Progression of LNCaP Prostate Tumor Cells during Androgen Deprivation: Hormone-Independent Growth, Repression of Proliferation by Androgen, and Role for p27Kip1 in Androgen-Induced Cell Cycle Arrest

John M. Kokontis, Nissim Hay1 and Shutsung Liao

The Ben May Institute for Cancer Research (J.M.K., N.H., S.L.) The Departments of Pharmacology and Physiology (N.H.) and Biochemistry and Molecular Biology (S.L.) The University of Chicago Chicago, Illinois 60637


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The molecular mechanism of androgen-independent growth of prostate cancer after androgen ablation was explored in LNCaP cells. An androgen-dependent clonal subline of the LNCaP human prostate carcinoma cell line, LNCaP 104-S, progressed to a slow growing stage (104-R1) and then to a faster growing stage (104-R2) during more than 2 yr of continuous culture in the absence of androgen. Androgen-induced proliferation of 104-S cells is inhibited by the antiandrogen Casodex, while proliferation of 104-R1 and 104-R2 cells is unaffected by Casodex. This indicates that proliferation of 104-R1 and 104-R2 cells is not supported by low levels of androgen in the culture medium. Compared with LNCaP 104-S cells, both 104-R1 and 104-R2 cells express higher basal levels of androgen receptor (AR), and proliferation of these two cell lines is paradoxically repressed by androgen. After continuous passage in androgen-containing medium, 104-R1 cells reverted back to an androgen-dependent phenotype. The mechanism of androgenic repression of 104-R1 and 104-R2 sublines was further evaluated by examining the role of critical regulatory factors involved in the control of cell cycle progression. At concentrations that repressed growth, androgen transiently induced the expression of the cyclin-dependent kinase (cdk) inhibitor p21waf1/cip1 in 104-R1 cells, while expression of the cdk inhibitor p27Kip1 was persistently induced by androgen in both 104-R1 and 104-R2 cells. Induced expression of murine p27Kip1 in 104-R2 cells resulted in G1 arrest. Specific immunoprecipitates of Cdk2 but not Cdk4 from androgen-treated 104-R1 cells contained both p21waf1/cip1 and p27Kip1. This observation was confirmed by in vitro assay of histone H1 and Rb (retinoblastoma protein) phosphorylation by the proteins associated with the immune complex. Furthermore, inhibition of Cdk2 activity correlated with the accumulation of p27Kip1 and not p21waf1/cip1. From these results we conclude that androgenic repression of LNCaP 104-R1 and 104-R2 cell proliferation is due to the induction of p27Kip1, which in turn inhibits Cdk2, a factor critical for cell cycle progression and proliferation.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The resistance to androgen ablation therapy (1) acquired by prostate tumor cells during androgen deprivation remains a severe obstacle to the effective treatment of metastatic prostate cancer (2). Tumor cells that were formerly sensitive to antiandrogen or androgen ablation strategies almost always emerge as androgen-independent tumors after 1–3 yr of treatment (3). Understanding the mechanism(s) by which hormonally sensitive tumor cells lose hormone dependency or otherwise acquire the ability to defeat hormone deprivation-based therapies has therefore been an important objective in the study of prostate cancer. To examine this phenomenon, investigators have derived androgen-independent cells from the Dunning tumor (4), the Shionogi mouse mammary carcinoma 115 (5, 6, 7), a mouse prostate carcinoma system (8), and the LNCaP cell line (9), among others.

We reported previously that adaptation of an androgen-dependent clonal isolate of the LNCaP human prostate carcinoma cell line, LNCaP 104-S, to androgen deprivation in vitro after 40–60 passages was accompanied by increased androgen receptor (AR) expression and activity (10). Cells with heightened sensitivity to androgen, called 104-R cells, proliferated at a much faster rate than 104-S cells in medium deficient in androgen, and their proliferation rate was, in fact, severely repressed by androgen even at low concentrations. This repression by androgen was accompanied by reduced c-myc expression and was blocked by ectopic overexpression of c-myc. We also found that tumors derived from LNCaP 104-R cells grown in athymic mice are repressed by testosterone and that this growth repression could be blocked by the 5{alpha}-reductase inhibitor finasteride (11). Others have also studied androgen-independent LNCaP cells arising as tumors after the inoculation of androgen-dependent cells into castrated nude mice (12, 13, 14, 15) or arising spontaneously in vitro (16, 17, 18, 19, 20). The LNCaP cell line, therefore, provides a good model for changes that occur in prostate tumor cells as the cells are subjected to androgen deprivation both in vitro and in vivo. In this paper we report on the continued adaptation of LNCaP 104-R cells, now designated 104-R1 cells, to androgen withdrawal and the progression of 104-R1 cells to more rapidly proliferating cells designated 104-R2. We also report on the ability of LNCaP 104-R1 cells to adapt to repressive levels of androgen and to reacquire positive proliferative responsiveness to androgen.

To study the mechanism of androgen repression, we examined the expression of several cyclin-dependent kinase inhibitors, including p21waf-1/cip-1 and p27Kip1, in LNCaP 104-S, 104-R1, and 104-R2 cells after androgen treatment. The cyclin-dependent kinase (cdk) inhibitors p21waf-1/cip-1 and p27Kip1 are members of a structurally related family of proteins that can mediate G1 cell cycle arrest (for review, see Ref. 21). Cell cycle arrest mediated by p21waf1/cip1 or p27Kip1 is thought to occur because these proteins inhibit the phosphorylation by cdks of members of the retinoblastoma protein (Rb) family of regulators (Rb, p107, and p130). Hypophosphorylated Rb family members form complexes with members of the E2F family (E2F-1 through 5) of transcription factors and the DP (DP-1, DP-2) family of associated proteins (22). Phosphorylation of Rb by cdks results in release of active E2F/DP complexes that transactivate genes required for G1 transit and progression through S phase, or in release of Rb/E2F complexes that can repress transcription of such genes. Results described in this paper show that, although androgen induces a transient elevation of p21waf1/cip1 in 104-R1 cells, it is the persistent androgen-induced accumulation of p27Kip1 in both 104-R1 and 104-R2 cells that correlates with the inhibition of Cdk2 activity and G1 cell cycle arrest.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Androgen-Dependent and -Independent Proliferation of LNCaP 104 Sublines
Previously we had shown that LNCaP 104-S cells adapt to androgen withdrawal after about 40 passages (PA- 40) in androgen-deficient medium and progress to 104-R1 cells (formally called 104-R; Ref. 10). This adaptation is manifested as a higher proliferation rate of 104-R1 cells in androgen-deficient medium as compared with 104-S cells and is accompanied by increases in AR expression and activity. However, the proliferation of LNCaP 104-R1 cells is repressed by androgen at concentrations that are optimal for LNCaP 104-S growth. After about 60 additional passages in androgen-deficient medium, 104-R1 cells attained proliferation rates in the absence of androgen equivalent to or higher than 104-S cells maximally stimulated by 0.1 nM 17ß-hydroxy-17-methylestra-4,9,11-trien-3-one (R1881) (Fig. 1Go). LNCaP 104 cells at this stage (>PA- 100) are designated 104-R2 to distinguish them from 104-R1 cells, which proliferate at a lower rate. The acquisition of higher proliferation rates occurred gradually so the boundary between 104-R1 and 104-R2 cells is somewhat arbitrary. LNCaP 104-R2 cells are somewhat less sensitive than 104-R1 cells to androgenic repression of growth.



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Figure 1. Proliferation of Various LNCaP Sublines in the Presence of R1881

For testing the effect of androgen on cell proliferation, LNCaP cells were plated in triplicate wells (3 x 104 cells per well) in 2 ml of DMEM supplemented with 10% CS-FBS and the indicated concentration of R1881 in a 12-well dish. After 6 days, cells were trypsinized and counted with a hemocytometer. PA- 80 and PA- 221 refer to the number of passages grown in the absence of androgen at the time of testing.

 
Proliferation of LNCaP cells, which express a mutant form of AR (23, 24), is stimulated by antiandrogens such as hydroxyflutamide and cyproterone acetate (23, 24, 25) but is inhibited by Casodex (26). Casodex at a concentration of 5 µM severely repressed LNCaP 104-S cell proliferation induced by 0.1 nM R1881, as expected, but had little or no effect on LNCaP 104-R1 or 104-R2 cell proliferation in the absence of R1881, indicating that the proliferation of these cells is not dependent on residual androgen that may be present in the medium (Fig. 2Go). On the other hand, Casodex efficiently blocked the repressive effect of R1881 on 104-R1 and 104-R2 cell proliferation. This shows that androgen repression of the proliferation of 104-R1 and 104-R2 cells is mediated through AR.



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Figure 2. Effect of R1881 and Casodex on Cell Proliferation of LNCaP 104-S, 104-R1, and 104-R2 Cells

LNCaP cells were plated in triplicate wells (3 x 104 cells per well) in a 12-well dish in 2 ml of DMEM supplemented with 10% CS-FBS and ethanol vehicle (control), 0.1 nM R1881, 5 µM Casodex, or both 0.1 nM R1881 and 5 µM Casodex, as indicated. After 6 days, cells were trypsinized and counted with a hemocytometer.

 
While the proliferation of LNCaP 104-R1 cells is severely inhibited by R1881 at concentrations of 0.1 nM and higher, these cells exhibited an ability to adapt over time and to grow in high concentrations of R1881. Starting at PA- 55, 104-R1 cells were grown continuously in DMEM with 10% dextran-coated, charcoal-stripped (CS)-FBS supplemented with 20 nM R1881. During the first 5–10 passages, the proliferation rate was low. Gradually, the proliferation rate climbed to levels similar to 104-R1 cells grown in the absence of androgen. When these adapted cells, termed 104-R1Ad, were grown over a range of R1881 concentrations, the optimal concentration of R1881 for growth was now 1–10 nM (Fig. 3Go). Casodex, at 5 µM, repressed R1881-induced proliferation, similar to its effect on 104-S cells. R1881 at 100 nM appeared to overcome the inhibition by Casodex. These observations indicate that 104-R1 cells can reacquire positive proliferative sensitivity to previously repressive levels of androgen after they are subjected to long-term exposure. The proliferative responses of the various LNCaP 104 sublines to androgen and Casodex are summarized in Table 1Go.



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Figure 3. Effect of R1881 and Casodex on Cell Proliferation of LNCaP 104-R1Ad Cells

LNCaP 104-R1 cells were cultured for 30 passages in 20 nM R1881 to generate 104-R1Ad cells. 104-R1Ad cells were passaged twice in medium supplemented with 20 nM DHT before testing to clear the cells of R1881. DHT was used because, unlike R1881, it is rapidly metabolized in LNCaP cells (49 ) and therefore is efficiently removed from cells before the proliferation assays. LNCaP 104-R1Ad cells were plated in triplicate wells (3 x 104 cells per well) in 2 ml of DMEM supplemented with 10% CS-FBS, the indicated concentration of R1881, and 5 µM Casodex (open circles) or ethanol vehicle (filled circles) in a 12-well dish. After 6 days, cells were trypsinized and counted with a hemocytometer.

 

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Table 1. Proliferation of LNCaP 104 Sublines in Medium Containing R1881 (0.1 nM) or R1881 and Casodex (5 µM)

 
Expression of AR and Prostate-Specific Antigen (PSA) in LNCaP 104-S, 104-R1, and 104-R2 Cells
AR expression in 104-R1 and 104-R2 cells was about 20-fold higher compared with 104-S cells as quantified by densitometric scans of AR immunoblots (Fig. 4Go). AR level in 104-R1Ad cells, however, was dramatically lower than the level of AR in 104-R1 cells and even lower than the level found in 104-S cells. If cells were cultured for 4 days in medium containing 0.1 or 10 nM R1881, AR levels in 104-S and 104-R1Ad cells were enhanced 2- to 3-fold but were still lower than AR levels in 104-R1 or 104-R2 cells. The increased amount of AR in 104-S and 104-R1Ad cells treated for 4 days with R1881 probably reflects stabilization of the AR protein by androgen (27), since AR mRNA is down-regulated by androgen in LNCaP cells (10, 28, 29). Probing of HindIII-digested genomic DNA from 104-S, 104-R1, and 104-R2 cells with an amino-terminal AR-specific cDNA probe revealed that the AR gene had not undergone amplification in these sublines (data not shown).



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Figure 4. AR Immunoblot of Total Cellular Proteins Extracted from LNCaP 104-S, 104-R1, 104-R2, or 104-R1Ad Cells after 4 Days of Growth in R1881 at 0, 0.1, and 10 nM R1881

Cells from 6-cm plates were lysed in 0.5 ml 2x Laemmli gel loading buffer lacking bromophenol blue. After quantification of protein concentration using the Bradford reagent, 40-µg aliquots were run on 10% SDS-PAGE gels. Blots were incubated with rabbit polyclonal antibody AN-21 raised against the N-terminal 21 amino acids of hAR (10 ), and bound antibody was visualized using peroxidase-conjugated goat antirabbit IgG (Stratagene) and an enhanced chemiluminescence procedure (15-sec exposure) AR migrated as a 110-kDa protein.

 
The ability of AR to stimulate transcription in these sublines was examined by measuring induction of PSA mRNA by androgen. The 5'-regulatory region of the PSA gene contains multiple androgen response elements (30). Induction of PSA mRNA by androgen was higher in 104-R1 cells and 104-R2 cells than in 104-S cells (Fig. 5Go). The level of PSA mRNA in LNCaP 104-R1 and 104-R2 cells increased 10- to 15-fold within 48 h after treatment with 1 nM R1881, while the PSA mRNA level in 104-S cells increased only about 5-fold. Therefore, AR in LNCaP 104-R1 and 104-R2 cells is functional and transcriptionally activated by androgen, even though these cells are proliferatively repressed by androgen.



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Figure 5. Induction of PSA mRNA by Androgen in LNCaP 104 Sublines

RNAse protection assay using 32P-labeled antisense PSA and ß2-microglobulin (ß2-MG) RNA probes (10 ) was employed to measure the induction of PSA mRNA expression by R1881 in LNCaP 104-S, 104-R1 (PA- 68), and 104-R2 (PA- 210) cells. Total RNA was isolated from LNCaP 104-S, 104-R1, and 104-R2 cells grown in DMEM supplemented with 10% CS-FBS in the presence or absence of 1 nM R1881 for 48 h. Ordinate values were calculated by dividing the amount of radioactivity present in PSA mRNA bands by the amount of radioactivity present in ß2-MG mRNA bands and setting the ratio of one of the untreated 104-S points to an arbitrary value of 1.

 
Androgen Induction of G1 Arrest in 104-R1 and 104-R2 Cells
Flow cytometric analysis of LNCaP 104-S, 104-R1, and 104-R2 cells grown in the presence of R1881 demonstrated that androgen induces cell cycle arrest at the G1 phase in 104-R1 and 104-R2 cells, but not in 104-S or 104-R1Ad cells (Fig. 6Go, A and B). Flow cytometric analysis of the time course of G1 arrest induced by R1881 in 104-R1 cells showed that arrest was first apparent 24 h after addition of hormone (Fig. 6CGo). The percentage of cells in S phase declined to the minimum by 72 h after addition of hormone. The decline in the percentage of untreated control cells in S phase over the last 2 days of the 4-day culture period can be attributed to the gradual overcrowding of cells in the culture dishes, even though medium was changed on day 2.



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Figure 6. Androgen Induction of G1 Arrest in 104-R1 and 104-R2 Cells

A, Representative histograms of 104-R1 cells incubated for 4 days in the absence or presence of 10 nM R1881. B, Flow cytometry of 104-S, 104-R1, 104-R2, and 104-R1 cells adapted to growth in 20 nM R1881 after 4 days of growth in 0, 0.1, and 10 nM R1881. Cells (3 x 105) were plated on 6-cm plates. The next day, medium supplemented with 10% CS-FBS was replaced with fresh medium containing the indicated concentration of R1881, and the cells were incubated for 4 days before trypsinization and fixation for flow cytometric analysis. C, Flow cytometric analysis of the time course of G1 arrest in 104-R1 cells treated with 10 nM R1881 for the indicated periods. Data in panels B and C represent the mean ± SE of three independent experiments.

 
Androgen Induction of Accumulation of the cdk Inhibitors p21waf1/cip1 and p27Kip1 in 104-R1 Cells and p27Kip1 in 104-R2 Cells
Because androgen specifically induced a G1 cell cycle arrest in 104-R1 and 104-R2 cells, we surveyed the expression of several cdk inhibitors that are known to elicit G1 arrest when overexpressed. From our time course studies of androgen-induced G1 arrest in 104-R1 cells, we knew that increased expression of pertinent cdk inhibitors should be apparent within 24 h after androgen treatment. Therefore we first examined the expression of cdk inhibitors in 104-R1 cells grown for increasing lengths of time in 10 nM R1881. Expression of p21waf1/cip1 was induced 2- to 3-fold 12 h and 24 h after addition of R1881, but declined to the basal level at the 48- and 72-h timepoints and was almost undetectable by 96 h (Fig. 7AGo). Expression of p27Kip1 was induced 2- to 3-fold in 104-R1 cells after 24 h of incubation in 10 nM R1881 and appeared to peak at 48–72 h. Unlike p21waf1/cip1, the level of p27Kip1 persisted at a high level up to 96 h of incubation with androgen. Expression of cdk inhibitors p57Kip2, p16INK4A, and p15INK4B were not affected significantly by androgen in any of the cell types (data not shown).



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Figure 7. Effect of Androgen on Accumulation of p21waf1/cip1 and p27Kip1 in 104-R1 and 104-R2 Cells

A, Time course analysis of p21waf1/cip1 and p27Kip1 protein expression in 104-R1 cells treated with 10 nM R1881 for the indicated periods. B, Western blot of p21waf1/cip1 and p27Kip1 expression in 104-S, 104-R1, 104-R2, and 104-R1Ad cells after 24 h (p21waf1/cip1) or 48 h (p27Kip1) days of growth in R1881 at 0, 0.1, 1, and 10 nM R1881. Cells from 6-cm plates were lysed in 0.5 ml 2x Laemmli gel loading buffer lacking bromophenol blue. After quantification of protein concentration using the Bradford reagent, 40-µg (p21waf1/cip1) or 20-µg (p27Kip1) aliquots were run on 12% SDS-PAGE gels. Blots shown are representative of three independent experiments.

 
Next we examined the effect of a range of R1881 concentrations on p21waf1/cip1 and p27Kip1 expression in 104-S, 104-R1, 104-R2, and 104-R1Ad cells at 24 h (p21waf1/cip1) and 48 h (p27Kip1) of incubation (Fig. 7BGo). R1881 at 0.1 nM induced only p27Kip1 and not p21waf1/cip1 in 104-R1 cells, suggesting that p27Kip1 is more sensitive to androgen stimulation than p21waf1/cip1. In 104-R2 cells, R1881 induced p21waf1/cip1 slightly only at the highest concentration. To check the possibility that the kinetics of p21waf1/cip1 induction are delayed in 104-R2 cells compared with 104-R1 cells, a time course experiment similar to that shown in Fig. 6AGo was performed with 104-R2 cells. It revealed that p21waf1/cip1 does not accumulate in 104-R2 cells at any time within 96 h (data not shown). The basal level of p27Kip1 expression in these cells was higher than in 104-R1 cells, and accumulation of p27Kip1 appeared to be less sensitive to R1881. In 104-S and 104-R1Ad cells, R1881 did not induce accumulation of p21waf1/cip1 or p27Kip1. Rather, R1881, at the proliferatively optimal concentration of 0.1 nM, reduced the expression of both p21waf1/cip1 and p27Kip1 in 104-S cells. In 104-R1Ad cells, R1881 reduced the level of p21waf1/cip1 in a dose-dependent manner, while R1881 at all concentrations only slightly reduced the level of p27Kip1. These observations suggest that 1) in 104-R1 cells, p27Kip1 appears to be more sensitive to low concentrations of R1881, but accumulates with slower kinetics than p21waf1/cip1 and is persistently expressed; 2) in 104-R2 cells, accumulation of p27Kip1 appears to be less sensitive to androgen than in 104-R1 cells, and accumulation of p21waf1/cip1 is insensitive to androgen except at the highest dose used, consistent with the generally lower sensitivity of 104-R2 cells to androgen-induced G1 arrest (Fig. 6BGo); and 3) in androgen-dependent 104-S cells and in 104-R1Ad cells, accumulation of p21waf1/cip1 and p27Kip1 may have a role in growth arrest after androgen deprivation, opposite to the effect of androgen in 104-R1 and 104-R2 cells.

We then measured the levels of p21waf1/cip1 and p27Kip1 mRNA in 104-R1 cells incubated in 10 nM R1881 over a time course spanning 96 h. The message levels of each were increased slightly after 12 h of incubation in 10 nM R1881, but this increase was not significant (Fig. 8Go) and did not correlate with the protein levels of p21waf1/cip1 or p27Kip1, suggesting that the induction of p21waf1/cip1 and p27Kip1 proteins by androgen occurred mainly by a posttranscriptional mechanism.



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Figure 8. RNase Protection Analysis Using 32P-Labeled 419-Base and 355-Base Antisense RNA Probes for p21waf1/cip1 and p27Kip1 mRNA Expression, Respectively, in 104-R1 Cells Grown for 24 h (p21waf1/cip1) or 48 h (p27Kip1) in the Presence of 10 nM R1881

A 109-base antisense GAPDH probe was used as internal control for the total amount of RNA present. The lower panel represents the means of normalized values from two independent experiments. The values were calculated by dividing the amount of radioactivity present in protected p21waf1/cip1 or p27Kip1 mRNA bands by the amount of radioactivity present in GAPDH mRNA bands and setting the ratio of one of the untreated points to an arbitrary value of 1. The same probe preparations were used for each experiment.

 
Induced Expression of Exogenous p27kip1 Results in G1 Arrest
To demonstrate that induced expression of p27Kip1 can elicit G1 cell cycle arrest independently of androgen, a clone of 104-R2 cells expressing a modified lac repressor protein (31) was infected with LNXRO2 retrovirus (32) containing a cDNA encoding murine p27Kip1. LNCaP 104-R1 cells were not used because we were unable to obtain 104-R1 clones that stably expressed the lac repressor protein. Expression of p27Kip1 was induced in three selected clones by incubation in 4 mM isopropyl ß-D-thiogalactoside (IPTG) for 48 h (Fig. 9AGo). Murine p27Kip1 had a faster mobility on Western blots than endogenous human p27Kip1 and was induced at three different levels in the three clones, with the lowest level corresponding roughly to the induction of endogenous p27Kip1 by androgen. Interestingly, endogenous human p27Kip1 was induced in IPTG-treated clones as well as the retroviral mouse p27Kip1. Flow cytometry of retrovirally infected cells incubated in the presence or absence of IPTG showed that expression of p27Kip1 was sufficient to arrest cells in the G1 stage, and the extent of G1 arrest correlated with the level of p27Kip1 expression in the three clones (Fig. 9BGo). IPTG had no effect on parental 104-R2 cells expressing only the lac repressor. A higher level of murine p27Kip1 was required to elicit the same degree of G1 arrest observed by androgen induction of endogenous p27Kip1. This suggests that murine p27Kip1, which has 87% amino acid identity to human p27Kip1, is not as active in LNCaP cells as human p27Kip1. However, the observation that endogenous p27Kip1 is elevated after IPTG treatment may mean that murine p27Kip1 acts to cause G1 arrest indirectly by raising endogenous human p27Kip1 levels through an unknown mechanism. Since p27Kip1 is thought to be degraded through the ubiquitin/proteosome pathway (33), it is possible that overexpressed murine p27Kip1 competes with human p27Kip1 at steps along this pathway.



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Figure 9. Induction of G1 Arrest by Exogenous Expression of p27Kip1

A, IPTG induces expression of murine p27Kip1 in three 104-R2 clones (5 11 16 ) infected with LNXRO2-murine p27Kip1 retrovirus. Parental 104-R2 cells not infected with the LNXRO2-murine p27Kip1 retrovirus are designated as 3'SS. Cells (5 x 105) were plated in 6-cm plates and grown for 48 h. Medium was then replaced with control medium or medium containing 4 mM IPTG and cells were incubated for an additional 48 h. Cells were then lysed by the addition of 0.5 ml 2x Laemmli gel loading buffer lacking bromophenol blue. Aliquots containing 20 µg of protein were run on 12% SDS-PAGE gels. B, IPTG induces G1 arrest in 104-R2 clones infected with LNXRO2-murine p27Kip1 retrovirus. Flow cytometric analysis was performed on cells treated with IPTG as described in panel A. Data represent the mean ± SE of three independent experiments.

 
p21waf1/cip1 and p27Kip1 Inhibit Cdk2 but Not Cdk4 Activity in 104-R1 Cells Arrested by Androgen
To identify the targets of p21waf1/cip1 and p27Kip1 in androgen-repressed LNCaP cells, 104-R1 cells were incubated for 24 or 48 h in the absence or presence of R1881, and whole cell extracts were immunoprecipitated with either Cdk2 or Cdk4 polyclonal antibody that had been preadsorbed to Protein A-agarose beads. Washed immunoprecipitates were separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with p21waf1/cip1 or p27Kip1 antibodies. Both p21waf1/cip1 and p27Kip1 proteins coimmunoprecipitated with Cdk2 but not Cdk4, and androgen increased the amount of p21waf1/cip1 and p27Kip1 complexed with Cdk2 by roughly the same extent as was seen in total cell extracts (Fig. 10AGo). Under identical conditions, the Cdk4 antibody was able to coimmunoprecipitate p21waf-1/cip-1 and Cdk4 in PC-3 prostate carcinoma cells overexpressing p21waf1/cip1 (data not shown). The total amount of Cdk2 and Cdk4 in cell lysates was not altered by androgen (Fig. 10BGo). Cdk2 and Cdk4 immunoprecipitates were used in in vitro assays of kinase activity using histone H1 and a glutathione-S-transferase (GST)-Rb fusion protein (Rb residues 769–921) as substrates. Cdk2 activity in immunoprecipitates from R1881-treated 104-R1 cells was reduced more than 95% compared with untreated cells, but had no effect on Cdk4 activity (Fig. 10CGo). In a time course study, Cdk2 activity, as measured by histone H1 phosphorylation, was not reduced by androgen treatment of cells until 24 h after addition of androgen and was almost complete by 48 h (Fig. 10DGo). This result mirrors the onset of G1 arrest and the accumulation of p27Kip1 but not the induction of p21waf1/cip1 (Figs. 6CGo and 7AGo). Therefore, in LNCaP 104-R1 cells, although androgen transiently induces the accumulation of p21waf1/cip1 before that of p27Kip1, it is the accumulation of p27Kip1 and not p21waf1/cip1 that correlates directly with inhibition of Cdk2 activity and onset of G1 arrest.



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Figure 10. Effect of Androgen on Cdk2 and Cdk4 Activity in 104-R1 Cells

A, Androgen induces association of p21waf1/cip1 and p27Kip1 with Cdk2 but not Cdk4. 104-R1 cells were grown for 24 h (p21waf1/cip1) or 48 h (p27Kip1) in the absence or presence of 10 nM R1881. Whole cell lysates were made, and Cdk2 and Cdk4 were immunoprecipitated from lysate (2 mg protein) with Protein A-agarose preloaded with anti-Cdk2 or anti-Cdk4 antibody. After washing, immunoprecipitated protein was eluted by boiling in Laemmli SDS-PAGE loading buffer, separated on 12% SDS-PAGE gels, blotted onto nitrocellulose, and probed with anti-p21waf1/cip1 or anti-p27Kip1 antibody. B, Immunoblot showing that the total amount of Cdk2 and Cdk4 in 104-R1 cells is unaffected by R1881. C, Inhibition of Cdk2 activity by androgen in 104-R1 cells. Cdk2 and Cdk4 immunoprecipitates from 104-R1 cells grown in the absence or presence of 10 nM R1881 for 48 h were used in histone H1 and GST-Rb phosphorylation reactions. D, Time course assay of Cdk2 activity, using histone H1 as substrate, from Cdk2 immunoprecipitates obtained from 104-R1 cells treated with 10 nM R1881 for the indicated periods. Blots and autoradiograms are representative of three independent experiments.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The findings reported in this paper support the contention that androgen-dependent prostate tumor cells can adapt to an androgen-depleted environment and give rise to androgen-independent cells that can grow unchecked by antiandrogen strategies. These hormone-independent cells instead are paradoxically repressed by androgen through a mechanism involving induction of the cdk inhibitor p27Kip1. Because the 104-S cell line is clonally derived, the possibility that preexistent androgen-independent cells emerge by selection (34) is excluded. LNCaP 104-S cells, while not strictly androgen-dependent in vitro, are extremely slow growing in the absence of androgen and must undergo more than 2 yr of passage in androgen-depleted medium during the progression to first the 104-R1 stage and then to the 104-R2 stage. 104-R2 cells proliferate in androgen-depleted medium at a rate equivalent to 104-S cells grown in an optimal concentration of androgen. LNCaP 104-S tumors grown in male athymic mice rapidly regress after castration, suggesting that 104-S cells are strictly androgen-dependent in vivo (11). The insensitivity of 104-R1 and 104-R2 cells to Casodex inhibition shown in this report indicates that 104-R1 and 104-R2 cells do not scavenge residual androgen from the culture medium but are truly proliferatively independent of androgen. If an extremely low concentration of androgen was supporting 104-R1 and 104-R2 cell proliferation, then a many log-fold excess of Casodex should have repressed proliferation even more effectively than in 104-S cells growing in 0.1 nM R1881. Hypersensitivity to estradiol was observed in MCF-7 human breast cancer cells after hormone deprivation, although the growth of hypersensitive cells was not repressed by estradiol (35). The change was characterized as an adaptive response to low levels of hormone.

Significantly, LNCaP 104-R1 and 104-R2 cells, although proliferatively independent of androgen, continue to express high levels of functional AR. LNCaP 104-R1 and 104-R2 cells may therefore be classified as both androgen-independent and androgen-sensitive. This is consistent with the finding that most androgen-independent metastatic prostate tumors retain AR expression. When the AR status of distant androgen-independent metastatic prostatic tumors in 18 patients was analyzed, all were found to express AR (36). Earlier studies had also found that the majority of prostate tumors from patients experiencing relapse exhibited widespread AR expression (37, 38). Additionally, amplification of the AR gene was observed in 30% of metastatic prostate tumors after androgen ablation (39, 40). In the Shionogi mammary carcinoma, progression to androgen independence does not correlate with loss of AR expression or functionality (41, 42). It is possible that functional AR is required for the proliferation of androgen-independent cells if AR is activated independently of ligand, e.g. through peptide growth factors (43, 44), or through activation of a protein kinase A-signaling pathway (45). However, activation of AR by insulin-like growth factor-I or through activation of protein kinase A is inhibited by Casodex (43, 45), while proliferation of LNCaP 104-R1 and 104-R2 cells is clearly insensitive to Casodex in the absence of androgen. Additionally, LNCaP 104-R1 and 104-R2 cells do not exhibit androgen-independent PSA expression, which might be expected if AR could activate transcription in the absence of androgen. Androgen-dependent PSA expression in LNCaP 104-R1 and 104-R2 cells stands in contrast to the results of Gleave et al. (46) and Sato et al. (14), who found that LNCaP tumors grown in castrated athymic mice can acquire androgen-independent growth and androgen-independent PSA production. The recently established human prostate tumor cell line, ARCaP, is repressed by androgen (47). Unlike LNCaP 104-R1 and 104-R2 cells, however, ARCaP cells exhibit androgen-repressible PSA expression, which coincides with the repressive effect of androgen on ARCaP cell proliferation.

LNCaP 104-R1 and 104-R2 cells appear to be hypersensitive to the repressive effects of 0.1 nM R1881 on proliferation, which can be seen in 104-S cells (grown in medium supplemented with untreated FBS; Ref. 10) and most other LNCaP cells (48, 49, 50, 51) only at higher concentrations of R1881 or other androgens. Soto et al. (20) observed similar hypersensitivity with LNCaP-LNO cells. The heightened sensitivity in 104-R1 and 104-R2 cells is probably due to heightened AR expression as compared with 104-S cells. Consistent with this is the recent observation that 1{alpha},25-dihydroxyvitamin D3 up-regulates AR expression and synergizes with 5{alpha}-dihydrotestosterone (DHT) in the repression of LNCaP cell proliferation (52). LNCaP 104-R1 and 104-R2 cells, however, can also adapt to high concentrations of androgen so that previously repressive concentrations are required for optimal proliferation rate. This adaptation is accompanied by reduced AR expression, providing further evidence that the level of AR expression may be related to whether a positive or a negative androgenic effect on proliferation is seen. Langeler et al. (19) and Soto et al. (20) had found previously that LNCaP-FGC and LNCaP-LNO cells can also adapt to repressively high androgen concentration.

Androgen treatment of 104-R1 cells results in the transient elevation of p21waf1/cip1 levels. The significance of this transient androgen-induced elevation is not clear at the present time. This elevation does not correlate with the kinetics of G1 arrest or inhibition of Cdk2 activity. Forty-eight hours after addition of androgen, the level of p21waf1/cip1 has returned to basal levels, while Cdk2 activity is maximally repressed. Further, p21waf1/cip1 is induced only minimally by androgen at a 10 nM concentration in 104-R2 cells, but these cells are substantially arrested at both 0.1 and 10 nM R1881 concentrations. Unlike p21waf1/cip1, elevated p27Kip1 levels are sustained for prolonged periods in both 104-R1 and 104-R2 cells treated with androgen. This sustained level of p27Kip1 expression coincides with the repression of Cdk2. Moreover, expression of exogenous p27Kip1 induced by IPTG clearly elicited G1 arrest in 104-R2 cells in the absence of androgen, suggesting that p27Kip1 accumulation by itself can account for all of the repressive effects of androgen. Therefore, we conclude that androgen-induced G1 arrest of 104-R1 and 104-R2 cells is mediated by the accumulation of p27Kip1. We cannot, however, rule out the participation of cdk inhibitors for which we have not screened. Both p21waf1/cip1 and p27Kip1 have recently been implicated in progesterone-induced growth arrest of T47D breast cancer cells (53). These investigators also found that p21waf1/cip1 induction precedes induction of p27Kip1 and is more transient in duration.

The observation that p21waf1/cip1 or p27Kip1 steady state mRNA levels are not significantly affected by androgen leads to the conclusion that elevation of these cdk inhibitors occurs by a translational or posttranslational mechanism. While regulation of p21waf1/cip1 is known to occur principally at the transcriptional level, interference with protein degradation rate has been reported to induce its accumulation (54). Posttranscriptional control is consistent with previous findings that p27Kip1 is regulated at the level of rate of translation and protein turnover (33, 55, 56). Recently, Peng, et al. (57) reported that the antiproliferative activity of an antibody directed against epidermal growth factor receptor in DU145 prostate carcinoma cells coincided with elevation of both p27Kip1 mRNA and protein. As in the present study, Cdk2 and not Cdk4 (nor Cdk6) was found to be inhibited by p27Kip1 in DU145 cells.

It is possible that androgen regulates p27Kip1 through a mechanism involving c-myc. Previous studies by others have shown that c-Myc overexpression can block p27Kip1-induced cell cycle arrest (58), and c-Myc activation or overexpression leads to the loss of p27Kip1 protein in Rat1 fibroblasts (59) and in rat embryo fibroblasts when co-expressed with activated H-ras (60). Expression of c-myc is repressed by androgen in 104-R1 cells (10), and others have reported that androgen represses c-myc expression in LNCaP cells through a rapid transcriptional mechanism (61). Therefore, it is possible that p27Kip1 level rises as a result of a drop in c-myc expression. The observation that constitutive retroviral c-myc overexpression in 104-R1 cells blocks the repression of cell proliferation by androgen (10) is consistent with this mechanism. Preliminary data show that c-myc overexpression blocks androgen-induced G1 arrest in 104-R1 cells and reduces the accumulation of p27Kip1 in 104-R1 cells after androgen treatment (our unpublished observations).

LNCaP 104-R1Ad cells that have adapted to growth in androgen no longer accumulate high levels of either p21waf1/cip1 or p27Kip1 in response to androgen, suggesting that the androgen-signaling pathway leading to the accumulation of these inhibitors in these cells has somehow become altered. It is possible that a second, distinct proliferation pathway (or set of pathways), that is activated in 104-S cells growing in 0.1 nM R1881 and is masked in hypersensitive 104-R1 and 104-R2 cells by the repressive p27Kip1 pathway, is now revealed in these cells. It is unclear, however, why 104-R1Ad cells lose some of their previously acquired ability to grow independently of androgen. This particular phenomenon may be related to the observation that prostate tumor cells subjected to intermittent androgen deprivation remain androgen-dependent for a longer time than cells subjected to continuous androgen deprivation (14, 62, 63).

Recently, expression of the cdk inhibitor p16INK4a was reported to be down-regulated in LNCaP-FGC cells induced to proliferate by androgen (64). In LNCaP 104-S and 104-R1Ad cells, down-regulation of both p21waf1/cip1 and p27Kip1 was much stronger than the slight down-regulation of p16INK4a we observed after androgen treatment. In addition, we did not observe changes in Cdk2 or Cdk4 expression after androgen treatment, in contrast to the report of Lu et al. (64). This variability among LNCaP sublines suggests that cdk and cdk inhibitor expression may be far downstream of the primary sites of androgen action in the induction of cell proliferation. The mechanism(s) by which androgen induces cell proliferation in 104-S cells is probably fundamentally more complex than the mechanism by which androgen induces G1 arrest in 104-R1 and 104-R2 cells. However, both mechanisms appear to be subject to reversible, epigenetic adaptation driven by changes in the hormonal environment. Further identification of gene products and pathways that mediate androgen-dependent growth, androgen-independent growth, and androgen repression of growth in LNCaP cells in different hormonal environments may lead to a better general understanding of how hormone-dependent prostate tumor cells become independent and ultimately, how to better treat hormone-independent prostate cancer.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
The LNCaP 104-S and 104-R1 (formerly called 104-R) cell lines were described previously (10). [{alpha}-32P]UTP (800 Ci/mmol), [{alpha}-32P]dCTP (3000 Ci/mmol), and [{gamma}-32P]ATP (5000 Ci/mmol) was purchased from Amersham (Arlington Heights, IL). A Super-Signal chemiluminescence detection kit was from Pierce Chemical Co. (Rockford, IL). Restriction endonucleases and other enzymes were purchased from Boehringer Mannheim (Indianapolis, IN), New England Biolabs (Beverly, MA), Stratagene (La Jolla, CA), and Life Technologies (Gaithersburg, MD). AN-21, a polyclonal rabbit antibody raised against a 21-residue peptide corresponding to the first 21 amino acids of human and rat AR was described previously (10). Polyclonal antibodies against p21waf-1/cip-1 (C-20), p27Kip1 (C-19), p57Kip2 (C-20), p16INK4a (C-20) and p15INK4b (C-20), Cdk2 (M2), and Cdk4 (C-22) and a GST-Rb fusion protein (residues 769–921) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The 3'SS vector and anti-lac repressor antibody were purchased from Stratagene, and the LNXRO2 vector was generously provided by I. Roninson at the University of Illinois at Chicago. Histone H1 from calf thymus was purchased from Boehringer Mannheim (Indianapolis, IN). R1881 was from New England Nuclear (Boston, MA). Casodex (ICI 176, 334; (2RS)-4'-cyano-3-(4-fluorophenylsulfonyl)-2-hydroxy-2-methyl-3'-(trifluoromethyl)-propionanilide; bicalutamide) was a generous gift from Zeneca Pharmaceuticals (Wilmington, DE).

Cell Culture and Stable Retroviral Infection
LNCaP 104-S, 104-R1, and 104-R2 cells were passaged and maintained as described previously (10). One passage consisted of cells split 1:10 and grown for 5 days with a medium change on day 3. Cells were infected with amphotropic pMV7 control retrovirus and pMV7 c-myc retrovirus generated in the packaging cell line PA317 as described previously (10) and selected by growth in 400 µg/ml G418 (Geneticin, Life Technologies). One hundred to 200 colonies were pooled and maintained in 400 µg/ml G418. For proliferation assays, cells were trypsinized and resuspended in DMEM supplemented with 10% dextran-coated CS-FBS (65) (Summit Biotechnology, Ft. Collins, CO). Cells (3 x 104 cells per well) were added to wells in a 12-well dish containing 2 ml DMEM supplemented with 10% CS-FBS and the appropriate concentration of R1881 and/or Casodex. The amount of ethanol added as vehicle never exceeded 0.1% of the total volume.

RNA Analysis
Isolation of total RNA and RNAse protection analysis of PSA mRNA were carried out as described previously (10). For RNAse protection assay of p21waf1/cip1 mRNA, an antisense 32P-labeled riboprobe that protects a 419-base p21waf1/cip1 mRNA fragment was synthesized by using T7 RNA polymerase and PstI-digested p21waf1/cip1 cDNA (66) inserted into pBluescript SK+ (pBS; Stratagene). Template for a 355-base human p27Kip1 antisense mRNA was isolated by PCR amplification from an LNCaP 104-R1 UniZAP cDNA library (10) using Pfu polymerase (Stratagene) and the primers 5'-ACCACGAAGAGTTAACCCGG and 5'-GGTCGCTTCCTTATTCCTGC, which correspond to nucleotides 110–129 and 464–445 of human p27Kip1 (67). Template for a 109-base glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antisense mRNA probe was isolated by PCR amplification from the same library using the primers 5'-CCATGGGGAAGGTGAAGGTCGGAG and 5'-GGGTCATTGATGGCAACAATA-TCC, which correspond to nucleotides 59–82 and 167–144 of human GAPDH (68). The amplification products were ligated into EcoRV-digested pBS. For generation of antisense probes, pBS-p27Kip1 was linearized with HindIII and transcribed with T3 RNA polymerase; pBS-GAPDH was linearized with EcoRI and transcribed with T7 RNA polymerase. The amount of radioactivity in protected bands was quantified by scanning dried gels in an AMBIS radioanalytic imaging system (AMBIS Systems, San Diego, CA).

Immunoblot Analysis
Extraction of cellular proteins and immunoblot analysis with polyclonal anti-AR (AN-21) antibodies were performed essentially as described previously (10). Prestained molecular weight markers were from BRL Life Technologies. For analysis of p21waf1/cip1 and p27Kip1 protein levels by SDS-PAGE, 12% polyacrylamide gels were used. Protein concentration in cell lysates was determined with the Bradford reagent using BSA standards. All antibodies were used at a concentration of 0.5 µg/ml.

Flow Cytometric Analysis
LNCaP 104 cell lines and retrovirally infected LNCaP 104-R1 cells were plated (5 x 105 cells per 6-cm dish) in DMEM supplemented with 10% CS-FBS and were incubated overnight. The next day, medium was replaced with fresh medium containing R1881 at various concentrations or ethanol vehicle, and cells were grown for either 4 days (with a change of medium on day 2) or for varying lengths of time in the time course experiment. Cells were trypsinized, resuspended in complete medium, pelleted, and fixed in 70% ethanol/30% PBS overnight at -20 C. Cells were then washed in PBS, incubated for 30 min with 100 µg/ml RNAse A, and stained with 50 µg/ml propidium iodide in PBS. Cell cycle profiles and distributions were determined by flow cytometric analysis of 104 cells using the LysisII program on a FACScan flow cytometer (Becton-Dickinson, San Jose, CA). Clumped cells were excluded from cell cycle distribution analysis by gating.

Induced Expression of Exogenous p27Kip1
LNCaP 104-R2 cells were transfected with the 3'SS vector (Stratagene) encoding a modified lac repressor protein (31). Clones expressing large amounts of repressor were selected in hygromycin B and screened with anti-lac repressor antibody (Stratagene). One clone, clone 8, was infected with retrovirus generated as described above from the vector LNXRO2 (32) containing a cDNA encoding mouse p27Kip1, and clones were selected in G418. The LNXRO2 vector contains an Rous sarcoma virus (RSV) promoter with two lac operator sequences upstream of the cDNA insert site. Three clones, 5, 11 and 17, exhibited induced expression of p27Kip1 after 48 h of incubation in 4 mM IPTG and were used in subsequent cell cycle analysis.

Immunoprecipitation and Kinase Assays
LNCaP 104-R1 cells were plated and treated with R1881 as described above. After 48 h of incubation in the presence or absence of R1881, cells were washed twice in PBS, lysed, and scraped from the dishes in 1 ml of buffer containing 50 mM Tris HCl (pH 8.0), 100 mM NaCl, 0.5% NP-40, 0.5 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, and 1 µg/ml leupeptin. Lysate was agitated for 15 min at 4 C and centrifuged for 10 min at 4 C to pellet debris. Protein concentration of supernatant was determined with Bradford reagent, and an aliquot of the supernatant (2 mg of protein) was preincubated for 1 h at 4 C with 1 µg rabbit IgG and Protein A-agarose beads (25 µl packed volume; Santa Cruz Biotechnology) equilibrated with lysis buffer. Beads were removed by centrifugation, and lysates were incubated for 1 h at 4 C with Protein A-agarose beads (25 µl packed volume) loaded with 1 µg rabbit anti-Cdk2 or anti-Cdk4 polyclonal antibody. Beads were washed three times with lysis buffer, and immunocomplexes were eluted by boiling in 25 µl 2x Laemmli gel loading buffer. After electrophoresis and transfer, cdk inhibitors were detected using goat anti-p21waf1/cip1 or anti-p27Kip1 antibody and horseradish peroxidase-conjugated donkey anti-goat IgG (Santa Cruz Biotechnology). In vitro cdk phosphorylation assays were performed according to Serrano et al. (69). Cdk2 and Cdk4 were immunoprecipitated as described above. Protein A-agarose beads were washed twice in kinase buffer (20 mM Tris-HCl, 10 mM MgCl2, 1 mM dithiothreitol, 1 mM EDTA, pH 8.0) after which beads were resuspended in 25 µl kinase buffer containing 10 µCi [{gamma}-32P]ATP, 1 µM unlabeled ATP, and 2.5 µg of either histone H1 or GST-Rb fusion protein. After 20 min at room temperature, reactions were terminated by adding 0.33 volumes of 4x Laemmli gel loading buffer and boiling. Aliquots were run on 10% SDS-PAGE gels, which were dried and analyzed by autoradiography.


    ACKNOWLEDGMENTS
 
The authors thank Dr. Richard Hiipakka for helpful comments on the manuscript and Dr. Igor Roninson (University of Illinois at Chicago) for the LNXRO2 vector.


    FOOTNOTES
 
Address requests for reprints to: Shutsung Liao, The Ben May Institute for Cancer Research, Box MC6027, The University of Chicago, 5841 South Maryland Avenue, Chicago, Illinois 60637.

This work was supported by NIH Grants CA-58073 and DK-41670 to S.L. and by NIH Grant CA-71874 and American Chemical Society Grant CB-133 to N.H.

Results described in this paper were presented at the Schilling Research Conference, Hormones and Cancer in Santa Cruz, CA, September 18–21, 1997.

1 Present address: Department of Molecular Genetics, The University of Illinois College of Medicine at Chicago, 900 South Ashland Avenue, Chicago, Illinois 60607. Back

Received for publication January 28, 1998. Revision received March 17, 1998. Accepted for publication March 25, 1998.


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