Antiproliferative B cell translocation gene 2 protein is down-regulated 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

1 Department of Urology,
2 Department of Pathology and
3 Department of Biochemistry, New York University School of Medicine, 540 First Avenue, New York, NY 10016 and
4 Department of Pathology, University of Michigan, Ann Arbor, MI, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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 p53-dependent 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.

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.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The coordinated expression of proliferative genes (proto-oncogenes) and antiproliferative genes (tumor suppressor 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 (13). 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 (711). 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 damage-inducible 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 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 (1618), 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). Predisposition 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
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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 2x SSC at 65°C and autoradiographed. 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 {Psi}+ 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 2x104 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 2x106 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
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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 hyperproliferative 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 1AGo). 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).



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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).

 
Six cases were evaluated with benign glands containing regions of hyperplasia in the basal cell layer. An example, shown in Figure 1BGo, 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.Go)

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 <=3) varied from absent to very weak cytoplasmic staining (Figure 1FGo). BTG2 protein expression was undetectable in higher grade tumors (Gleason grade >=4) (n = 19) and in bone metastases (n = 4). These 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 BGo). The weak nucleolar staining pattern persisted in Gleason grade 3 frozen prostate cancer tissue (Figure 2CGo), 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.



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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.

 
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 3Go. Taken together, these results indicate that the cytoprotective function of BTG2 is not activated in all 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.



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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.

 
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 1FGo) 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, androgen-insensitive), 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 4AGo). In contrast, BTG2 transcripts were not present (or present at low levels) in PC-3 cells (Figure 4AGo). 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.



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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.

 
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 4BGo). Etoposide or adriamycin failed to induce detectable expression of BTG2 mRNA in PC-3 cells (Figure 4BGo) 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 growth-associated proteins c-Myc and c-Fos that respond immediately to growth factor stimulation (4446). 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 5Go). The effect of cycloheximide was more pronounced in PC-3 cells compared with LNCaP cells (Figure 5Go). These data suggest that there is increased transcription of BTG2 mRNA and/or increased stabilization of BTG2 mRNA when protein synthesis is inhibited.



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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.

 
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 wild-type 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 4Go) 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 6AGo). In contrast, cell contact, even at high density, did not result in detectable accumulation of BTG2 protein in LNCaP cells (Figure 6BGo). Further analyses revealed that BTG2 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.



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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).

 
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 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 4Go), 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-Leu-Met-al (55). Lactacystin, but not N-acetyl-Leu-Leu-Met-al, caused increased accumulation of BTG2 protein in both PE and LNCaP cells (Figure 7Go), 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.



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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.

 
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 4Go). 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 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 8AGo). 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 8BGo). 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).



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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 ({circ}) or presence (•) 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.

 
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 9Go). 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.



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Fig. 9. In vivo growth of PC-3 cells transfected with BTG2 cDNA under the control of an inducible promoter. PC-3 cells (2x106) transfected with BTG2 cDNA under the control of an inducible promoter (see Figure 8Go) 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
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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 G1checkpoint 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 expression 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 p53-dependent 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 p53-transactivated 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 damage-induced 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.

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.


    Notes
 
5 To whom correspondence should be addressedEmail: paul.walden{at}nyu.edu Back


    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.).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received January 23, 2001; revised April 14, 2001; accepted April 19, 2001.