| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Molecular Oncology, Markers, Clinical Correlates |
Division of Experimental Therapeutics [A. K., A. O., W. Y., R. D., D. S.] and Department of Radiation Oncology, Urology Department [B. L. L.], Sylvester Cancer Center, University of Miami School of Medicine, Miami, Florida 33136
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
In view of its importance as a possible clinical marker, a precise and reliable assessment of AR expression in prostate cancer cells may be important for diagnostic and prognostic studies in human prostatic disease. Biochemical methods for quantitation of AR expression in cytosolic or nuclear fractions of frozen tissue homogenates cannot discriminate between AR expression of the tumor cells and that of the nonmalignant epithelial and stromal cells. As a result, studies correlating AR expression with response to hormonal therapy have been limited and controversial and cannot assess the heterogeneity of receptor expression in a mixture of tumor and nonmalignant cells. Recently, immunohistochemical techniques have been developed for the study of AR expression in human prostate tumors (15, 16, 17) . However, standard IHC methods in general are not quantitative and cannot simultaneously determine the expression of other cellular markers. In some of the recent publications, automated and color video image analysis has been used for quantitation of AR expression and determination of receptor heterogeneity (18, 19, 20) .
Laser flow cytometry offers a means for rapid analysis of cellular marker expression in a heterogeneous tumor cell population where multiparametric and simultaneous analysis of several markers can be used to correlate marker expression with diagnostic and prognostic indicators. We have recently described flow cytometric methods for the simultaneous determination of ER and PgR expression and DNA content in human breast tumor cells (21 , 22) . The present study was undertaken with the aim of developing a similar method for multiparametric analysis of AR expression in human prostate tissues by flow cytometry.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-reductase
activity and low AR expression (as determined by IHC). The LNCaP cell
line established from a metastatic lymph node has high acid
phosphatase, prostate-specific antigen, and AR expression. Monolayer cultures were harvested from T-75 tissue culture flasks by treatment with 0.05% trypsin/0.53% EDTA solution. Cells retrieved by centrifugation at 130 x g for 5 min were resuspended in PBS containing 2% FBS and 2% HEPES buffer and fixed in 0.5% paraformaldehyde at 4°C for 15 min. After centrifugation, the cell pellets were resuspended in 2 ml of 0.1% Triton X-100 at 4°C for 5 min.
Formalin-fixed and paraffin-embedded human prostatic tissues were analyzed in the present study by both IHC and flow cytometry. H&E-stained sections were used to confirm the presence of tumor or BPH cells in the block and to select areas to be processed for the flow cytometric analysis. Data included in the present report are from: (a) a patient with BPH; (b) a BPH patient with an atrophied prostate gland; (c) a patient with ductal Gleason grade 4 prostate carcinoma; (d) seminal vesicles of patient 3 with invasive cerviform tumor; and (e) a positive lymph node from a patient with Gleason score 9 prostatic carcinoma. A H&E-stained slide from each paraffin block was used to confirm the presence of tumor cells in the sections processed for IHC and flow analysis.
Specimen Preparation.
Sections (25 µm thick) were deparaffinized and rehydrated in a
descending ethanol/water series, followed by two washes in distilled
water. For nuclear isolation, deparaffinized sections were digested in
pepsin [0.05% in normal saline (pH 1.65)] for 60 min at 37°C.
After 60 min of incubation, samples were vortexed every 5 min for an
additional 30 min. The proteolytic reaction was terminated by the
addition of chilled 10% FBS in PBS. The resulting digest was filtered
through a 40-µm nylon mesh (Small Parts, Inc., Miami, FL), washed in
PBS, and centrifuged at 200 x g for 10 min. Antigen
unmasking was achieved by heating the nuclear suspension in citrate
buffer for 15 min at 90°C in a water bath (21)
.
Sections (6 mm thick) were deparaffinized and rehydrated in an ethanol/water series, washed in two changes of distilled water, and incubated in 70% methanol at -20°C for 10 min. Antigen unmasking was performed in a pressure cooker placed in a 600 W microwave oven for 30 min.
Incubation with Antibodies.
Cells and nuclei (from suspension cultures or enzyme digests of
sections from paraffin blocks) were incubated with 150 µl of anti-AR
MAb (F39.4.1; BioGenex, San Ramon, CA) at a 1:25 dilution (in PBS) for
2 h at 37°C. The negative isotype control used for the anti-AR
antibody was normal mouse IgG (Sigma, St. Louis, MO) adjusted to the
same concentration as anti-AR MAb. After incubation with the primary
MAb, cells were washed twice with 2 ml of 0.1% Triton X-100 and
stained with 150 µl of FITC-conjugated antimouse IgG antibody (Sigma)
at a 1:80 dilution (in PBS) for 35 min at 37°C. After incubation, the
cells were washed with 3 ml of 0.1% Triton X-100 and centrifuged at
200 x g for 10 min. The pellets were resuspended in 1
ml of PBS for flow cytometric analysis. For simultaneous monitoring of
AR expression and nuclear DNA content, PI (final concentration, 25
µg/ml + 0.5 mg/ml RNase) was added to the AR FITC-stained samples for
15 min at 37°C.
For determining AR immunoreactivity by the immunoperoxidase staining method, the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA) was used. Background nonspecific binding was blocked by incubation with 1.5% normal horse serum (20 min), and endogenous peroxidase activity was quenched by the addition of 3% hydrogen peroxide (4 min). Cytospin preparations and histological sections were incubated with MAb F39.4.1 (BioGenex) at a 1:50 dilution in PBS for 2 h at 37°C. Mouse IgG (Sigma) was used as a negative control. Sections were incubated with the biotinylated secondary antimouse antibody (Vector Laboratories) for 10 min. After washing with PBS, the slides were treated with horseradish peroxidase-conjugated avidin-biotin complex for 5 min. The immunoperoxidase complexes were visualized by incubation with 3,3'-diaminobenzidine tetrahydrochloride (Sigma) for 5 min.
Flow Cytometric Analysis.
Samples were analyzed on a Coulter Electronics XL/MCL flow cytometer
with the standard argon ion laser excitation and filter configuration
for the FITC/PI dye combination. The percentage of receptor-positive
cells/nuclei was determined by setting electronic gates to exclude 95%
of positive cells (fluorescent) in the isotype controls. The relative
fluorescence intensity was determined by dividing the MFC value of the
antibody-reacted samples by that of the isotype control. Overton’s
modified histogram subtraction method was also used for comparison of
flow cytometric data (22)
. Modfit Software (Verity
Software House, Topsham, ME) was used for cell cycle analysis.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
To determine the effect of cell density on AR expression, cells were
harvested from monolayer cultures at different levels of confluence.
Data in Fig. 1
compare the AR expression
of the PC-3 cells with that of the LNCaP cells. PC-3 cells, which have
a short doubling time (approximately 24 h), show a
prominent population of cells with S-phase DNA content (Fig. 1, A–C)
. In contrast, LNCaP cells are slow growing (with a
2–3-fold longer doubling time than PC-3 cells), which results in the
absence of a major and distinct population of cells with S-phase DNA
content (data not shown). Histograms in Fig. 1
are of human prostate
PC-3 (Fig. 1, A–F)
and LNCaP cells (Fig. 1, G–I)
stained with PI for DNA content/cell cycle analysis
(Fig. 1, A–C)
and with the anti-AR/FITC antibody (Fig. 1, D–I)
. In Fig. 1, D–I
, the shaded
histograms are of isotype controls, whereas the overlays
(unshaded histograms) are of AR-stained cells. Histograms in
the top row (Fig. 1, A, D, and G)
show cells from
highly confluent cultures (80–100% confluence), histograms in the
middle row Fig. 1, B, E, and H)
show cells from
50–70% confluenct cultures, and histograms in the bottom row (Fig. 1, C, F, and I)
show cells from cultures with low
confluence (25–40%).
|
. As shown in the overlay histogram (Fig. 1, D–F)
of AR expression, the decrease in S
phase was accompanied by a corresponding increase in the number
of AR-positive cells (from 10% to 19%), and the antigen density (as
measured by the ratio of the MFC value of the isotype:the MFC value of
the antibody-treated sample) increased from 1.38 (Fig. 1D)
to 1.65 (Fig. 1F)
. In contrast to PC-3 cells, the LNCaP
cells shown in overlay histograms (Fig. 1, G–I)
had a much
higher reactivity (38–94% AR-positive cells with a MCF of
2.48–11.14). In cultures with low confluence (25%; Fig. 1I)
, the percentage of AR-positive cells and the MCF ratio
were approximately double those of the most reactive low-density PC-3
cells (Fig. 1F)
. In contrast, the number of
AR-positive cells was the highest (94%) in the 70% confluent LNCaP
cultures (Fig. 1G)
, with more than a log difference in the
MFC value of the isotype and the AR MAb-stained samples.
As shown in Fig. 2
, the number of
AR-positive PC-3 cells was less than 20% by flow cytometry, and the
MFC ratio was less than 2. In contrast, in the AR-positive LNCaP cells,
the number of gated positive cells and the MFC increased with increased
confluence and reached a maximum of 94% positive cells and a MFC of 11
at 70% or greater confluence.
|
. In samples analyzed
by laser flow cytometry for DNA content, most of the population had
diploid content, with a small number of cells in S phase and
G2-M phase of the cell cycle.
|
shows overlay
histograms of nuclei from this BPH specimen stained with the anti-AR
antibody and the isotype controls (shaded histogram). In
this specimen, 93% of the cells had an AR expression greater than that
of the gated isotype controls, with a MFC ratio of 17 (1.7 log). In
contrast to this specimen with active BPH, the prostate biopsy from a
patient with atrophied tissue had small acini, and in IHC slides,
immunoreactivity was medium (++). In flow cytometric analysis (Fig. 4B)
, 35% of the cells in this specimen had positive AR
expression, and the MFC ratio was 3 (approximately one-third of a log).
|
, a DNA distribution histogram
(Fig. 5A)
, two parameter scattergrams (DNA and FITC
fluorescence, Fig. 5, B and C
), and overlay
histograms (isotype versus anti-AR antibody, Fig. 5D)
of a prostate adenocarcinoma are shown. As seen in
photomicrographs (Fig. 3C)
, loss of acini in this tumor was
accompanied by heterogenous immunostaining with the anti-AR antibody
(Fig. 3D)
. DNA distribution histograms of this tumor and its
metastasis in the seminal vesicle (Fig. 5, A and E)
indicate the presence of a small population with
hypertetraploid DNA content (arrows). This population had a
peak channel value of 500 in contrast to the G1
and G2 peak channel values of 200 and 400,
respectively. Scattergrams (Fig. 5, B, C, F, and G)
and overlay histograms (Fig. 5, D and H)
of the primary tumor (Fig. 5, B–D)
and the
seminal vesicle (Fig. 5, F–H)
show that 13–26% of cells
in these samples had positive AR reactivity, with MFC ratios of 1.5 and
2.2. We used electronic gated analysis to determine the AR reactivity
of the hypertetraploid population in this tumor. As shown in Fig. 6
, the immunoreactivity of this gated
population (Fig. 6, A and E)
was greater than
that of the total (ungated) cell population. The percentage of positive
cells increased from 13% to 18.7% in the prostate and from 26%
to 44.3% in the seminal vesicle. The MFC values also increased in the
gated population from 1.5 to 2.1 (in prostate tumor) and from 2.2 to
4.32 in the seminal vesicle.
|
|
. As shown in a flow histogram (Fig. 7E)
, 90% of cells in this
lymph node had positive AR expression, and the ratio of the MFC of the
isotype:MFC of MAb-treated cells was 11. In DNA histograms of cells
from this lymph node (Fig. 7A)
, a predominant population of
cells with tetraploid DNA content was seen. To determine whether the
tetraploid tumor cells had a different AR expression than the diploid
cells in this tumor, gated analysis of subpopulations in this specimen
was carried out. Fig. 7D
shows forward angle scatter
(fS) and DNA scattergram of nuclei isolated from this lymph
node. In Fig. 7F
, AR expression of the R1 gated
population in Fig. 7D
shows 80% of cells with AR-positive
expression and a MFC ratio of 7.7. In contrast, the tetraploid
population (R2 gated in Fig. 7D)
had 97%
AR-positive cells with a higher MFC ratio of 12.2 (Fig. 7G)
.
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
In general, data from the immunohistochemical studies have shown that BPH and normal prostatic epithelial cells have high AR expression; in advanced PIN and prostate tumors of higher Gleason score and T value, AR expression is reduced, and metastatic prostatic tumor cells in lymph nodes have positive AR expression (27 , 28) .
The role of mutations and other genetic alterations in hormone-refractory AR-positive tumors has been discussed by several investigators. Culig et al. (9) concluded that the adaptation of prostate tumor cells to a low androgen-containing environment involves mutations that generate receptors with a wider activation spectrum and increased expression and activation by other signaling pathways. Several studies have discussed the "field effect" phenomena in which normal cells in the vicinity of the malignant cells may have altered phenotypes. In a recent study, Olapade-Olaopa et al. (29) found that whereas AR expression was decreased in dedifferentiated tumors, the decrease in immunoreactivity of the stromal nuclei was far more pronounced. They suggested the presence of a unique field effect that resulted in decreased AR expression in adjacent benign glands and its total loss in the surrounding stroma.
Most of the earlier studies on AR expression in human prostate tissues used radioligand binding assays. These assays cannot distinguish between receptor expression of the different normal and malignant subpopulations, nor can one determine the antigen density on individual tumor cells. With the availability of AR-specific antibodies, it has been possible to monitor cellular AR expression by IHC, with the added advantage that one can determine the heterogeneity of receptor expression and correlate expression with histology and tissue architecture. To add a quantitative dimension to IHC data, the use of image analysis has been recently introduced to measure antigen expression and correlate it with histology (18, 19, 20) . In general, these studies have shown that AR expression in human xenografts of castrated mice is one-half that of intact mice or animals injected with testosterone. In human tumors, epithelial cells have higher average AR immunostaining than BPH cells. In a recent article, Kim et al. (20) used an automatic video color image analysis system to measure AR antigen density in human prostate tumor xenografts in hormone-manipulated (castration/testosterone stimulated) animals and in clinical specimens obtained by transurethral resection of prostate. Their results indicate that this methodology could be standardized for accurate quantitation of AR immunostaining in human prostate tissues. These authors also showed that an average transurethral resection of prostate specimen of BPH or prostate tumors had significantly higher immunostaining intensity than specimens obtained by prostatectomy, thus suggesting that rapid fixation was essential for preserving the AR content.
Although flow cytometry has become a standard and universal method for phenotypic analysis of marker expression in human leukemia and lymphoma, its use for the analysis of human solid tumors has been hindered by a variety of technical reasons. The difficulty in obtaining a suspension of single cells with intact cell membranes and the antigen-masking effects of the fixatives have contributed to some of these problems. Based on the observation that antibodies currently used for the immunohistochemical determination of ER, PgR, and AR expression specifically stain the nuclear antigens, we have used nuclear isolation methods to prepare specimens for flow cytometric analysis of hormone receptors. In contrast to the isolation of intact single cells, the isolation of nuclei from fresh or paraffin-embedded tissues for flow analysis is relatively easy. Fixation of isolated nuclei from fresh tissues with low concentrations of paraformaldehyde avoids the problem of antigen masking. As shown in the present study, with the use of antigen retrieval methods for processing of nuclei isolated by enzyme digestion from formalin-fixed, paraffin-embedded tissues, one can perform multiparametric analysis of AR expression in human prostate tumors.
In contrast to ligand binding assays and IHC, laser flow cytometry
offers several advantages for the analysis of hormone receptor
expression in human tumors. As shown in our earlier study on ER and PgR
analysis by flow cytometry (21
, 22)
and the present report
on AR expression, laser flow cytometry can provide multiparametric data
in which several markers and their coexpression can be determined
simultaneously. For example, in breast tumors, one can examine estrogen
versus progesterone or estrogen/progesterone
versus DNA content of a heterogenous tumor population. By
using electronic gates for the selection of subpopulations
(e.g., DNA content of diploid or aneuploid cells), one can
determine the expression of a second marker (e.g., ER or AR
expression) in a selected population. We have illustrated this useful
feature of flow analysis in Figs. 6
and 7
, in which the near tetraploid
tumor cells in the primary tumor, seminal vesicle, and the lymph node
have more positive AR-expressing cells and higher antigen density than
the total population. By using electronic gates and comparing the ratio
of the MFC value of the isotype control:MFC value of the
antibody-treated samples, we can get a fair quantitative estimate of
the percentage of cells with positive expression and the mean antigen
density of the subpopulations. An additional advantage of flow
cytometric analysis is that one can sort the selected populations for
further biochemical or molecular analysis. However, it should be noted
that unless one uses a tumor-specific marker (e.g.,
prostate-specific antigen and cytokeratin), flow cytometric analysis
cannot differentiate between the receptor expression of a normal and a
malignant cell in a heterogeneous tumor.
|
FOOTNOTES |
|---|
1 Supported by Research Pilot Grant F99-UM-3 from
the American Cancer Society, Florida Division. A. O. was supported by
an American Cancer Society (Florida Division) John Forman Memorial
Summer Research Fellowship. D. S. is a Summer Research Fellow
supported by the Staley Fund for Cancer-related Research, Wellesley
College Science Center, Waltham, Massachusetts. 
2 To whom requests for reprints should be
addressed, at Division of Experimental Therapeutics (R-71), P. O. Box
016960, Miami, FL 33101.
3 The abbreviations used are: AR, androgen
receptor; IHC, immunohistochemistry; BPH, benign prostatic hyperplasia;
FBS, fetal bovine serum; MAb, monoclonal antibody; PI, propidium
iodide; MFC, mean fluorescence channel; PIN, prostatic intraepithelial
neoplasia; PgR, progesterone receptor; ER, estrogen receptor.
Received 10/21/99; revised 1/24/00; accepted 1/31/00.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  B. Wegiel, A. Bjartell, J. Tuomela, N. Dizeyi, M. Tinzl, L. Helczynski, E. Nilsson, L. E. Otterbein, P. Harkonen, and J. L. Persson Multiple Cellular Mechanisms Related to Cyclin A1 in Prostate Cancer Invasion and Metastasis J Natl Cancer Inst, July 16, 2008; 100(14): 1022 - 1036. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. R. Sterbis, C. Gao, B. Furusato, Y. Chen, S. Shaheduzzaman, L. Ravindranath, D. J. Osborn, I. L. Rosner, A. Dobi, D. G. McLeod, et al. Higher Expression of the Androgen-Regulated Gene PSA/HK3 mRNA in Prostate Cancer Tissues Predicts Biochemical Recurrence-Free Survival Clin. Cancer Res., February 1, 2008; 14(3): 758 - 763. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. V. Litvinov, D. J. Vander Griend, L. Antony, S. Dalrymple, A. M. De Marzo, C. G. Drake, and J. T. Isaacs Androgen receptor as a licensing factor for DNA replication in androgen-sensitive prostate cancer cells PNAS, October 10, 2006; 103(41): 15085 - 15090. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Li, K. Passebosc-Faure, C. Lambert, A. Gentil-Perret, F. Blanc, E. Oosterwijk, J.-F. Mosnier, C. Genin, and J. Tostain The Expression of G250/MN/CA9 Antigen by Flow Cytometry: Its Possible Implication for Detection of Micrometastatic Renal Cancer Cells Clin. Cancer Res., January 1, 2001; 7(1): 89 - 92. [Abstract] [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
