility. In the case of Cyp6a8 gene,
-648 (-) 0.850 0.834 tcttaTGTCAaa CREB
the -199/-109 DNA gave a 4-fold and the -491/-199 DNA
-647 (-) 0.967 0.878 cttaTGTCAaa AP-1
gave about a 6-fold rise in basal promoter activity. Caffeine- -646 (-) 0.818 0.842 ttaTGTCA CREB
-549 (+) 0.831 0.891 TTACTtaa CRE-BP1
inducibility of the Cyp6a8 upstream DNA, however, increased
-489 (-) 0.955 0.850 tTGAATgac AP-1
progressively as its length was increased from position -109 to
-486 (+) 0.962 0.918 aaTGACCgtgt AP-1
-761. The -109/-11 DNA of Cyp6a8 did not show any
-482 (-) 0.967 0.927 accgTGTCAtt AP-1
caffeine-inducibility. These observations suggest that sequences
-371 (+) 0.811 0.802 aaTGAGTgata AP-1
present in the longest upstream of each Cyp6 gene act -370 (-) 0.989 0.880 aTGAGTgat AP-1
-182 (+) 0.967 0.899 caTGACAacaa AP-1
synergistically and give the highest basal and caffeine-induced
-161 (-) 1.000 0.920 gcgtAGTCAtg AP-1
expression. It is not known whether the same sequences are
-120 (-) 0.967 0.896 atgcTGTCAtg AP-1
involved in basal and caffeine-induced activity.
-99 (+) 1.000 0.975 gcAGGGGa STRE
Stonehouse et al. (2003) showed that -117/-75 region of rat
Cyp6a8 -749 (+) 1.000 0.999 taAGGGGg STRE
dopamine 2 receptor (D2R) gene containing putative binding -695 (+) 0.818 0.823 TAACGtag CREB
-524 (+) 0.967 0.945 aaTGACAcact AP-1
sites for dopamine receptor regulating factor (DRRF) and SP1
-523 (-) 0.804 0.852 aTGACAcac AP-1
showed a 1.9-fold induction in caffeine-treated cells. It has been
-510 (-) 1.000 0.919 atagAGTCAtg AP-1
hypothesized that stimulation of the D2R gene by caffeine is
-441 (-) 0.962 0.889 gtctGGTCAac AP-1
probably mediated by alleviating the repressive activity of DRRF
-390 (+) 0.811 0.813 cgTGAGTaagc AP-1
(Stonehouse et al., 2003). Hwang et al. (2001) have shown that -389 (-) 0.989 0.898 gTGAGTaag AP-1
-328 (-) 0.839 0.839 tgataAGTCAca CREB
DRRF, a zinc finger type transcription factor, binds to GTand GC
-327 (-) 1.000 0.962 gataAGTCAca AP-1
boxes and displaces the Sp1 and Sp3 transcription factors.
-326 (+) 1.000 0.884 ataAGTCAc AP-1
Although Alibaba2.1 program (http://www.gene-regulation.
-318 (-) 1.000 0.927 cagttCGTCAct CREB
com/pub/programs/alibaba2/index.html) identified putative Sp1
-317 (-) 0.967 0.954 agttCGTCAct AP-1
sites at -944/-934 and -17/-8 positions of Cyp6a2, and at -316 (-) 1.000 0.889 gttCGTCActgt CREB
-292 (-) 0.811 0.807 cattATTCAtc AP-1
-736/-737 and -641/-631 positions of Cyp6a8 genes, in SL-2
-291 (+) 0.955 0.880 attATTCAt AP-1
cells these sites are probably not involved in the expression of
-133 (-) 0.831 0.815 taaAGTAA CRE-BP1
Cyp6a2 and Cyp6a8 genes because Sp1 proteins are not found in
-95 (-) 1.000 0.849 tgcatCGTCAtg CREB
this cell line (Courey and Tjian, 1988). Therefore, the constitutive
-94 (-) 0.967 0.880 gcatCGTCAtg AP-1
and caffeine-induced expression found in SL-2 cells must be -93 (-) 1.000 0.888 catCGTCAtgca CREB
-81 (-) 0.839 0.800 aagtaAGTCAat CREB
mediated by factors other than Sp1. However, involvement of the
-80 (-) 1.000 0.958 agtaAGTCAat AP-1
putative Sp1 sites in the regulation of Cyp6 gene promoter in the
-80 (-) 1.000 0.957 agtaAGTCAat AP-1
adult flies cannot be ruled out.
-79 (+) 1.000 0.881 gtaAGTCAa AP-1
In two studies (Goasduff et al., 1996; Svenningsson et al.,
-79 (-) 0.818 0.819 gtaAGTCA CREB
-39 (+) 1.000 0.849 TTACGgaa CRE-BP1
1995), caffeine has been shown to increase the steady-state level
a
of two CYP mRNAs, and RNAs for FOS and JUN family
The capital letters indicate the positions in the sequence which match with
proteins. The homo- and heterodimer of these proteins are called the core sequence of the matrix.
64 S. Bhaskara et al. / Gene 377 (2006) 56 64
Fredholm, B.B., Battig, K., Holmen, J., Nehlig, A., Zvartau, E.E., 1999. Actions
caffeine-inducibility (Fig. 7), the AP-1 and CREB sites found
of caffeine in the brain with special reference to factors that contribute to its
within about -100 bp upstream DNA of both genes may not be
widespread use. Pharmacol. Rev. 51, 83 133.
involved in caffeine induction. However, the CREB-BP and AP-
Goasduff, T., Dreano, Y., Guillois, B., Menez, J.F., Berthou, F., 1996. Induction
1 sites found at -133 position of the Cyp6a8, and at -161 and
of liver and kidney CYP1A1/1A2 by caffeine in rat. Biochem. Pharmacol.
-182 positions of the Cyp6a2 (Table 2) may play a role in 52, 1915 1919.
Guengerich, F.P., 2004. Cytochrome P450: what have we learned and what are
caffeine induction because 0.2luc-A8 and 0.26luc-A2 DNA
the future issues? Drug Metab. Rev. 36, 159 197.
carrying these elements gave about 2- and 4.5-fold caffeine
Hankinson, O., 2005. Role of coactivators in transcriptional activation by the
induction, respectively. Similarly, the putative AP-1 and CREB
aryl hydrocarbon receptor. Arch. Biochem. Biophys. 433, 379 386.
sites found in the DNA upstream of position -549 of the Cyp6a2
Hwang, C.K., et al., 2001. Dopamine receptor regulating factor, DRRF: a zinc
and -510 of the Cyp6a8 genes (Table 2) may be important for finger transcription factor. Proc. Natl. Acad. Sci. U. S. A. 98, 7558 7563.
Kel, A.E., Gossling, E., Reuter, I., Cheremushkin, E., Kel-Margoulis, O.V.,
caffeine induction because the longest reporter constructs
Wingender, E., 2003. MATCH: a tool for searching transcription factor
(0.98luc-A2 and 0.8luc-A8) carrying these putative AP1 and
binding sites in DNA sequences. Nucleic Acids Res. 31, 3576 3579.
CREB sites gave further increase in fold-induction (Fig. 7).
Lorist, M.M., Tops, M., 2003. Caffeine, fatigue, and cognition. Brain Cogn. 53,
Cyp6a2 and Cyp6a8 DNA upstream of positions -549 and
82 94.
-510, respectively, also have a sequence that matches with the Maibeche-Coisne, M., Monti-Dedieu, L., Aragon, S., Dauphin-Villemant, C.,
2000. A new cytochrome P450 from Drosophila melanogaster, CYP4G15,
stress response element (STRE) found in many yeast genes
expressed in the nervous system. Biochem. Biophys. Res. Commun. 273,
(Parrou et al., 1999). Since caffeine is thought to cause cellular
1132 1137.
stress, the putative STREs may also play a role in caffeine
Maitra, S., Dombrowski, S.M., Waters, L.C., Ganguly, R., 1996. Three second
induction of the Cyp6a2 and Cyp6a8 genes. Refined mapping
chromosome-linked clustered Cyp6 genes show differential constitutive and
coupled with site-directed mutagenesis will be necessary to barbital-induced expression in DDT-resistant and susceptible strains of
Drosophila melanogaster. Gene 180, 165 171.
determine the role of putative AP-1, CREB and STRE elements.
Maitra, S., Dombrowski, S.M., Basu, M., Raustol, O., Waters, L.C., Ganguly,
If AP-1 and/or CREB are indeed involved in caffeine-induced
R., 2000. Factors on the third chromosome affect the level of Cyp6a2 and
transcription, it may be hypothesized that caffeine-induction of
Cyp6a8 expression in Drosophila melanogaster. Gene 248, 147 156.
Cyp6 genes is mediated via cAMP signaling pathway. This
Maitra, S., Price, C., Ganguly, R., 2002. Cyp6a8 of Drosophila melanogaster:
hypothesis is currently being investigated. gene structure, and sequence and functional analysis of the upstream DNA.
Insect Biochem. Mol. Biol. 32, 859 870.
Mandal, P.K., 2005. Dioxin: a review of its environmental effects and its aryl
Acknowledgements
hydrocarbon receptor biology. J. Comp. Physiol., B 175, 221 230.
Parrou, J.L., Enjalbert, B., Francois, J., 1999. STRE- and cAMP-independent
The project was supported by the National Research
transcriptional induction of Saccharomyces cerevisiae GSY2 encoding
Initiative of the USDA Cooperative State Research, Education glycogen synthase during diauxic growth on glucose.
Sahir, N., Evrard, P., Gressens, P., 2004. Caffeine induces sonic hedgehog gene
and Extension Service, grant number 2002-35302-12281 to RG.
expression in cultured astrocytes and neurons. J. Mol. Neurosci. 24, 201 206.
Schneider, I., 1972. Cell lines derived from late embryonic stages of Drosophila
References
melanogaster. J. Embryol. Exp. Morphol. 27, 353 365.
Scott, J.G., 1999. Cytochrome P450 and insecticide resistance. Insect Mol. Biol.
Bassett, M.H., McCarthy, J.L., Waterman, M.R., Sliter, T.J., 1997. Sequence and
29, 757 777.
developmental expression of Cyp18, a member of a new cytochrome P450
Sng, J.C., Taniura, H., Yoneda, Y., 2004. A tale of early response genes. Biol.
family from Drosophila. Mol. Cell. Endocrinol. 131, 39 49.
Pharm. Bull. 27, 606 612.
Brandt, A., et al., 2002. Differential expression and induction of two Drosophila
Stonehouse, A.H., Adachi, M., Walcott, E.C., Jones, F.S., 2003. Caffeine regulates
cytochrome P450 genes near the Rst(2)DDT locus. Insect Mol. Biol. 11,
neuronal expression of the dopamine 2 receptor gene. Mol. Pharmacol. 64,
337 341.
1463 1473.
Courey, A.J., Tjian, R., 1988. Analysis of Sp1 in vivo reveals multiple
Sueyoshi, T., Negishi, M., 2001. Phenobarbital response elements of
transcriptional domains, including a novel glutamine-rich activation motif.
cytochrome P450 genes and nuclear receptors. Annu. Rev. Pharmacol.
Cell 55, 887 898.
Toxicol. 41, 123 143.
Daborn, P., Boundy, S., Yen, J., Pittendrigh, B., ffrench-Constant, R., 2001.
Svenningsson, P., Strom, A., Johansson, B., Fredholm, B.B., 1995. Increased
DDT resistance in Drosophila correlates with Cyp6g1 over-expression and
expression of c-jun, junB, AP-1, and preproenkephalin mRNA in rat striatum
confers cross-resistance to the neonicotinoid imidacloprid. Mol. Genet.
following a single injection of caffeine. J. Neurosci. 15, 3583 3593.
Genomics 266, 556 563.
Wang, H., Negishi, M., 2003. Transcriptional regulation of cytochrome p450 2B
Dombrowski, S.M., et al., 1998. Constitutive and barbital-induced expression of
genes by nuclear receptors. Curr. Drug Metab. 4, 515 525.
the Cyp6a2 allele of a high producer strain of CYP6A2 in the genetic
background of a low producer strain. Gene 221, 69 77.