Insect Control: Biological and Synthetic Agents - Index of

y specific proteins in signaling pathways, cell
differentiation, and development (Allgood and
Eastman, 1997). Other applications include largescale
production of proteins in cells, cell-based highthroughput
screening assays, and regulation of traits
in both plants and animals. Unlike all other previous
and current insecticides, commercial nonsteroidal
ecdysone agonists are the first class of insect toxic
chemistry that has led to such a high level of interest
and utility outside the insecticide arena. The following
is a brief overview of the use of bisacylhydrazine
insecticides and EcRs as gene switches in mammalian
and plant systems.
4.2.9.1. Gene switch in mammalian systems
Christopherson et al. (1992) demonstrated that
DmEcR could function as an ecdysteroid-dependent
transcription factor in human 293 cells cotransfected
with an RSV-based expression vector that
encoded DmEcR and a reporter gene, which
contained four copies of Drosophila Hsp27 linked
to a MTV promoter-CAT construct. They demonstrated
that the activity of DmEcR could not be
activated by any of the mammalian steroid hormones
tested. Even amongst the ecdysteroids, the
phytoecdysteroid muristerone A was the best transactivator
of the reporter gene, while 20E was not as
effective. These authors further demonstrated that
chimeric receptors containing the LBD of DmEcR
and the DBD of glucocorticoid receptor could also
function when cotransfected with plasmids containing
glucocorticoid receptor response elements fused
to a reporter gene. Subsequently, No et al. (1996)
demonstrated that an EcR-based gene switch consisting
of DmEcR, Homo sapiens RXR, and appropriate
response elements fused to a reporter gene
in mammalian cells or transgenic mice could be
transactivated with micromolar levels of muristerone
A. Muristerone was shown to maintain its
activity when injected into mice and was not found
to be toxic, teratogenic, or inactivated by serum
binding proteins. Moreover, overexpression of modified
DmEcR and RXR did not appear to to be
toxic, at least in transfected cells. This study, like
an earlier one (Yao et al., 1995), demonstrated that
HsRXR could substitute for its insect homolog and
heterodimer partner for EcR, USP. However, in all
these studies, the range of activating ligands was
limited to ecdysteroids and, in particular, to muristerone
A. In addition, RXR, which is present in
almost all mammalian cells, was found not to be as
good a partner for EcR as USP. Hence, very high
endogenous levels were necessary for stimulation
with muristerone A to occur.
4: Insect Growth- and Development-Disrupting Insecticides 143
With the availability of EcR and USPs from other
insects, especially lepidopteran insects, and understanding
the high affinity of nonsteroidal ecdysone
agonists like tebufenozide and methoxyfenozide for
lepidopteran EcRs/USPs, Suhr et al. (1998) demonstrated
that the B. mori EcR (BmEcR), unlike
DmEcR, could be transactivated to very high levels
in mammalian cells with tebufenozide without adding
an exogenous heterodimer partner like RXR.
The endogenous levels of RXR were enough to
provide the high transactivation levels. The work
by Suhr et al. (1998) further demonstrated that
while the D domain (hinge region) of EcR was necessary
for high-affinity heterodimerization with
USP, both D and E (LBD) domains were necessary
for high-affinity interaction with RXR. By creating
chimeric EcR using parts of E-domains from BmEcR
and DmEcR, these investigators showed that a region
in the middle of the E-domain (amino acids 402
to 508) in BmEcR constituted the region conferring
high specificity for tebufenozide. Subsequent research
by others has mainly focused on using EcRs and
USPs from other insects as well as using different
promoters for expression of the transfected genes
(Kumar et al., 2002; Dhadialla et al., 2002; Palli
and Kapitskaya, 2002; Palli et al., 2002, 2003).
Based on EcR homology modeling studies and sitedirected
mutagenesis of selected amino acid
residues, Kumar et al. (2002) found that a CfEcR
mutant, which had an A110P mutation in the LBD,
was responsive only to nonsteroidal ecdysone agonists,
including methoxyfenozide, but not to any of
the ecdysteroids tested. This demonstrated that the
affinity and functional specificity of an EcR for a
ligand can be altered, thus offering the possibility of
using multiplexed EcR-based gene switches that
could regulate different traits with different ligands.
Palli et al. (2003) developed a two-hybrid EcR based
gene switch, which consisted of constructs containing
DEF domains of CfEcR fused to the GAL4 DNA
binding domain, and CfUSP or Mus musculus retinoid
X receptor (MsRXR) EF domains fused to the
VP16 activation domain. These constructs were
tested in mammalian cells for their ability to drive
a luciferase gene placed under the control of GAL4
response elements and synthetic TATAA promoter.
This combination gave very low level basal activation
of the reporter gene in the absence of the inducer.
In the presence of the inducer, there was a rapid
increase in the expression of the luciferase reporter
gene, reaching levels as high as 8942-fold greater
than basal level by 48 h. Withdrawal of the ligand
resulted in 50% and 80% reduction of the reporter
gene by 12 h and 24 h, respectively.