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DIFFERENTIAL RESPONSIVENESS OF PROSTATIC ACID PHOSPHATASE AND PROSTATE-SPECIFIC ANTIGEN mRNA TO ANDROGEN IN PROSTATE CANCER CELLS
Cell Biology International 2000, Vol. 24, No. 10, 681–689 doi:10.1006/cbir.2000.0433, available online at http://www.idealibrary.com on
DIFFERENTIAL RESPONSIVENESS OF PROSTATIC ACID PHOSPHATASE AND PROSTATE-SPECIFIC ANTIGEN mRNA TO ANDROGEN IN PROSTATE CANCER CELLS MING-FONG LIN1,2,3,4*, MING-SHYUE LEE1, RENEE GARCIA-ARENAS1 and FEN-FEN LIN1 1
Department of Biochemistry and Molecular Biology and 2Section of Urologic Surgery, College of Medicine, 3 Eppley Cancer Institute, University of Nebraska Medical Center, and 4Omaha VA Hospital, Omaha, NE 68198, U.S.A. Received 2 September 1999; accepted 3 March 2000
Androgens regulate the expression of both human prostatic acid phosphatase (PAcP) and prostate-specific antigen (PSA), two major prostate epithelium-specific differentiation antigens. Due to the important role of these two enzymes as prostate epithelium differentiation markers, we investigated their regulation of expression at the mRNA level in LNCaP human prostate carcinoma cells. Interestingly, phenol red, a pH indicator in the culture medium, promoted cell growth. To eliminate this non-specific effect, a phenol red-free, steroid-reduced medium was utilized. When high-density cells were grown in that medium, 5-dihydrotestosterone (DHT) suppressed PAcP but stimulated PSA. However, tumor promoter phorbol ester 12-otetradecanoyl phorbol-13-acetate (TPA) functioned as a potent inhibitor of both PAcP and PSA expression. Prolonged treatment with DHT as well as TPA resulted in a similar down-regulation of protein kinase C and cellular PAcP activities. Thus, the levels of PAcP and PSA mRNA are 2000 Academic Press differentially regulated by androgens in LNCaP cells. K: androgens; prostatic acid phosphatase; prostate-specific antigen; differentiation antigen.
INTRODUCTION Androgens play an important role in male physiology and pathology by regulating the expression of various genes in respective cells (Berger and Watson, 1989; Kallio et al., 1996). The prostate gland serves as a useful model in understanding this androgen action since the development, maintenance and differentiation of the prostate are dependent on androgens (Kallio et al., 1996; Udayakumar et al., 1998). Prostate cancer is the most common cancer affecting men in the United States (Landis et al., 1998). Androgens are thought to play an important role in the initiation and/or progression of prostate carcinogenesis, although the mechanism remains largely unknown (Gittes, 1991; Kallio et al., 1996; *To whom correspondence should be addressed: Ming-Fong Lin, Ph.D., Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, 984525 Nebraska Medical Center, Omaha, NE 68198-4525, U.S.A. E-mail: [email protected] 1065–6995/00/100681+09 $35.00/0
Koivisto et al., 1998). It is therefore interesting to delineate androgen action in the cells of the prostate. In the human prostate gland, there are two major prostate epithelium-specific differentiation antigens, prostatic acid phosphatase (PAcP) and prostate-specific antigen (PSA) (Chu and Lin, 1998; Kamoshida and Tsutsumi, 1990). Since their respective serum levels are frequently elevated in patients with prostate carcinomas and correlate with tumor progression, serum PAcP is an old ‘gold’ marker of prostate cancer. PSA is the new ‘standard’ in the diagnosis of prostate cancer and its level is used to monitor the response to therapy (Chu and Lin, 1998). Secretions of both PAcP and PSA are stimulated by androgens (Andrews et al., 1992; Bolton et al., 1981; Henttu and Vihko, 1992; Lin et al., 1993a; Udayakumar et al., 1998). Thus, the secretion of these two enzymes serves conventionally as a marker of androgen action on prostate cells (Andrews et al., 1992; Henttu et al., 1992; Lin 2000 Academic Press
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et al., 1993a). However, the molecular mechanisms by which these two enzymes are regulated, especially by androgens, are not completely understood. For example, it is not known whether the expression of these two enzymes is regulated by androgen via a similar mechanism (Andrews et al., 1992; Henttu et al., 1992; Lin et al., 1993a). The LNCaP human prostate cancer cell line is the only commercially available androgen- responsive cell line that expresses endogenous PAcP and PSA (Andrews et al., 1992; Horoszewicz et al., 1983; Lin et al., 1993a). In these cells, the expression and secretion of PAcP, as well as PSA, can be regulated by androgen (Andrews et al., 1992; Lin et al., 1993a). Interestingly, the tumor promoter phorbol ester 12-o-tetradecanoyl phorbol-13acetate (TPA) can also regulate PAcP and PSA secretion and/or expression in these cells (Andrews et al., 1992; Lin and Tsao, 1994). However, it is not known whether the expression of these two enzymes is coordinately regulated by TPA. Additionally, although TPA can down-regulate androgen-stimulated PSA mRNA (Andrews et al., 1992), the TPA action on the basal level of PSA expression remains to be studied. Recent results indicate that PAcP and PSA may be involved in one step in the multiple stages of prostate carcinogenesis. During tumor progression, it is proposed that the cellular form of PAcP interacts with c-ErbB-2/neu protein, resulting in the down-regulation of cell growth (Lin et al., 1998; Lin and Meng, 1996; Meng and Lin, 1998). Concurrently, PSA may interact with the insulin-like growth factor (IGF) signaling pathway (Cohen et al., 1992; Cohen et al., 1994). Due to the potential importance of these two enzymes in prostate carcinogenesis, we studied the regulation of PSA and PAcP mRNA expression. We examined whether the mRNA levels of these two enzymes are coordinately regulated by an androgen, 5-dihydrotestosterone (DHT), and also the possible role of protein kinase C (PKC) in androgen action on PAcP gene expression in LNCaP cells. MATERIALS AND METHODS Cell culture The LNCaP-FGC (LNCaP) human prostate carcinoma cell line (Horoszewicz et al., 1983) was obtained from the American Type Culture Collection (ATCC, Rockville, MD, U.S.A.) and maintained in RPMI 1640 medium (Life Technologies, Grand Island, NY, U.S.A.) supplemented
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with 7% (v/v) fetal bovine serum (FBS), 1% glutamine and 0.5% gentamicin (Life Technologies) (Lin et al., 1992; Lin et al., 1993a). LNCaP cells are the only commercially available cells that are androgen-responsive and express PAcP and PSA (Andrews et al., 1992; Horoszewicz et al., 1983; Lin et al., 1993a). In this study, LNCaP cells that had passage numbers less than 33 were designated as clone-33, cells with passage numbers over 80 as clone-81, and passage numbers between 34 and 80 as clone-51; clone-33 cells represented the parental cells (Lin et al., 1998; Meng and Lin, 1998). This is because different passages of LNCaP cells exhibit different biochemical properties, including growth rate, and PAcP and PSA expression (Fig. 1). To examine hormone effects, a steroid-reduced medium, i.e. phenol red (PR)-free RPMI 1640 medium supplemented with 2% (v v) heatinactivated dialyzed FBS, was used. The final concentration of testosterone was less than 2 p (Lin et al., 1993a), while the Kd values of androgen to its receptor were in n ranges (Horoszewicz et al., 1983). Cells were harvested by scraping, then were rinsed and pelleted in 20 m N-[2hydroxyethyl]piperazine-N -[2-ethane sulfonic acid] (Hepes), 0.9% NaCl, pH 7.0. Cell pellets were lysed in 20 m Hepes (pH 7.0) containing 0.5% Nonidet P-40, 0.5 m dithiothreitol, and a battery of protease inhibitors (Lin et al., 1998; Meng and Lin, 1998) for quantifying protein levels and phosphatase activity. Protein determination The protein concentration in cell lysates was quantified by the Bio-Rad dye protein assay (BioRad, Hercules, CA, U.S.A.) using bovine serum albumin (BSA) as a standard (Lin et al., 1998). Acid phosphatase activity assay p-Nitrophenyl phosphate (PNPP) was used as a substrate to quantify acid phosphatase (AcP) activity at pH 5.5 by measuring the absorbance of released p-nitrophenol (PNP) at 410 n. In prostate epithelium cells, including LNCaP cells, L(+)tartrate-sensitive AcP activity is conventionally taken to represent PAcP activity (Lin et al., 1986). Protein kinase C assay PKC activity was quantified with the Protein Kinase C Assay System Kit from Life Technologies. The kinase assay was performed exactly
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purified by a DEAE-cellulose column and its activity was quantified in the presence and absence of a specific inhibitor of PKC (Lin and Tsao, 1994). The inhibitor-sensitive activity was used to represent specific PKC activity.
A 60
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To analyze the PAcP mRNA level, a PAcP cDNA containing a 294 bp EcoRI/PstI fragment of the coding region was used for RNA blot analyses (Lin et al., 1992; Lin et al., 1993a). The PSA cDNA probe was a polymerase chain reaction product, as described previously (Garcia-Arenas et al., 1995). The -actin cDNA probe was purchased from Clontech (Palo Alto, CA, U.S.A.), and a 0.78 Kb PstI/Xba1 fragment of glyceraldehyde-3phosphate dehydrogenase (GAPDH) cDNA was from ATCC. They were used as probes to examine the quality of RNA preparations.
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Fig. 1. PAcP and PSA expression in different LNCaP cells. A: Cell growth. Different LNCaP cells were seeded at a density of 3104 cells/well in RPMI 1640 medium containing 7% FBS. After growth for 3 days, cells were fed with fresh medium and harvested at days 0, 3, 5 and 7. Fresh medium (2 ml/well) was added to the remaining cells at days 3 and 5. The data shown is the average of triplicates from one set of experiments. Similar results were obtained from three sets of independent experiments. Clone-33, ——; clone-51, — —; clone-81, ——. B: Expression of AR protein. Total cell lysates were prepared from different LNCaP and DU 145 cells at the exponential growth phase. The AR protein level was analyzed by Western blotting with an anti-AR antibody and detected by an ECL method. Similar results were observed in three sets of independent experiments. C: Androgen effects on PAcP and PSA expression. LNCaP cells were seeded at high density (8104 cells/cm2) for 3 days. After growth in PR-free medium for an additional 2 days, cells were maintained in fresh PR-free medium in the presence or absence of 10 nM DHT for 3 days. Total RNA was prepared for Northern blot analyses. The same membrane filter was subsequently hybridized with PAcP, PSA, and GAPDH cDNA probes. Similar results were obtained from two sets of independent experiments.
as described by the manufacturer in the protocol accompanying the kit. Briefly, PKC was partially
Total RNA was prepared from LNCaP cells by the guanidine isothiocyanate method (Garcia-Arenas et al., 1995; Lin et al., 1992). Ten g each of total RNA samples were separated using electrophoresis on 1.2% agarose gels containing formaldehyde as a denaturing agent (Garcia-Arenas et al., 1995; Lin et al., 1992). After electrophoresis, the gel was stained with ethidium bromide (EtBr) and visualized to ensure that there were approximately equal amounts of RNA per lane, as well as to examine the quality of RNA preparation. The separated RNA was transferred to nitrocellulose membrane filters by standard techniques (Brown, 1993). Filters were hybridized and washed under stringent conditions as described in previous reports (Lin et al., 1992; Lin et al., 1993a). A commercially available random oligonucleotide-primed labeling system (Bethesda Research Laboratories, Grand Island, NY, U.S.A.) was used to label cDNA probes with (-32P) dCTP (Feinberg and Vogelstein, 1983). Between each hybridization, the membrane was stripped as described (Lin et al., 1993a). The intensity of hybridization band was semi-quantified by scanning autoradiograms relating to the corresponding GAPDH band, as described previously (Lin and Garcia-Arenas, 1994). Hormonal effects on expression of PAcP and PSA LNCaP cells were plated at a density of 8.0104 cells/cm2 in a PR-free RPMI 1640 medium containing 7% FBS and maintained in a 37C incubator
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Statistical analysis The significance of difference between two groups of data was analyzed by paired two-tailed or onetailed Student’s t test (P value) (Lin et al., 1992; Lin and Tsao, 1994). The value of P<0.05 is considered as significant.
160 150 *
130 *** 120 110 100 90 80 0
RESULTS Effect of phenol red on cellular growth We initially examined whether PR supplemented in the commercial culture medium could have an effect on cell growth, since the expression of PAcP is a cell growth-related process (Lin et al., 1992; Lin et al., 1994). Cells were seeded with a high density of 8104 cells/cm2 to mimic the cell differentiation stage (Lin and Garcia-Arenas, 1994; Lin et al., 1994). Interestingly, as shown in Figure 2, PR at a concentration of that in the commercial medium promoted growth by approximately 40% of that of untreated LNCaP cells. The same amount of growth stimulation was seen in LNCaP cells treated with 10 n DHT. When the PR concentration was doubled, the promoting activity decreased to approximately 20% stimulation. PR also stimulated cell growth in low density culture conditions (8103 cells/cm2; data not shown). Because PR at a concentration found in commercial media [5 mg/l] exhibited a similar stimulatory effect to that of 10 n DHT on the growth of LNCaP cells, a PR-free RPMI medium was utilized to elucidate the DHT effect. Effect of androgen on PAcP and PSA mRNA levels in different LNCaP cells The effects of DHT on the PAcP and PSA expression at the mRNA level were investigated in different subclones of LNCaP cells, including clone-33, -51, and -81 cells. These cells exhibited different growth rates (Fig. 1A). The doubling time of
**
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Cell growth (%)
(5% CO2) for 3 days without disturbance (Lin et al., 1992; Lin et al., 1993a). To examine hormonal effects, cells were maintained in a PR-free steroid-reduced medium for an additional 48 h (Lin et al., 1993a; Lin et al., 1998), and then fed with the same fresh medium in the presence of various reagents (or solvent alone) for 3 days (Lin et al., 1993a; Lin et al., 1998). Cells were harvested for preparation of total RNA as described above.
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C
1 × PR
2 × PR
DHT
Fig. 2. Effects of Phenol red and DHT on cell growth. Clone-33 LNCaP cells were seeded at a density of 8104 cells/cm2 in 6-well flasks in RPMI 1640 medium supplemented with 7% (v/v) FBS for 3 days. After growth in a PR-free steroid-reduced medium for 3 days, cells were exposed to 5 mg/l (1) or 10 mg/l (2) of (PR), or 10 n DHT in fresh medium, for 5 days. Control cells received the solvent alone. All cells were harvested and the total cell number was determined by cell counting. The data shown is the average of triplicates from one set of experiments. Similar results were obtained in two sets of independent experiments. *P<0.01 (n=3, two-tailed); **P<0.02 (n=3, two-tailed); ***P<0.002 (n=3, one-tailed).
clone-51 and -81 cells was approximately half that of clone-33 cells. Western blot analyses demonstrated that these three LNCaP cells expressed a similar level of androgen receptor (AR) protein, while DU 145 cells, used as controls, did not express AR protein (Fig. 1B). Interestingly, the PAcP mRNA level decreased in rapidly growing clone-81 cells, lower than that in clone-33 cells (Fig. 1C). DHT treatment resulted in an approximately 30% diminution of PAcP mRNA in all three subclone cells, regardless of the basal level of expression (Fig. 1C). Nevertheless, the basal level of PSA mRNA was elevated in clone-81 cells, higher than that in clone-33 and clone-51 cells (Fig. 1C). The androgen DHT induced a two- to four-fold elevation of mRNA in all three subclone cells, independent of their growth rates and the basal level of mRNA expression (Fig. 1C). Thus, androgen exhibited an opposite effect on PAcP compared to PSA expression at the mRNA level in LNCaP cells.
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C
TPA
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DHT
PAcP PSA GAPDH
Fig. 3. Effect of TPA and DHT on PAcP and PSA expression. Clone-33 LNCaP cells were seeded at a density of 8104 cells/cm2. After treatment with 10 n DHT or TPA for 3 days, total RNA was prepared for Northern blot analyses. Control cells (C) received the solvent alone. The same membrane was subsequently hybridized with PAcP, PSA, and GAPDH cDNA probes.
Effects of a time-course of DHT and TPA treatment
Effect of TPA on PAcP and PSA mRNA levels Since PKC has been demonstrated as being involved in mediating the action of several steroid hormones (Doolan and Harvey, 1996), we compared the effects of TPA, a PKC ligand, with DHT on PAcP and PSA expression in clone-33 cells that expressed a high basal level of PAcP (Fig. 1C). Initially, cells were exposed to each reagent for 3 days, due to a relatively slow response of PAcP to DHT in these cells (Fig. 1C). As shown in Figure 3, TPA caused a decrease in PAcP mRNA levels by A
more than 90%, and PSA mRNA levels by over 95% (Fig. 3). In fact in TPA-treated cells, PSA mRNA was barely detectable (Fig. 3). In the same set of experiments, DHT exposure resulted in an approximately 35% decrease in PAcP mRNA and a two-fold increase in PSA mRNA (Fig. 3), as in Figure 1C. The less pronounced effect of DHT on PAcP and PSA in Figure 3 compared to Figure 1C was due to the overexposure of the film for detecting PAcP and PSA by TPA regulation. Thus, it is clear that TPA effectively down-regulates the basal level of PAcP as well as PSA mRNA.
TPA can counteract androgen stimulation of PSA mRNA over a 6 h treatment (Andrews et al., 1992). We examined whether TPA can down-regulate the basal level of PSA mRNA expression within the same time period in the absence of androgen stimulation. Clone-33 cells were exposed to TPA over a concentration range of 1 n to 50 n for 6 h, and the total RNA was prepared for Northern blot analyses. At this time point, neither the PAcP nor the PSA mRNA levels were noticeably altered by TPA treatment (Fig. 4A).
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Fig. 4. Effect of TPA and DHT on PAcP and PSA mRNA levels. A: TPA effect. Clone-33 LNCaP cells were exposed to 0, 1, 10, 25 and 50 nM TPA. Control cells received the solvent alone (C) or 50 nM 4-Phorbol (4-P). After 6 h exposure, total RNA was prepared for Northern blotting. The same membrane was subsequently hybridized with PAcP, PSA, and GAPDH cDNA probes. Similar results were obtained from two sets of independent experiments. B: TPA and DHT effects. Clone-33 LNCaP cells were exposed to 10 nM DHT (D) or TPA (T) for 0, 16, 40 and 64 h. Control cells received the solvent alone (C). The same membrane filter was subsequently hybridized with PAcP, PSA, GAPDH, and -actin cDNA probes.
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Table 1. Effect of TPA and DHT on protein kinase C and cellular PAcP activitiesa Group I II III IV
Reagent
PKC Activityb
PAcP Activityc
None DHT TPA (TPA+DHT)
6.25 (100%) 4.51 (72%) 3.63 (58%) 3.13 (50%)
0.759 (100%) 0.618 (81%) 0.416 (55%) 0.380 (50%)
a
LNCaP cells in duplicate flasks were treated with DHT (50 n), TPA (50 n), DHT+TPA, or solvent alone. After 20 h, cells were harvested for quantifying PKC and PAcP activities in total cell lysate proteins. The data shown were the average from cells in duplicate flasks. b PKC activity was demonstrated as pmol phosphate incorporated by PKC in 1 mg of cellular protein with 1 min incubation. The differences in PKC activity between Groups III and IV were observed in 2 sets of independent experiments (n=22) with P<0.05 (two-tailed). c The phosphatase activity was the average of the absorbance at 410 nm of p-nitrophenol released by PAcP in 1 mg of cell lysate per min. The difference in PAcP activity between Groups III and IV were observed in 4 sets of independent experiments (n=24) with P<0.01 (two-tailed).
We further examined the effects of a time-course of DHT and TPA treatment on the expression of PAcP and PSA mRNA. As shown in Figure 4B, relating to GAPDH and -actin expression, DHT inhibition of the PAcP mRNA essentially followed a time-dependent course. After the 64 h exposure, DHT caused an approximately 30% inhibition of PAcP mRNA, as observed previously in Figures 1C and 3. Nevertheless, DHT up-regulation of PSA mRNA was observed after the 16 h treatment, and was still seen after 40 h. However, the degree of stimulation decreased after 64 h exposure. In comparison, TPA treatment caused an approximately 90% inhibition of PAcP mRNA in the same set of experiments with a 64 h exposure, and greater than 95% suppression of PSA mRNA after only 40 h exposure. Thus, DHT had an opposite effect on PAcP and PSA expression, while TPA inhibited both PSA and PAcP expression (although PSA was more susceptible to TPA than PAcP). Effect of DHT on PKC activity Since TPA and DHT function as potent suppressors of the expression of PAcP, a classically known androgen-regulated antigen, we examined whether DHT action on PAcP expression was possibly mediated in part by PKC, a TPA receptor (Doolan and Harvey, 1996; Gschwendt et al., 1991; Kikkawa and Nishizuka, 1986; Lin and Tsao,
1994). A 20 h treatment with TPA resulted in an approximately 40% down-regulation of PKC activity (Table 1), similar to previous reports (Kikkawa and Nishizuka, 1986). Surprisingly, DHT caused PKC activity to decrease by about 30% (Table 1). A simultaneous exposure to TPA and DHT only had a partially additive effect on the down-regulation of PKC activity. This downregulation by TPA and DHT together correlated with a decreased cellular PAcP activity (Table 1). Thus, DHT and TPA may execute the downregulation of PAcP via a similar molecular mechanism. DISCUSSION PAcP and PSA are the two major cell-specific differentiation antigens in normal prostate epithelia. Classically, these two enzymes serve as markers for androgen action, cancer diagnosis, and monitoring the response to therapy (Chu and Lin, 1998). Recent results further indicate the importance of these two enzymes in prostate cell growth regulation and carcinogenesis (Cohen et al., 1994; Lin et al., 1998; Lin and Meng, 1996; Meng and Lin, 1998). However, the mechanism of PAcP and PSA regulation of expression at the molecular level is not completely known. Furthermore, most studies on these two enzymes have been conducted independently in phenol red-containing medium. Our data, to the best of our knowledge, constitute the first report that clearly shows that the expression of these two enzymes is differentially regulated at the mRNA level in human prostate cancer cells, although several common factors, including androgens and TPA, are involved in their regulation. For example, in rapidly growing clone-81 LNCaP cells, the expression of PAcP is decreased and PSA is increased, in comparison with that expressed in clone-33 LNCaP cells (Fig. 1C). Although TPA can suppress both PAcP and PSA mRNA levels, DHT causes opposite effects on the mRNA levels of PAcP and PSA in high density-cultured slowgrowing cells, a growth condition mimicking the cell differentiation stage (Figs. 1C, 3, and 4B). Interestingly, PR exhibits a growth promoting activity in LNCaP cells. This growth stimulation is decreased at higher concentrations of PR (Fig. 2), similar to that reported for DHT on the same cells (Horoszewicz et al., 1983; Lin et al., 1992). The PR stimulation of prostate cell growth could be due to the fact that LNCaP cells express a mutant form of AR that binds to various steroids, including antiandrogens, resulting in a positive growth response
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(Veldscholte et al., 1992). Alternatively, it is possible that PR exhibits non-specific steroid activity, as it has been reported that PR exhibits an estrogenic activity (Berthois et al., 1986; Hubert et al., 1986). No matter what the mechanism of PR is, a PR-free medium for LNCaP cells is a better choice for eliminating the basal non-specific effect when studying androgen activity. Interestingly, clone-51 and clone-81 LNCaP cells can both grow rapidly yet have different PSA expressions in the PR-free medium. As shown in Figure 1, in clone-51 and clone-81 LNCaP cells the expression of PAcP decreases, while cell growth increases. In prostate cancer cells, cellular PAcP dephosphorylates the phosphotyrosine of c-ErbB-2 protein, leading to a diminished growth rate. Conversely, decreased cellular PAcP expression correlates with an elevated phosphotyrosine level of c-ErbB-2 and a rapid growth rate, as observed in clone-51 and clone-81 LNCaP cells. Since mitogenactivated protein kinases (MAP kinases) transduce the downstream signaling of ErbB-2, activated c-ErbB-2 leads to an elevated MAP kinase activity. Therefore, in the clone-51 and clone-81 LNCaP cells, a decreased PAcP expression concurs with an increased c-ErbB-2 activity (Meng and Lin, 1998) which can activate the downstream MAP kinases and, subsequently, the AR transcriptional activity which in turn up-regulates PSA gene expression (Yeh et al., 1999). Thus, there are higher levels of PSA mRNA in the clone-51 and clone-81 LNCaP cells than in the clone-33 LNCaP cells under androgen-depleted conditions. We therefore propose that, biochemically, clone-51 and clone-81 LNCaP cells resemble advanced hormonerefractory prostate cancers, since they express functional AR, as indicated by the up-regulation of PSA expression, but exhibit no growth response to androgens (Lin et al., 1998). Further, under androgen-depleted conditions, the expression of PSA is elevated (Fig. 1C). The cell density effect on gene expression is an interesting common phenomenon in epithelial cells. For example, cell culture density can influence the gene expression and protein levels of several biologically important molecules, including growth factors and/or their receptors (Hunter and Cooper, 1981; Monget et al., 1998; Pfeiffer et al., 1998; Singh et al., 1996). We have shown that cell culture density does not only affect the level of PAcP mRNA (Lin et al., 1994) but also modulates DHT regulation of PAcP mRNA (Lin and GarciaArenas, 1994). Therefore, in this communication, LNCaP cells have been maintained at a high density with a slow growth rate to eliminate the cell
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density effects on PAcP expression and to mimic the differentiated state of these cells (Lin et al., 1994). Under these growth conditions, DHT suppresses PAcP mRNA but stimulates PSA mRNA. However, stimulation of PAcP secretion is a classic indicator of androgen action in prostate epithelial cells, including LNCaP cells, whatever the cell culture conditions (Bolton et al., 1981; Lin et al., 1993b). Since DHT can regulate the PAcP mRNA level as well as its secretory pathway in prostate cells, the promotion of PAcP secretion by DHT in high density cultured cells is, at least in part, due to the activation of its secretory pathway (Lin et al., 1993a, 1993b). The differences in regulation of expression of PAcP and PSA by DHT may indicate that these two enzymes are involved in different cellular differentiation processes. The expression of PAcP is inversely correlated with the cell growth rate, while the PSA expression is directly correlated, increasing in rapidly growing cells (Fig. 1C). DHT stimulation of cell proliferation coincides with a decrease in PAcP and an increase in PSA mRNA (Figs 1C, 3, and 4). The difference in the effect of DHT on PAcP and PSA expression could be due to the fact that the cellular form of PAcP functions as a negative regulator by dephosphorylating c-ErbB-2/ neu oncoprotein, resulting in the down-regulation of cell proliferation (Lin et al., 1998; Lin and Meng, 1996; Meng and Lin, 1998), while PSA may activate an insulin-like growth factor by hydrolyzing its binding protein (IGF-BP), leading to the stimulation of cell growth (Cohen et al., 1992; Cohen et al., 1994). Alternatively, DHT up-regulation of PSA could be due to a direct, immediate interaction of the DHT/AR complex with the functional androgen-responsive element (ARE) in the promoter of the PSA gene (Cleutjens et al., 1997; Cleutjens et al., 1996), which is preferable to its down-regulation via PKC signaling. DHT down-regulation of PAcP could, in part, be via the PKC pathway, since the functional ARE of the PAcP gene has not been identified (Shan et al., 1997), and also since TPA has a more potent effect than DHT. This thus provides an explanation of why both DHT and TPA have a similar effect on PKC activity, but have different effects on PAcP and PSA expression. Alternatively, the different effects on PAcP and PSA by DHT and TPA could be due to the fact that DHT and TPA signalings are mediated by different PKC isoenzymes and/or different cofactors of PKC, since more than 11 isoenzymes of the PKC family have been identified (Mellor and Parker, 1998). This notion is supported by the observation that there is a significant
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difference in the down-regulation of PKC activity by TPA (Gr. III, Table 1), versus TPA plus DHT (Gr. IV, Table 1). Further investigation is required to understand this regulation. Unexpectedly, prolonged exposure to DHT correlates with decreased PKC activity (Table 1). Although the detailed molecular mechanism is still not known, it is possible that DHT mobilizes Ca2+ (Steinsapir et al., 1991), a cofactor of PKC, resulting in modulation of the PKC activity. Since DHT treatment can also induce increased tyrosine kinase activity in LNCaP cells (Lin et al., 1986; Lin et al., 1998), tyrosine kinase signaling and PKC thus coordinately mediate the diverse effects induced by DHT in prostate cells. For example, PKC may be involved indirectly in androgen stimulation of tyrosine phosphorylation by inhibiting cellular PAcP activity and its expression. Although PKC may directly down-regulate cellular PAcP activity (Lin and Tsao, 1994), the effect of DHT and TPA on the mRNA level of PAcP cannot be a direct effect by PKC. This is because their effects are only observed after prolonged treatment of at least 40 h (Fig. 4B). Other factors are involved in this mode of regulation. In summary, further studies are needed to elucidate the molecular mechanism of regulation and the functional role of these two cell-specific differentiation antigens. These results, however, provide new insight into androgen action on prostate epithelial cells, hopefully leading to a better understanding of androgen biology in prostate carcinogenesis. ACKNOWLEDGEMENTS This study was supported in part by CA72274 from the National Cancer Institute, National Institutes of Health, LB506, #2000-19, from Nebraska Cancer and Smoking Diseases Research Program, and Nebraska Research Initiative. We thank Dr James Seberger for his critical reading and suggestion. We also would like to dedicate this article to Dr T. Ming Chu for his devotion to improving the quality of life by the development of a better tumor marker, including the discovery of prostate-specific antigen (PSA) in prostate cancer. REFERENCES A PE, Y CY, M BT, T DJ, 1992. Tumor-promoting phorbol ester down-regulates the androgen induction of prostate-specific antigen in a human prostatic adenocarcinoma cell line. Cancer Res 52: 1525–1529.
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B FG, W G, 1989. Androgen-regulated gene expression. Annu Rev Physiol 51: 51–65. B Y, K JA, K BS, 1986. Phenol red in tissue culture media is a weak estrogen: implications concerning the study of estrogen-responsive cells in culture. Proc Natl Acad Sci USA 83: 2496–2500. B NJ, L R, V P, K M, V R, 1981. Androgens and prostate-specific acid phosphatase in whole tissue and in separated epithelium from human benign prostatic hypertrophic glands. Prostate 2: 409–416. B T, 1993. Analysis of RNA by Northern and slot blot hybridization. In: Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Amith JA, Struhl K, eds. Current Protocols in Molecular Biology. New York, Greene/WileyInterscience. 4.9.1–4.9.14. C TM, L MF, 1998. PSA and acid phosphatase in the diagnosis of prostate cancer. J Clin Ligand Assay 21: 24–34. C KB, V-D K HA, V-E CC, V-R HC, F PW, T J, 1997. An androgen response element in a far upstream enhancer region is essential for high, androgen-regulated activity of the prostate-specific antigen promoter. Mol Endocrin 11: 148–161. C KB, V-E CC, V-D-K HA, B AO, T J, 1996. Two androgen response regions cooperate in steroid hormone regulated activity of the prostate-specific antigen promoter. J Biol Chem 271: 6379–6388. C P, G HC, P DM, K M, G LC, R RG, 1992. Prostate-specific antigen (PSA) is an insulin-like growth factor binding protein-3 protease found in seminal plasma. J Clin Endocrinol Metab 75: 1046–1053. C P, P DM, G HC, R RG, 1994. Biological effects of prostate specific antigen as an insulinlike growth factor binding protein-3 protease. J Endocrinol 142: 407–415. D CM, H BJ, 1996. Modulation of cytosolic protein kinase C and calcium ion activity by steroid hormones in rat distal colon. J Biol Chem 271: 8763–8767. F AP, V B, 1983. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132: 6–13. G-A R, L FF, L DL, J LP, S CCY, C C, L MF, 1995. The expression of prostatic acid phosphatase is transcriptionally regulated in human prostate carcinoma cells. Mol Cell Endo 111: 29–37. G RF, 1991. Carcinoma of the prostate. N Engl J Med 324: 236–245. G M, K W, M F, 1991. Protein kinase C activation by phorbol esters: do cysteine-rich regions and pseudosubstrate motifs play a role? Trends Biochem Sci 16: 167–169. H P, L S, V P, 1992. Androgens up-regulate the human prostate-specific antigen messenger ribonucleic acid (mRNA), but down-regulate the prostatic acid phosphatase mRNA in the LNCaP cell line. Endocrinology 130: 766–772. H P, V P, 1992. Steroids inversely affect the biosynthesis and secretion of human prostatic acid phosphatase and prostate-specific antigen in the LNCaP cell line. J Steroid Biochem Mol Biol 41: 349–360. H JS, L SS, K E, K JP, R H, C TM, M EA, M GP, 1983. LNCaP model of human prostatic carcinoma. Cancer Res 43: 1809–1818.
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H JF, V A, L F, 1986. Estrogenic activity of phenol red in rat anterior pituitary cells in culture. Biochem Biophys Res Commun 141: 885–891. H T, C JA, 1981. Epidermal growth factor induces rapid tyrosine phosphorylation of proteins in A431 human tumor cells. Cell 24: 741–752. K PJ, P JJ, J OA, 1996. Genetic regulation of androgen action. Prostate Suppl 6: 45–51. K S, T Y, 1990. Extraprostatic localization of prostatic acid phosphatase and prostate-specific antigen: distribution in cloacogenic glandular epithelium and sexdependent expression in human anal gland. Hum Pathol 21: 1108–1111. K U, N Y, 1986. The role of protein kinase C in transmembrane signalling. Annu Rev Cell Biol 2: 149–178. K P, K M, V T, K OP, 1998. Androgen receptor gene and hormonal therapy failure of prostate cancer. Am J Pathol 152: 1–9. L SH, M T, B S, W PA, 1998. Cancer statistics, 1998. Ca Cancer J Clin 48: 6–29. L MF, D J, G-A R, 1992. Expression of Human Prostatic Acid Phosphatase Activity and the growth of Prostste Carcinoma Cells. Cancer Res 52: 4600–4607. L MF, G-A R, 1994. Effect of cell density on androgen regulation of the mRNA level of human prostatic acid phosphatase. Mol Cell Endo 99: R21–R24. L MF, G-A R, C YC, L MMC, P PC, X XZ, 1993a. Regulation of prostatic acid phosphatase expression and secretion by androgen in LNCaP human prostate carcinoma cells. Arch Biochem Biophys 300: 384–390. L MF, G-A R, K M, L FF, 1993b. Regulation of the expression of prostatic acid phosphatase in LNCaP human prostate carcinoma cells. Cell Mol Biol Res 39: 739–750. L MF, G-A R, X XZ, B B, L FF, 1994. The cellular level of prostatic acid phosphatase and the growth of human prostate carcinoma cells. Differentiation 57: 143–149. L MF, L CL, C GM, 1986. Tyrosyl kinase activity is inversely related to prostatic acid phosphatase activity in two human prostatic carcinoma cell lines. Mol Cell Biol 6: 4753–4757. L MF, M TC, R PS, C C, S AH, L FF, 1998. Expression of human prostatic acid phosphatase correlates with androgen-stimulated cell proliferation in prostate cancer cell lines. J Biol Chem 273: 5939–5947. L MF, M TC, 1996. Tyrosine phosphorylation of a p185 phosphoprotein (pp185) inversely correlates with the cellular
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activity of human prostatic acid phosphatase. Biochem Biophy Res Comm 226: 206–213. L MF, T JL, 1994. Tumor promoter, 12-o-Tetradecanoyl Phorbol-13-Actate, downregulates the cellular activity of human prostatic acid phosphatase. Mol Cell Diff 2: 127–140. M H, P PJ, 1998. The extended protein kinase C superfamily. Biochem J 332: 281–292. M TC, L MF, 1998. Tyrosine phosphorylation of c-ErbB-2 is regulation by the cellular form of prostatic acid phosphatase in human prostate cancer cells. J Biol Chem 273: 22096–22104. M P, P C, M D, 1998. Expression of insulin-like growth factor binding protein-5 by ovine granulosa cells is regulated by cell density and programmed cell death in vitro. J Cell Physiol 177: 13–25. P D, K R, H J, R M, F EA, S P, J A, S H, P A, 1998. Epidermal-growth-factor receptor correlates negatively with cell density in cervical squamous epithelium and is downregulated in cancers of the human uterus. Int J Cancer 79: 49–55. S JD, P K, R M, K A, L V, H P, O J, V P, 1997. Steroidinvolved transcriptional regulation of human genes encoding prostatic acid phosphatase, prostate-specific antigen, and prostate-specific glandular Kallikrein. Endocrinology 138: 3764–3770. S RK, L N, B CD, S R, K A, F IJ, 1996. Cell density-dependent regulation of basic fibroblast growth factor expression in human rental cell carcinoma cells. Cell Growth Diff 7: 397–404. S J, S R, R P, 1991. Effects of androgen on intracellular calcium of LNCaP cells. Biochem Biophys Res Commun 179: 90–96. U TS, T A, R M, D SN, H S, B JS, 1998. Changes in structure and functions of prostate by long-term administration of an androgen, testosterone enanthate, in rhesus monkey (Macaca mulatta). Anat Rec 252: 637–645. V J, B CA, B AO, G JA, M E, 1992. Anti-androgens and the mutated androgen receptor of LNCaP cells: differential effects on binding affinity, heat-shock protein interaction, and transcription activation. Biochemistry 31: 2393–2399. Y S, L HK, K HY, T TH, L MF, C C, 1999. From HER2/Neu signal cascade to androgen receptor and its coactivators: A novel pathway by induction of androgen target genes through MAP kinase in prostate cancer cells. Proc Natl Acad Sci USA 96: 5458–5463.
DIFFERENTIAL RESPONSIVENESS OF PROSTATIC ACID PHOSPHATASE AND PROSTATE-SPECIFIC ANTIGEN mRNA TO ANDROGEN IN PROSTATE CANCER CELLS MING-FONG LIN1,2,3,4*, MING-SHYUE LEE1, RENEE GARCIA-ARENAS1 and FEN-FEN LIN1 1
Department of Biochemistry and Molecular Biology and 2Section of Urologic Surgery, College of Medicine, 3 Eppley Cancer Institute, University of Nebraska Medical Center, and 4Omaha VA Hospital, Omaha, NE 68198, U.S.A. Received 2 September 1999; accepted 3 March 2000
Androgens regulate the expression of both human prostatic acid phosphatase (PAcP) and prostate-specific antigen (PSA), two major prostate epithelium-specific differentiation antigens. Due to the important role of these two enzymes as prostate epithelium differentiation markers, we investigated their regulation of expression at the mRNA level in LNCaP human prostate carcinoma cells. Interestingly, phenol red, a pH indicator in the culture medium, promoted cell growth. To eliminate this non-specific effect, a phenol red-free, steroid-reduced medium was utilized. When high-density cells were grown in that medium, 5-dihydrotestosterone (DHT) suppressed PAcP but stimulated PSA. However, tumor promoter phorbol ester 12-otetradecanoyl phorbol-13-acetate (TPA) functioned as a potent inhibitor of both PAcP and PSA expression. Prolonged treatment with DHT as well as TPA resulted in a similar down-regulation of protein kinase C and cellular PAcP activities. Thus, the levels of PAcP and PSA mRNA are 2000 Academic Press differentially regulated by androgens in LNCaP cells. K: androgens; prostatic acid phosphatase; prostate-specific antigen; differentiation antigen.
INTRODUCTION Androgens play an important role in male physiology and pathology by regulating the expression of various genes in respective cells (Berger and Watson, 1989; Kallio et al., 1996). The prostate gland serves as a useful model in understanding this androgen action since the development, maintenance and differentiation of the prostate are dependent on androgens (Kallio et al., 1996; Udayakumar et al., 1998). Prostate cancer is the most common cancer affecting men in the United States (Landis et al., 1998). Androgens are thought to play an important role in the initiation and/or progression of prostate carcinogenesis, although the mechanism remains largely unknown (Gittes, 1991; Kallio et al., 1996; *To whom correspondence should be addressed: Ming-Fong Lin, Ph.D., Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, 984525 Nebraska Medical Center, Omaha, NE 68198-4525, U.S.A. E-mail: [email protected] 1065–6995/00/100681+09 $35.00/0
Koivisto et al., 1998). It is therefore interesting to delineate androgen action in the cells of the prostate. In the human prostate gland, there are two major prostate epithelium-specific differentiation antigens, prostatic acid phosphatase (PAcP) and prostate-specific antigen (PSA) (Chu and Lin, 1998; Kamoshida and Tsutsumi, 1990). Since their respective serum levels are frequently elevated in patients with prostate carcinomas and correlate with tumor progression, serum PAcP is an old ‘gold’ marker of prostate cancer. PSA is the new ‘standard’ in the diagnosis of prostate cancer and its level is used to monitor the response to therapy (Chu and Lin, 1998). Secretions of both PAcP and PSA are stimulated by androgens (Andrews et al., 1992; Bolton et al., 1981; Henttu and Vihko, 1992; Lin et al., 1993a; Udayakumar et al., 1998). Thus, the secretion of these two enzymes serves conventionally as a marker of androgen action on prostate cells (Andrews et al., 1992; Henttu et al., 1992; Lin 2000 Academic Press
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et al., 1993a). However, the molecular mechanisms by which these two enzymes are regulated, especially by androgens, are not completely understood. For example, it is not known whether the expression of these two enzymes is regulated by androgen via a similar mechanism (Andrews et al., 1992; Henttu et al., 1992; Lin et al., 1993a). The LNCaP human prostate cancer cell line is the only commercially available androgen- responsive cell line that expresses endogenous PAcP and PSA (Andrews et al., 1992; Horoszewicz et al., 1983; Lin et al., 1993a). In these cells, the expression and secretion of PAcP, as well as PSA, can be regulated by androgen (Andrews et al., 1992; Lin et al., 1993a). Interestingly, the tumor promoter phorbol ester 12-o-tetradecanoyl phorbol-13acetate (TPA) can also regulate PAcP and PSA secretion and/or expression in these cells (Andrews et al., 1992; Lin and Tsao, 1994). However, it is not known whether the expression of these two enzymes is coordinately regulated by TPA. Additionally, although TPA can down-regulate androgen-stimulated PSA mRNA (Andrews et al., 1992), the TPA action on the basal level of PSA expression remains to be studied. Recent results indicate that PAcP and PSA may be involved in one step in the multiple stages of prostate carcinogenesis. During tumor progression, it is proposed that the cellular form of PAcP interacts with c-ErbB-2/neu protein, resulting in the down-regulation of cell growth (Lin et al., 1998; Lin and Meng, 1996; Meng and Lin, 1998). Concurrently, PSA may interact with the insulin-like growth factor (IGF) signaling pathway (Cohen et al., 1992; Cohen et al., 1994). Due to the potential importance of these two enzymes in prostate carcinogenesis, we studied the regulation of PSA and PAcP mRNA expression. We examined whether the mRNA levels of these two enzymes are coordinately regulated by an androgen, 5-dihydrotestosterone (DHT), and also the possible role of protein kinase C (PKC) in androgen action on PAcP gene expression in LNCaP cells. MATERIALS AND METHODS Cell culture The LNCaP-FGC (LNCaP) human prostate carcinoma cell line (Horoszewicz et al., 1983) was obtained from the American Type Culture Collection (ATCC, Rockville, MD, U.S.A.) and maintained in RPMI 1640 medium (Life Technologies, Grand Island, NY, U.S.A.) supplemented
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with 7% (v/v) fetal bovine serum (FBS), 1% glutamine and 0.5% gentamicin (Life Technologies) (Lin et al., 1992; Lin et al., 1993a). LNCaP cells are the only commercially available cells that are androgen-responsive and express PAcP and PSA (Andrews et al., 1992; Horoszewicz et al., 1983; Lin et al., 1993a). In this study, LNCaP cells that had passage numbers less than 33 were designated as clone-33, cells with passage numbers over 80 as clone-81, and passage numbers between 34 and 80 as clone-51; clone-33 cells represented the parental cells (Lin et al., 1998; Meng and Lin, 1998). This is because different passages of LNCaP cells exhibit different biochemical properties, including growth rate, and PAcP and PSA expression (Fig. 1). To examine hormone effects, a steroid-reduced medium, i.e. phenol red (PR)-free RPMI 1640 medium supplemented with 2% (v v) heatinactivated dialyzed FBS, was used. The final concentration of testosterone was less than 2 p (Lin et al., 1993a), while the Kd values of androgen to its receptor were in n ranges (Horoszewicz et al., 1983). Cells were harvested by scraping, then were rinsed and pelleted in 20 m N-[2hydroxyethyl]piperazine-N -[2-ethane sulfonic acid] (Hepes), 0.9% NaCl, pH 7.0. Cell pellets were lysed in 20 m Hepes (pH 7.0) containing 0.5% Nonidet P-40, 0.5 m dithiothreitol, and a battery of protease inhibitors (Lin et al., 1998; Meng and Lin, 1998) for quantifying protein levels and phosphatase activity. Protein determination The protein concentration in cell lysates was quantified by the Bio-Rad dye protein assay (BioRad, Hercules, CA, U.S.A.) using bovine serum albumin (BSA) as a standard (Lin et al., 1998). Acid phosphatase activity assay p-Nitrophenyl phosphate (PNPP) was used as a substrate to quantify acid phosphatase (AcP) activity at pH 5.5 by measuring the absorbance of released p-nitrophenol (PNP) at 410 n. In prostate epithelium cells, including LNCaP cells, L(+)tartrate-sensitive AcP activity is conventionally taken to represent PAcP activity (Lin et al., 1986). Protein kinase C assay PKC activity was quantified with the Protein Kinase C Assay System Kit from Life Technologies. The kinase assay was performed exactly
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purified by a DEAE-cellulose column and its activity was quantified in the presence and absence of a specific inhibitor of PKC (Lin and Tsao, 1994). The inhibitor-sensitive activity was used to represent specific PKC activity.
A 60
5
Cell numbers (¥ 10 )
50 40
Preparation of cDNA probe
30
To analyze the PAcP mRNA level, a PAcP cDNA containing a 294 bp EcoRI/PstI fragment of the coding region was used for RNA blot analyses (Lin et al., 1992; Lin et al., 1993a). The PSA cDNA probe was a polymerase chain reaction product, as described previously (Garcia-Arenas et al., 1995). The -actin cDNA probe was purchased from Clontech (Palo Alto, CA, U.S.A.), and a 0.78 Kb PstI/Xba1 fragment of glyceraldehyde-3phosphate dehydrogenase (GAPDH) cDNA was from ATCC. They were used as probes to examine the quality of RNA preparations.
20 10
0
2
B
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8
6
LNCaP C-33
C-51
C-81
DU
AR
Northern blot analysis
C
Clone-33 DHT
+
Clone-51
+
Clone-81
+
PAcP PSA GAPDH
Fig. 1. PAcP and PSA expression in different LNCaP cells. A: Cell growth. Different LNCaP cells were seeded at a density of 3104 cells/well in RPMI 1640 medium containing 7% FBS. After growth for 3 days, cells were fed with fresh medium and harvested at days 0, 3, 5 and 7. Fresh medium (2 ml/well) was added to the remaining cells at days 3 and 5. The data shown is the average of triplicates from one set of experiments. Similar results were obtained from three sets of independent experiments. Clone-33, ——; clone-51, — —; clone-81, ——. B: Expression of AR protein. Total cell lysates were prepared from different LNCaP and DU 145 cells at the exponential growth phase. The AR protein level was analyzed by Western blotting with an anti-AR antibody and detected by an ECL method. Similar results were observed in three sets of independent experiments. C: Androgen effects on PAcP and PSA expression. LNCaP cells were seeded at high density (8104 cells/cm2) for 3 days. After growth in PR-free medium for an additional 2 days, cells were maintained in fresh PR-free medium in the presence or absence of 10 nM DHT for 3 days. Total RNA was prepared for Northern blot analyses. The same membrane filter was subsequently hybridized with PAcP, PSA, and GAPDH cDNA probes. Similar results were obtained from two sets of independent experiments.
as described by the manufacturer in the protocol accompanying the kit. Briefly, PKC was partially
Total RNA was prepared from LNCaP cells by the guanidine isothiocyanate method (Garcia-Arenas et al., 1995; Lin et al., 1992). Ten g each of total RNA samples were separated using electrophoresis on 1.2% agarose gels containing formaldehyde as a denaturing agent (Garcia-Arenas et al., 1995; Lin et al., 1992). After electrophoresis, the gel was stained with ethidium bromide (EtBr) and visualized to ensure that there were approximately equal amounts of RNA per lane, as well as to examine the quality of RNA preparation. The separated RNA was transferred to nitrocellulose membrane filters by standard techniques (Brown, 1993). Filters were hybridized and washed under stringent conditions as described in previous reports (Lin et al., 1992; Lin et al., 1993a). A commercially available random oligonucleotide-primed labeling system (Bethesda Research Laboratories, Grand Island, NY, U.S.A.) was used to label cDNA probes with (-32P) dCTP (Feinberg and Vogelstein, 1983). Between each hybridization, the membrane was stripped as described (Lin et al., 1993a). The intensity of hybridization band was semi-quantified by scanning autoradiograms relating to the corresponding GAPDH band, as described previously (Lin and Garcia-Arenas, 1994). Hormonal effects on expression of PAcP and PSA LNCaP cells were plated at a density of 8.0104 cells/cm2 in a PR-free RPMI 1640 medium containing 7% FBS and maintained in a 37C incubator
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Statistical analysis The significance of difference between two groups of data was analyzed by paired two-tailed or onetailed Student’s t test (P value) (Lin et al., 1992; Lin and Tsao, 1994). The value of P<0.05 is considered as significant.
160 150 *
130 *** 120 110 100 90 80 0
RESULTS Effect of phenol red on cellular growth We initially examined whether PR supplemented in the commercial culture medium could have an effect on cell growth, since the expression of PAcP is a cell growth-related process (Lin et al., 1992; Lin et al., 1994). Cells were seeded with a high density of 8104 cells/cm2 to mimic the cell differentiation stage (Lin and Garcia-Arenas, 1994; Lin et al., 1994). Interestingly, as shown in Figure 2, PR at a concentration of that in the commercial medium promoted growth by approximately 40% of that of untreated LNCaP cells. The same amount of growth stimulation was seen in LNCaP cells treated with 10 n DHT. When the PR concentration was doubled, the promoting activity decreased to approximately 20% stimulation. PR also stimulated cell growth in low density culture conditions (8103 cells/cm2; data not shown). Because PR at a concentration found in commercial media [5 mg/l] exhibited a similar stimulatory effect to that of 10 n DHT on the growth of LNCaP cells, a PR-free RPMI medium was utilized to elucidate the DHT effect. Effect of androgen on PAcP and PSA mRNA levels in different LNCaP cells The effects of DHT on the PAcP and PSA expression at the mRNA level were investigated in different subclones of LNCaP cells, including clone-33, -51, and -81 cells. These cells exhibited different growth rates (Fig. 1A). The doubling time of
**
140
Cell growth (%)
(5% CO2) for 3 days without disturbance (Lin et al., 1992; Lin et al., 1993a). To examine hormonal effects, cells were maintained in a PR-free steroid-reduced medium for an additional 48 h (Lin et al., 1993a; Lin et al., 1998), and then fed with the same fresh medium in the presence of various reagents (or solvent alone) for 3 days (Lin et al., 1993a; Lin et al., 1998). Cells were harvested for preparation of total RNA as described above.
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C
1 × PR
2 × PR
DHT
Fig. 2. Effects of Phenol red and DHT on cell growth. Clone-33 LNCaP cells were seeded at a density of 8104 cells/cm2 in 6-well flasks in RPMI 1640 medium supplemented with 7% (v/v) FBS for 3 days. After growth in a PR-free steroid-reduced medium for 3 days, cells were exposed to 5 mg/l (1) or 10 mg/l (2) of (PR), or 10 n DHT in fresh medium, for 5 days. Control cells received the solvent alone. All cells were harvested and the total cell number was determined by cell counting. The data shown is the average of triplicates from one set of experiments. Similar results were obtained in two sets of independent experiments. *P<0.01 (n=3, two-tailed); **P<0.02 (n=3, two-tailed); ***P<0.002 (n=3, one-tailed).
clone-51 and -81 cells was approximately half that of clone-33 cells. Western blot analyses demonstrated that these three LNCaP cells expressed a similar level of androgen receptor (AR) protein, while DU 145 cells, used as controls, did not express AR protein (Fig. 1B). Interestingly, the PAcP mRNA level decreased in rapidly growing clone-81 cells, lower than that in clone-33 cells (Fig. 1C). DHT treatment resulted in an approximately 30% diminution of PAcP mRNA in all three subclone cells, regardless of the basal level of expression (Fig. 1C). Nevertheless, the basal level of PSA mRNA was elevated in clone-81 cells, higher than that in clone-33 and clone-51 cells (Fig. 1C). The androgen DHT induced a two- to four-fold elevation of mRNA in all three subclone cells, independent of their growth rates and the basal level of mRNA expression (Fig. 1C). Thus, androgen exhibited an opposite effect on PAcP compared to PSA expression at the mRNA level in LNCaP cells.
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C
TPA
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DHT
PAcP PSA GAPDH
Fig. 3. Effect of TPA and DHT on PAcP and PSA expression. Clone-33 LNCaP cells were seeded at a density of 8104 cells/cm2. After treatment with 10 n DHT or TPA for 3 days, total RNA was prepared for Northern blot analyses. Control cells (C) received the solvent alone. The same membrane was subsequently hybridized with PAcP, PSA, and GAPDH cDNA probes.
Effects of a time-course of DHT and TPA treatment
Effect of TPA on PAcP and PSA mRNA levels Since PKC has been demonstrated as being involved in mediating the action of several steroid hormones (Doolan and Harvey, 1996), we compared the effects of TPA, a PKC ligand, with DHT on PAcP and PSA expression in clone-33 cells that expressed a high basal level of PAcP (Fig. 1C). Initially, cells were exposed to each reagent for 3 days, due to a relatively slow response of PAcP to DHT in these cells (Fig. 1C). As shown in Figure 3, TPA caused a decrease in PAcP mRNA levels by A
more than 90%, and PSA mRNA levels by over 95% (Fig. 3). In fact in TPA-treated cells, PSA mRNA was barely detectable (Fig. 3). In the same set of experiments, DHT exposure resulted in an approximately 35% decrease in PAcP mRNA and a two-fold increase in PSA mRNA (Fig. 3), as in Figure 1C. The less pronounced effect of DHT on PAcP and PSA in Figure 3 compared to Figure 1C was due to the overexposure of the film for detecting PAcP and PSA by TPA regulation. Thus, it is clear that TPA effectively down-regulates the basal level of PAcP as well as PSA mRNA.
TPA can counteract androgen stimulation of PSA mRNA over a 6 h treatment (Andrews et al., 1992). We examined whether TPA can down-regulate the basal level of PSA mRNA expression within the same time period in the absence of androgen stimulation. Clone-33 cells were exposed to TPA over a concentration range of 1 n to 50 n for 6 h, and the total RNA was prepared for Northern blot analyses. At this time point, neither the PAcP nor the PSA mRNA levels were noticeably altered by TPA treatment (Fig. 4A).
4a-P C
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TPA 1
10
25
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PAcP PSA GAPDH B
0h C
16 h C
D
40 h T
C
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64 h T
C
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PAcP PSA GAPDH b-actin
Fig. 4. Effect of TPA and DHT on PAcP and PSA mRNA levels. A: TPA effect. Clone-33 LNCaP cells were exposed to 0, 1, 10, 25 and 50 nM TPA. Control cells received the solvent alone (C) or 50 nM 4-Phorbol (4-P). After 6 h exposure, total RNA was prepared for Northern blotting. The same membrane was subsequently hybridized with PAcP, PSA, and GAPDH cDNA probes. Similar results were obtained from two sets of independent experiments. B: TPA and DHT effects. Clone-33 LNCaP cells were exposed to 10 nM DHT (D) or TPA (T) for 0, 16, 40 and 64 h. Control cells received the solvent alone (C). The same membrane filter was subsequently hybridized with PAcP, PSA, GAPDH, and -actin cDNA probes.
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Table 1. Effect of TPA and DHT on protein kinase C and cellular PAcP activitiesa Group I II III IV
Reagent
PKC Activityb
PAcP Activityc
None DHT TPA (TPA+DHT)
6.25 (100%) 4.51 (72%) 3.63 (58%) 3.13 (50%)
0.759 (100%) 0.618 (81%) 0.416 (55%) 0.380 (50%)
a
LNCaP cells in duplicate flasks were treated with DHT (50 n), TPA (50 n), DHT+TPA, or solvent alone. After 20 h, cells were harvested for quantifying PKC and PAcP activities in total cell lysate proteins. The data shown were the average from cells in duplicate flasks. b PKC activity was demonstrated as pmol phosphate incorporated by PKC in 1 mg of cellular protein with 1 min incubation. The differences in PKC activity between Groups III and IV were observed in 2 sets of independent experiments (n=22) with P<0.05 (two-tailed). c The phosphatase activity was the average of the absorbance at 410 nm of p-nitrophenol released by PAcP in 1 mg of cell lysate per min. The difference in PAcP activity between Groups III and IV were observed in 4 sets of independent experiments (n=24) with P<0.01 (two-tailed).
We further examined the effects of a time-course of DHT and TPA treatment on the expression of PAcP and PSA mRNA. As shown in Figure 4B, relating to GAPDH and -actin expression, DHT inhibition of the PAcP mRNA essentially followed a time-dependent course. After the 64 h exposure, DHT caused an approximately 30% inhibition of PAcP mRNA, as observed previously in Figures 1C and 3. Nevertheless, DHT up-regulation of PSA mRNA was observed after the 16 h treatment, and was still seen after 40 h. However, the degree of stimulation decreased after 64 h exposure. In comparison, TPA treatment caused an approximately 90% inhibition of PAcP mRNA in the same set of experiments with a 64 h exposure, and greater than 95% suppression of PSA mRNA after only 40 h exposure. Thus, DHT had an opposite effect on PAcP and PSA expression, while TPA inhibited both PSA and PAcP expression (although PSA was more susceptible to TPA than PAcP). Effect of DHT on PKC activity Since TPA and DHT function as potent suppressors of the expression of PAcP, a classically known androgen-regulated antigen, we examined whether DHT action on PAcP expression was possibly mediated in part by PKC, a TPA receptor (Doolan and Harvey, 1996; Gschwendt et al., 1991; Kikkawa and Nishizuka, 1986; Lin and Tsao,
1994). A 20 h treatment with TPA resulted in an approximately 40% down-regulation of PKC activity (Table 1), similar to previous reports (Kikkawa and Nishizuka, 1986). Surprisingly, DHT caused PKC activity to decrease by about 30% (Table 1). A simultaneous exposure to TPA and DHT only had a partially additive effect on the down-regulation of PKC activity. This downregulation by TPA and DHT together correlated with a decreased cellular PAcP activity (Table 1). Thus, DHT and TPA may execute the downregulation of PAcP via a similar molecular mechanism. DISCUSSION PAcP and PSA are the two major cell-specific differentiation antigens in normal prostate epithelia. Classically, these two enzymes serve as markers for androgen action, cancer diagnosis, and monitoring the response to therapy (Chu and Lin, 1998). Recent results further indicate the importance of these two enzymes in prostate cell growth regulation and carcinogenesis (Cohen et al., 1994; Lin et al., 1998; Lin and Meng, 1996; Meng and Lin, 1998). However, the mechanism of PAcP and PSA regulation of expression at the molecular level is not completely known. Furthermore, most studies on these two enzymes have been conducted independently in phenol red-containing medium. Our data, to the best of our knowledge, constitute the first report that clearly shows that the expression of these two enzymes is differentially regulated at the mRNA level in human prostate cancer cells, although several common factors, including androgens and TPA, are involved in their regulation. For example, in rapidly growing clone-81 LNCaP cells, the expression of PAcP is decreased and PSA is increased, in comparison with that expressed in clone-33 LNCaP cells (Fig. 1C). Although TPA can suppress both PAcP and PSA mRNA levels, DHT causes opposite effects on the mRNA levels of PAcP and PSA in high density-cultured slowgrowing cells, a growth condition mimicking the cell differentiation stage (Figs. 1C, 3, and 4B). Interestingly, PR exhibits a growth promoting activity in LNCaP cells. This growth stimulation is decreased at higher concentrations of PR (Fig. 2), similar to that reported for DHT on the same cells (Horoszewicz et al., 1983; Lin et al., 1992). The PR stimulation of prostate cell growth could be due to the fact that LNCaP cells express a mutant form of AR that binds to various steroids, including antiandrogens, resulting in a positive growth response
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(Veldscholte et al., 1992). Alternatively, it is possible that PR exhibits non-specific steroid activity, as it has been reported that PR exhibits an estrogenic activity (Berthois et al., 1986; Hubert et al., 1986). No matter what the mechanism of PR is, a PR-free medium for LNCaP cells is a better choice for eliminating the basal non-specific effect when studying androgen activity. Interestingly, clone-51 and clone-81 LNCaP cells can both grow rapidly yet have different PSA expressions in the PR-free medium. As shown in Figure 1, in clone-51 and clone-81 LNCaP cells the expression of PAcP decreases, while cell growth increases. In prostate cancer cells, cellular PAcP dephosphorylates the phosphotyrosine of c-ErbB-2 protein, leading to a diminished growth rate. Conversely, decreased cellular PAcP expression correlates with an elevated phosphotyrosine level of c-ErbB-2 and a rapid growth rate, as observed in clone-51 and clone-81 LNCaP cells. Since mitogenactivated protein kinases (MAP kinases) transduce the downstream signaling of ErbB-2, activated c-ErbB-2 leads to an elevated MAP kinase activity. Therefore, in the clone-51 and clone-81 LNCaP cells, a decreased PAcP expression concurs with an increased c-ErbB-2 activity (Meng and Lin, 1998) which can activate the downstream MAP kinases and, subsequently, the AR transcriptional activity which in turn up-regulates PSA gene expression (Yeh et al., 1999). Thus, there are higher levels of PSA mRNA in the clone-51 and clone-81 LNCaP cells than in the clone-33 LNCaP cells under androgen-depleted conditions. We therefore propose that, biochemically, clone-51 and clone-81 LNCaP cells resemble advanced hormonerefractory prostate cancers, since they express functional AR, as indicated by the up-regulation of PSA expression, but exhibit no growth response to androgens (Lin et al., 1998). Further, under androgen-depleted conditions, the expression of PSA is elevated (Fig. 1C). The cell density effect on gene expression is an interesting common phenomenon in epithelial cells. For example, cell culture density can influence the gene expression and protein levels of several biologically important molecules, including growth factors and/or their receptors (Hunter and Cooper, 1981; Monget et al., 1998; Pfeiffer et al., 1998; Singh et al., 1996). We have shown that cell culture density does not only affect the level of PAcP mRNA (Lin et al., 1994) but also modulates DHT regulation of PAcP mRNA (Lin and GarciaArenas, 1994). Therefore, in this communication, LNCaP cells have been maintained at a high density with a slow growth rate to eliminate the cell
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density effects on PAcP expression and to mimic the differentiated state of these cells (Lin et al., 1994). Under these growth conditions, DHT suppresses PAcP mRNA but stimulates PSA mRNA. However, stimulation of PAcP secretion is a classic indicator of androgen action in prostate epithelial cells, including LNCaP cells, whatever the cell culture conditions (Bolton et al., 1981; Lin et al., 1993b). Since DHT can regulate the PAcP mRNA level as well as its secretory pathway in prostate cells, the promotion of PAcP secretion by DHT in high density cultured cells is, at least in part, due to the activation of its secretory pathway (Lin et al., 1993a, 1993b). The differences in regulation of expression of PAcP and PSA by DHT may indicate that these two enzymes are involved in different cellular differentiation processes. The expression of PAcP is inversely correlated with the cell growth rate, while the PSA expression is directly correlated, increasing in rapidly growing cells (Fig. 1C). DHT stimulation of cell proliferation coincides with a decrease in PAcP and an increase in PSA mRNA (Figs 1C, 3, and 4). The difference in the effect of DHT on PAcP and PSA expression could be due to the fact that the cellular form of PAcP functions as a negative regulator by dephosphorylating c-ErbB-2/ neu oncoprotein, resulting in the down-regulation of cell proliferation (Lin et al., 1998; Lin and Meng, 1996; Meng and Lin, 1998), while PSA may activate an insulin-like growth factor by hydrolyzing its binding protein (IGF-BP), leading to the stimulation of cell growth (Cohen et al., 1992; Cohen et al., 1994). Alternatively, DHT up-regulation of PSA could be due to a direct, immediate interaction of the DHT/AR complex with the functional androgen-responsive element (ARE) in the promoter of the PSA gene (Cleutjens et al., 1997; Cleutjens et al., 1996), which is preferable to its down-regulation via PKC signaling. DHT down-regulation of PAcP could, in part, be via the PKC pathway, since the functional ARE of the PAcP gene has not been identified (Shan et al., 1997), and also since TPA has a more potent effect than DHT. This thus provides an explanation of why both DHT and TPA have a similar effect on PKC activity, but have different effects on PAcP and PSA expression. Alternatively, the different effects on PAcP and PSA by DHT and TPA could be due to the fact that DHT and TPA signalings are mediated by different PKC isoenzymes and/or different cofactors of PKC, since more than 11 isoenzymes of the PKC family have been identified (Mellor and Parker, 1998). This notion is supported by the observation that there is a significant
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difference in the down-regulation of PKC activity by TPA (Gr. III, Table 1), versus TPA plus DHT (Gr. IV, Table 1). Further investigation is required to understand this regulation. Unexpectedly, prolonged exposure to DHT correlates with decreased PKC activity (Table 1). Although the detailed molecular mechanism is still not known, it is possible that DHT mobilizes Ca2+ (Steinsapir et al., 1991), a cofactor of PKC, resulting in modulation of the PKC activity. Since DHT treatment can also induce increased tyrosine kinase activity in LNCaP cells (Lin et al., 1986; Lin et al., 1998), tyrosine kinase signaling and PKC thus coordinately mediate the diverse effects induced by DHT in prostate cells. For example, PKC may be involved indirectly in androgen stimulation of tyrosine phosphorylation by inhibiting cellular PAcP activity and its expression. Although PKC may directly down-regulate cellular PAcP activity (Lin and Tsao, 1994), the effect of DHT and TPA on the mRNA level of PAcP cannot be a direct effect by PKC. This is because their effects are only observed after prolonged treatment of at least 40 h (Fig. 4B). Other factors are involved in this mode of regulation. In summary, further studies are needed to elucidate the molecular mechanism of regulation and the functional role of these two cell-specific differentiation antigens. These results, however, provide new insight into androgen action on prostate epithelial cells, hopefully leading to a better understanding of androgen biology in prostate carcinogenesis. ACKNOWLEDGEMENTS This study was supported in part by CA72274 from the National Cancer Institute, National Institutes of Health, LB506, #2000-19, from Nebraska Cancer and Smoking Diseases Research Program, and Nebraska Research Initiative. We thank Dr James Seberger for his critical reading and suggestion. We also would like to dedicate this article to Dr T. Ming Chu for his devotion to improving the quality of life by the development of a better tumor marker, including the discovery of prostate-specific antigen (PSA) in prostate cancer. REFERENCES A PE, Y CY, M BT, T DJ, 1992. Tumor-promoting phorbol ester down-regulates the androgen induction of prostate-specific antigen in a human prostatic adenocarcinoma cell line. Cancer Res 52: 1525–1529.
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