Noninvasive imaging of cell death using an Hsp90 ligand

Article abstract of DOI:10.1021/ja110226y

Cell death plays a central role in normal physiology and in disease. Common to apoptotic and necrotic cell death is the eventual loss of plasma membrane integrity. We have produced a small organoarsenical compound, 4-(N-(S-glutathionylacetyl)amino)phenylarsonous acid, that rapidly accumulates in the cytosol of dying cells coincident with loss of plasma membrane integrity. The compound is retained in the cytosol predominantly by covalent reaction with the 90 kDa heat shock protein (Hsp90), the most abundant molecular chaperone of the eukaryotic cytoplasm. The organoarsenical was tagged with either optical or radioisotope reporting groups to image cell death in cultured cells and in murine tumors ex vivo and in situ. Tumor cell death in mice was noninvasively imaged by SPECT/CT using an ¹¹¹In-tagged compound. This versatile compound should enable the imaging of cell death in most experimental settings.

Full text of DOI:10.1021/ja110226y

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Noninvasive Imaging of Cell Death Using an Hsp90 Ligand
Danielle Park,† Anthony S. Don,† Tania Massamiri,‡ Amol Karwa,‡ Beth Warner,‡ Jan MacDonald,‡
Christine Hemenway,‡ Arati Naik,‡ Kah-Tiong Kuan,‡ Pierre J. Dilda,† Jason W. H. Wong,†
Kevin Camphausen,§ Lori Chinen,‡ Mary Dyszlewski,‡ and Philip J. Hogg†,*
†Lowy Cancer Research Centre and POW Clinical School, University of New South Wales, Sydney, NSW 2052, Australia
‡Covidien Imaging Solutions, 675 McDonnell Boulevard, Hazelwood, Missouri 63042, United States
§Imaging and Molecular Therapeutics Section, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, United States
S Supporting Information
b
ABSTRACT: Cell death plays a central role in normal
physiology and in disease. Common to apoptotic and
necrotic cell death is the eventual loss of plasma membrane
integrity. We have produced a small organoarsenical com-
pound, 4-(N-(S-glutathionylacetyl)amino)phenylarsonous
acid, that rapidly accumulates in the cytosol of dying cells
coincident with loss of plasma membrane integrity. The com-
pound is retained in the cytosol predominantly by covalent
reaction with the 90 kDa heat shock protein (Hsp90), the most
Figure 1. Structure of GSAO. The different reporter groups are linked
through the amine of the γ-glutamyl residue.
abundant molecular chaperone of the eukaryotic cytoplasm. The
organoarsenical was tagged with either optical or radioisotope
Jurkat A3 cells13 were used to identify at what point in
reporting groups to image cell death in cultured cells and in
murine tumors ex vivo and in situ. Tumor cell death in mice was
noninvasively imaged by SPECT/CT using an 111In-tagged
compound. This versatile compound should enable the imaging
of cell death in most experimental settings.
the death pathway cells are labeled with GSAO conjugates.
GSAO linked to either Oregon Green (GSAO-OG) or Cy5.5
(GSAO-Cy5.5) was used to demonstrate the independence of
the outcome on the reporting group. The control compound,
4-(N-(S-glutathionylacetyl)amino)benzoic acid (GSCA), con-
tained an inert carboxylic acid group in place of the chemically
reactive trivalent arsenic in GSAO. Three different cell-death
inducers were employed to demonstrate the independence of
the cell-death detection on the mechanism of cell death. A
monoclonal antibody that binds the Fas/CD95 death receptor
was used to trigger the extrinsic apoptotic pathway,14 while the
microbial alkaloid staurosporine, a broad-spectrum protein ki-
nase inhibitor, was used to activate the intrinsic or mitochondrial-
mediated pathway.15,16 The third cell-death mediator was the
chemotherapeutic agent doxorubicin, which triggers G2/M
growth arrest and apoptotic cell death and is used to treat various
solid tumors, including soft-tissue sarcomas, aggressive lympho-
mas, and tumors of the breast, esophagus, and liver.17,18
Jurkat A3 cells were treated with either Fas antibody
staurosporine, or doxorubicin to induce apoptosis and then labeled
with GSAO-OG or GSAO-Cy5.5; annexin V-APC, a marker of
early-stage apoptosis; and propidium iodide (PI) or Sytox Blue,
DNA binding agents that signal loss of plasma membrane
integrity.19 Flow cytometry analysis showed that label-
ing with GSAO-OG or GSAO-Cy5.5 is coincident with that of
PI or Sytox Blue (Figure 2a,b), indicating that the GSAO-
fluorophore conjugates label cells at the point where the integrity
of the plasma membrane is compromised. There was no labeling of
cells with control GSCA conjugates.
ell death plays an integral role in physiology, including
C
turnover of cells in the gastrointestinal tract,1 the menstrual
cycle,2 and the immune system.3 Excessive cell death is char-
acteristic of vascular disorders,4 neurodegenerative diseases,5
myelodysplastic syndromes,6 ischemia/reperfusion injury,7 and
organ transplant rejection,8 among others. Cell death also plays a
role in the treatment of disease. In cancer, for example, most
chemotherapeutics, radiation treatments, and antihormonal
agents act by inducing the death of cancer cells.9,10
In view of the prevalence of cell death in normal physiology
and disease, noninvasive imaging of this process is likely to
have wide application in biological research and patient diagno-
sis and management. Here we describe a peptide trivalent
arsenical, 4-(N-(S-glutathionylacetyl)amino)phenylarsonous acid
(GSAO), that selectively labels dying and dead cells when conjugated
to reporter groups through the γ-glutamyl amine (Figure 1).
Trivalent arsenicals cross-link two cysteine thiols in close
proximity, forming stable cyclic dithioarsinites in which both
sulfur atoms of the cysteine thiols are complexed to arsenic.11
This metalloid has very low reactivity for single protein thiols.
Very few proteins react with trivalent arsenic in the extracellular
milieu, as closely spaced protein cysteine thiols are usually
oxidized to a cystine disulfide bond in this environment. The
intracellular milieu, in contrast, contains a number of proteins
that react with trivalent arsenic.12 Conjugates of GSAO with
different reporter groups very selectively label dying cells.
Received: November 14, 2010
Published: February 15, 2011
r
2011 American Chemical Society
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Figure 3. GSAO-fluorophore labeling of apoptotic cells is rapid and
saturable. (a) Time dependence of the labeling of apoptotic vs viable
HT1080 cells by 1 μM GSAO-F. (b) Concentration dependence of
the labeling of apoptotic HT1080 cells in a 15 min incubation.
(c) Competition for GSAO-F labeling of apoptotic HT1080 cells by
unconjugated GSAO and the GSAA control. Labeling is expressed as the
fraction of the mean fluorescence with no unconjugated GSAO or
GSAA. Each data point and error bar represents the mean and range of
two separate experiments.
unreactive As(V) in place of As(III) did not (Figure 3c). Half-
maximal inhibition of labeling of 1 μM GSAO-F over a 5 min
interval occurred with ∼16 μM unconjugated GSAO. GSAO label-
ing, therefore, is rapid, saturable, and highly specific for dying cells.
To determine the subcellular localization of GSAO-F in apoptotic
cells, camptothecin-treated HT1080 cells were incubated with GSAO-
F and annexin V-Alexa 594 and then imaged by confocal microscopy.
GSAO-F was distributed in the cytoplasm of annexin V-positive cells
(Figure 4a, panels a-f). There was negligible GSAO-F fluorescence
in cells that did not stain with annexin V (Figure 4a, panel g).
To identify the proteins that react with GSAO conjugates in the
cytosol, staurosporine-treated Jurkat A3 cells were incubated with-
out or with the biotinylated compound (GSAO-biotin), and the
labeled proteins were collected on streptavidin beads, resolved on
SDS-PAGE, and stained with Sypro Ruby (Figure 4b). The major
labeled band had an Mr of ∼90 kDa and was identified as Hsp90 by
mass spectrometry. In addition, an LC-MS/MS analysis of the in-
solution tryptic digest of all GSAO-biotin-labeled proteins showed
on the basis of mass-spectral counts20 that Hsp90 is the most
abundant protein. It is roughly twice as abundant as the next most
abundant proteins, eukaryotic translation elongation factor 2 and
filamin A (110 spectral counts vs 58 and 57, respectively). The
reaction of GSAO-biotin with Hsp90 was confirmed in assays
using the purified protein (Figure 4c). The reaction was dependent
on the presence of cysteine thiols in Hsp90, as prior alkylation of the
thiols blocked the interaction with GSAO-biotin.
Hsp90 plays a central role in a number of fundamental cellular
pathways by mediating the folding, stabilization, activation, and assem-
bly of a variety of “client” proteins.21,22 Notably, tumor cell malignancy
is dependent on higher than normal activity of Hsp90, which assists the
folding/stabilization of oncoproteins and other unstable mutant or
overexpressed proteins.23 Hsp90 is the most abundant chaperone of the
eukaryotic cytoplasm and contains a highly conserved Cys-Cys motif
in the C-terminal domain.24 This pair of cysteines (Cys719 and Cys720
in the human protein) are involved in redox reactions in the cytoplasm
and are cross-linked by the trivalent arsenical arsenite.24 The effective-
ness of an imaging agent is determined in part by how much of the
agent accumulates in a given volume. A high concentration of imaging
agent at the target results in better limits of detection and resolution.
The abundance of Hsp90 in the cytosol allows for high levels of GSAO
conjugates in apoptotic cells and therefore superior detection and
resolution of cell death.
Figure 2. GSAO-fluorophore conjugates label cells during the mid-to-
late stage of apoptosis, coincident with the loss of plasma membrane
integrity. (a) Jurkat A3 cells were untreated or treated with Fas antibody
(Ab) for 24 h to induce apoptosis, incubated with the GSCA-OG control
(upper panels) or GSAO-OG (lower panels), annexin V-APC, and PI
(all panels), and analyzed by flow cytometry. GSAO-OG-labeled cells
(lower right panel) were also positive for annexin V-APC or PI (top right
quadrant). The GSCA-OG control (upper panels) did not label any
cells. The numbers on the histograms or scatter plots are the percentage
of cells in the gate or quadrant, respectively. (b) Time courses of
GSCA-OG and GSAO-OG labeling of Jurkat A3 cells treated with Fas
antibody or staurosporine and GSCA-Cy5.5 and GSAO-Cy5.5 label-
ing of Jurkat A3 cells treated with doxorubicin. GSAO-OG or GSAO-
Cy5.5 labeling was coincident with that of PI (Fas antibody and
staurosporine) or Sytox Blue (doxorubicin), respectively, both markers
of loss of plasma membrane integrity. The GSCA-OG and GSCA-
Cy5.5 controls did not label any cells. Each data point and error bar
represents the mean ( SD of three separate experiments.
Human fibrosarcoma HT1080 cells and a conjugate of GSAO with
fluorescein (GSAO-F) were used to characterize the specificity and
kinetic parameters of labeling. The time and concentration depen-
dence of GSAO-F labeling of apoptotic versus viable HT1080 cells
was measured. The mean fluorescence of the viable or apoptotic cell
population was used to quantify the GSAO-F labeling.
The time required for half-maximallabeling of annexin V-positive
cells with 1 μM GSAO-F was ∼1.6 min (Figure 3a), and half-
maximal labeling of GSAO-F over 15 min occurred at ∼3.4 μM
(Figure 3b). There was ∼1000-fold less labeling of viable cells
(Figure 3a). Unconjugated GSAO competed for GSAO-F labeling
of apoptotic cells, but the unconjugated 4-(N-(S-glutathionylace-
tyl)amino)phenylarsonic acid (GSAA) control containing chemically
Solid tumors generally contain large numbers of dying and dead
cells. This is thought to be due to the high rate of cell death in tumors
coupled with restricted rate of access of macrophages to the dying cells.
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Figure 5. GSAO-fluorophore conjugate labels apoptotic tumor cells
in mice. (a) Time course of GSAO-Cy5.5 labeling of Jurkat A3 cells
treated with doxorubicin in the absence or presence of the caspase
inhibitor Z-VAD-FMK. Blocking doxorubicin-mediated apoptosis with
the caspase inhibitor inhibited GSAO-Cy5.5 labeling. Each data point
and error bar represents the mean ( SD of three separate experiments.
(b) Balb/c mice bearing CT26 colorectal carcinoma tumors in the flank
were treated with doxorubicin and the following day were administered
1 mg/kg GSCA-Cy5.5 or GSAO-Cy5.5. After 1 h, the tumors were
excised, sectioned, stained for activated caspase 3, and counterstained
with a nucleic acid stain (DAPI). Fluorescence confocal microscopy
showed that cells that were positive for activated caspase 3 (green) were
also positive for GSAO-Cy5.5 (red). There was no detectable GSCA-
Cy5.5 in any tumor section analyzed.
Figure 4. GSAO-fluorophore conjugates are retained in the cytosol
predominantly by covalent reaction with Hsp90. (a) Fluorescence con-
focal image of a camptothecin-treated HT1080 cell incubated with
GSAO-F (green) and annexin V-Alexa 594 (red). Panels a-f are serial
transverse sections through a cell that labeled with annexin V. Panel g
shows a cell that did not label with annexin V. (b) Staurosporine-treated
Jurkat A3 cells were incubated without or with GSAO-biotin. The
labeled proteins were collected on streptavidin beads, resolved on SDS-
PAGE, and stained with Sypro Ruby. The major labeled band was
identified as Hsp90 by mass spectrometry. The positions of Mr markers
are indicated at the left. (c) GSAO-biotin reacts with cysteine thiols in
Hsp90. Purified Hsp90 was incubated without or with the small thiol
alkylators iodoacetamide (IAM), N-ethylmaleimide (NEM), and methyl
methanethiolsulfonate (MMTS) and then labeled with GSAO-biotin.
Complex formation was measured by blotting with streptavidin-
peroxidase. To control for nonspecific blotting with streptavidin-
peroxidase, GSAO-biotin was omitted in the experiment shown in
the last lane. The positions of Mr markers are indicated at the left.
* indicates an Hsp90 degradation product.
treated Jurkat cells were incubated with GSAO-111In to test the
labeling of apoptotic cells with this conjugate. The labeling of
treated cells with the 111In conjugate was 6.1-fold greater than
that of untreated cells, and the labeling was comparable to that of
untreated cells when caspases were inhibited (Figure 6a). There
was no labeling of the cells with the GSCA-111In control.
To determine whether GSAO was suitable as a noninvasive cell-
death imaging agent, we examined its labeling of organs in mice bearing
solid tumors. Mice bearing subcutaneous murine Lewis lung tumors
were injected with the GSCA-111In control or GSAO-111In in the tail
vein, and after 5 h the blood, kidneys, livers, lungs, spleens, and tumors
were harvested to examine the biodistribution of the compounds.
GSAO-111In was mostly present in the kidneys, where the compound
is excreted. Fluorophore conjugates of GSAO were also observed in the
urine within 30 min of intravenous or subcutaneous administration. A
few percent of the compound was found in the liver, lungs, spleen, and
tumor (Figure 6b; also see the Supporting Information). Very low
levels of the GSCA-111In control were found in the organs. There
were no signs or symptoms of toxicity of the compounds in mice.
Mice bearing subcutaneous murine Lewis lung tumors were injected
with GSAO-111In and annexin V-99mTc in the tail vein, and dual
SPECT/CT images were collected 5 h later. GSAO-111In was observed
in the kidneys and tumor, while annexin V-99mTc was found in the
kidneys, tumor, and liver (Figure 6c). The liver localization of annexin V
is in accordance with the site of metabolism of this protein.26,27 The
GSAO-111In image of a subcutaneous murine CT26 colorectal
carcinoma was comparable to that of the Lewis lung tumor image
(Figure 6d). The GSCA-111In control was not detectable when
used in place of GSAO-111In in these tumor models.
GSAO was conjugated to the near-IR fluorescent dye Cy5.5 to image
doxorubicin-mediated tumor cell death in murine colon tumors.
GSAO-Cy5.5 detected the doxorubicin-mediated death of cultured
cells (Figure 2b), which is caspase-dependent (Figure 5a). The caspase
inhibitor Z-VAD-FMK25 inhibited labeling of the cells by both
GSAO-Cy5.5 and Sytox Blue (Figure 5a).
Mice bearing subcutaneous murine CT26 colorectal carcinoma
tumors were treated with doxorubicin by tail-vein injection to
induce tumor cell death. GSAO-Cy5.5 was injected in the tail vein
the day after treatment, and the tumors were excised 1 h later. Cell
death was detected by staining of tumor sections with an antibody to
activated caspase 3. Cells that were positive for activated caspase 3
were also positive for GSAO-Cy5.5 (Figure 5b). GSAO-Cy5.5
did not label caspase 3-negative cells. The GSCA-Cy5.5 control
was not detected in any of the tumor sections analyzed.
There was a punctuate distribution of the radiolabeled com-
pounds in the tumor, which is consistent with labeling of patches
of dying or dead cells. This pattern of tumor cell death is what is
typically observed histologically.
Annexin V is the best-studied agent for in vitro and in vivo
imaging of apoptosis. It is a 35 kDa protein that detects
GSAO was conjugated to a DTPA-111In chelate (DTPA =
diethylenetriaminepentaacetic acid) for noninvasive imaging of
tumor cell death in murine Lewis lung tumors. Staurosporine-
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’ ACKNOWLEDGMENT
We thank Mark Raftery and Gavin Mackenzie for assistance with mass
spectrometry and microscopy, respectively. This study was supported by
grants from the National Health and Medical Research Council of
Australia, the New South Wales Cancer Council, and a fellowship (K.C.)
from the American Society of Therapeutic Radiation Oncology.
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GSAO conjugates have the advantages of chemical stability, exquisite
selectivity for dying cells, and versatility with respect to the reporting
group. The nature and abundance of its cytosolic target, Hsp90, also
bodes well for this compound’s suitability as a cell-death imaging agent.
’ ASSOCIATED CONTENT
S
Supporting Information. Experimental procedures and
characterization data. This material is available free of charge via
b
the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
p.hogg@unsw.edu.au
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