Carcinogenesis vol.22 no.8 pp.1271–1279, 2001
Antiproliferative B cell translocation gene 2 protein is downregulated post-transcriptionally as an early event in prostate
carcinogenesis
Michael A.Ficazzola1, Mitchell Fraiman1, Jordan Gitlin1,
Kenneth Woo1, Jonathan Melamed2, Mark A.Rubin4 and
Paul D.Walden1,3,5
1Department
of Urology, 2Department of Pathology and 3Department of
Biochemistry, New York University School of Medicine, 540 First Avenue,
New York, NY 10016 and 4Department of Pathology, University of
Michigan, Ann Arbor, MI, USA
5To
whom correspondence should be addressed
Email: [email protected]
B cell translocation gene 2 (BTG2) is a p53 target that
negatively regulates cell cycle progression in response to
DNA damage and other stress. The objective of this study
was to examine the expression, regulation and tumor
suppressor properties of BTG2 in prostate cells. By
immunohistochemistry BTG2 protein was detected in ~50%
of basal cells in benign glands from the peripheral zone of
the human prostate. BTG2 was expressed in all hyperproliferative atrophic peripheral zone lesions examined
(simple atrophy, post-atrophic hyperplasia and proliferative
inflammatory atrophy), but was undetectable or detectable
at very low levels in the hyperproliferative epithelial cells
of HGPIN and prostate cancer. BTG2 mRNA was detected
in non-malignant prostate epithelial (PE) cells and in
LNCaP cells, but not in PC-3 cells, consistent with p53dependent regulation. In PE cells BTG2 protein was
detected in areas of cell confluence by immunohistochemistry. BTG2 protein in LNCaP cells was undetectable
by immunohistochemistry but was detected by immunoblotting at 8- to 9-fold lower levels than in PE cells. BTG2
protein levels were shown to be regulated by the ubiquitin–
proteosome system. Forced expression of BTG2 in PC-3
cells was accompanied by a decreased rate of cell proliferation and decreased tumorigenicity of these cells in vivo.
Taken together, these findings suggest that BTG2 functions
as a tumor suppressor in prostate cells that is activated by
cell quiescence, cell growth stimuli as part of a positive
feedback mechanism and in response to DNA damage or
other cell stress. The low steady-state levels of BTG2 protein
in HGPIN and prostate cancer, a potential consequence of
increased proteosomal degradation, may have important
implications in the initiation and progression of malignant
prostate lesions. Furthermore, these findings suggest that
a significant component of the p53 G1 arrest pathway might
be inactivated in prostate cancer even in the absence of
genetic mutations in p53.
Introduction
The coordinated expression of proliferative genes (protooncogenes) and antiproliferative genes (tumor suppressor
Abbreviations: BPH, benign prostatic hyperplasia; BTG, B cell translocation
gene; CDK, cyclin-dependent kinase; DHT, dihydrotestosterone; FBS, fetal
bovine serum; PAH, post-atrophic hyperplasia; PE, prostate-derived epithelial;
PIA, proliferative inflammatory atrophy; pRb, retinoblastoma protein; PSA,
prostate-specific antigen.
© Oxford University Press
genes) regulate cell cycle progression, thereby controlling cell
growth, differentiation and apoptosis. Retinoblastoma protein
(pRb) and p53 are two central regulators of the cell cycle that
function as tumor suppressors. The activity of pRb is modulated
during the cell cycle by phosphorylation, with hypophosphorylated forms predominating in the G0 and G1 phases and
hyperphosphorylated forms predominating in the G2, S and M
phases (1–3). The cyclin-dependent kinases (CDKs) phosphorylate pRb, which in turn dictates the biological activity
of the E2F family of transcription factors, thereby controlling
a checkpoint in G1 (reviewed in ref. 4). Hypophosphorylated
pRb associates with and impairs the activity of E2F. Conversely,
hyperphosphorylated pRb is unable to bind E2F, enabling
transactivation of genes required for progression into S phase.
The G1 checkpoint is also controlled by cell stress and DNA
damage through the action of p53. p53 protein functions to
arrest the cell cycle at G1 in response to DNA damage or
genotoxic stress, allowing DNA repair to occur (for reviews see
refs 5,6). A G1 arrest function of p53 involves transactivation of
p21CIP/WAF, which inhibits the activity of CDKs, thereby
preventing pRb phosphorylation (7–11). If this growth arrest
function fails p53 can activate apoptosis (6). Also, p53 has
been implicated in a further checkpoint control at G2/M (12).
Recently members of the B cell translocation gene (BTG)
family of antiproliferative genes have emerged as important
regulators of the cell cycle by acting as both affectors of pRb
action and effectors of p53 action. The human BTG family of
antiproliferative genes is continually growing in number. The
protein products of these genes negatively regulate cell growth,
promote differentiation and share common structural motifs,
including two highly conserved domains (BTG boxes A and
B) separated by 20–25 non-conserved amino acids (13). Among
the BTG proteins, BTG1 and BTG2 display the greatest
degree of amino acid sequence homology. BTG2 is a p53
transcriptional target gene (14), a feature that distinguishes
BTG2 from BTG1 even though both are DNA damageinducible genes (15). Furthermore, inactivation of BTG2
expression in embryonic stem cells results in apoptosis in
response to DNA damage because of a failure in growth arrest
(14), indicating that BTG2 may promote cell cycle arrest (and
inhibit apoptosis) in a manner similar to p21CIP/WAF. This is
particularly relevant considering that the G1 checkpoint is not
entirely absent in mice lacking p21CIP/WAF (8).
Human BTG2 has homologs in the rat (PC3) and mouse
(TIS21). Levels of BTG2/PC3/TIS21 rise in response to cellular
growth stimuli (16,17), suggesting that these early response
effectors function as cytoprotective ‘growth brakes’ or ‘promotion suppressors’ (18). Many data have accumulated concerning
the antiproliferative mechanism of action of this family of
proteins. The BTG1 and BTG2 proteins have been shown to
bind to and positively modulate the activity of a protein
arginine methyltransferase (PRMT1) (19). The substrates of
PRMT1 include the histones and the heterogeneous ribonucleoproteins, implicating BTG1/2 in the modulation of chromatin
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M.A.Ficazzola et al.
structure and maturation of mRNA precursors, which may
augment the transcriptional regulatory function of BTG1/2.
The N-terminus (amino acids 1–38) of BTG1/2 physically
interacts with Hox9B and enhances its transcriptional activation
(20). Box B of the BTG1 and BTG2 proteins can associate
with CAF1 (CCR-4 associated factor 1) (21,22), while CCR4
(carbon catabolite repressor) protein is a component of the
general transcription multisubunit complex which can affect
transcription either positively or negatively. These findings
implicate BTG1 and BTG2 in the transcriptional regulation of
genes involved in control of cell growth. Since BTG1 and
BTG2 possess no consensus DNA-binding domains, these
proteins are unlikely to have any direct effects on transcription
and most likely act as adaptors that regulate the activity of
transcription factors. Cyclin D1 has been shown to be an
important transcriptional target negatively influenced by PC3/
BTG2 (23). The D-type cyclins are short-lived proteins that
bind and activate CDK4, CDK5 and CDK6 in G1 leading to
Rb phosphorylation and G1 to S progression (24,25). Inhibition
of cyclin D1 expression may therefore represent a major
mechanism by which BTG2 causes G1 arrest. In summary, the
available data indicate that the BTG2/PC3/TIS21 proteins
activate the G1 checkpoint at cell quiescence and in response to
mitogenic stimuli, DNA damage and other stressful situations.
Our interest in the regulation of prostate cell cycle dynamics
by BTG2 stemmed from our finding of abundant BTG2
expression by mRNA differential display in human prostate
transition zone tissue containing foci of histologically atrophic
appearing glands (i.e. crowded glands with irregular nuclei,
visible nucleoli and an increased nuclear–cytoplasmic ratio)
(26). Further, we showed that BTG2 mRNA was regulated in
a growth cycle-dependent manner in primary cultures of
human prostate stromal and epithelial cells, being expressed
at maximal levels in quiescent cultures and at lowest levels
2–4 h following entry into the growth cycle (26). Abundant
expression of BTG2 protein in foci of atrophic prostate glands
(simple atrophy) initially made sense given the histological
atrophic appearance of these glands and the known antiproliferative properties of BTG2 (26). However, since publication of
this study we have become aware that, relative to benign
prostate glands, these focal atrophic lesions are actually associated with increased cell proliferation (as assessed by Ki-67
staining) (27,28). Finding BTG2 expression in such hyperproliferative prostate lesions suggest that, as in other systems (16–
18), BTG2/PC3/TIS21 functions as a tumor suppressor in
prostate cells by acting as a ‘growth brake’ or ‘promotion
suppressor’ in response to cellular growth signals. Given that
our initial studies had focused on lesions of the transition zone
of the prostate, where benign prostatic hyperplasia (BPH)
originates, one objective of the present study was to examine
BTG2 expression in lesions of the peripheral zone of the
prostate where prostate cancer most often originates. Simple
atrophy is seen in the peripheral zone, although less frequently
than in the transition zone. Post-atrophic hyperplasia (PAH) is
seen at much greater frequency in the peripheral zone compared
with the transition zone (29). PAH describes an epithelial
hyperplasia occurring within atrophic acini or ducts (30).
De Marzo et al. (31) recently coined the term proliferative
inflammatory atrophy (PIA) to describe focal hyperproliferative
lesions that are frequently associated with chronic inflammation
and that have the appearance of simple atrophy (28) or PAH
(32). Furthermore, the morphology of PIA is consistent with
McNeal’s description of post-inflammatory atrophy (33). Pre1272
disposition to carcinoma of the liver, large bowel, urinary
bladder and gastric mucosa appears to occur in the presence
of proliferation in the setting of long-standing chronic inflammation. The proposed mechanism of carcinogenesis involves
repeated tissue damage and regeneration in the presence of
highly reactive oxygen and nitrogen species. These findings
have led to the speculation that PIA may give rise to prostate
cancer (31,34).
Prostate cancer is the most commonly diagnosed cancer and
the second leading cause of cancer death in men (35). Effective
treatment strategies for prostate cancer necessarily depend
upon elucidation of the molecular mechanisms underlying
prostate cancer progression. While the multistep nature of
carcinogenesis has been demonstrated for many human cancers,
the individual ‘hits’ involved in prostate carcinogenesis remain
elusive (36). Because of the involvement of BTG2 in the DNA
damage repair pathway, functional inactivation of BTG2 in
prostate cells could therefore also conceivably increase the
potential for accumulating genetic damage leading to prostate
carcinogenesis. A second objective of this study was to examine
the regulation and functional consequences of BTG2 expression
in non-tumorigenic prostate epithelial cells and in prostate
carcinoma cell lines.
Materials and methods
Tissue procurement and processing
The Institutional Board of Research Associates at NYU School of Medicine
approved all procedures involving human tissue. Human prostate tissue was
obtained from male patients undergoing radical retropubic prostatectomy for
prostate cancer at our institution. Peripheral zone tissue was dissected away
from the gland and was either used for generation of primary cell cultures or
cut into cubes ⬍5 mm in dimensions, snap frozen in liquid nitrogen and
stored frozen at –80°C for mRNA isolation and histochemistry.
Cell culture
Primary monolayer cultures of non-tumorigenic prostate-derived epithelial
(PE) cells were derived from mixed explants of human prostate peripheral
zone tissue as described (37). Primary cultures in this study were used after
2–4 passages. LNCaP (FGC) and PC-3 cells were obtained from the American
Type Culture Collection. LNCaP cells were grown in 90% RPMI 1640, 10%
fetal bovine serum (FBS). PC-3 cells were grown in 90% F12, 10% FBS. For
immunohistochemistry cells were cultured in Nunc Lab-Tek chamber slides
(Nalgene, Rochester, NY). For genotoxicity studies exponentially growing
cell monolayers were incubated for 16 h in regular growth medium containing
either adriamycin (0.1 µg/ml) or etoposide (0.5 µg/ml) and expression of
BTG2 mRNA and protein were examined by northern and western analysis,
respectively. For androgen sensitivity studies exponentially growing LNCaP
cell monolayers were incubated in phenol red-free RPMI 1640 culture medium
containing 10% charcoal-stripped FBS and the following concentrations of
either dihydrotestosterone (DHT) or the synthetic androgen R-1881; 0, 10–12,
10–11, 10–10, 10–9, 10–8 or 10–7 M. Cells were harvested at 0, 1, 2, 3, 4, 6, 8,
10, 14, 18, 22, 26, 30, 36 and 40 h following addition of DHT or R-1881 and
BTG2 mRNA and protein expression were examined by northern and western
analysis. Prostate-specific antigen (PSA) levels in the conditioned medium of
androgen-treated LNCaP cells was measured using an AIA-600 automated
enzyme immunoassay system (Tosoh Medics, Foster City, CA). For studies
involving cycloheximide exponentially growing cell monolayers were incubated for 2 or 4 h with medium containing cycloheximide (5 µg/ml). For
studies involving protein degradation inhibitors exponentially growing cell
monolayers were incubated for 16 h with medium containing lactacystin
(100 µM) or N-acetyl-Leu-Leu-Met-al (100 µM).
RNA isolation and northern analysis
RNA from prostate tissue samples and prostate cell cultures was isolated by
the acid guanidine thiocyanate/phenol/chloroform extraction procedure (38).
For northern analysis 10 µg RNA was electrophoresed in 1% (v/v) agarose,
2.2 M formaldehyde gels and either stained with ethidium bromide to assess
RNA quality and to ascertain equivalency of gel loading or transferred to
Duralon nylon membranes (Stratagene, La Jolla, CA) and probed with a
radiolabeled BTG2 3⬘-untranslated region cDNA probe (26). Membranes were
washed to a final stringency of 2⫻ SSC at 65°C and autoradiographed.
BTG2 in prostate cancer
Membranes were re-probed with a human glyceraldehyde 3-phosphate dehydrogenase probe.
Generation of specific antibodies to human BTG2
Specific antibodies to the BTG2 protein product were generated as described
(26). Briefly, the full-length BTG2 cDNA was engineered by PCR to contain
six histidine codons fused in-frame at the C-terminus, followed by a stop
codon. The resultant modified BTG2 cDNA was ligated into the bacterial
expression plasmid pQE-60 (Qiagen, Santa Clarita, CA). This construct was
introduced into the Escherichia coli host M15(pREP4) and the recombinant
protein produced from this expression construct was purified on nickel–NTA
(nitrilo triacetic acid) resin under denaturing conditions (urea). The recombinant
protein was further purified on a preparative (1 cm thick) SDS–15% polyacrylamide gel and the 17 kDa BTG2 protein was used for antibody production in
rabbits. The resulting antibodies were tested for reactivity with the protein
used as immunogen and absence of reactivity with poly-L-histidine (Sigma,
St Louis, MO) by dot blotting. By PCR we also generated a cDNA for BTG1
that was similarly expressed in E.coli and the protein product purified. The
BTG1 and BTG2 proteins were coupled to solid supports for use as affinity
matrices. Immune serum from rabbits immunized with BTG2 protein was
affinity purified by negative adsorption onto the BTG1 column and followed
by adsorption to and elution from the BTG2 column. In this manner we
obtained antibodies that specifically interacted with BTG2 and not with other
members of the BTG family (as assessed by western blotting). These specific
antibodies were used in all of our subsequent experiments.
Immunohistochemistry
Immunohistochemistry was performed on sections of radical retropubic
prostatectomy tissue. The appropriate dilutions of affinity purified BTG2
antibody for frozen sections and formalin-fixed, paraffin-embedded sections
were determined empirically using simple atrophy tissue as a control. Immunohistochemistry was performed on frozen 5 µm tissue sections as previously
described (26,39). Frozen tissue was obtained from 23 patients with an average
age of 66.9 ⫾ 7.5 years.
Immunohistochemistry was performed on formalin-fixed, paraffin-embedded
3 µm prostate tissue sections according to standard procedures following
antigen retrieval by microwave boiling in citrate buffer and using the ABC
kit from Vector Laboratories (Burlingame, CA). The tissue sections were
counterstained with hematoxylin. Formalin-fixed tissues were obtained from
138 patients with an average age of 65.7 ⫾ 9.0 years.
Immunohistochemistry for the proliferation-associated antigen Ki-67 was
performed on adjacent sections to those used for BTG2 immunohistochemistry.
Tissue microarrays of prostate atrophic lesions were also stained for BTG2
and Ki-67, to confirm the staining pattern with a set of tissues from another
institution and to provide a direct comparison of ⬎300 tissue specimens
stained under identical conditions. The slides were examined using a Zeiss
Axiophot microscope equipped with a CCD camera and DEI-470 control box
(Optronics Corp., Goleta, CA) coupled to a PC with a CG-7 frame grabber
board (Scion Corp., Frederick, MD). With the exception of basal cell
hyperplasia, staining of BTG2 and Ki-67 were assessed in lesions from ⬎20
different patients (see Results). The proportion of Ki-67-positive nuclei was
quantified using the Scion Image software (Scion Corp.). In all cases the
lesions were evaluated by M.A.R. or J.M.
Immunoblotting
Protein lysates were prepared from cell monolayers by dissolution directly in
Laemmli sample buffer (40). Protein lysates were resolved on SDS–15%
(w/v) polyacrylamide gels (40) and either stained with Coomassie blue for
quantitative densitometry using a GS-710 calibrated imaging densitometer
(Bio-Rad, Hercules, CA) or transferred to Immobilon P membranes (Millipore,
Bedford, MA). For immunoblotting the ECL reagent system from Amersham
(Arlington Heights, IL) was used in conjunction with 2 µg affinity purified
antiserum to BTG2. Blots were reprobed with antibodies to mitogen-activated
protein kinase (Santa Cruz Biotechnology, Santa Cruz, CA) to assess equivalency of loading.
Generation of cell lines that inducibly express BTG2
The pRetro-Off vector (Clontech, Palo Alto, CA) is a Moloney murine
leukemia virus-derived retroviral vector that expresses the tetracycline-controlled transactivator from the SV40 promoter and contains a tetracycline
response element controlling expression of the gene of interest, which is
cloned into the multiple cloning site. The pRetro-Off vector also contains the
ψ⫹ packaging signal and the puromycin resistance gene, allowing selection
of cell lines stably transfected with the recombinant retroviral vector.
The full-length BTG2 cDNA was ligated with BamHI linkers and cloned
into the BamHI site of the pRetro-Off vector in the sense orientation with
respect to the tetracycline promoter. This construct was transfected (using
Lipofectamine; Life Technologies) into the amphotropic Phoenix A packaging
cell line (obtained from the American Type Culture Collection, under the
authority of Garry P.Nolan, Stanford University). Stably transfected Phoenix
A cells were selected in the presence of puromycin (the concentration of
puromycin used for selection was determined empirically for each experiment)
and the potent tetracycline analog doxycycline (1.0 µg/ml), ensuring tight
repression of the promoter. Forty-one puromycin-resistant clones were isolated
and propagated for further study. The retroviral-containing tissue culture
supernatants were collected from each of the 41 clones, filtered through a
0.45 µm Durapore membrane (Millex-HV; Millipore) and polybrene (hexadimethrine bromide) was added to a final concentration of 4 µg/ml. The
resulting filtrates were titered using exponentially growing NIH 3T3 cells.
Four cell lines with titers of ⬎105 p.f.u./ml were used to infect actively
growing PC-3 cells. Stably transfected PC-3 cells were selected in the presence
of puromycin and doxycycline as above and 42 puromycin-resistant PC-3
clones were isolated using cloning cylinders and propagated and tested for
inducible expression of BTG2 mRNA and protein by northern and western
analysis, respectively.
Analysis of the growth properties of BTG2 transfected cells in vitro and in vivo
For growth studies in vitro PC-3 cells that inducibly expressed BTG2 were
plated in 12-well plates at a density of 2⫻104 cells/well. Parallel groups of
cells were grown at 37°C in regular growth medium containing either no
additions or 1 µg/ml doxycycline. Cell growth was determined by measurement
of cell numbers. Apoptosis was determined in adherent cells using the TUNEL
method using the Apoptag kit (Intergen Co., Purchase, NY) and in adherent
floating cells by measurement of caspase 3 activity using the FluorAce
Apopain assay kit and a Versafluor fluorometer (Bio-Rad). The proportion of
cells in the G1 and S phases of the cell cycle was determined by flow
cytometry analysis using the core facility at the Skirball Institute of NYU
School of Medicine.
For growth studies in vivo 2⫻106 PC-3 cells that inducibly expressed BTG2
were implanted s.c. into two groups of six 8-week-old athymic BALB/c
(nu/nu) mice. (The Institutional Animal Care and Use Committee at NYU
School of Medicine approved all procedures involving laboratory animals.)
The first group of mice received doxycycline (200 µg/ml) in their drinking
water 1 week prior to cell implantation and thereafter for the duration of
the experiment. The second group of animals received no doxycycline in the
drinking water. After 38 days the animals were examined for tumor growth,
killed and the tumors excised, weighed and analyzed for BTG2 mRNA and
protein expression by northern and western analysis.
Results
Our finding that BTG2 was activated in atrophic lesions from
the transition zone of the human prostate (26) that were
subsequently shown to be hyperproliferative (28) led us to
speculate that BTG2 expression is activated as part of a
hierarchical positive feedback mechanism that attempts to keep
cell proliferation in check. The objective of the present study
was to gain some insight into the regulation of BTG2 expression
and the functional consequences of BTG2 expression in
prostate cancer cells. The first series of experiments were
designed to obtain information on the types and proliferative
properties of the lesions within the prostate where BTG2 is
expressed. The second series of experiments were designed to
gain some insight into the mechanisms that regulate BTG2
expression and steady-state levels of BTG2 in prostate cells.
The final series of experiments were aimed at determining the
effects of forced BTG2 expression in prostate cancer cells that
normally do not express this gene.
BTG2 protein expression in hyperproliferative lesions of the
prostate peripheral zone
Our previous study, which examined only the transition zone
of the human prostate, revealed that BTG2 protein was most
abundantly expressed in focal atrophic lesions (simple atrophy)
(26). The transition zone represents the predominant region of
origin of BPH (41). While BPH does represent a benign
neoplasm, proliferative indices in the stroma and epithelium
of BPH are low, consistent with an estimated doubling time
of 20 years (42). We evaluated BTG2 expression in hyperprolif1273
M.A.Ficazzola et al.
Fig. 2. Immunolocalization of BTG2 protein in fresh frozen prostate tissue
containing HGPIN and Gleason grade 3 adenocarcinoma. (A) Area of
HGPIN showing weak nucleolar staining (red/brown) of circumferential
epithelial cell layer. (B) Higher magnification of (A). (C) Area of Gleason
grade 3 prostate cancer. Nucleolar staining for BTG2 persists, but is not
seen in all cells.
Fig. 1. Immunolocalization of BTG2 protein in peripheral zone prostate
tissue. Thin (3 µm) tissue sections of human radical prostatectomy
peripheral zone tissue were immunohistochemically stained with affinity
purified antibodies to BTG2 following antigen retrieval in citrate buffer. A
brown coloration depicts positive staining for BTG2. Representative images
are shown. (A) Benign tissue showing staining of ~50% of basal cells in
glands (arrow). (B) Basal cell hyperplasia (arrow) occurring within a benign
gland. The arrowhead shows the luminal epithelial layer surmounting a
region of basal cell hyperplasia. (C) Simple atrophy showing staining of
epithelial cells (arrow). (D) PAH (arrow) and simple atrophy (arrowhead).
(E) PIA (arrow) with abundant lymphocytic infiltrate. (F) HGPIN (larger
glands in upper portion of image) and Gleason grade 3 cancer (smaller
glands in lower portion of image).
erative lesions of the peripheral zone of the human prostate,
the predominant region of origin of prostate cancer (41).
BTG2 protein expression was consistently seen at moderate
to high expression levels in ~50% of the basal cells in benign
glands (Figure 1A). No significant differences in Ki-67 staining
were observed in the basal cell layer of glands that stained for
BTG2 compared with those that did not stain. In our previous
study we observed no staining of the basal layer of benign
glands in fresh frozen tissue from the transition zone (26).
Occasional patchy staining of the luminal cell layer was seen
in benign glands from both the transition zone (26) and
peripheral zone (present study).
Six cases were evaluated with benign glands containing
regions of hyperplasia in the basal cell layer. An example,
shown in Figure 1B, shows BTG2 staining in the basal cell
layer and in the adjacent region of basal cell hyperplasia.
Luminal cells that do not stain for BTG2 surmount a portion
of the hyperplastic basal cell layer.
Our studies revealed that BTG2 protein is expressed at
moderate to high levels in all hyperproliferative atrophic
lesions of the peripheral zone examined, simple atrophy
(n ⫽ 63), PAH (n ⫽ 31) and PIA (n ⫽ 22) (Figure 1C–E).
HGPIN represents the probable precursor lesion for at least
a subset of prostate carcinomas. The transition of normal
prostatic epithelial cells to HGPIN cells is associated with
increased cellular proliferation and increased apoptosis, thereby
increasing the risk of accruing genetic changes (36). The
progression of HGPIN to prostate cancer involves a decrease
in apoptosis, resulting in net cell proliferation (36). BTG2
expression in archival HGPIN lesions (n ⫽ 38) and low grade
carcinomas (n ⫽ 42) (Gleason grade 艋3) varied from absent
to very weak cytoplasmic staining (Figure 1F). BTG2 protein
expression was undetectable in higher grade tumors (Gleason
grade 艌4) (n ⫽ 19) and in bone metastases (n ⫽ 4). These
1274
Fig. 3. Proliferative indices of selected human prostate peripheral zone
lesions. Data are derived from the percentage of nuclei staining positive for
the proliferation-associated antigen Ki-67 and are represented as the mean
proliferative index ⫾ SEM. Data are based on analysis of 20–50 confirmed
foci from different patients. Data from basal cell hyperplastic lesions were
not included due to the small number of confirmed lesions.
findings are consistent with our studies on prostate cancer cell
lines, derived from metastases, described later in Results. At
high power very weak nucleolar staining for BTG2 could
occasionally be discerned in the enlarged nuclei of HGPIN.
This nucleolar staining was more evident in fresh frozen
sections of HGPIN and prostate cancer tissue. In fresh frozen
sections of HGPIN tissue weak nucleolar staining was seen in
the circumferential cell layer at high magnification, but not in
intraluminal cells (Figure 2A and B). The weak nucleolar
staining pattern persisted in Gleason grade 3 frozen prostate
cancer tissue (Figure 2C), however, the number of cells with
nucleoli that stained positively for BTG2 was reduced. (The
fact that we observed a similar nucleolar staining pattern for
BTG2 in both HGPIN and to a lesser extent in prostate cancer
is further evidence of the pre-malignant nature of HGPIN.)
We cannot rule out the possibility that nucleolar staining was
present in benign lesions (and in cultured PE cells), however,
if present, it was likely obscured by the strong nuclear staining
in these lesions.
The mean proliferative indices (based on staining for the
proliferation-associated antigen Ki-67) of the various human
prostate peripheral zone lesions examined in this study are
shown in Figure 3. Taken together, these results indicate that
the cytoprotective function of BTG2 is not activated in all
BTG2 in prostate cancer
Fig. 4. BTG2 mRNA is expressed in a p53-dependent manner in prostate
cells. (A) BTG2 mRNA expression was examined by northern analysis in
actively growing LNCaP and PC3 prostate adenocarcinoma cells and in
primary cultures of non-tumorigenic PE cells. (B) BTG2 mRNA expression
was examined by northern analysis in actively growing LNCaP and PC3
cells in response to adriamycin (A) (0.1 µg/ml) and etoposide (E)
(0.5 µg/ml). V, vehicle used to dissolve drugs.
hyperproliferative lesions of the prostate peripheral zone and
that a reduction in, or loss of, BTG2 expression is coincident
with malignant progression. These findings are of interest in
following the progression of early prostate cancer precursor
lesions, especially if PIA lesions do in fact turn out to be
precursors to HGPIN or prostate cancer as suggested (31). If
PIA is a prostate cancer precursor then loss of BTG2 expression
would represent the earliest known cell cycle regulator lost in
prostate carcinogenesis. Given the involvement of BTG2 in
the DNA repair pathway this would have major implications
for the accrual of genetic damage and disease progression.
In summary, our findings indicate that benign basal cells,
simple atrophy, PAH and PIA represent the lesions where
BTG2 protein is most abundantly expressed. In contrast, BTG2
protein does not significantly accumulate in the hyperproliferative epithelial cells of HGPIN and prostate cancer. Despite
these findings, BTG2 is transcribed in foci of HGPIN and
prostate cancer, as evidenced by our own unpublished
observations and by the existence of BTG2 cDNA clones in
microdissected HGPIN and invasive tumor libraries forming
part of the NCI Cancer Genome Anatomy Project. Possible
reasons for the discrepancy between BTG2 mRNA and BTG2
protein expression in prostate cancer are addressed below.
Induction of BTG2 mRNA expression by genotoxic stress in
prostate cells that contain wild-type p53 expression
The next series of experiments were designed to provide further
insight into the above immunohistochemical observations by
examining regulation of BTG2 expression in prostate cells.
BTG2 is a p53 transcriptional target gene (14). Inactivating
genetic mutations in p53 are rare in localized prostate tumors,
being more prevalent in metastatic lesions (43). Failure to
observe BTG2 expression in localized prostate cancer lesions
(Figure 1F) was therefore unlikely to represent a failure to
transactivate BTG2 expression by p53. We examined BTG2
mRNA expression in primary cultures of non-tumorigenic
human PE cells (wild-type p53, wild-type pRb, androgeninsensitive), in LNCaP cells (wild-type p53, wild-type pRb,
androgen-sensitive, isolated from a lymph node metastasis)
and in PC-3 cells (inactivating mutations in both p53 alleles,
wild-type pRb, androgen-insensitive, isolated from a bone
metastasis). Expression of BTG2 mRNA was detected by
northern analysis in actively growing cultures of LNCaP cells
and at higher levels in PE cells (Figure 4A). In contrast, BTG2
transcripts were not present (or present at low levels) in PC3 cells (Figure 4A). As expected, BTG2 mRNA was not
present (or present at low levels) in the DU145 cell line
(inactivating mutations in both p53 and both pRb alleles,
androgen-insensitive, isolated from a brain metastasis) (data
not shown). Thus BTG2 mRNA expression correlates with the
existence of wild-type p53 in both non-malignant prostate cells
and in prostate cancer cells.
BTG2 mRNA can be induced in response to genotoxic
stress by etoposide or adriamycin in PE cells (data not shown)
and in LNCaP cells (Figure 4B). Etoposide or adriamycin
failed to induce detectable expression of BTG2 mRNA in
PC-3 cells (Figure 4B) or in DU145 cells (data not shown).
Genotoxic stress therefore results in superinduction of BTG2
mRNA levels in prostate cells that contain wild-type p53 and
that genotoxic stress is unable to affect BTG2 mRNA levels
in cells that have inactivating mutations in both p53 alleles.
p53-independent stimulation of BTG2 mRNA expression by
cycloheximide
Many genes expressed at the G1–S phase boundary of the cell
cycle are often sensitive to the effects of protein synthesis
inhibitors. For example, the protein synthesis inhibitor cycloheximide stimulates accumulation of mRNA for the growthassociated proteins c-Myc and c-Fos that respond immediately
to growth factor stimulation (44–46). Cycloheximide also
induces cellular apoptosis in vivo in rats (47,48) and causes
increased expression of mRNAs for clusterin (47) and Fas
antigen (48). We have shown that treatment of both LNCaP
and PC-3 cells with cycloheximide caused abundant accumulation of BTG2 mRNA (Figure 5). The effect of cycloheximide
was more pronounced in PC-3 cells compared with LNCaP
cells (Figure 5). These data suggest that there is increased
transcription of BTG2 mRNA and/or increased stabilization
of BTG2 mRNA when protein synthesis is inhibited.
BTG2 expression in LNCaP cells is not significantly affected
by androgens or by cell quiescence
Despite observing the well-documented effects of androgens
on PSA levels and the biphasic effects of androgens on LNCaP
cell growth (49,50), androgens had no significant effect on
BTG2 mRNA and protein levels in these cells (data not
shown). Furthermore, BTG2 mRNA levels in LNCaP cells
were not markedly affected in cells made quiescent by either
serum starvation or addition of lovastatin to the culture medium
(data not shown). This is in contrast to the situation with
primary cultures of prostatic stromal and epithelial cells,
which show marked growth cycle regulation of BTG2 mRNA
expression (26), indicating inherent differences in the regulation of BTG2 expression comparing PE cells containing wildtype p53 and LNCaP cells. This was addressed further below.
Cell–cell contact results in detectable BTG2 protein accumulation in PE cells but not in LNCaP cells
The detection of BTG2 mRNA in actively growing cultures
of PE and LNCaP cells (Figure 4) prompted us to evaluate
expression of the protein in these cells. We examined BTG2
protein expression by immunohistochemistry in confluent and
subconfluent cultures of PE and LNCaP cells. BTG2 protein
expression was detected by immunohistochemistry in PE cells
in regions where the cells were in contact with each other
(Figure 6A). In contrast, cell contact, even at high density, did
not result in detectable accumulation of BTG2 protein in
LNCaP cells (Figure 6B). Further analyses revealed that BTG2
1275
M.A.Ficazzola et al.
Fig. 7. Degradation of BTG2 protein in prostate cells is regulated by the
ubiquitin–proteosome system. Confluent monolayers of PE and LNCaP cells
were treated for 16 h with vehicle (1), N-acetyl-Leu-Leu-Met-al (2) or
lactacystin (3). Protein extracts prepared from the cells were analyzed by
western blotting using affinity purified BTG2 antibodies. C, full-length
bacterially expressed BTG2 protein used as a positive control.
Fig. 5. Induction of BTG2 mRNA independently of p53 by cycloheximide.
PE, LNCaP and PC-3 cells were treated with vehicle (0) for 4 h or with
cycloheximide (CX) for 2 and 4 h. Northern analysis was performed using
10 µg total RNA isolated from each cell line. Equivalent amounts of RNA
were loaded as assessed by ethidium bromide staining of 28S RNA (not
shown). The gel content was transferred to a nylon membrane and probed
with a 32P-labeled BTG2 cDNA probe.
Fig. 6. Detectable accumulation of BTG2 protein in PE cells, but not in
LNCaP cells, in areas of cell–cell contact. Primary cultures of (A) PE cells
and (B) LNCaP cells were grown as monolayers in chamber slides. Cells
were stained by immunohistochemistry with affinity purified antibodies to
BTG2 protein (red/brown) and counterstained with hematoxylin (blue).
mRNA was bound to polysomes in both PE and LNCaP cells,
suggesting that BTG2 mRNA was being translated in both
cell types. Comparing PE and LNCaP cells, these findings
suggest that there are differences in BTG2 protein translation,
protein modification or protein stability in prostate cancer cell
lines even on a background of wild-type p53.
BTG2 protein represents a greater proportion of the total
cellular protein in PE cells compared to LNCaP cells and BTG2
degradation is regulated by the ubiquitin–proteosome system
Since immunohistochemistry is relatively insensitive, we examined expression of BTG2 in actively growing and confluent
1276
PE and LNCaP cell cultures by immunoblotting. BTG2 protein
could be detected in cultures of PE and LNCaP cells by
immunoblotting following overnight incubation with primary
antibody. Confluent cultures of PE cells expressed more BTG2
than subconfluent PE cultures, whereas cell density had no
detectable effects on BTG2 expression in LNCaP cells.
Approximately 9-fold more LNCaP total cellular protein (based
on densitometry of Coomassie blue stained gels) was required
to give BTG2 staining equivalent to that seen in subconfluent
PE cells by immuoblotting. Since BTG2 mRNA is present in
both cell types (Figure 4), post-transcriptional mechanisms
likely account for differences in BTG2 protein levels comparing
LNCaP and PE cells.
Many important regulators of the cell cycle are short-lived
proteins. The proteosome is involved in both normal turnover
of cellular proteins and degradation of cell cycle regulators
(51,52). In order to determine whether the proteosome system
was involved in regulation of steady-state BTG2 protein levels,
confluent and subconfluent cultures of PE and LNCaP cells
were treated with the specific 26S proteosome inhibitor lacatcystin (53,54) or with the calpain inhibitor N-acetyl-Leu-LeuMet-al (55). Lactacystin, but not N-acetyl-Leu-Leu-Met-al,
caused increased accumulation of BTG2 protein in both PE
and LNCaP cells (Figure 7), suggesting that the ubiquitin–
proteosome pathway is a major effector of degradation and
steady-state levels of BTG2 protein in both PE and LNCaP
cells. We might expect that the half-life of BTG2 protein in
non-malignant prostate epithelial cells is greater than that in
LNCaP cells. However, because our antibodies do not work
well in immunoprecipitation, we have been unable to unequivocally determine this in pulse–chase labeling experiments.
BTG2 inhibits cell proliferation when introduced into
PC-3 cells
To examine the consequences of BTG2 expression in prostate
cancer cells we forcibly expressed BTG2 in PC-3 cells, which
do not normally express detectable levels of BTG2 mRNA
(see Figure 4). Our attempts to generate stable cell lines
that constitutively express BTG2 failed. The antiproliferative
properties of BTG2 may have resulted in selection of stable
lines with methylation of the exogenous promoter, as in the
case of constructs designed to express BRCA1 in cells (56).
A retroviral expression system (Retro-Off) designed to
express full-length BTG2 under the control of an inducible
tetracycline promoter was subsequently used to circumvent
these difficulties. Retrovirally infected cells were selected
in the presence of puromycin (selectable marker) and the
tetracycline analog doxycycline (to repress the promoter).
Despite the previous observation that the tetracycline promoter
is more leaky in PC-3 than in LNCaP cells (57), we were able
to obtain three of 42 PC-3 cell lines that inducibly expressed
BTG2 (as assessed by northern and western blotting) with
BTG2 in prostate cancer
Fig. 8. In vitro growth of PC3 cells transfected with BTG2 cDNA under the
control of an inducible promoter. (A) Cells were plated at equal densities
and were maintained in the absence (s) or presence (d) of doxycycline
(transcriptional inhibitor) and counted (using a Coulter counter) at the times
indicated. (B) Differences in the proportion of cells in the G1 and S phases
of the cell cycle were determined by flow cytometry in BTG2-expressing
and non-expressing cells after 16 h.
undetectable basal transcription in the presence of doxycycline.
One of these cell lines was used for further study. Induction
of BTG2 expression in this cell line was accompanied by a
reduction in the cell proliferation rate (Figure 8A). Inhibition
of cell growth was greatest 1–2 days following promoter
activation. Thereafter the cells continued to grow at a slower
rate, with reduced expression of BTG2. Cell cycle analysis by
flow cytometry revealed that BTG2 expression resulted in an
increased number of cells in G1 phase and a corresponding
reduction in the number of cells in S phase (Figure 8B). BTG2
expression in PC-3 cells did not significantly affect the rate
of cell apoptosis, although there was a trend towards reduced
rates of apoptosis (1.52 ⫾ 0.38 U caspase 3/mg protein in
non-expressing cells versus 1.10 ⫾ 0.32 U caspase 3/mg
protein in BTG2-expressing cells). Furthermore, PC-3 cells
expressing BTG2 were more irregular in shape, larger and
reached lower saturation densities than non-expressing counterparts (data not shown). Lim et al. made similar observations
in 293 cells overexpressing TIS21 (58).
We achieved high level expression of LacZ in PC-3 cells
using a control vector and this protein had no effect on cell
growth. Induced expression levels of BTG2 using this system
were very low and did not exceed the basal level of expression
of BTG2 seen in PE cells. However, these low levels of BTG2
expression caused a significant reduction in cell proliferation
rate. One specific reason for using the Retro-Off as opposed
to the Retro-On system was that doxycycline had previously
been reported to cause cell growth inhibition (albeit at much
higher concentrations than needed to activate the promoter).
Activation of antiproliferative BTG2 in the absence of doxycycline therefore avoids these potential artefacts. In fact, we
determined that the concentration of doxycycline used to
switch off the promoter (1 µg/ml) had no effect on the rate of
proliferation of PC-3 cells.
PC-3 cells expressing BTG2 under the control of the
inducible tetracycline promoter were also injected s.c. into
athymic nude mice. In a series of experiments involving six
nude mice, tumor growth occurred to a greater extent in those
mice given doxycycline in the drinking water, where BTG
expression would not be expected due to repression of the
promoter. Tumor volume was reduced by ⬎6-fold (P ⫽ 0.002)
in tumors expressing BTG2 (Figure 9). In these mice the
influence of BTG2 expression was more evident early on as
it took longer for the BTG2-expressing tumors to take hold.
Fig. 9. In vivo growth of PC-3 cells transfected with BTG2 cDNA under
the control of an inducible promoter. PC-3 cells (2⫻106) transfected with
BTG2 cDNA under the control of an inducible promoter (see Figure 8)
were implanted s.c. into 12 athymic nude mice. Six mice received drinking
water containing doxycyline (⫹) and six mice received drinking water with
no additions (–). After 38 days the tumors were excised. Data shown are
mean tumor volumes ⫾ SEM.
Discussion
Prostate cancer represents the most frequently diagnosed cancer
and second leading cause of cancer death in men. Unfortunately
however, very little is known about the molecular events
involved in the transformation of normal prostate epithelial
cells into prostate cancer cells. In the first part of our study
we have shown that antiproliferative BTG2 protein is localized
in ~50% of benign prostatic basal cells (the major proliferative
compartment of the human prostate) and in the hyperproliferative lesions simple atrophy, PAH and PIA. These findings
suggest that BTG2 expression might be activated as part of a
positive feedback loop to keep prostate cell growth in check
in the presence of growth stimuli. Although BTG2 protein
was present in ~50% of basal cells of benign glands, in PIA
and associated lesions BTG2 expression was present in most
if not all cells, including those of basal and luminal origin.
Thus, BTG2 expression in the luminal cells correlates with a
shift in the topographical fidelity of proliferation in PIA and
associated lesions.
De Marzo et al. (31) showed distinct histological and
pathophysiological relationships between the cells found in
PIA, HGPIN and prostate cancer, leading to the suggestion
that PIA develops into HGPIN or prostate cancer. In addition,
they showed that the phenotype of many of the cells in PIA
was most consistent with that of an immature secretory type
cell. BTG2 protein expression was undetectable or detectable
at very low levels in the hyperproliferative epithelial cells of
HGPIN and prostate cancer. Loss of BTG2 protein expression
would render cells more sensitive to the effects of oxidative
DNA damage. Because of the involvement of BTG2 in the
DNA damage repair pathway, loss of BTG2 expression could
lead to further genetic damage and disease progression.
BTG2/PC3/TIS21 is intricately involved in the G1
checkpoint of the cell cycle, both as an affector of pRb
phosphorylation (correlated with its ability to repress cyclin
D1 expression) (23) and as a p53-transactivated gene (14). In
the light of the immunohistochemical staining pattern of BTG2
in human prostate tissue we initiated a series of experiments
aimed at gaining some insight into regulation of BTG expres1277
M.A.Ficazzola et al.
sion in prostate cells. Since inactivating genetic mutations in
p53 are rare in localized prostate tumors (43), we anticipated
that failure to observe BTG2 expression in HGPIN and
localized prostate cancer was unlikely to represent a failure to
transactivate BTG2 expression by p53. Indeed, we showed
that BTG2 mRNA expression could be induced in a p53dependent manner by genotoxic stress, indicating that BTG2
is a p53 target gene in prostate cancer cells, as in other systems
(14). We also showed induction of BTG2 mRNA expression
in non-tumorigenic prostate cell cultures and in prostate
carcinoma cell lines by cycloheximide, suggesting that inadequate protein synthesis increases BTG2 expression, either by
increasing transcription from the BTG2 promoter (supporting
the concept that a labile, rapidly turning over, transcriptional
inhibitory factor binds to the BTG2 promoter) and/or by
stabilization of BTG2 mRNA. Since cycloheximide induced
BTG2 mRNA in PC-3 cells, which contain inactivating
mutations in both p53 alleles and normally express undetectable
levels of BTG2 mRNA, the induction of BTG2 mRNA by
cycloheximide occurs independently of p53 (at least in PC-3
cells). Wild-type but not mutant p53 has been shown to inhibit
ribosomal gene transcription by indirectly inhibiting the Pol I
transcriptional machinery (59). Therefore, in addition to directly influencing gene transcription, it is possible that p53 may
also indirectly induce BTG2 expression through inhibition of
cellular protein synthesis.
BTG2 likely represents a significant component of the p53transactivated G1 arrest function for two reasons. Firstly,
inactivation of BTG2 expression in embryonic stem cells
resulted in apoptosis in response to DNA damage because of
a failure in growth arrest (14). Secondly, the G1 checkpoint is
not entirely absent in mice lacking p21CIP/WAF (8). The consistent staining pattern observed with the BTG2 antibody and the
fact that p53 gene mutations are rare in localized prostate
cancer suggest that there could be defects in the DNA damageinduced cytoprotective pathway in prostate cancer that may
be independent of p53 mutations. These defects in the DNA
repair pathway could potentially arise from decreased
stability of cytoprotective proteins in prostate cancer cells. We
showed that the endogenous steady-state levels of BTG2
protein in LNCaP cells are 8- to 9-fold lower than in PE
cells. The proteosome inhibitor lactacystin promotes inhibition
of cell cycle progression and increased differentiation (54).
Lactacystin caused increased accumulation of BTG2 protein
in PE and LNCaP cells, indicating that this protein is degraded
by the ubiquitin–proteosome pathway. It would be interesting
to speculate that in LNCaP cells there is either increased
proteosome activity in general or that BTG2 protein is
destabilized in particular. At present we are unable to address
this issue because our antibodies do not work well in immunoprecipitation, preventing us from determining the half-life of
BTG2 in pulse–chase labeling experiments. The lack of growth
cycle regulation of BTG2 mRNA that we observed in LNCaP
cells may therefore be a consequence of a failure of regulation
of this growth inhibitory mechanism in prostate cancer cells
(possibly due to decreased stability of BTG2 protein in LNCaP
compared with normal epithelial cells).
Finally, we showed that forced expression of BTG2 in the
PC-3 cell line, which does not normally express BTG2, was
accompanied by a reduction in cell proliferation without an
effect on cell apoptosis. Furthermore, these cells were less
tumorigenic in athymic nude mice. These findings demonstrate
the functional growth suppressor properties of BTG2.
1278
In summary, the BTG2 protein product forms part of a
hierarchical cascade that has a tumor suppressive function in
prostate cell by causing growth arrest in response to DNA
damage or as part of a positive feedback loop in response to
growth stimuli. The influence of this tumor suppressor function
is significantly reduced or absent in the hyperproliferative
epithelial cells of HGPIN and prostate cancer. These results
have additional significance in that they indicate that a
significant component of the p53 regulatory pathway can be
inactivated in the absence of inactivating genetic mutations
in p53.
Acknowledgments
The authors acknowledge Isadora Quarles for technical assistance and Marie
Monaco for critically reading the manuscript. This work was supported by
NIH grant R01 CA 84441 (P.D.W.), NCI Specialized Program of Research
Excellence in Prostate Cancer Grant #CAP50-69568 (to M.A.R.) and by a
Yamanouchi Research Award (K.W.).
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Received January 23, 2001; revised April 14, 2001; accepted April 19, 2001
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