From the Ludwig Institute for Cancer Research, the
§ Department of Medicine, the ¶ Center for Molecular
Genetics, and the
Cancer Center, University of California at San
Diego, La Jolla, California 92093-0660
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ABSTRACT |
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Prostatic epithelial cells and most primary prostate tumors are dependent on androgen for growth, but how androgen regulates cellular proliferation remains unsolved. Using poorly understood mechanisms, recurrent tumor cells evade the androgen requirement. We utilized androgen-dependent prostatic tumor cells to demonstrate that androgen exerts its effect on the cell cycle by influencing specific aspects of G1-S progression. Androgen depletion of these cells results in early G1 arrest, characterized by reduced cyclin-dependent kinase activity, and underphosphorylated retinoblastoma tumor suppressor protein (RB). The reduction in kinase activity was partially attributed to reduction of specific G1 cyclins and alternate regulation of cyclin-dependent kinase inhibitors. Using this information, we developed a reliable assay to assess the ability of specific G1 regulatory proteins to circumvent these controls and promote androgen-independent growth. As expected, inactivation of RB was required for progression through the cell cycle. Surprisingly, overexpression of G1 cyclins, which drives RB phosphorylation, was insufficient to promote androgen-independent cell cycle progression. Introduction of viral oncoproteins did promote G1-S progression in the absence of androgen, dependent on their ability to sequester RB and related proteins. These results provide the first evidence that multiple elements governing the G1-S transition dictate androgen-dependent growth, and the formation of androgen-independent prostatic tumors may be because of misregulation of these processes.
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INTRODUCTION |
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Prostatic epithelial cells are dependent on androgen for survival and enter programmed cell death following hormone ablation (1-3), resulting in a massive involution of the prostate gland (4). Hormone ablation also results in the death of most primary prostate tumor cells, but this relief is transient, because recurrent tumors usually develop, and the cells of these recurrent tumors are able to proliferate and metastasize in the absence of serum androgen (5).
Despite the importance of androgens for the growth of prostatic epithelia, the mechanisms of their effect on the stimulation of proliferation and inhibition of cell death remain obscure. Similarly, little is known about how recurrent prostate tumor cells are able to proliferate in the absence of serum androgen. It is known that androgen exerts its biological effects through the androgen receptor, a member of the nuclear steroid hormone receptor family (6). However, target genes of the androgen receptor that promote proliferation remain unknown. In this report, we sought to identify downstream targets of the androgen-responsive proliferative pathway by determining the effects of androgen withdrawal on the cell cycle machinery of androgen-dependent cells. In addition, we determined how the requirement for androgen might be circumvented by misregulation of specific cell cycle pathways.
While in G1, mammalian cells evaluate growth-promoting and/or growth-inhibitory environmental cues to either progress through the mitotic cell cycle or enter into quiescence (7, 8). Extracellular signals ultimately impinge on cyclin-dependent kinases (CDKs),1 which dictate transitions into and within the proliferative cell cycle (9, 10). The activity of these kinases is regulated at many levels, including (i) both inhibitory and stimulatory phosphorylation events; (ii) binding to cyclins; and (iii) binding to CDK inhibitors (11, 12).
Cell cycle progression in G1 is regulated by the activity of cyclin D-, E-, and A-associated CDKs. D-type cyclins (cyclins D1, D2, and D3) are the earliest of the cyclins to accumulate in the cell cycle, and once produced, they associate with and promote the activation of their CDK partners, CDK4 or CDK6 (13, 14). Subsequent to CDK4(6)-cyclin D activation, cyclin E accumulates and activates CDK2 for progression through G1. Cyclin A, whose expression is initiated temporally after cyclin E in late G1, associates with both CDK2 (S phase) and CDC2 (late S and G2). Cyclin A-associated kinases are required not only for progression through late G1 but also for completion of S phase (10). All three G1 cyclins shorten the G1 phase when ectopically expressed and are required for progression from G1 to S phase (15, 16).
In addition to regulation by cyclin association and phosphorylation, mammalian CDKs are regulated by cyclin-dependent kinase inhibitors (CDIs). These molecules attenuate the kinase activity of CDKs by binding directly with active CDK-cyclin complexes or by competing with cyclin for binding to CDKs (12). The CDIs can be divided into two families, the INK4 family (p16ink4a, p15ink4b, p18ink4c, and p19ink4d), which act early in G1 to inhibit the activity of CDK4 and CDK6, and the CIP family (p21cip1, p27kip1, and p57kip2), which inhibit a large number of CDK-cyclin complexes (17).
The principal substrates for G1 cyclin-associated kinase
activity appear to be the retinoblastoma tumor suppressor protein, RB,
and the related proteins p107 and p130 (collectively deemed the
"pocket proteins") (18). Prior to their phosphorylation by
G1 CDKs, RB, p107, and p130 are believed to prevent
progression to S phase by sequestering key proteins required for DNA
synthesis, such as the E2F family of heterodimeric transcription
factors (19). E2F regulates the expression of a number of genes
required for cell cycle expression, for example, cyclin E, cyclin A,
and DNA polymerase (20, 21). Binding of RB to specific E2F family members results in repression of these E2F-responsive genes, thus preventing S phase initiation. Upon phosphorylation of RB, E2F and
other S phase promoting proteins are released and activated (19-22).
Although cyclin E and cyclin A-associated complexes are hypothesized to
phosphorylate substrates in addition to RB, a large body of evidence
supports the notion that the main function of CDK4-cyclin D or
CDK6-cyclin D complexes is to phosphorylate RB (13). Specifically,
CDK4-associated kinase activity is no longer required for cell cycle
progression in RB-deficient cells (23). Although initiation of RB
phosphorylation is initially catalyzed by the G1
cyclin-associated CDKs, cyclin B-associated complexes (CDC2-cyclin B),
which promote G2/M transition, are thought to also
contribute to the post-S phase maintenance of RB phosphorylation. In
fact, phosphorylation of RB is maintained until the completion of
mitosis, at which time RB is dephosphorylated to allow the cells to
enter a new G1 phase or to exit the cell cycle (22).
Importantly, although phosphorylation of RB by the G1 CDKs is required to traverse the G1-S transition, it is not sufficient for S phase progression. This fact is evident in that (i) RB-negative cells still require growth factors for cell cycle progression (24); (ii) premature G1 cyclin expression shortens G1 but does not eliminate the requirement of the cell for growth factors (15, 25); and (iii) specific late-G1 arrested cells contain hyperphosphorylated RB (26). As such, mechanisms separate from the regulation of RB phosphorylation must contribute to the control of cell cycle progression.
Here, we sought to determine the effects of androgens on the regulation of key cell cycle regulatory proteins and to elucidate cell cycle pathways that may be deregulated to achieve androgen-independent growth. We utilized cultured LNCaP human prostatic adenocarcinoma cells (27), which retain most of the features common to androgen-responsive tumor cells, especially: (i) the expression of both the androgen receptor and prostate-specific antigen (28, 29); (ii) stimulation of proliferation by androgens in vitro (30); (iii) formation of tumors in male mice but only weakly in castrated male mice or female mice (27, 31); and (iv) growth arrest upon androgen removal (32). The results presented provide the first evidence that multiple G1 elements are responsive to the presence of androgen and act to prevent G1-S progression in its absence. Surprisingly, these regulatory mechanisms cannot be circumvented through the ectopic expression of G1 cyclins. However, oncoproteins capable of sequestering pocket proteins rendered prostatic carcinoma cells androgen-independent for proliferation. Thus, although androgen acts as a growth stimulus for prostatic cells, it does so in a manner that is distinct from that of many mitogens.
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EXPERIMENTAL PROCEDURES |
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Cell Culture-- The human prostatic adenocarcinoma cell line, LNCaP, was obtained from the American Type Culture Collection, and passages 25-39 were used in the experiments described. For regular passage, cells were grown in Improved Minimum Essential Medium (Biofluids) containing 10% heat-inactivated fetal bovine serum (Hyclone) supplemented with 100 units/ml penicillin-streptomycin and 2 mM L-glutamine at 37 °C in a humidified atmosphere of 5% CO2. For growth in androgen-depleted media, cells were propagated in improved minimum essential medium containing 5% charcoal dextran-treated fetal bovine serum (Hyclone). Dihydrotestosterone (Steraloids), when added, was diluted in ethanol and then medium and used at a final concentration of 0.1 nM (ethanol was less than 0.1% in the medium). Medium containing hormone and/or ethanol was changed every 48 h. For growth analysis, cells were seeded at a density of 2-3 × 104 cells/well in poly-L-lysine-coated 6-well dishes, and medium was changed every 48 h. At the times indicated, cells were washed with Dulbecco's phosphate-buffered saline lacking calcium and magnesium (MediaTech) and harvested using 0.05% trypsin-EDTA solution (Life Technologies, Inc.). Cell numbers were each determined using a hemocytometer and trypan blue exclusion. All conditions were tested in triplicate, and the averages/deviations are shown. Poly-L-lysine coating was carried out by incubating freshly diluted 0.001% poly-L-lysine (Sigma) in H2O on the desired substrate for 20-30 min in a sterile tissue culture hood. After incubation, poly-L-lysine was completely aspirated, and substrates were thoroughly dried. Poly-L-lysine had no effect on the growth or cell cycle profile of LNCaP cells, under all conditions used (data not shown).
Plasmids--
Plasmid expressing the green fluorescent protein,
pGreen Lantern, was obtained from Life Technologies, Inc.
pRSV-T-antigen was a gift of Dr. Suresh Subramani (University of
California at San Diego). Plasmids expressing the SV40 early region and
corresponding mutants (WT-T-Ag, PVU1-T-Ag, K1-T-Ag, and 2831-T-Ag) were
kindly provided by Dr. Charles Cole (Dartmouth University) and have
been previously described (33). Plasmids WT-E1A, CXDL-E1A, and
30-85-E1A, which express E1A constructs from the cytomegalovirus
promoter, were obtained from Dr. David Livingston (Dana-Farber Cancer
Institute). Plasmids expressing the E1A splice variants 12 S and 13 S
from the cytomegalovirus promoter were obtained from Dr. Gilbert Morris (Tulane University). Plasmids expressing WT-LP, PSM.7-LP, cyclin A, and
p16ink4a from the cytomegalovirus promoter were generous gifts
of Dr. Jean Wang (University of California at San Diego) and have been previously described (34). Cyclin D1 and cyclin D3 expression constructs (expressed from the Rous sarcoma virus promoter) were obtained from Dr. Charles Sherr (St. Jude's Children's Research Hospital) (35). The cyclin E expression construct (expressed from a
long terminal repeat) was obtained from Dr. James Roberts (Fred
Hutchison Cancer Research Center) (36).
Transfections-- For transfection, LNCaP cells were seeded at a density of 3 × 105 cells/well in poly-L-lysine-coated wells of 6-well dishes (or poly-L-lysine-coated coverslips resting in 6-well dishes). After 48 h, wells were washed one time with serum-free and antibiotic-free improved minimum essential medium. Transfection substrate (1 ml of serum-free/antibiotic-free improved minimum essential medium, 30 µg of Lipofectin (Life Technologies, Inc.), and 5 µg of DNA) was then applied and allowed to incubate for 6 h, after which the serum-containing medium indicated was added back. To monitor protein expression, cells were harvested 48 h post-transfection. Co-transfection of pCMV-LP and the cyclin expression constructs were performed with a 1:2 ratio of plasmid DNA. To monitor S phase progression, co-transfections with pGreen Lantern (1.0 µg) and a secondary effector expression plasmid (4.0 µg) were carried out. Cells were labeled 48 h after transfection with cell proliferation labeling reagent (Amersham Pharmacia Biotech), according to manufacturer's recommended protocol. Pulse labeling was continued for 14 h, at which time cells were fixed and processed for immunofluorescence.
Flow Cytometry-- LNCaP cells were propagated under the conditions described and harvested at the times shown by trypsinization, washed once with phosphate-buffered saline, fixed with ethanol, and stained with propidium iodide as described previously (37). Analysis of the stained cells was carried out using a FACSort (Becton Dickinson) and the CELLFit software program. At least 10,000 forward scatter gated events were collected for each sample, and each experiment was performed in triplicate.
Immunofluorescence-- To monitor BrdUrd incorporation of both transfected and untransfected cells, cells were fixed and subjected to indirect immunofluorescence as described previously (38). Cells were visualized using a Zeiss Axiophot with a 40× objective, and pictures were captured using a Color Chilled 3CCD Camera (Hamamatsu).
Immunoblotting--
For immunoblotting, 2-4 × 106 cells were harvested at the times indicated by
trypsinization and washed with phosphate-buffered saline. Cells were
lysed in ice-cold RIPA buffer containing protease inhibitors (10 µg/ml 1, 10 phenanthroline, 10 µg/ml aprotinin, 10 µg/ml
leupeptin, and 1 mM phenylmethylsulfonyl fluoride) and phophatase inhibitors (10 mM sodium fluoride, 10 mM sodium pyrophosphate, 0.1 mM sodium
vanadate, 15 mM p-nitrophenylphosphate, and 10 mM -glycerophosphate) for 15 min on ice. Following brief
sonication, lysates were clarified at 10,000 × g
(4 °C for 15 min). Protein concentration of the clarified lysates
was determined using the Bio-Rad DC Protein Assay Reagent. Lysates were
denatured by boiling in SDS loading buffer, and 20 µg of each sample
was resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and
transferred to Immobilon-P (Millipore) by standard methods. Blots were
probed for the following proteins with polyclonal antibodies: RB and LP
(851, gift of Dr. Jean Wang, University of California at San Diego);
p130 (C-20, Santa Cruz Biotechnology); CDK4 (H-22, Santa Cruz
Biotechnology); cyclin D3 (C-16, Santa Cruz Biotechnology); CDK2 (M2,
Santa Cruz Biotechnology); cyclin A (BF683, PharMingen); cyclin E
(HE12, PharMingen); p21 (C-19, Santa Cruz Biotechnology); p27 (N-20,
Santa Cruz Biotechnology); p16 (G175-405, PharMingen); T-Ag (Pab 101, Santa Cruz Biotechnology); and E1A (13 S-5, Santa Cruz
Biotechnology). Goat anti-mouse horseradish peroxidase or protein
A-horseradish peroxidase (Bio-Rad) was used for antibody visualization
via enhanced chemiluminescence (Amersham Pharmacia Biotech).
Immunoprecipitations and Kinase Assays-- For in vitro kinase assays, 2-4 × 106 cells from each condition indicated were harvested by trypsinization and washed in phosphate-buffered saline. To analyze CDK2-associated kinase activity, cells were lysed as in ice-cold IPCDC2 buffer (10 mM Tris-HCl, pH 7.5, 5 mM EDTA, pH 8.0, 130 mM sodium chloride, and 1% Triton X-100) containing the above mentioned protease and phosphatase inhibitors. After sonication and clarification, 200 µg of lysate was incubated for 2 h (4 °C with rotation) with anti-CDK2 antiserum (Santa Cruz Biotechnology) or anti-VEGF antiserum (Santa Cruz Biotechnology) as a negative control. Immunocomplexes were recovered with protein A-Sepharose (Amersham Pharmacia Biotech). Washes and kinase reactions were carried out as described previously (39). Histone H1 substrate was purchased from Amersham Pharmacia Biotech. To analyze CDK4-associated kinase activity, cells were lysed in ice-cold cyclin D lysis buffer (50 mM HEPES, pH 7.5, 150 mM sodium chloride, 1 mM EDTA, pH 8.0, 2.5 mM EGTA, pH 8.0, 80% glycerol, 0.5 mM dithiothreitol, and 0.1% Tween 20) containing the above mentioned protease and phosphatase inhibitors. After brief sonication and clarification, 200 µg of lysate was precleared using normal rabbit serum and protein A-Sepharose. After preclearing, lysates were incubated for 2 h (4 °C with rotation) with anti-CDK4 antiserum (Santa Cruz Biotechnology) or anti-VEGF antiserum (Santa Cruz Biotechnology) as a negative control. Washes and kinase reactions were carried out as described previously (40). A glutathione S-transferase fusion of the C terminus of RB was used as substrate and was generously provided by Dr. Erik Knudsen (University of California at San Diego). Relative kinase activity signals were determined using a PhosphorImager (Molecular Dynamics).
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RESULTS |
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LNCaP Cells Deprived of Androgen Arrest in G1-- Although LNCaP cells have been shown to be dependent on androgen for growth (27, 31), the precise mechanisms for this have not been determined. To address this, we first characterized the growth of LNCaP cells in the presence or absence of serum androgen (Fig. 1). Subconfluent, exponentially growing cultures of LNCaP cells propagated in complete serum (FBS) were harvested and equally seeded into media containing one of three serum conditions: (i) 5% FBS; (ii) 5% charcoal dextran-treated FBS (CDT); or (iii) 5% CDT supplemented with 0.1 nM dihydrotestosterone (DHT). As shown in Fig. 1A, cells grown in complete serum exhibited a generation time of approximately 48 h. However, cells cultured in serum stripped of steroid hormone (CDT) demonstrated no increase in cell number up to 8 days post-treatment. This growth arrest was partially reversed by addition of nearly physiological levels (41, 42) of DHT, as expected based on previous reports (29, 32, 43). Also in agreement with previous reports, the proliferation rate of cells propagated in CDT serum supplemented with DHT is not as robust as that of cells propagated in complete serum (32, 43). Similar results are observed when comparing the growth of estrogen-dependent breast carcinoma cells in CDT supplemented with estrogen versus complete serum (44). Although it is possible that optimal cellular proliferation could be achieved upon readdition of multiple steroids or factors, only DHT was added back to allow for clear study of androgen-dependent proliferation. Cells grown in CDT supplemented with DHT demonstrated a doubling time of approximately 72 h when medium changes were carried out every 48 h. Growth of "androgen-independent" human prostate tumor cell lines DU-145, PC-3, and TSU-Pr1 demonstrated no decrease in their growth rates when cultured in the absence of androgen (data not shown), confirming that the growth arrest of LNCaP cells cultured in CDT is unique to prostate cells still dependent on androgen for growth (29, 32, 43).
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Pocket Protein Phosphorylation Is Reduced in the Absence of Androgen-- Because phosphorylation of the retinoblastoma tumor suppressor protein, RB, is required in mammalian cells for the G1-S transition (34, 45), we analyzed the phosphorylation state of RB and the related pocket protein, p130, in LNCaP cells. Hyperphosphorylated RB and p130 migrate at a slower mobility by SDS-PAGE than do the corresponding underphosphorylated forms. As monitored by immunoblot, both RB and p130 existed in all phosphorylation states after 48 h of propagation in androgen-containing and androgen-deprived media (Fig. 2, lanes 1-3). However, by 72 h of treatment, the underphosphorylated forms of RB and p130 predominated in cells grown without androgen (Fig. 2, compare lanes 4 and 5). This trend was even more dramatic by 96 h without androgen (Fig. 2, compare lanes 7 and 8), when G1 arrest was maximal (Fig. 1B).
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CDK4 Kinase Activity Is Diminished in Cells Deprived of Androgen-- Phosphorylation of RB and p130 is catalyzed by the G1 cyclin-dependent kinases, CDK4 and CDK2 (18, 46). As such, the increase in underphosphorylated RB and p130 associated with androgen depletion (Fig. 2) indicated that the G1 arrest observed by fluorescence-activated cell sorting analysis (Fig. 1B) was likely to be correlated with a reduction in G1 CDK-cyclin kinase activities.
CDK4-cyclin D complexes assemble early in G1 and are believed to initiate phosphorylation of RB (18, 19). By immunoblot, CDK4 protein expression patterns remained constant in LNCaP cells for all serum conditions tested (Fig. 3A, top panel). However, cyclin D3 protein levels were diminished in cells grown in the absence of androgen by 72 h of treatment (Fig. 3A, compare lanes 1 and 2 and compare lanes 3 and 4) and were only weakly detected after 96 h of treatment (Fig. 3A, compare lanes 7 and 8). We were unable to detect the expression of the other D-type cyclins, cyclin D1 and cyclin D2, under any growth condition (data not shown) and therefore suggest that cyclin D3 is the principal D-type cyclin in these cells.
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CDK2 Kinase Activity Is Also Reduced upon Androgen Depletion-- After initial phosphorylation of RB by the CDK4-cyclin D complexes, G1 CDK2 kinase complexes are required to maintain RB phosphorylation and to enter S phase (18, 46). We therefore sought to determine the levels and activation state of CDK2-associated complexes in LNCaP cells. Similar to CDK4, CDK2 protein levels did not significantly fluctuate in accordance with the presence or absence of androgen (Fig. 4A, top panel). Likewise, levels of cyclin E, which migrated as a series of species, did not fluctuate with androgen levels (Fig. 4A, middle panel). However, cyclin A protein levels were responsive to the presence of androgen (Fig. 4A, bottom panel). After 48 h of treatment, cyclin A levels were significantly reduced (8-fold) (compare lanes 1-2). This reduction in cyclin A levels continued throughout treatment (Fig. 4A, compare lanes 4 and 5 and compare lanes 7 and 8).
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p21cip1 and p27kip1 Are Alternately Regulated in Response to Androgen Removal-- To determine what role the CDK inhibitors may play in the G1 arrest induced by androgen withdrawal, p21cip1 and p27kip1 were analyzed. The p16ink4a protein was only weakly expressed in LNCaP cells grown under any condition, and levels of p16ink4a were not affected by the presence or absence of androgen (data not shown). However, p21cip1 protein levels were correlated directly with cell growth in LNCaP cells (Fig. 5, top panel). Cells proliferating in complete serum exhibited relatively constant levels of p21cip1 for all times tested (Fig. 5, compare lanes 1, 4, and 7). By contrast, p21cip1 levels diminished by 72 h of propagation in the absence of androgen and continued to diminish through 96 h of treatment (compare lanes 2, 5, and 8). These data indicate that in LNCaP cells, p21cip1 is retained in cells that exhibit high levels of G1 CDK kinase activity, similar to a variety of other systems (40, 47). They also corroborate the suggestion (40, 47) that p21cip1 can serve as an assembly factor for G1 CDKs and, in some instances, promotes G1 CDK activity.
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Ectopic Expression of G1 Cyclin Does Not Rescue the G1 Arrest Associated with Androgen Depletion-- The data presented in Figs. 1-5 indicate that the G1 arrest associated with androgen withdrawal is characterized by underphosphorylated RB and reduced G1 CDK kinase activities. Because G1 cyclins are normally limiting for G1-S progression (15), we hypothesized that their forced overexpression might restore cell cycle progression in the absence of androgen. To test this, we co-transfected plasmids expressing the green fluorescent protein (GFP) and individual G1 cyclins (at a 1:4 ratio) into LNCaP cells that had been propagated for 48 h in the absence of androgen. The ability of these cells to incorporate BrdUrd was assessed 48 h later by indirect immunofluorescence. Experiments were repeated at least two or three times, and at least 500 transfected (GFP-positive) and untransfected (GFP-negative) cells were counted for each experiment. Only 2-5% of cells transfected with vector alone in the absence of androgen incorporated BrdUrd (Fig. 6, , A and C, left panel), and approximately equal numbers of nontransfected cells on the same coverslip incorporated BrdUrd (Fig. 6A). Surprisingly, cells transfected with expression constructs for the G1 cyclins cyclin D1, cyclin D3, cyclin E, or cyclin A were not induced to incorporate BrdUrd in the absence of androgen (Fig. 6, A and C). To affirm that functional cyclins were being expressed from these plasmids, cells were co-transfected with plasmids expressing the G1 cyclin and the large pocket region of RB, deemed LP (48, 49). The LP protein contains the A, B, and C pockets of RB, and as such contains 10 of the 16 consensus CDK phosphorylation sites present in full-length RB (49). Co-transfections were carried out with a 2:1 ratio of cyclin plasmids to LP, and the phosphorylation state of LP was ascertained by immunoblot (Fig. 6B). Cells transfected with vector alone lacked the LP protein, which migrated at an apparent molecular mass of 60 kDa, as opposed to endogenous RB protein, which migrated as a species between 105 and 110 kDa (48). Cells co-transfected with plasmids expressing LP and empty vector demonstrated only minimal phospho-LP because of limiting endogenous G1 CDK-cyclin activity (Fig. 6B, lane 2). However, cells co-transfected with both LP and plasmids encoding any of the G1 cyclins (cyclin D1, D3, E, or A) demonstrated substantial increases in phospho-LP (Fig. 6B, lanes 3-6). These data indicate not only that the G1 cyclins were expressed by transient transfection, but that they formed active complexes with endogenous CDKs. Thus, it appears that overexpression of G1 cyclins was insufficient to induce S phase progression in the absence of androgen.
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RB Phosphorylation Is Required for S Phase Progression in LNCaP Cells-- Oncoproteins such as T-Ag act in part by sequestration of cellular RB, thus eliminating the requirement for CDK4 kinase activity and allowing premature release and activation of S phase promoting genes such as E2F (18, 46). As such, we wanted to determine the requirement of RB phosphorylation for S phase progression in LNCaP cells. To test this, LNCaP cells propagated in complete serum were co-transfected with plasmids expressing GFP and proteins that modulate RB activity at a 1:4 ratio (Fig. 7B). More than 1500 transfected cells and an equal number of untransfected cells were microscopically scored. Approximately 50% of cells transfected with vector alone supported BrdUrd incorporation (Fig. 7, A and C), and a similar proportion of untransfected (GFP-negative) cells also incorporated BrdUrd (Fig. 7A), demonstrating that transfection of GFP bears no influence on the ability of LNCaP cells to incorporate BrdUrd. In contrast, only 4-6% of cells transfected and expressing the CDK inhibitor p16ink4a were able to incorporate BrdUrd, suggesting that these cells were inhibited from completing the G1-S transition. Untransfected cells from the same experiment retained the ability to incorporate BrdUrd.
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Pocket-Protein Binding Is Required for Oncoproteins to Initiate G1-S Progression in the Absence of Androgen-- Having observed that RB phosphorylation was required in LNCaP cells for the G1-S transition and that ectopic expression of T-Ag was able to initiate S phase progression in the absence of androgen, we hypothesized that the ability of oncoproteins to initiate androgen-independent cell cycle progression may be linked to their ability to sequester the activity of RB pocket proteins. To test this, we co-transfected plasmids expressing GFP and mutants of viral oncoproteins in the absence of androgen. The ability of cells to complete the G1-S transition was measured by incorporation of BrdUrd. Cells transfected with DNA encoding the entire early region of SV40 (Figs. 6A and 8A) or the large T-Ag cDNA (data not shown) incorporated BrdUrd in the absence of androgen. By contrast, T-Ag constructs carrying mutations affecting the LXCXE motif (mutants Pvu-1 or K1) lost the ability to promote androgen-independent S phase progression (Fig. 8, A and B); this LXCXE motif has been shown to be required for binding to the pocket proteins (50, 51). A mutant of T-Ag defective in the N-terminal J domain function (mutant 2831) also lacked the ability to promote S phase progression. The J domain is thought to serve several different functions, including binding to Hsc 70, ATPase activity, and cellular transformation (52, 53). Recent evidence suggests that the J domain also plays a role in the ability of T-Ag to modulate the activities of pocket proteins (54). Thus, the ability of T-Ag to promote androgen-independent G1-S transition appears to be bestowed through its ability to modulate pocket proteins.
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ISCUSSION |
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In this report, we provide the first evidence that androgen-responsive growth signals are mediated through regulation of multiple elements controlling the G1-S cell cycle transition. We have also devised a reliable system through which specific proteins can be individually introduced into androgen-dependent cells and assayed for their abilities to promote androgen-independent cell cycle progression and have identified pathways that can be disrupted to convert prostatic epithelial cells to androgen independence.
Androgen Withdrawal Causes a Specific Early G1 Arrest-- Androgen-dependent prostatic cells depleted of hormone arrested in early G1 phase (Fig. 1), as characterized by a global decrease in hyperphosphorylated RB and p130 (Fig. 2) and a reduction in CDK4-cyclin D3-associated RB kinase activity (Fig. 3). This reduction in kinase activity was at least partially accounted for by a decrease in cyclin D3 protein levels. A reduction in CDK2-associated kinase activity was also observed in response to androgen withdrawal (Fig. 4). Interestingly, cyclin E protein levels were unaffected by the presence or absence of androgen, whereas cyclin A accumulation was drastically diminished in the absence of androgen. These observations suggest that a determination of the mechanisms through which androgen regulates the synthesis or stability of cyclin D3 and cyclin A will be of interest, because androgen regulates their accumulation.
In addition to monitoring the expression and activities of CDKs and associated cyclins, we also determined the effect of androgen withdrawal on the CIP family of CDIs. p16ink4a was not expressed at high levels in these cells, and its level of expression was unaffected by the presence or absence of androgen (data not shown). Interestingly, the CDIs, p21cip1 and p27kip1, were found to be alternately regulated in response to androgen depletion (Fig. 5). p27kip1 exhibited the expected characteristics of a CDI, because its protein levels increased in response to androgen depletion and were directly correlated with inhibition of CDK4 and CDK2. p27kip1 is known to be regulated at both the translational and post-translational level and is expressed at high levels in quiescent cells (56). As such, it is perhaps not surprising that p27kip1 levels increased in cells arrested at the androgen checkpoint and likely contribute to the reduction of CDK4 and/or CDK2-associated kinase activity upon androgen depletion. In contrast, p21cip1 correlated with cell growth, because its protein levels diminished upon androgen depletion. Although this observation is inconsistent with the role of p21cip1 as a CDK inhibitor, it is entirely consistent with recent observations that specific CIP family members promoted the association of CDK4-cyclin D complexes both in vitro and in vivo (40). It is known that cyclin D and CDK4 do not readily associate to form active kinase complexes, and the CIP family members may act not only to assemble CDK4-cyclin D complexes but to target these complexes to the nucleus. In addition, p21cip1 has been shown to increase CDK4-cyclin D kinase activity when present at low levels (40); the p21cip1 in LNCaP cells may be fulfilling this function.Ectopic G1 Cyclin Expression Does Not Bypass the Androgen Requirement-- Because G1 cyclins are normally limiting for progression past the G1-S transition and because the G1 arrest associated with androgen depletion was characterized by a reduction of G1 cyclins and G1 cyclin-associated kinase activity, we tested whether ectopic expression of individual G1 cyclins would be sufficient to induce cell cycle progression in the absence of androgen. Strikingly, expression of no single G1 cyclin was sufficient to induce cell cycle progression (Fig. 6). These data emphasize the strength of the androgen requirement, because ectopic expression of cyclin E has been shown to even rescue other cell types that have been blocked by expression of p16ink4a or constitutively active RB (45, 57). However, Alevizopoulos et al. (57) have shown that cyclin overexpression cannot bypass a p27kip1-mediated cell cycle arrest. Because we observed slightly increased levels of p27kip1 in androgen-depleted cells (Fig. 5), this may partially explain the inability of excess cyclins to promote cellular proliferation in the absence of androgen.
Phosphorylation of RB is required in most cell types to traverse the G1-S transition. We examined the requirement of RB phosphorylation in androgen-dependent cells directly by transiently transfecting LNCaP cells with constructs expressing inhibitors of RB inactivation in the presence of androgen (Fig. 7). Specifically, transfection of p16ink4a or PSM.7-LP (a constitutively active mutant of RB) substantially inhibited cell cycle progression in the presence of androgen. Thus, RB phosphorylation appears to be required for LNCaP cells to traverse the G1-S transition, and down-regulation of CDK4-cyclin activity and dephosphorylation of RB likely contribute to the G1 arrest imposed by androgen withdrawal. Thus, although RB phosphorylation was required by LNCaP cells to progress through the G1-S transition, simple cyclin overexpression, which can overcome p16ink4a or RB-mediated cell cycle arrest (45, 57), was not sufficient to induce androgen-independent cell cycle progression. This suggests that the G1 block associated with androgen depletion of androgen-dependent cells must be regulated by mechanisms more complex than attenuation of individual G1 CDK activity, such as down-regulation of cyclin D3-associated activity. In this regard, it is of interest to note that amplification of the genes for cyclin D1 or CDK4 have yet to be reported in prostatic adenocarcinoma. This directly contrasts with observations in breast cancers, where cyclin D1 is amplified; transgenic mice expressing murine mammary tumor virus promoter-driven cyclin D1 also exhibit mammary hyperplasia and carcinogenesis (59, 60). Induction of cyclin D1 in G1-arrested breast cancer cells has also been shown to promote cell cycle progression (61). These observations, together with the present data, imply that distinct mechanisms may be employed by breast and prostate cells for hormonal growth control.Viral Oncoproteins Capable of Sequestering Pocket Proteins
Alleviate Androgen Dependence--
Because inactivation of RB was
shown to be required for G1-S progression in LNCaP cells
but overexpression of G1 cyclins was incapable of reversing
the G1 arrest associated with androgen depletion, we sought
to identify additional cell cycle pathways that might be disrupted to
allow androgen-independent growth. We chose the viral oncoproteins SV40
T-Ag and adenovirus E1A, because they have been shown in other systems
to partially bypass the regulation of the G1-S transition
but not necessarily allow for growth in the absence of serum (51). We
observed that wild-type T-Ag and E1A supported androgen-independent
cell cycle progression (Fig. 7-9). To determine the mechanisms whereby
this occurred, we used specific mutants of T-Ag and E1A. Such mutants
disrupted in the conserved LXCXE motif (mutants
K1-T-Ag, PvuI-T-Ag, and CXDL-E1A) were unable to promote
proliferation. The LXCXE motif is known to be the
region through which both T-Ag and E1A bind and sequester the pocket
proteins RB, p107, and p130 (50, 51). A mutant of E1A defective in a
secondary region of binding to pocket proteins (30-85) was also
incapable of driving the S phase transition. Lastly, mutants disrupting
the J domain of T-Ag also eliminated proliferation potential. The J
domain shares homology with the Escherichia coli DnaJ
protein, is responsible for several functions, including binding to Hsc
70 and DNA replication (52, 53, 62), and has recently been shown to be
important for inactivation of p107 and p130 (54). Taken together, these
data suggest that inactivation of RB (and other pocket proteins) may be
required for the transition to an androgen-independent state. This
observation is likely the underlying mechanism whereby transgenic mice
carrying a probasin-promoter-SV40 large T-antigen fusion develop
prostatic carcinomas capable of making the transition to an
androgen-independent state upon castration of the animal (63).
Additionally, our observations agree well with what has been observed
in human tumorigenesis. For example, mutations of the RB1
gene have been shown to correlate with poor prognosis of prostatic
adenocarcinomas (64) and have been shown to occur as frequent, early
events in prostate cancers (65); between 20-60% of prostate cancers
have mutations of the RB1 gene (65-67). Moreover,
inactivation of p16ink4a occurs at a relatively high frequency
(40-60%) in advanced prostate carcinomas (68, 69). It has been well
documented that the growth inhibitory activity of p16ink4a is
dependent on functional RB, and p16ink4a inactivation simulates
RB phosphorylation and subsequent inactivation by G1 CDKs
(45, 58).
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ACKNOWLEDGEMENTS |
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We thank Drs. C. Cole, D. Livingston, C. Sherr, G. Morris, S. Subramani, and J. Y. J. Wang for generously supplying reagents, Dr. E. S. Knudsen for technical assistance and critical ongoing discussions, and Dr. W. Biggs and R. Gordon for advice and critical reading of the manuscript.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed. Tel.: 619-534-7802; Fax: 619-534-7750; E-mail: wcavenee{at}ucsd.edu.
The abbreviations used are: CDK, cyclin-dependent kinase; CDI, cyclin-dependent kinase inhibitor; BrdUrd, bromodeoxyuridine; PAGE, polyacrylamide gel electrophoresis; FBS, fetal bovine serum; CDT, charcoal dextran-treated FBS; DHT, dihydrotestosterone; GFP, green fluorescent protein; T-Ag, T-antigen; RB, retinoblastoma tumor suppressor protein; LP, large pocket of RB.
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REFERENCES |
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