Induction of two cytochrome P450 genes, Cyp6a2 and Cyp6a8,

of Drosophila melanogaster by caffeine

in adult flies and in cell culture

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

Cytochrome P450 monooxygenases or CYPs comprise a

superfamily of enzymes that are involved in the biosynthesis of
many biologically important compounds and metabolism of a
variety of xenobiotic (foreign) chemicals to which living organ-
isms are exposed to on a daily basis (

Guengerich, 2004

). CYPs

have been studied in various organisms from different biological

perspectives. One such perspective is the induction and
regulation of mammalian and human CYP genes, especially in
relation to the metabolism of different toxic chemicals,
carcinogenic compounds and drugs including barbiturate com-
pounds such as phenobarbital and barbital (

Wang and Negishi,

2003; Mandal, 2005

). Interestingly, many of these xenobiotic

compounds have been used as tools to identify the cis-regulatory
elements, receptors and other factors that regulate CYP gene
expression in mammals (

Sueyoshi and Negishi, 2001; Hankin-

son, 2005

).

Besides drugs and environmental toxicants, caffeine is

probably another xenobiotic compound to which most people
are exposed everyday. Caffeine is naturally found in berries,
seeds and leaves of many plants including coffee, cocoa and tea.

Gene 377 (2006) 56

–64

www.elsevier.com/locate/gene

Abbreviations: bp, base pair(s); CYP, cytochrome P450 monooxygenase;

Cyp (or CYP), CYP-encoding gene; kb, kilobase(s) or 1000 bp; LUC,
Luciferase; luc, LUC-encoding gene; nt, nucleotide(s).

⁎ Corresponding author. Tel.: [phone]; fax: [phone].

E-mail address:

[e-mail]

(R. Ganguly).

0378-1119/$ - see front matter © 2006 Elsevier B.V. All rights reserved.
doi:

10.1016/j.gene.2006.02.032

A variety of commercial food and drinks also contain a high
quantity of caffeine. Because caffeine is consumed heavily and
it is chemically very similar to other purines, extensive studies
have been done on the effect of caffeine on various biological
processes in humans and other mammals such as brain motor
activity, cognitive functions, sleep and hypertension (

Fredholm

et al., 1999; Lorist and Tops, 2003

). However, not much work

has been done on the effect of caffeine on gene expression
except for three studies. One study showed that caffeine
treatment increases CYP1A1 and CYP1A2 mRNA levels in rat
liver and kidney (

Goasduff et al., 1996

). In the other two studies

caffeine was found to increase the levels of c-fos, c-jun, and
junB mRNAs in rat striatum (

Svenningsson et al., 1995

), and

sonic hedgehog mRNA in primary murine neuronal and
astroglial cells in culture (

Sahir et al., 2004

).

In insects, CYPs are known to be involved in conferring

insecticide resistance, and insecticide resistance-associated
overexpression of one or more CYP genes has been observed
in many insect species (see

Scott, 1999

for review). In

Drosophila also, at least five Cyp genes, including Cyp6a2
and Cyp6a8, show overexpression in DDT resistant strains
(

Maitra et al., 1996; Dombrowski et al., 1998; Daborn et al.,

2001; Brandt et al., 2002

). However, the mechanism of

overexpression or the regulation of insect CYP genes in general
is not well-understood. Since xenobiotic compounds have been
successfully used to understand mammalian CYP gene regula-
tion, we surmised whether caffeine could also be used to study
insect CYP gene regulation. Therefore, in the present study we
used Drosophila as a model insect and examined the effect of
caffeine on the Cyp6a2 and Cyp6a8 gene expression in adult fly
and also in Drosophila cells in culture. The results showed that
both genes are induced by caffeine, and Cyp6a8 shows
differential constitutive and caffeine-induced expression in
different tissues of the adult flies.

2. Materials and methods

2.1. Fly strains and cell culture

The 91-C strain and the following five reporter transgenic lines

of Drosophila melanogaster created by

Maitra et al. (2002)

in the

ry

506

host strain were used: 0.2-luc30-4 (also referred as 0.2-luc4),

0.8-luc110, 0.8-luc14, 0.8-luc121 and 3.1-luc2. Each of these
transgenic lines has a single reporter transgene with a firefly
luciferase (luc) reporter gene under the control of 0.2- (

−11/

−199), 0.8- (−11/−761) or 3.1-kb (−11/−3100) upstream DNA of
the Cyp6a8 gene. The chromosomal locations of the transgene in
different lines have been described earlier (

Maitra et al., 2002

). The

0.2-luc4 and 0.8-luc110 strains were made homozygous for their
2nd chromosome-linked transgene by using appropriate balancer
stocks (

Maitra et al., 2000

) and two genetic crossing schemes. One

scheme gave 0.2-luc4H and 0.8-luc110H stocks. Genotypes of
these flies are +/+; 0.2-luc/0.2-luc; +/+ and +/+; 0.8-luc/0.8-luc; +/
+, respectively. The transgene carrying 2nd chromosomes of these
stocks are from the ry

506

strain that was used for transformation.

The other scheme produced 0.2-luc4H-ry and 0.8-luc110H-ry
stocks. Besides the transgene carrying 2nd chromosomes, the X
and 3rd chromosomes in these stocks are also from the ry

506

strain

(indicated with r). Genotypes of these stocks are X

r

/X

r

; 0.2-luc/

0.2-luc; 3

r

/3

r

and X

r

/X

r

; 0.8-luc/0.8-luc; 3

r

/3

r

, respectively. The

source of the 4th chromosome in any of these stocks is not known.
Other reporter transgenic lines, 0.8-luc14, 0.8-luc121 and 3.1-
luc2, carrying 3rd chromosome-linked reporter transgenes are the
original transgenic lines made in the ry

506

host strain. These strains

have been maintained by crossing ry

+

transgenic males and

females in every generation. Flies were reared at 25 °C on standard
cornmeal-agar-molasses Drosophila medium.

Schneider line 2 or SL-2 cells of D. melanogaster (

Schneider,

1972

), obtained from Invitrogen (Carlsbad, CA), were used for

promoter assays. Cells were maintained at 25 °C in Schneider's
Drosophila medium (Gibco-BRL) supplemented with 10% heat-
inactivated bovine calf serum and 0.01% penicillin

–streptomy-

cin (Sigma). Every fourth day the cells were transferred to fresh
media.

2.2. Construction of reporter plasmids for Cyp6a2 and Cyp6a8
promoter assay in SL-2 cells

Recombinant plasmids carrying 0.9-kb (

−983/−1) and 0.8-

kb (

−761/−11) upstream DNA of Cyp6a2-91R and Cyp6a8-91R

alleles of 91-R strain respectively (

Dombrowski et al., 1998;

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

Genes

Primer names

Regions amplified

Primer sequences and regions they are identical to

a

T

m

(°C)

Cyp6a2

b

A2-C

−983/−1

5

′-ctcacgcgtTTCATTCGTTTTATCGCCG-3′ (−983/965)

61

A2R-1M

−522/−1

5

′-ccacgcgtCAAGTGGGATCGTCCTGTAC-3′ (−522/−503)

53

A2R-2M

−265/−1

5

′-ccacgcgtGCAGTGAAGCGTATGAGTATC-3′ (−265/−245)

52

A2R-3M

−129/−1

5

′-ccacgcgtGCTAGCTAGCTCACATGCTGTC-3′ (−129/−112)

56

Cyp6a8

c

A8-5F

−491/−11

5

′-ggcctcgagGTTGTGGTAGGTTAGTAGC-3′ (−491/−473)

72.1

A8-1F

−109/−11

5

′-ggcctcgagCTAGCGACTGAGAATGCATC-3′ (−109/−90)

78

A8-0.06F

−67/−11

5

′-ggcctcgagCAGCGTTTAAAAGCAGTTTGC-3′ (−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 (5

′-ctcgtcgacTTTGCGTAGCTGCTCCC-3′), 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. T

m

= 63 °C.

c

G10 proximal primer (5

′-cgttcgaaATGATCTTCGAATACG-3′), 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. T

m

= 70 °C.

57

S. Bhaskara et al. / Gene 377 (2006) 56

–64

regions of the upstream DNA of Cyp6a2 and Cyp6a8 genes via
PCR.

Table 1

shows the sequences of the distal and proximal

primers used for each Cyp6 gene. Appropriate restriction
enzyme sites that were added to these primers for cloning
purpose are detailed in

Table 1

. PCR amplifications and

purification of the amplified DNAs were done by using Easy-
Start PCR kit (Molecular Bioscience, Boulder) and kits from
Qiagen (CA), respectively. Four amplified DNA fragments
representing the

−983/−1, −522/−1, −265/−1 and −129/−1

regions (ATG at + 1) of the Cyp6a2 gene were digested with MluI
and SalI, and cloned into MluI

–XhoI cut pGL3-basic vector

(Promega, WI). The resulting recombinant promoter-reporter
plasmids were named 0.98luc-A2, 0.52luc-A2, 0.26luc-A2 and
0.12luc-A2, respectively. In case of Cyp6a8 gene, three
amplified upstream DNAs, representing

−491/−11, −109/−11

and

−67/−11 regions (ATG at +1) of Cyp6a8 were digested with

XhoI and HindIII and cloned into XhoI

–HindIII cut pGL2(N)

vector which is the pGL2-basic vector (Promega, WI) with SalI
site modified to NotI (

Maitra et al., 2002

). The recombinant

promoter-reporter plasmids were named 0.5luc-A8, 0.1luc-A8
and 0.06luc-A8, respectively. In addition to these plasmids,
0.8luc-A8 and 0.2luc-A8 plasmids carrying

−766/−11 and

−199/−11 upstream DNA of Cyp6a8 (

Maitra et al., 2002

) were

also used in the present study. All PCR products were sequenced
and found to match with the reported sequence of Cyp6a2 and
Cyp6a8 upstream DNAs (

Dombrowski et al., 1998; Maitra et al.,

2002

).

2.3. Treatment of flies, fly extract preparation, and luciferase assay

Transgenic female flies, 2

–4 days old, were allowed to

feed for 24 h on instant Drosophila food made with aqueous
solution of 2.0 mM Vivarin caffeine (obtained from local
store) or 16 mM pure caffeine (Sigma, St. Louis), a dose
found to be optimum for induction. Food made with water
was used as a control. After treatments, fly extracts were
prepared from groups of ten females for luciferase and
protein assays. Details of fly handling, extract preparation,
firefly luciferase and protein assays have been described
previously (

Maitra et al., 2002

). Luciferase activity and total

protein in the fly extract were determined by using a
luciferase assay reagent kit (Promega, Madison, WI) and
BCA protein assay kit (Pierce), respectively. Luciferase
activity in fly extracts was expressed as average Registered
Light Units per

μg of total protein (RLU/μg). For each

treatment, extracts prepared from three independent groups of
flies were assayed.

2.4. SL-2 cell transfection, extract preparation and dual
luciferase assay

For transient transfection of SL-2 cells, 1.0

μg of Cyp6a2

or Cyp6a8 firefly luciferase (F-luc) reporter plasmid and
0.5

μg of internal control Renilla luciferase (R-luc) plasmid

(Promega, Madison) were dissolved in sterile 0.1 M CaCl

2

(Invitrogen, CA) and mixed with equal volume of 2× HEPES
buffer (Invitrogen, CA) at pH 7.1 to make calcium phosphate-

DNA mix. Cells to be transfected were resuspended in fresh
SL-2 medium at 1 × 10

6

cells/ml density. To each well of a

48-well microtiter plate, 500

μl of cells were dispensed and to

each well, 84

μl of calcium phosphate-DNA mix was added

in a drop-wise manner. After 18 h incubation at 25 °C, the
medium was replaced with 500

μl of fresh medium and

incubated at 25 °C for 24 h. The cells were then spun down
and resuspended in 500

μl of fresh SL-2 medium containing

8 mM caffeine (Sigma, St. Louis). Following 24 h incubation
at 25 °C, cells were pelleted and 250

μl 1× passive lysis

buffer (Promega, WI) was added to each well. The plates
were agitated on a plate shaker for 15 min at room
temperature to lyse the cells and then centrifuged at
1500 rpm for 5 min at 4 °C. The clear supernatant was
collected from each well and assayed immediately for F-luc
and R-luc activities by using a dual luciferase assay kit
(Promega, WI). Briefly, to 20

μl cell lysate, 100 μl luciferase

assay reagent II was mixed, and placed in a luminometer
within 10 s to measure the firefly luciferase (F-luc) activity.
Immediately after recording the F-luc activity, 100

μl Stop

and Glo reagent (Promega, WI) was added, vortexed gently
and the Renilla luciferase (R-luc) activity was measured
within 10 s. R-luc activity was used to normalize the data,
which were expressed as the ratio of F-luc to R-luc activity.
In each experiment, all reporter constructs of both Cyp6 genes
were used to transfect the same batch of cells. For each
reporter construct, triplicate transfection was done. Two such
experiments were done with two different isolates of each
reporter plasmid DNA to obtain the final mean F-luc/R-luc
ratio.

2.5. Northern blot hybridization

The details of RNA blotting, probe synthesis and

hybridization have been described previously (

Maitra et al.,

2000

). Briefly, total RNA was isolated from caffeine-treated

and untreated adult female flies by using Tri-reagent (Sigma,
MO). For analysis, 20

μg of each RNA sample was dried,

dissolved in 20

μl formaldehyde loading dye solution

(Ambion, Austin, TX) and denatured by incubating at
65 °C for 15 min. Samples were electrophoresed on a
2.2 M formaldehyde

−1.2% agarose gel and blotted onto

nylon membrane. The blots were cut into two pieces to probe
CYP RNA in the upper half and the control RP49 mRNA in
the lower half as described (

Maitra et al., 2000

). Upper

halves of three blots made from three independent RNA
samples and lower halves of these blots were prehybridized
in separate bottles for 2 h at 37 °C in Northern Max
Prehybridization/Hybridization buffer (Ambion, TX). After
prehybridization,

32

P-labeled CYP (5 × 10

9

cpm/

μg) and RP49

(1.2 × 10

8

cpm/

μg) cDNA probes were added to the buffer in

respective bottles and hybridization was done at 37 °C for
22

–24 h. The blots were washed under high stringent

conditions and scanned with a Packard Radioanalytical
Imager as described (

Dombrowski et al., 1998

). Data were

normalized using RP49 hybridization signal as the internal
control.

58

S. Bhaskara et al. / Gene 377 (2006) 56

–64

2.6. Statistics

Data were analyzed using ANOVA program.

3. Results

3.1. Vivarin and caffeine induce Cyp6a8 promoter activity

To examine whether caffeine would induce Drosophila

Cyp6a8 gene, adult females of 0.2-luc4H and 0.8-luc110H
transgenic lines were exposed to instant Drosophila medium
made in aqueous solution of over-the-counter caffeine tablet
called Vivarin. The final concentration of caffeine in the me-
dium was 2.0 mM. The results (

Fig. 1

A) showed that in both

transgenic lines, luciferase activity was significantly higher in
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. 1

B), 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
ry

506

host strain used for germ line transformation, and the F1

ry

+

(transgene marker) females, heterozygous for the transgene,

were selected and treated with 16 mM caffeine, which was found
to be an optimum concentration in dose response experiments
(data not shown). While a six-fold induction was observed in
0.2-luc4 line, in three 0.8-luc lines the level of induction ranged

from four- to six-fold (

Fig 2

). In another transgenic line (0.2-

luc7), the 0.2-luc transgene located at different chromosomal
location (

Maitra et al., 2002

) also showed about 5-fold induction

when treated with caffeine (data not shown). Since different
transgenic lines carrying reporter transgene at different chro-
mosomal position showed similar levels of induction, it may be
concluded that caffeine induction is not a result of chromosomal
position effect; it is the function of the Cyp6a8 upstream DNA
driving the luciferase reporter gene.

3.2. Caffeine induces the endogenous Cyp6a8 and Cyp6a2
genes in transgenic and wild type strains

To rule out the possibility that induction of the luc reporter

gene by caffeine is not due to some peculiarity of the reporter

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 ± S.D. of three independent experiments. ANOVA p

b0.0001 for A

and B.

Fig. 2. Effect of pure caffeine on the expression of luciferase reporter transgene
in different transgenic lines. Each transgenic line was crossed to the ry

506

host

strain, and the ry

+

(red-eyed) F1 females carrying the reporter transgene were

allowed to feed on water or 16 mM caffeine containing medium for 24 h.
Extracts prepared from these flies were assayed for luciferase activity. Data
represent mean ± S.D. of three independent determinations. ANOVA p

b0.0001.

59

S. Bhaskara et al. / Gene 377 (2006) 56

–64

transgenes or the host ry

506

strain, RNA isolated from caffeine-

treated and untreated 0.8-luc110 (H-ry) transgenic flies
homozygous for the reporter transgene and a wild type strain
named 91-C were examined for endogenous Cyp6a8 gene
expression by Northern blot analysis. We also examined
Cyp6a2, another Cyp6 gene of Drosophila that appears to be
under the same gene regulatory mechanism that controls the
Cyp6a8 gene (

Maitra et al., 2000

). Results showed that in both

0.8-luc110 (H-ry) transgenic line and 91-C strain endogenous
Cyp6a8 and Cyp6a2 genes were induced by caffeine at the
steady-state mRNA level, although the level of induction of
these two genes varied between the two strains (

Fig. 3

).

3.3. The 0.2- and 0.8-kb upstream DNAs of Cyp6a8 show
differential promoter activity in different tissues

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. 5

A), gut (

Fig. 5

B), abdominal

cuticle plus fat body (

Fig. 6

A) and malpighian tubules (

Fig. 6

B)

of caffeine-treated and untreated adult female flies. In both
transgenic lines, expression of the reporter gene was very low in
the ovaries of the untreated flies (

Fig. 5

A) and caffeine treatment

did not induce the reporter gene in the 0.2-luc4 (H-ry) line
(p = 0.34, Student's t-test). However, in the 0.8-luc110 (H-ry)

line about a 12-fold induction of the reporter gene was observed
in caffeine-treated flies (

Fig. 5

A). In the gut (

Fig. 5

B), con-

stitutive expression of the reporter transgenes in the untreated
transgenic flies was very low; it was more than one order of
magnitude lower than that found in the ovaries. In fact the
specific activities of luciferase enzyme in treated and untreated
flies in the 0.2-luc4 (H-ry) line were too low to be considered as
an expression of the transgene. In the 0.8-luc110 (H-ry) line,
constitutive expression was also very low but, approximately a
10-fold induction was observed in the caffeine-treated flies
(

Fig. 5

B). In the cuticle plus fat body (

Fig. 6

A) and in the

malpighian tubules (

Fig. 6

B) of the untreated flies of both trans-

genic lines, luciferase activities were found to be several orders
magnitude higher than the activities observed in the ovary and
gut (

Fig. 5

). Both transgenes also showed a significant induction in

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 ± S.D. of three independent experiments done with three different RNA samples.
ANOVA p

b0.0001 for A and B.

Fig. 4. Expression of the luciferase reporter transgene in different body parts of
caffeine-treated and untreated female flies of 0.2-luc4H-ry and 0.8-luc110H-ry
transgenic lines. Female flies of each transgenic line were fed with 16 mM
caffeine or water for 24 h and luciferase activity was measured as described. The
results shown are the means ± S.D. of three independent experiments. ANOVA
p

b0.0001.

60

S. Bhaskara et al. / Gene 377 (2006) 56

–64

the cuticle plus fat body of the caffeine-treated flies (

Fig. 6

A).

However, 0.8-luc110 (H-ry) line showed much higher induction
than the 0.2-luc4 (H-ry) line. Two transgenic lines showed the
most striking difference when the reporter gene activities in the
malpighian tubules of the untreated flies were compared (

Fig. 6

B).

In the untreated flies, constitutive expression of the reporter gene
in the 0.8luc-A8 transgenic line was about 350-fold greater than
the expression found in the 0.2luc-A8 line (

Fig. 6

B). Although

caffeine treatment induced the reporter gene in both transgenic
lines, the induced activity was still about 160-fold higher in the
0.8-luc110 (H-ry) than that observed in the 0.2-luc4 (H-ry) line.

3.4. Analysis of the Cyp6a2 and Cyp6a8 upstream DNAs for
constitutive and caffeine-induced expression in SL-2 cells

To map the cis-regulatory DNA for constitutive and caffeine-

induced expression, luc reporter plasmids carrying different
lengths of Cyp6a2 or Cyp6a8 upstream DNA were used to
transfect SL-2 cells. The results showed that the basal activity of
0.12luc-A2 and 0.06luc-A8 plasmids carrying the shortest

upstream DNA of Cyp6a2 and Cyp6a8 genes were about 12.6
and 3.3 times greater than the basal activity of the empty pGL3
vector, respectively (

Fig. 7

). This high basal activity may be due

to the presence of a putative TATA element in these DNA
fragments (

Maitra et al., 2002

). When 0.1luc-A8 reporter plas-

mid was used, the basal activity increased about 36% relative to
the activity of the 0.06luc-A8 plasmid and its final activity was
about 4.5-fold higher than the activity of the empty vector
(

Fig. 7

B). The basal promoter activity increased another 4- and

5-fold relative to the 0.12luc-A2 and 0.1luc-A8 reporter
plasmids respectively, when 0.26luc-A2 and 0.2luc-A8 plasmids
were used. Compared to the empty vector, these plasmids
showed about a 46- and 21-fold higher basal activity, re-
spectively (

Fig. 7

). These data suggest that

−265/−129 Cyp6a2

DNA and

−199/−109 Cyp6a8 have a strong positive effect on

the basal transcription.

In the case of Cyp6a2 gene, increasing the length of the

upstream DNA from

−265 to −522 did not elevate the basal

promoter activity further; instead the promoter activity
decreased by about 42% (

Fig. 7

A). Nevertheless, the activity

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 ± S.D. of three independent experiments are shown.
ANOVA p

b0.0001.

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 ± S.D. of three independent
experiments. ANOVA p

b0.0001.

61

S. Bhaskara et al. / Gene 377 (2006) 56

–64

of the 0.52luc-A2 plasmid was still about 26-fold greater than
the activity of the empty vector. Another 4-fold rise in the basal
promoter activity was observed when the upstream DNA length
was increased from

−522 to −983 (

Fig. 7

A). Thus, the region

between

−522 and −983 of the Cyp6a2 gene also contains

sequences that have a strong positive effect on the basal
transcription. In the case of Cyp6a8, such sequences appear to
be located between regions

−199 and −491 because the basal

activity of the 0.5luc-A8 reporter plasmid carrying

−491/−11

upstream DNA was about 5.5-fold greater than the activity of
the 0.2luc-A8 reporter plasmid with the shorter (

−199/−11)

upstream DNA.

When transfected SL-2 cells were treated with 8 mM

caffeine, all reporter plasmids, except 0.12luc-A2, 0.1-luc-A8
and 0.06luc-A8, showed significant induction (

Fig. 7

). For these

three plasmids, induction level ranged between 1.07- and 1.38-
fold, suggesting that about first 100 bp upstream DNA of both
Cyp6 genes probably lacks caffeine-responsive element.
However, fold-induction increased steadily as the length of
the upstream DNA was increased. The fold-inductions with
0.2luc-A8, 0.5luc-A8 and 0.8luc-A8 reporter plasmids were
1.94, 2.56 and 3.82, respectively (

Fig. 7

B). In the case of

Cyp6a2 upstream DNA, the fold-induction profile was a little

different. While the 0.12luc-A2 plasmid did not show much
caffeine induction, a 4.5-fold caffeine induction was observed
with the 0.26luc-A2 plasmid. Similar to the basal activity,
caffeine-induced activity dropped when the 0.52luc-A2 plasmid
was used, but it increased again when the 0.98luc-A2 reporter
plasmid with longer upstream DNA was used. Cells transfected
with this plasmid showed about a 6-fold induction in
transcriptional activity following caffeine treatment, suggesting
that the upstream DNA sequence located between

−522 and

−983 regions plays a strong positive role in caffeine-induced
expression of the Cyp6a2 gene.

4. Discussion

The present investigation clearly demonstrated that the 0.2-

and 0.8-kb upstream DNA of Cyp6a8 show differential
promoter activity in different body parts of adult female D.
melanogaster. The basal promoter activity of both DNA
fragments was found to be highest in the head than in the
body (thorax + abdomen) or the whole fly. When individual
tissues were examined, both DNA fragments showed three and
four orders of magnitude higher promoter activities in the
cuticle plus fat body and malpighian tubules than in the ovaries

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 (

▾) of Cyp6a2 and Cyp6a8 are located at −85 and −64 positions, respectively.

ANOVA p

b0.0003 for fold-induction and b0.0001 for basal activity.

62

S. Bhaskara et al. / Gene 377 (2006) 56

–64

and gut, respectively. However, in all tissues, body parts or
whole flies, the 0.8-kb upstream DNA always showed signi-
ficantly higher basal promoter activity than the 0.2-kb upstream
DNA. In the malpighian tubules, the activity of the 0.8-kb DNA
was unusually higher (

∼350-fold) than the activity of the 0.2-kb

DNA. When caffeine-inducibility was compared, both DNA
fragments showed similar fold-induction in head, body and
whole fly. However, in the ovaries, gut and cuticle plus fat body,
0.8-kb DNA showed much higher caffeine-inducibility than the
0.2-kb DNA. Thus, the additional DNA (

−761/−199) present in

the 0.8-kb DNA not only gives a higher basal promoter activity,
but it also gives a higher caffeine-inducibility in ovaries, gut and
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

).

In SL-2 cells, the longest upstream DNA of both Cyp6 genes

also showed the highest basal and caffeine-induced promoter
activity. However, basal promoter activity increased in two
steps when the length of the upstream DNA of Cyp6 gene was
increased. In the case of Cyp6a2 gene, sequences giving two-
step increase of promoter activity are located in

−265/−129 and

−983/−522 regions of the upstream DNA. These sequences
also increased caffeine-inducibility. In the case of Cyp6a8 gene,
the

−199/−109 DNA gave a 4-fold and the −491/−199 DNA

gave about a 6-fold rise in basal promoter activity. Caffeine-
inducibility of the Cyp6a8 upstream DNA, however, increased
progressively as its length was increased from position

−109 to

−761. The −109/−11 DNA of Cyp6a8 did not show any
caffeine-inducibility. These observations suggest that sequences
present in the longest upstream of each Cyp6 gene act
synergistically and give the highest basal and caffeine-induced
expression. It is not known whether the same sequences are
involved in basal and caffeine-induced activity.

Stonehouse et al. (2003)

showed that

−117/−75 region of rat

dopamine 2 receptor (D2R) gene containing putative binding
sites for dopamine receptor regulating factor (DRRF) and SP1
showed a 1.9-fold induction in caffeine-treated cells. It has been
hypothesized that stimulation of the D2R gene by caffeine is
probably mediated by alleviating the repressive activity of DRRF
(

Stonehouse et al., 2003

).

Hwang et al. (2001)

have shown that

DRRF, a zinc finger type transcription factor, binds to GT and GC
boxes and displaces the Sp1 and Sp3 transcription factors.
Although Alibaba2.1 program (

http://www.gene-regulation.

com/pub/programs/alibaba2/index.html

) identified putative Sp1

sites at

−944/−934 and −17/−8 positions of Cyp6a2, and at

−736/−737 and −641/−631 positions of Cyp6a8 genes, in SL-2
cells these sites are probably not involved in the expression of
Cyp6a2 and Cyp6a8 genes because Sp1 proteins are not found in
this cell line (

Courey and Tjian, 1988

). Therefore, the constitutive

and caffeine-induced expression found in SL-2 cells must be
mediated by factors other than Sp1. However, involvement of the
putative Sp1 sites in the regulation of Cyp6 gene promoter in the
adult flies cannot be ruled out.

In two studies (

Goasduff et al., 1996; Svenningsson et al.,

1995

), caffeine has been shown to increase the steady-state level

of two CYP mRNAs, and RNAs for FOS and JUN family
proteins. The homo- and heterodimer of these proteins are called

activator protein or AP1, which binds with the AP1 element and
enhances promoter activity. The AP1 proteins are products of
immediate early genes, which are induced by various extracel-
lular stimuli via cAMP pathway (

Sng et al., 2004

). Many cAMP

responsive gene promoters are known to have AP1 and cAMP
response element or CRE, which binds with CRE binding
protein called CREB or CRE-BP (

Sng et al., 2004

and references

therein). Analysis of the Cyp6a2 and Cyp6a8 upstream DNAs
with MATCH

™ program (

Kel et al., 2003

) at 0.8 core and matrix

similarities identified 598 transcriptionally important sequence
motifs including many potential AP-1 and CREB sites (

Table 2

).

Since 0.12luc-A2 and 0.1luc-A8 DNA did not show any

Table 2
Locations of putative sequence motifs in the upstream DNA of Cyp6a2 and
Cyp6a8 that participate in cAMP-mediated transcriptional regulation

Gene

Position
(strand)

Core
match

Matrix
match

Sequence

a

(+ strand)

Factor
name

Cyp6a2

−865 (−)

0.850

0.834

aattaTGTCAtt

CREB

−864 (−)

0.967

0.940

attaTGTCAtt

AP-1

−863 (−)

0.848

0.865

ttATGTCa

CRE-BP1

−741 (−)

0.800

0.804

gCTCCTta

STRE

−648 (−)

0.850

0.834

tcttaTGTCAaa

CREB

−647 (−)

0.967

0.878

cttaTGTCAaa

AP-1

−646 (−)

0.818

0.842

ttaTGTCA

CREB

−549 (+)

0.831

0.891

TTACTtaa

CRE-BP1

−489 (−)

0.955

0.850

tTGAATgac

AP-1

−486 (+)

0.962

0.918

aaTGACCgtgt

AP-1

−482 (−)

0.967

0.927

accgTGTCAtt

AP-1

−371 (+)

0.811

0.802

aaTGAGTgata

AP-1

−370 (−)

0.989

0.880

aTGAGTgat

AP-1

−182 (+)

0.967

0.899

caTGACAacaa

AP-1

−161 (−)

1.000

0.920

gcgtAGTCAtg

AP-1

−120 (−)

0.967

0.896

atgcTGTCAtg

AP-1

−99 (+)

1.000

0.975

gcAGGGGa

STRE

Cyp6a8

−749 (+)

1.000

0.999

taAGGGGg

STRE

−695 (+)

0.818

0.823

TAACGtag

CREB

−524 (+)

0.967

0.945

aaTGACAcact

AP-1

−523 (−)

0.804

0.852

aTGACAcac

AP-1

−510 (−)

1.000

0.919

atagAGTCAtg

AP-1

−441 (−)

0.962

0.889

gtctGGTCAac

AP-1

−390 (+)

0.811

0.813

cgTGAGTaagc

AP-1

−389 (−)

0.989

0.898

gTGAGTaag

AP-1

−328 (−)

0.839

0.839

tgataAGTCAca

CREB

−327 (−)

1.000

0.962

gataAGTCAca

AP-1

−326 (+)

1.000

0.884

ataAGTCAc

AP-1

−318 (−)

1.000

0.927

cagttCGTCAct

CREB

−317 (−)

0.967

0.954

agttCGTCAct

AP-1

−316 (−)

1.000

0.889

gttCGTCActgt

CREB

−292 (−)

0.811

0.807

cattATTCAtc

AP-1

−291 (+)

0.955

0.880

attATTCAt

AP-1

−133 (−)

0.831

0.815

taaAGTAA

CRE-BP1

−95 (−)

1.000

0.849

tgcatCGTCAtg

CREB

−94 (−)

0.967

0.880

gcatCGTCAtg

AP-1

−93 (−)

1.000

0.888

catCGTCAtgca

CREB

−81 (−)

0.839

0.800

aagtaAGTCAat

CREB

−80 (−)

1.000

0.958

agtaAGTCAat

AP-1

−80 (−)

1.000

0.957

agtaAGTCAat

AP-1

−79 (+)

1.000

0.881

gtaAGTCAa

AP-1

−79 (−)

0.818

0.819

gtaAGTCA

CREB

−39 (+)

1.000

0.849

TTACGgaa

CRE-BP1

a

The capital letters indicate the positions in the sequence which match with

the core sequence of the matrix.

63

S. Bhaskara et al. / Gene 377 (2006) 56

–64

caffeine-inducibility (

Fig. 7

), the AP-1 and CREB sites found

within about

−100 bp upstream DNA of both genes may not be

involved in caffeine induction. However, the CREB-BP and AP-
1 sites found at

−133 position of the Cyp6a8, and at −161 and

−182 positions of the Cyp6a2 (

Table 2

) may play a role in

caffeine induction because 0.2luc-A8 and 0.26luc-A2 DNA
carrying these elements gave about 2- and 4.5-fold caffeine
induction, respectively. Similarly, the putative AP-1 and CREB
sites found in the DNA upstream of position

−549 of the Cyp6a2

and

−510 of the Cyp6a8 genes (

Table 2

) may be important for

caffeine induction because the longest reporter constructs
(0.98luc-A2 and 0.8luc-A8) carrying these putative AP1 and
CREB sites gave further increase in fold-induction (

Fig. 7

).

Cyp6a2 and Cyp6a8 DNA upstream of positions

−549 and

−510, respectively, also have a sequence that matches with the
stress response element (STRE) found in many yeast genes
(

Parrou et al., 1999

). Since caffeine is thought to cause cellular

stress, the putative STREs may also play a role in caffeine
induction of the Cyp6a2 and Cyp6a8 genes. Refined mapping
coupled with site-directed mutagenesis will be necessary to
determine the role of putative AP-1, CREB and STRE elements.
If AP-1 and/or CREB are indeed involved in caffeine-induced
transcription, it may be hypothesized that caffeine-induction of
Cyp6 genes is mediated via cAMP signaling pathway. This
hypothesis is currently being investigated.

Acknowledgements

The project was supported by the National Research

Initiative of the USDA Cooperative State Research, Education
and Extension Service, grant number 2002-35302-12281 to RG.

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