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of Biological and Environmental Research
DOE
Lowdose Radiation Program Workshop IV
Abstract
Title:
Bioluminescent imaging of Clusterin (CLU) transcriptional activity
after low dose ionizing radiation: a possible approach in biodosimetry
Authors: Dmitry Klokov1, Lakshmi Sampath2,
Tracy Criswell1, David L. Wilson2, David A. Boothman1
Institutions: 1Department of Radiation Oncology,
Case Western Reserve University, Ohio, USA; and 2Department
of Biomedical Engineering, Case Western Reserve University,
Ohio, USA, Ireland Comprehensive Cancer Center, 10900 Euclid
Avenue, BRB-326 East Cleveland, OH 44106-4942. dab30@po.cwru.edu
Various
cytotoxic stresses, including ionizing radiation (IR), can
cause substantial changes in gene expression. However, few
genes are altered in expression after low doses (<10 cGy)
of IR (Criswell et al., Oncogene, 2003). Alterations
in genes and proteins induced by IR can be used as molecular
markers of IR exposure, and can ultimately be used to understand
low dose-inducible signal transduction processes. Thus, detecting
and understanding low dose IR-inducible changes could significantly
improve our elucidation of the long-term biological effects
of environmental or man-made exposures. Moreover, certain
changes in gene expression can be early markers of cancer.
The clusterin (CLU) gene has been implicated in a number of
pathological diseases, including aging, Alzheimer’s
disease, and cancer. There are two forms of the CLU protein
produced by cells, one nuclear form of the protein (nCLU)
that binds Ku70 and is pro-cell death (i.e., nuclear CLU
(Yang et al., PNAS, 2000; Leskov et al., 2001; Leskov et al.,
JBC, 2003; Sawada et al NCB, 2003)), and another secretory
form (i.e., sCLU), involved in protecting cells from cytotoxic
stress. sCLU levels are very responsive to low doses of IR.
Biodosimetry, originally developed as the retrospective assessment
of chromosomal aberrations in human lymphocytes, could be
significantly improved on by utilizating combined molecular
biology-bioimaging technology. Transcriptional changes in
levels of certain genes can be monitored using reporter gene
technology that utilizes easily detectable molecules, such
as green or yellow fluorescence proteins (GFP or YFP, rewpectively),
luciferase, ß-galactosidase, and others. Improvements
in imaging technology and molecular biology during the past
few years has allowed us to overcome a number of limitations,
the outcome of which has allowed a dramatic improvement in
sensitivity (Klokov et al., 2003).
Our lab previously showed that the Clusterin (CLU) protein
(and transcript) levels were induced by as low as 2 cGy in
log-phase human MCF-7 breast cancer cells, making it the only
known gene induced at both the protein and transcript levels
at
this low dose of IR. Since CLU induction was observed in a
variety of normal and tumor cells after low doses of IR, our
goal in this study was to develop a cellular biological dosimetry
system utilizing the human CLU promoter-luciferase gene
construct and a liquid nitrogen cooled CCD camera-based imaging
system (Fig. 1). After cloning the 1403 base pair human CLU
promoter, we generated MCF-7 cells that stably contained the
human CLU promoter-luciferase cassette with a
neomycin-selectable resistance gene. MCF-7 clone 1403 was
isolated, wherein the regulation of luciferase protein expression
exactly mimicked the endogenous CLU gene, both in basal and
IR-inducible expression, and was therefore chosen for subsequent
imaging experiments. Using standard luciferase activity detection
by
luminometer assays, statistically significant induction of
CLU gene expression (i.e., promoter activity) was detected
by IR doses of ~50 cGy (lower limit), which represented poor
sensitivity for biodosimetry. In contrast, slight changes
in CLU promoter activity were detectable using the liquid
nitrogen-cooled CCD
camera imaging system (Roper Scientific, Fig. 1) after 10
cGy. At these low doses of IR, optimization of factors, such
as integration time, binning, cell density, cell buffers,
and other growth factors played significantly in the sensitivity
of detection of the CLU promoter, and all of these factors
had to be optimized in detailed studies. Dose-response experiments
showed that CLU promoter-luciferase activity in 1403 MCF-7
cells was induced in a dose-dependent manner (Fig.2), identical
to changes in endogenous transcript and protein levels after
low doses of IR (Criswell et al., 2003). Interestingly, maximum
CLU promoter induction, transcript and secretory protein levels
occurred 48-96 h post-IR exposures, and did not vary with
dose.
One potential
application of the IR-inducibility of the CLU-luciferase promoter
detected by bioimaging will be the generation of an MCF-7
1403 xenograft mouse model in athymic nude mice, wherein responses
to low doses of IR can be accurately monitored. Experiments
were, therefore, performed in which the IR
responses of luciferase were examined in large cell aggregates,
rather than in single cell monolayers of MCF- 7 1403 cells,
in the event that masses of cells squelch bioimaging signals
and limit detection. Under these experimental conditions that
may mimic a xenograft mouse model, we were able to detect
changes in
luciferase expression induced by 10 cGy. We have also generated
a transgenic mouse with stable integration of the 1403 human
CLU promoter-luciferase cassette in every cell of the body,
and these mice are currently being evaluated. Since CLU gene
expression has been implicated in stress responses, aging,
Alzheimer’s
disease and cancer, as well as being under the negative control
of the p53 tumor suppressor protein (Criswell et al.,
2003) and positively regulated by the TGF-ß1 cytokine
(Klokov et al, In Prep.), studies using this transgenic
animal alone. and after being crossed with p53, TGF-ß1,
and CLU knockout mice, should yield interesting results and
help clarify the role of this gene/protein in these pathologic
diseases.
Our results show that: (1) bioimaging using
a CCD camera is appropriate and more
sensitive
than standard luminometer luciferase assays commonly used
for imaging and reporter gene regulation via a promoter under
weak induction conditions (i.e., after low-dose IR);
(2) examination of CLU promoter-driven luciferase
levels in
irradiated MCF-7 1403 cells by CCD camera imaging is a promising
approach for
applications in biodosimetry; and (3) further
studies are focusing on the generation of xenograft and transgenic
mouse model systems using IRinducible CLU promoter-driven
luciferase reporter gene technology.
This
work was supported by a grant from NASA to D.L.W., and by
DOE grant DE-FG-022179 to D.A.B.
References:
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PNAS, USA, 97:5907-5912
