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The Prostate 30:65–68 (1997)
RAPID COMMUNICATION
Monoclonal Antibody 7E11.C5 Staining of Viable
LNCaP Cells
Robert J. Barren III,1 Eric H. Holmes,2* Alton L. Boynton,3 S. Leslie Misrock,4
and Gerald P. Murphy1
1
Cancer Research Division, Pacific Northwest Cancer Foundation, Northwest Hospital,
Seattle, Washington
2
Division of Cell Surface Biochemistry, Pacific Northwest Cancer Foundation, Northwest
Hospital, Seattle, Washington
3
Department of Molecular Medicine, Northwest Hospital, Seattle, Washington
4
Pennie and Edmonds, New York, New York
Background: Prostate-specific membrane antigen (PSMA) is a transmembrane glycoprotein
defined by the monoclonal antibody 7E11.C5. The 7E11.C5 antibody forms the basis of an in
vivo diagnostic imaging agent (ProstaScint, Cyt-356) for identification of metastatic prostate
cancer. The epitope on PSMA recognized by 7E11.C5 has been determined to be the first 6
amino acids from the N-terminal, expressed on the cytoplasmic side of the plasma membrane.
Thus, the basis for 7E11.C5 specificity in imaging studies remains unclear.
Methods: Fluorescence-activated cell sorter (FACS) analysis of fixed and viable cultured cells
was used to determine the staining intensity with FITC-labeled antibodies.
Results: The results indicate that FITC-labeled 7E11.C5 antibody is taken up and specifically
labels viable LNCaP cells in vitro. Labeling intensity of viable cells after 2 hr of antibody
incubation was similar to that of fixed cells. No labeling of cells that do not express PSMA was
observed, nor was labeling observed with LNCaP cells treated with an isotype-matched
irrelevant antibody.
Conclusions: Uptake and labeling of PSMA by FITC-labeled 7E11.C5 in viable cells in vitro
strongly suggest that this is a major basis for effectiveness of the 7E11.C5 antibody during in
vivo imaging applications with 111In-labeled antibody (ProstaScint, Cyt-356).
© 1997 Wiley-Liss, Inc.
KEY WORDS:
LNCaP, prostate-specific membrane antigen, 7E11.C5, flow cytometry
INTRODUCTION
7E11.C5 is a mouse monoclonal IgG1 antibody
raised against a membrane fraction of LNCaP cells.
Immunohistochemical studies demonstrated 7E11.C5
specificity for prostate epithelial cells [1]. In addition,
a weak expression has been shown in tissue extracts of
salivary glands, smooth muscle, and lower intestine
[2]. The resulting target of the antibody has been
termed prostate-specific membrane antigen (PSMA).
The coding sequence for this protein has been cloned
[3]. PSMA is expressed as a 120-kD transmembrane
glycoprotein whose physiological function in prostatic
cells is unclear. Portions of the extracellular domain
have homology with transferrin receptor [3]. More re© 1997 Wiley-Liss, Inc.
cently, PSMA was shown to have enzymatic activity
as a peptidase (N-acetylated a-linked acidic dipeptidase) capable of cleaving the bioactive peptide Nacetylaspartyl-glutamate [4] and as a novel folate hydrolase enzyme [5].
The 7E11.C5 antibody has been used in a competitive enzyme-linked immunosorbent assay (ELISA)
and in a Western blot assay to demonstrate elevated
Sponsored by the CaPCURE Foundation; the Phi Beta Psi sorority.
*Correspondence to: Eric H. Holmes, Ph.D., Pacific Northwest Cancer Foundation, Division of Cell Surface Biochemistry, 120 Northgate Plaza, Suite 218, Seattle, WA 98125.
Received 1 November 1996; Accepted 7 November 1996
66
Barren et al.
PSMA levels in prostate cancer patients [6–9]. In addition, 7E11.C5 labeled with 111Indium has been used
as a radioimmunoscintigraphy agent in the detection
of distant metastatic tumors in prostate cancer patients
(ProstaScint, Cyt-356) [10]. Recent studies of the epitope specificity for the 7E11.C5 antibody indicated
that the antibody bound to the first 6 amino acids from
the N-terminal, a portion of the protein within the
cytoplasmic domain of the protein [11]. These results
were consistent with earlier observations from electron microscopic studies with gold-labeled 7E11.C5
antibody, which showed antibody binding to the cytoplasmic side of the plasma membrane of LNCaP
cells [12]. Taken together, these results have led to
questions concerning the basis for the use of the
7E11.C5 antibody as an effective in vivo imaging agent
[11]. That is, does the antibody bind to accessible epitopes of nonviable cells or debris, or does it have some
ability to enter cells in vivo and bind to the intracellular epitope of viable cells? The results presented in
this paper indicate that viable LNCaP cells are able to
uptake fluorescently labeled 7E11.C5 antibody, which
binds to the intracellular epitope of PSMA. Thus, it is
reasonable to conclude that the 7E11.C5 antibody most
probably also has the ability to bind to the intracellular
epitope of PSMA expressed in viable prostatic tumor
cells during in vivo imaging applications.
MATERIALS AND METHODS
Cell Lines and Reagents
Prostatic cancer cell lines DU-145, PC-3, and LNCaP
were obtained from the American Type Culture Collection (Rockville, MD). Both DU-145 and PC-3 cells
do not express the PSMA antigen found in LNCaP
cells [1]. All three cell lines were grown in RPMI 1640
medium supplemented with 5% fetal calf serum (FCS)
(Gibco-BRL), and maintained at 37°C in a 5% CO2–
95% air incubator. The monoclonal antibody 7E11.C5
(CYT-356) was obtained from Cytogen Corporation
(Princeton, NJ). In some experiments, LNCaP cells
were exposed to Eagle’s Basal Medium (BME; GibcoBRL) containing only 0.025 mM calcium. It should be
noted that neoplastic cells in general maintain viability
and cellular proliferation in calcium-deficient medium
[13].
Direct Labeling of FITC to 7E11.C5
7E11.C5 was labeled with fluorescein isothiocyanate (FITC), using the FluoroTag Conjugation Kit
(Sigma Chemical Company, St. Louis, MO). In brief,
0.25 mg of FITC was added to 5 mg of 7E11.C5 in 0.1
M sodium carbonate–bicarbonate buffer pH = 9.0. The
conjugation reaction was allowed to take place for 2 hr
at room temperature in the dark with continual mixing. The reaction mixture was then placed over a
Sephadex G-25 column (9.2 ml resin bed volume)
equilibrated in phosphate-buffered saline (PBS) to
separate the FITC-bound antibody from the unbound,
free FITC. Antibody concentration was determined by
absorbance at 280 minus 35% of the absorbance at 495,
to correct for the FITC absorbance. The antibody was
diluted to the stock concentration of 1 mg/ml in PBS.
For FACS staining, the stock antibody solution was
diluted 1:100 in RPMI or BME medium containing 5%
FCS.
Cell Staining For Flow Cytometry Analysis
Cells were grown to approximately 80% confluence
on 150-mm tissue culture dish (Falcon, Franklin Lakes,
NJ). The media was then replaced with either RPMI
5% fetal calf serum or under low calcium conditions in
BME with 1% EDTA-treated FCS. The cells were allowed to incubate with the respective medium for 24
hr. After 24 hr, 35 mg of propidium iodide (PI) (Sigma,
St. Louis, MO) in 7 ml of RPMI containing 5% FCS or
BME containing 1% EDTA-treated FCS was added directly onto the cells. The cells were incubated at 37°C
in the dark for 15 min. The stain was removed from
the plates and the cells washed extensively with PBS
(0.1 M sodium phosphate, 0.15 M NaCl pH = 7.4). The
cells were then incubated with 70 mg of FITC-labeled
7E11.C5 or FITC-labeled IgG1 mouse monoclonal antibody to keyhole limpet hemocyanin (KLH) (Becton
Dickinson, San Jose, CA) as a negative control. The
antibodies were diluted in a solution composed of 7
ml RPMI containing 5% FCS or BME containing 1%
EDTA-treated FCS and the cells treated for 15 min or
2 hr. The cells were then harvested by scraping, pelleted by centrifugation for 5 min at 400g, washed with
PBS, and passed through a 35-mm strainer cap (Falcon,
Franklin Lakes, NJ) to prepare a single-cell suspension. The cells were then subjected to FACS analysis.
Control LNCaP and PC-3 were harvested by scraping, fixed with 2 ml of 3% paraformaldehyde for 10
min on ice, and collected by centrifugation for 5 min at
400g. The cell pellets were resuspended in 0.2 ml of PI
solution and incubated for 15 min on ice. The cells
were then washed with PBS, collected by centrifugation for 5 min at 400g, and 0.2 ml of either the negative
control, isotype-matched FITC-labeled anti-KLH, or
7E11.C5 antibody was added for a 30-min incubation
period on ice. At the end of this period, 2 ml of PBS
was added to the cells and centrifuged for 5 min at
400g. The pellets were resuspended in PBS and passed
through a 35-mm strainer cap.
The FACSCaliber flow cytometer (Becton Dickin-
LNCaP Staining by 7E11.C5
67
son, San Jose, CA) was used to collect the sample information, forward, side scatter, and fluorochrome
measurements. The data were collected in list mode.
The analysis was done using Cell Quest software on a
Macintosh 7600.
RESULTS
Figure 1 shows the results as three-dimensional
graphs comparing cell number with staining intensity
from FITC-labeled 7E11.C5 and propidium iodide (PI)
treatments. In all treatment conditions, elimination of
the initial PI staining step had no effect on the uptake
and intensity of staining with FITC-labeled 7E11.C5
(results not shown). The results indicate that no staining of either DU-145 or PC-3 cells was observed using
FITC-labeled 7E11.C5 (Fig. 1A,B) or with LNCaP cells
using the isotype-matched negative control FITClabeled anti-KLH antibody (Fig. 1C). All three had
greater than 95% of the cells, which were negatively
stained with the respective antibody. Positive staining
with 7E11.C5 (representing about 57% of the population) was observed for both the 15-min (Fig. 1D) and
the 2-hr (Fig. 1E) incubations. In addition, 63% of both
populations were positively stained with PI. The largest percentage of the population of these two time
points were the double positive population at 54% for
15 min and 43% for the 2-hr incubation. The positive
7E11.C5 staining, negative PI staining cells made up
5% of the population at 15 min and 14% of the population at 2 hr. The positive control paraformaldehydefixed LNCaP cells (Fig. 1F) showed strong PI and
7E11.C5 staining with greater than 95% of the cells
staining positive for both PI and 7E11.C5–FITC. This
was expected, since paraformaldehyde compromises
the cellular membrane, allowing efficient staining
with both PI and 7E11.C5. The intensity of the 7E11.C5
staining was similar in the positive control and the
2-hr incubation, with mean channel values of 98 and
101, respectively. The 15-min incubation showed
reduced staining intensity with a mean channel value
of 67.
LNCaP cells were next incubated in calciumdeficient medium (BME, containing 0.025 mM calcium). Previous reports indicate that this treatment
results in plasma membrane permeability to calmodulin antibodies [14] and under this condition neoplastic cells maintain viability and proliferate in calciumdeficient medium when compared to their nonneoplastic counterparts [13]. LNCaP cells were
exposed to calcium-deficient medium for 24 hr and
then stained with PI and FITC-labeled 7E11.C5 for 15
min (Fig. 1G) or 2 hr (Fig. 1H). Under conditions of
low calcium, LNCaP cells positively stain for both
7E11.C5 and internalization of PI (Fig. 1G) equivalent
Fig. 1. FACS analysis of cells. Three dimensional representations of cell number, FITC intensity, and PI intensity are shown on
the indicated axes. Panel A) DU-145 cells labeled with PI for 15
min followed by 7E11.C5-FITC for 2 hours; Panel B) PC-3 cells
labeled with PI for 15 min followed by 7E11.C5 for 2 hours; Panel
C) LNCaP cells labeled with PI for 15 min followed by the isotype
matched negative control Anti-KLH-FITC antibody for 2 hours;
Panel D) LNCaP cells labeled with PI for 15 min followed by
7E11.C5-FITC for 15 min; Panel E) LNCaP cells labeled with PI for
15 min followed by 7E11.C5-FITC for 2 hours; Panel F) Positive
control fixed LNCaP cells labeled with PI for 15 min followed by
7E11.C5 for 30 min; Panel G) LNCaP cells labeled with PI for 15
min followed by 7E11.C5-FITC (in BME) for 15 min; Panel H)
LNCaP cells labeled with PI for 15 min followed by 7E11.C5-FITC
(in BME) for 2 hours. The conditions of the analysis were as
described under ‘‘Materials and Methods.’’
to the fixed control cells (Fig. 1F). Similar results were
observed after the 2-hr incubation period, except that
the binding of 7E11.C5 and PI was increased above
that of the fixed control cells (Fig. 1H).
It is clear that calcium-deficient medium enables PI
increased access to the cellular interior and that the
7E11.C5 antibody also gains access to its cytoplasmic
epitope. The observation that a rapid 15-min exposure
68
Barren et al.
to the 7E11.C5 antibody stains a significant fraction of
the cell population when maintained in either calcium-containing RPMI medium (Fig. 1D) or calciumdeficient BME medium (Fig. 1G), compared to the control irrelevant anti-KLH antibody (Fig. 1C) indicates
membrane permeability and specific binding of the
7E11.C5 antibody to its intracellular epitope.
DISCUSSION
The finding that the 7E11.C5 epitope on PSMA is
composed of the first 6 amino acid residues from the
N-terminal of the protein, a portion of the protein
found on the cytoplasmic side of the plasma membrane, has led to questions concerning the basis for
targeting of the radiolabeled antibody to prostatic tumors during in vivo imaging with ProstaScint (Cyt356) [11]. It has been postulated that the labeled antibody binds to dead cells and cellular debris present in
metastatic tumor sites, as opposed to labeling viable
cells within the tumor. Such a requirement would
limit the value and applicability of an immunoscintigraphic scan. To address this question experimentally,
we have conducted a FACS analysis of viable LNCaP
cells, which are high expressors of the 7E11.C5 antigen, PSMA.
The results presented clearly indicate that viable
prostatic cancer cells uptake and specifically bind
FITC-labeled 7E11.C5. Prostatic cancer cell lines that
do not express PSMA (DU-145 and PC-3 cells) were
not stained by FITC-labeled 7E11.C5. Furthermore,
LNCaP cells were not stained by the isotype-matched
irrelevant FITC-labeled anti-KLH antibody. The staining intensity of viable LNCaP cells with 7E11.C5 increased with time of incubation from 15 min to 2 hr to
the point where the staining intensity resembled that
of fixed cells. Uptake of PI was also observed throughout the cell populations despite the fact that these
were viable cells initially in log phase of growth. These
results suggest that the LNCaP cell membranes are
highly permeable. Further, 7E11.C5 uptake was independent of the initial PI staining step, since elimination of it had no impact on 7E11.C5 uptake and labeling of the intracellular epitope of PSMA.
The results in this paper indicate that antibodies
such as 7E11.C5 are accessible to intracellular epitopes
of prostatic cancer cells in vitro. This finding strongly
suggests that similar processes are occurring during in
vivo imaging with ProstaScint (Cyt-356) aimed at
identification of metastatic prostatic cancer lesions.
CONCLUSION
The FITC-labeled anti-PSMA antibody 7E11.C5 enters and specifically binds to PSMA in viable cells in
vitro. This data strongly suggests that radiolabeled
7E11.C5 conjugates also enters viable tumor cells during applications of in vivo imaging of metastatic prostatic cancer tumors.
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