inhibitors of viruses such as human immunodefi- ciency virus HIV-1 and herpes simplex virus
HSV Gnabre et al., 1995a,b, 1996; Hwu et al., 1998. Studies on the mechanism of action of these
drugs determined that they inhibited the cellular transactivator Sp1 protein from binding to sites on
the viral promoters and interfered with viral gene expression thus inhibiting viral replication Gnabre
et al., 1995b. These derivatives form part of a new class of anti-viral agents in that they are active
against a cellular protein functioning on a viral promoter and inhibit its activity and virus repro-
duction well before they damage the cell itself Daelemans et al., 1999.
Human papillomaviruses HPVs are small DNA tumor viruses of which specific genomic
types have been found to be involved in the induction of uterine cervical and other cancers
worldwide Howley, 1991; zur Hausen, 1991, 1996. The HPV types closely associated to cervical
cancer and ano-genital warts have a highly con- served Sp1 DNA binding site within their major
early promoter region O’Conner et al., 1995. This site has been previously shown to be important in
HPV early gene expression Gloss and Bernard, 1990; Parker et al., 1997 and so it became of
interest to test if the lignan derivatives could interfere with HPV replication. However, since the
systems to study HPV in vitro Meyers et al., 1992; Parker et al., 1997 are technically difficult and not
convenient for screening the utility of potential anti-viral drugs at an early stage of development,
other simpler methods were used in this study. The experiments reported here show the effects of the
lignan derivatives on HPV gene expression as measured by viral promoter luciferase reporter
recombinant vector constructs and DNA transfec- tion into cells grown in tissue culture.
The methodology used to determine drug effects on viral gene expression as measured by a luci-
ferase reporter gene was also tested on non-papil- lomavirus viral promoters isolated from viruses
previously known to have viral replication inhib- ited by the lignan derivatives Gnabre et al.,
1995b. The results indicated that the drugs were inhibitory in a dose dependent manner on the
tested viral promoters that also inhibited viral replication in tissue culture. These experiments
suggested that the studies using the papillomavirus early promoter luciferase reporter constructs
would be a reasonable approach to screen for potential anti-viral and anti-cancer activities of the
lignan derivatives.
The results in this paper show that the isolated plant lignan 3-O-methyl NDGA Mal.4 and two
other derivatives of its parent compound, NDGA M
4
N and tetra-acetyl NDGA are substantial inhibitors of the HPV type 16 early promoter, P
97
. These results further suggest that such drugs may
be useful anti-viral agents against the sexually transmitted papillomaviruses and their induced
tumors.
2. Materials and methods
2
.
1
. Reagents The main reagents used in this study were plant
lignan derivatives of NDGA that have been iso- lated and synthesized as described previously
Gnabre et al., 1995a, 1996; Hwu et al., 1998. The drugs used in this study are: 3-O-methyl NDGA
Mal.4, tetra methyl NDGA M4N, and tetra acetyl NDGA Fig. 1. Pure drug compounds were
typically dissolved in 40 – 100 dimethyl sulfoxide DMSO. Dilutions of the stock solutions were
made in Dulbecco’s modified Eagles DME medium and added to the cell culture growth DME
medium with the final DMSO concentration being
Fig. 1. Molecular structures of a plant lignan, nordihydrogua- iaretic acid and its derivatives. NDGA = nordihydroguaiaretic
acid 1,4-bis-3,4-dihydroxyphenyl-2,3-dimethyl
butane, C
18
H
22
O
4
, mol. wt. 302.37; R
1
, R
2
, R
3
, R
4
are all OH; M
4
N = derivative of NDGA; R
1
, R
2
, R
3
, R
4
are all CH
3
O; Tetra acetyl NDGA = R
1
, R
2
, R
3
, R
4
are all CH
3
COO; Mal.4 = derivative of NDGA R
1
, R
2
, R
3
, are independently OH, R
4
is CH
3
O.
Fig. 2. Reporter expression vectors. PV16P97 contains the critical sequences for early gene expression and the entire
enhancer element for human papillomavirus HPV 16. Simian virus 40 SV40Enh contains the SV40 early promoter, Sp1
DNA binding sites, and enhancer elements. SV40 contains only the early promoter and the Sp1 DNA binding sites.
HIVLTR contains the cDNA of the U3 and R regions of the 3 human immunodeficiency virus HIV long terminal repeat
LTR and is known to be responsive to the tat protein. Adenovirus major late promoter AdMLP contains all the
essential sequences for the adenovirus 2 major late promoter. Cytomegalovirus CMV-b-gal contains the sequences for the
immediate early promoter and enhancer for hCMV. The symbol represents the location of the Sp1 DNA binding sites
within the viral promoter and enhancer elements.
were obtained from the American Type Culture Collection ATCC no. HTB31. The cells were
grown and maintained in DME supplemented with 5 fetal bovine serum, 0.075 sodium bicar-
bonate, and 50 mgml gentamicin sulfate Sigma.
2
.
3
. Recombinant plasmids Several of the recombinant plasmids used in
this study were constructed in the laboratory see Fig. 2. The reporter vector containing the luci-
ferase gene into which the various promoters were inserted was pGL2 Promega. Briefly, the HPV
16 long control region LCR containing the P
97
promoter and the enhancer elements was sub- cloned out of a plasmid containing the entire
genome of HPV16 gifts from Drs zur Hausen and Du¨rst by using restriction enzymes EcoO109
PstI and ligated into pGL2. The HIV-1 3 long terminal repeat LTR was removed from pU3R-
III CAT AIDS Research Reference Reagents Program no. 128 by XhoI and HindIII and was
then ligated into pGL2. The adenovirus type 2 major late promoter was removed from pADb
Clontech by XhoI and SmaI and ligated into pGL2. The SV40 promoter pGL2-promoter and
the SV40 promoterenhancer pGL2-control con- structs were from Promega. They were used with-
out
any modifications.
The pCMV-b-galactosidase
containing vector
was from Clontech.
2
.
4
. DNA transfection and drug addition Purified recombinant plasmid DNA containing
the various viral promoter luciferase reporter con- structs were introduced into tissue culture cells by
the calcium phosphate method Ausubel et al., 1994. The cells to be transfected were seeded at
5 × 10
5
cells in a 35 mm well of a six well culture plate Falcon. Generally 1 – 3 mg of DNA and
CaCl
2
solution were added to HEPES buffered saline and the resulting precipitate was permitted
to sit at room temperature for 5 min. The DNA phosphate precipitate was distributed over the
cells and not removed until 18 h later. As the DNA was removed, various drug derivatives were
2. Nordihydroguaiaretic acid NDGA and DMSO were purchased from Sigma, St. Louis,
MO. Other molecular biological reagents such as restriction endonuclease enzymes and T4 DNA
ligase were from New England Biolabs and Promega. Plasmid DNA was isolated and purified
from bacterial cells by a procedure using alkaline lysis and column chromatography Clontech.
2
.
2
. Cells The major cell line used in these studies is
C33A, derived from a cervical carcinoma, and
added at different concentrations with fresh medium. At 30 h post DNA removal the cells
were lysed, cellular debris was centrifuged, and the supernatant was assayed for luciferase activ-
ity. The basic transfection experiments were com- pleted at least three times with each drug
concentration completed in triplicate. Protein measurements were made by the Bradford method
Biorad and the luciferase activity was divided by protein concentration for a specific activity mea-
surement. Further separate experiments were con- ducted with a co-transfection of a control plasmid
pCMV-b-galactosidase assayed as described by methods from Promega in order to normalize for
any apparent transfection efficiency differences and it was determined that repeated trials without
co-transfection of control DNA gave comparable results to the normalized results.
2
.
5
. Luciferase assay The luciferase activity in the cell lysate was
measured with the addition of substrate luciferin and the resulting light production was measured
with the use of a scintillation counter Nguyen et al., 1988. Each luciferase assay is the average of
at least three transfections and each experiment was repeated at least three times. Protein mea-
surements were accomplished by the Bradford method BioLabs and divided into the luciferase
activity to provide specific luciferase activity. A number of experiments were conducted to exam-
ine for any transfection efficiency differences be- tween cells in separate wells as measured by the
addition of a second plasmid containing the b- galactosidase gene. The cell lysate was measured
for luciferase, protein, and b-galactosidase. How- ever, the main results of the drug effects were
derived from repeated experiments and not nor- malized for transfection efficiency for all the data.
Purified luciferase Roche was used to deter- mine the linear range of counting for the scintilla-
tion counter. Also, purified luciferase was used in experiments to rule out any effect of the drug
derivatives interfering directly on enzyme activity under the conditions that are used to measure
luciferase in the cell lysate.
2
.
6
. Cell toxicity studies C33A cells were exposed to various concentra-
tions of drug derivatives dissolved in DMSO. Treated cell cultures were observed and trypan
blue dye exclusion assays were performed on the cell cultures Strober, 1997. Cell viability was
determined and as some cell death B 15 was noted at concentrations of 100 mgml the experi-
ments were all conducted at levels below this concentration. An additional measurement of cell
viability was determined by the use of the tetra- zolium
salt 3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyltetrazolium bromide MTT Sigma. The assay for the product formazan produced by liv-
ing cells when treated with MTT was performed as in Carmichael et al. 1987. Spectrophotometric
readings were performed using a plate reader, Spectra Max Molecular Devices.
3. Results
