Differential Requirements for Ras and the Retinoblastoma ... · Differential Requirements for Ras and the Retinoblastoma Tumor Suppressor Protein in the Androgen Dependence of Prostatic

Differential Requirements for Ras and the RetinoblastomaTumor Suppressor Protein in the Androgen Dependenceof Prostatic Adenocarcinoma Cells1

Anne F. Fribourg, Karen E. Knudsen,Matthew W. Strobeck, Clint M. Lindhorst, andErik S. Knudsen2

Department of Cell Biology, University of Cincinnati College ofMedicine, Cincinnati, Ohio 45267-0521

AbstractProstate cells are dependent on androgen forproliferation, but during tumor progression prostatecancer cells achieve independence from the androgenrequirement. We report that androgen withdrawal failsto inhibit cell cycle progression or influence theexpression of cyclin-dependent kinase (CDK)/cyclins inandrogen-independent prostate cancer cells, indicatingthat these cells signal for cell cycle progression in theabsence of androgen. However, phosphorylation of theretinoblastoma tumor suppressor protein (RB) is stillrequired for G1-S progression in androgen-independentcells, since the expression of constitutively active RB(PSM-RB) or p16ink4a caused cell cycle arrest andmimicked the effects of androgen withdrawal ondownstream targets in androgen-dependent LNCaPcells. Since Ras is known to mediate mitogenicsignaling to RB, we hypothesized that active V12Raswould induce androgen-independent cell cycleprogression in LNCaP cells. Although V12Ras was ableto stimulate ERK phosphorylation and induce cyclin D1expression in the absence of androgen, it was notsufficient to promote androgen-independent cell cycleprogression. Similarly, ectopic expression ofCDK4/cyclin D1, which stimulated RB phosphorylationin the presence of androgen, was incapable ofinactivating RB or driving cell cycle progression in theabsence of androgen. We show that androgenregulates both CDK4/cyclin D1 and CDK2 complexesto inactivate RB and initiate cell cycle progression.Together, these data show that androgenindependence is achieved via deregulation of theandrogen to RB signal, and that this signal can only be

partially initiated by the Ras pathway in androgen-dependent cells.

IntroductionTumorigenic growth is characterized by proliferation occur-ring under inappropriate environmental conditions (1, 2). Thisphenomenon is clearly observed during the progression ofprostate cancer. Initially, prostatic epithelia or early-stageprostatic cancer cells are androgen-dependent and thus re-quire androgen to proliferate (3–5). In the most commonlyused therapy for prostate cancer, androgens are removedeither by chemical or surgical means, leading to the cessa-tion of proliferation (6). Despite this initial remission, thetumor cells rapidly become androgen-independent, render-ing hormonal treatment ineffective (7, 8). Strikingly, there isno efficient treatment for androgen-independent tumors.Furthermore, little is known about how these recurrent tumorcells are able to proliferate in the absence of androgen.

Androgen is a known growth factor for prostatic epithelialcells (9). However, the mechanism through which androgensignals to promote proliferation is poorly understood (10). Ingeneral, mitogenic factors act through the Ras/ERK3 path-way to activate a host of CDK/cyclin complexes, which arerequired for cell cycle progression and cellular proliferation(1, 2, 11, 12). Early in the G1 phase of the cell cycle, mitogenicsignals activate CDK4/cyclin D complexes, which are re-quired to initiate phosphorylation of RB (2, 13). Reagents thatblock RB phosphorylation (e.g., the CDK4 inhibitor p16ink4a)or constitutively active RB proteins (PSM-RB) arrest cells inG1 (2, 14, 15). Thus, inactivation of RB is typically required forentry into S-phase, although specific cell types acquire re-sistance to the actions of these reagents, either through theattenuation of downstream signaling or through the expres-sion of viral oncoproteins of DNA tumor viruses (16, 17). Weand others have shown recently that RB inhibits cell cycleprogression by attenuating cyclin A expression, thus dimin-ishing CDK2 activity (16, 18–20). Because both CDK2/cyclinE and CDK2/cyclin A activities are required for entry intoS-phase, inhibition of cyclin A expression is a critical com-ponent of the antiproliferative action of RB (18, 21, 22).

The LNCaP (androgen-dependent) tumor cell line is a modelsystem for the study of androgen-dependent proliferation (23,24). Androgen deprivation causes a documented reduction incyclin D protein levels and CDK4/cyclin D kinase activity. As aresult, RB remains underphosphorylated/active, and cyclin A

Received 1/4/00; revised 5/11/00; accepted 5/11/00.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indi-cate this fact.1 This work was supported in part by Grant R01-CA82525-01 from theNational Cancer Institute-NIH and a grant from the Sidney Kimmel CancerFoundation (to E. S. K.). K. E. K. is supported by Postdoctoral FellowshipCA82034 from the National Cancer Institute.2 To whom requests for reprints should be addressed, at Department of CellBiology, Neurobiology, and Anatomy, University of Cincinnati College ofMedicine, P. O. Box 670521, 3125 Eden Avenue, Cincinnati, OH 45267-0521.Phone: (513) 558-6849; Fax: (513) 558-4454; E-mail: [email protected].

3 The abbreviations used are: ERK, extracellular signal-regulated kinase;CDK, cyclin-dependent kinase; RB, retinoblastoma tumor suppressorprotein; BrdUrd, bromodeoxyuridine; CDT, charcoal dextran treated; GFP,green fluorescent protein; HPV, human papillomavirus; LP, large pocket;FBS, fetal bovine serum.

361Vol. 11, 361–372, July 2000 Cell Growth & Differentiation

protein levels (as well as CDK2 activity) are attenuated (25).Thus, these cells arrest in G1 when androgen is removed. Con-version to androgen independence in LNCaP cells can beachieved by ectopic expression of viral oncoproteins E1A andT-Ag, which functionally disrupt RB (25). Together, these resultssuggest that RB plays an important role in maintaining andro-gen dependence in prostatic tumor cells. Furthermore, thissuggests that androgen independence occurs through signalsthat disrupt RB function, either through upstream or down-stream signaling.

In an effort to understand the molecular changes that underlieandrogen independence, we evaluated the ability of androgen-independent cells to transmit signals to RB and for RB to signalto its downstream effectors. We show that androgen-indepen-dent cells remained responsive to RB activation and that RBtargets were invoked in an appropriate fashion. By contrast, RBinactivation in these cells was deregulated and occurred in theabsence of androgen. Thus, signals upstream of RB are mis-regulated in androgen-independent prostate cells. BecauseRas is known to link growth factor signaling to the cyclin D/RBnetwork, we investigated the function of activated Ras in an-drogen-dependent cells. Surprisingly, although Ras did induce

cyclin D1 in the absence of androgen, Ras had an overallnegative effect on cell cycle progression that was p53 depend-ent. Even when the growth-inhibitory activity of Ras was dis-rupted with HPV-E6 or when CDK4/cyclin D1 was ectopicallyexpressed, androgen-independent cell cycle progression failedto occur. These data indicate that androgen must regulate morethan CDK4/cyclin D to initiate cell cycle progression. In fact, wedemonstrate that androgen also regulates CDK2/cyclin activity,and the collective activities of CDK4/cyclin D and CDK2/cyclincomplexes are required to inactivate RB and initiate cell cycleprogression in the absence of androgen. These data demon-strate that maintenance of the androgen to RB pathway re-quires factors distinct from Ras and involves the combinedregulation of CDK4/cyclin D and CDK2/cyclin complexes.

ResultsAndrogen Signaling to RB Is Deregulated in Androgen-independent Cells. The LNCaP cell line is androgen de-pendent and fails to incorporate BrdUrd when cultured inCDT serum that lacks androgen (Fig. 1A). We have shownpreviously that cell cycle progression in LNCaP cells is re-

Fig. 1. Androgen-independent cellsdo not arrest and fail to regulate RB inthe absence of androgen. A, JCA-1,LNCaP, and TSUPr-1 cells were prop-agated for 96 h in the presence of either10% CDT FBS (CDT) or 10% FBS. Cellswere then labeled with BrdUrd for aperiod of ;14 h. Subsequent to thepulse, cells were fixed and processedto monitor BrdUrd incorporation via di-rect immunofluorescence. Data shownare the average of at least three inde-pendent experiments in which at least150 cells/experiment were analyzed;bars, SD. Representative immunofluo-rescence from the experiment de-scribed are shown in the inset. B,JCA-1, TSUPr-1, and LNCaP cells werepropagated for 96 h in the presence ofeither 10% CDT (Lanes 2, 4, and 6) or10% FBS (Lanes 1, 3, and 5), har-vested, and clarified, and protein ly-sates were prepared. Equal protein wasresolved by SDS-PAGE, and the en-dogenous cyclin A, cyclin E, cyclin D1,and RB proteins were detected by im-munoblotting with the respective anti-bodies. pRB, underphosphorylated RB;ppRB, hyperphosphorylated RB. C, ly-sates from JCA-1, TSUPr-1, andLNCaP cells as described in B wereresolved by SDS-PAGE, and the andro-gen receptor (AR) was detected by im-munoblotting.

362 RB Regulates the Androgen Requirement

stored by supplementing CDT with dihydrotestosterone (25).In contrast, the androgen-independent cell lines JCA-1 andTSUPr-1 continue to incorporate BrdUrd when cultured inCDT (Fig. 1A). Consistent with the BrdUrd incorporation re-sults, whereas androgen-independent cells proliferate in theabsence of androgen, androgen-dependent LNCaP fail toproliferate, as determined by growth curves (not shown).When deprived of androgen, LNCaP cells exhibited de-creased levels of cyclin D1, and as a consequence RB wasdephosphorylated (Fig. 1B, compare Lanes 5 and 6). CyclinE expression remained constant in the presence or absenceof androgen, whereas cyclin A protein levels were diminishedin the absence of androgen (Fig. 1B, Lanes 5 and 6). Thesedata are consistent with a model wherein androgen actsthrough cyclin D1 to stimulate RB phosphorylation, whichthen inactivates RB and derepresses cyclin A expression.Thus, we hypothesize that androgen-independent cells haveeither lost this signal from androgen to RB or from RB to itsknown downstream effectors. To distinguish between thesepossibilities, JCA-1 and TSUPr-1 cells were propagated ineither the presence or the absence of androgen. Regardlessof androgen status, these cells maintained high levels ofcyclin D1 protein and demonstrated RB hyperphosphoryla-tion, indicating that CDK4 activity was also maintained (Fig.1B, compare Lanes 1 and 2, Lanes 3 and 4). Cyclin A proteinlevels remained unchanged, consistent with the phosphoryl-ation status of RB. Cyclin E protein levels also remainedconstant (Fig. 1B, compare Lanes 1 and 2, Lanes 3 and 4).These data indicate that androgen-independent cells havelost the ability to regulate RB in response to androgen with-drawal. A possible explanation for this finding would be thatthe androgen receptor in these cell lines is constitutivelyactive and thus functions in the absence of androgen. How-ever, immunoblot analysis of the androgen receptor showedthat JCA-1 and TSU-PR1 cells do not express detectablereceptor (Fig. 1C, Lanes 1–4), whereas in LNCaP cells thereceptor was readily detectable (Fig. 1C, Lanes 5 and 6).Thus, these androgen-independent cell lines have subvertedthe requirement for the androgen receptor to promote cellcycle progression.

RB Signaling Is Retained in Androgen-independentCells. The finding that RB phosphorylation was observed inthe androgen-independent cells in the absence of androgensuggested that signals upstream of RB (i.e., cyclin D-asso-ciated kinase activity) must be deregulated for ongoing cellcycle progression. However, it is known that disruption of RBby the oncoproteins of DNA tumor viruses or deregulation ofdownstream targets leads to inappropriate cell cycle pro-gression (17, 18). Therefore, we designed experiments todetermine whether RB signaling is intact in androgen-inde-pendent cell lines. JCA-1 cells were cotransfected in com-plete serum with a GFP expression plasmid and either pa-rental vector, p16ink4a, or PSM-RB expression plasmids,and cell cycle progression of transfected cells (GFP positive)was monitored by BrdUrd incorporation. Cells transfectedwith vector showed approximately the same level of BrdUrdincorporation as nontransfected cells (GFP negative) fromthe same coverslip (Fig. 2A). By contrast, cells transfectedwith p16ink4a or PSM-RB showed significant reduction in

BrdUrd incorporation in comparison to vector-transfectedcells or nontransfected cells from the same coverslip. To-gether with our previous observation that PC3 and TSU-PR1cells also arrest in response to active RB (17), these resultsindicate that signaling of RB to its downstream targets isintact in androgen-independent tumors.

To assess the importance of RB signaling in these cells, wecompared the molecular response of RB-arrested androgen-independent cells (JCA-1 and TSU-PR1) with that observedupon androgen withdrawal in androgen-dependent cells(LNCaP). JCA-1 and TSU-PR1 cells were cotransfected inthe presence of androgen with a puromycin-resistance plas-mid and either parental vector, p16ink4a, or PSM-RB. Twenty-four h after transfection, cells were then selected with puro-mycin for 48 h, at which time the majority of untransfectedcells were eliminated by puromycin (data not shown). Trans-fected cells were harvested and protein levels were exam-ined by immunoblot analyses. Cyclin D1 and cyclin E proteinlevels remained constant in each cell line whether the cellswere transfected with p16ink4a, PSM-RB, or vector (Fig. 2B,Lanes 1–6). By contrast, when the androgen-independentprostatic tumor cells were either transfected with p16ink4a orPSM-RB, cyclin A protein levels were reduced in comparisonwith the same cells transfected with parental vector (Fig. 2B,compare Lanes 1 and 3 with Lane 2 or Lanes 4 and 6 withLane 5). In addition, the endogenous RB maintained highlevels of phosphorylation when transfected with PSM-RB(Fig. 2B, Lanes 3 and 6), whereas after transfection withp16ink4a, only the underphosphorylated form of RB waspredominant in JCA-1 and TSUPr-1 (Fig. 2B, Lanes 1 and 4).This indicates that as reported previously (16), PSM-RB doesnot feed back to modify the phosphorylation of endogenousRB, whereas p16ink4a blocks the CDK4 kinase activity thatis required to initiate RB phosphorylation. Taken together,these data show that the signaling pathway from androgen toRB is lost in androgen-independent cells, but these cellshave not lost the ability to arrest in response to RB. More-over, active RB or p16ink4a transfected into androgen-inde-pendent cells mimics the effect of androgen withdrawal inandrogen-dependent cells (i.e., down-regulation of cyclin Abut not cyclin E). Thus, androgen-independent cells haveachieved independence not by subverting RB activity di-rectly or by downstream disruption of RB signaling but ratherby dysregulation of upstream signaling pathways.

Ras Induction of Cyclin D Is Insufficient to PromoteAndrogen-independent Proliferation. These data suggestthat androgen acts through cyclin D1 to stimulate RB phos-phorylation/inactivation, thus leading to induced cyclin Aexpression and cell cycle progression. Consistent with thisidea, a kinetic study of androgen stimulation in LNCaP cellsrevealed that increased cyclin D1 expression correlates withincreased RB phosphorylation that preceded the accumula-tion of cyclin A protein (Fig. 3A, Lanes 1–3). It is known thatgrowth factors can act through the Ras signaling pathway toactivate cyclin D1 expression (1, 2, 11, 12, 26). We thereforesurmised that androgen acts through Ras to stimulate cyclinD1 expression and inactivate RB. To test whether activationof Ras is sufficient to promote proliferation in LNCaP cells,we used activated V12Ras. To confirm that the V12Ras

363Cell Growth & Differentiation

(activated Ras) expression plasmid expressed active proteinwhen transfected into LNCaP cells in androgen-deprivedmedia, cells were transfected in CDT with either V12Rasexpression plasmid or parental vector. Cells were harvested48 h after transfection, and immunoblot analyses were per-formed. As shown in Fig. 3B, Ras protein was overexpressedwhen the expression plasmid was transfected into LNCaPcells and could be readily detected through its HA epitope(Fig. 3B, compare Lanes 1 and 2). To test V12Ras activity,ERK phosphorylation was also monitored, because ERK is amajor downstream target of Ras (27–29). No significant dif-ference in ERK protein expression was observed betweenthe two transfections, although ERK1 and ERK2 phospho-rylation was dramatically induced when LNCaP cells ex-pressed V12Ras (Fig. 3B, compare Lanes 1 and 2). Further-more, V12Ras led to an increase in the expression of cyclinD1 in CDT cultured LNCaP cells (Fig. 3D, compare Lanes1–4). Thus, these data confirmed that V12Ras is expressedand active when transfected into the LNCaP cells in theabsence of androgen and acts to promote the accumulationof cyclin D1.

To determine whether Ras signaling is sufficient to pro-mote androgen-independent cell cycle progression, LNCaPcells cultured in CDT were cotransfected with a GFP expres-sion plasmid and either parental vector, V12Ras, or E1A

expression plasmids, and BrdUrd incorporation was moni-tored. As shown in Fig. 3C, cells transfected with vectoralone failed to incorporate BrdUrd in the absence of andro-gen. Strikingly, LNCaP cells transfected with activated Rasalso failed to demonstrate androgen-independent cell cycleprogression. By contrast, E1A rescued cell cycle progressionof LNCaP cells to approximately the same level as vector-transfected cells in the presence of androgen. Using a similarprotocol, V12Ras promoted cell cycle progression in serum-starved Rat-1 cells (Fig. 3E). Thus, V12Ras signaling is notsufficient to promote cell cycle progression in LNCaP cells.Consistent with this finding, although we observed an in-crease in cyclin D1 expression, we did not observe an in-crease in cyclin A expression (Fig. 3D), nor did we observeRB phosphorylation (not shown). Cyclin E levels were unaf-fected by the expression of V12Ras (Fig. 3D).

V12Ras Inhibits Cell Cycle Progression in LNCaP Cellsvia p53. Although the expression of cyclin D1 was stimu-lated when V12Ras was transfected into LNCaP cells cul-tured in CDT, neither increased RB phosphorylation nor wascyclin A expression detected. These data suggested thatV12Ras was either incapable of acting to activate the cyclinD1-associated kinase activity, or alternatively that V12Raswas stimulating an antiproliferative pathway. Because it hasbeen shown recently that V12Ras can inhibit proliferation of

Fig. 2. Androgen-independent cells arrested by PSM-RB mimic that of androgen-dependent cells deprived of androgen. A, JCA-1 cells were cotrans-fected with a GFP expression plasmid (0.12 mg) and the indicated plasmids (4 mg). BrdUrd was added 24 h after transfection for a total labeling of ;14h. Cells were stained with anti-BrdUrd antibody, and the percentage of GFP-positive and GFP-negative cells exhibiting BrdUrd incorporation wasdetermined by indirect immunofluorescence. The average and deviation values shown are from two to three independent experiments with at least 150GFP-positive cells counted/experiment. A, representative immunofluorescence from the experiment; arrows, GFP positive (transfected) cells. Bars, SD. B,JCA-1 and TSUPr-1 cells were cotransfected with a puromycin-resistance plasmid (1 mg) and the indicated expression plasmids (15 mg) in 10-cm dishesby the calcium phosphate method. Twenty-four h after transfection, JCA-1 and TSUPr-1 cells were selected with 2 mg/ml of puromycin for 48 h. Then, thecells were harvested, and clarified lysates were prepared. Total equal protein was resolved by SDS-PAGE, and cyclin A, cyclin E, cyclin D1, RB, and PSM-RBproteins were detected by immunoblotting with the respective antibodies.


primary fibroblasts (30), we determined whether V12Ras ex-erted a growth-inhibitory effect in LNCaP cells. To test this,LNCaP cells cultured in FBS were cotransfected with a GFPexpression plasmid and either vector control or V12Ras ex-pression plasmids (Fig. 4B). Cells were labeled with BrdUrd,and the incorporation of BrdUrd in GFP-positive cells wasdetermined. Although the vector had no influence on BrdUrdincorporation, transfection of V12Ras significantly inhibitedBrdUrd incorporation, thus demonstrating that V12Ras in-hibits cell cycle progression in LNCaP cells.

Because the growth-inhibitory activity of V12Ras is knownto act through the p53/p21Cip1 signaling axis (30), the abilityof V12Ras to stimulate expression of p53 and p21Cip1 (a p53target) was determined. In cells transfected with V12Ras,there was a consistent increase in p53 and p21Cip1 proteinlevels, regardless of androgen status, suggesting that thegrowth-inhibitory activity of V12Ras is mediated via p53 (Fig.4A, compare Lanes 1 and 2 or Lanes 3 and 4). To test thisdirectly, LNCaP cells cultured in FBS were cotransfected

with GFP expression plasmid and either V12Ras or V12Rasand HPV-E6 expression plasmids. HPV-E6 is known to pro-mote p53 degradation and thereby overcome the antiprolif-erative activity associated with p53 (Fig. 4B). Although theGFP-positive cells cotransfected with V12Ras expressionplasmid arrested, coexpression of HPV-E6 partially reversedthis effect of V12Ras (Fig. 4B).

Cyclin D Expression Is Not Sufficient for AndrogenIndependence. Because the growth-inhibitory effects ofV12Ras can be overcome by HPV-E6, we reasoned that thiscombination should be sufficient to render LNCaP cells an-drogen independent. LNCaP cells cultured in CDT were co-transfected with GFP expression plasmid and either Vector,E1A, or V12Ras 1 HPV-E6 expression plasmids. Consistentwith our earlier observations, expression of E1A promotedBrdUrd incorporation in CDT (Fig. 4C). Surprisingly, cotrans-fection of V12Ras with HPV-E6 was not sufficient to driveandrogen-independent cell cycle progression (Fig. 4C). How-ever, E1A rendered LNCaP cells androgen independent in

Fig. 3. E1A but not V12Ras is sufficient to promote androgen-independent cell cycle progression. A, LNCaP cells were seeded and grown in 10% CDTfor 96 h; after which, medium was changed to 10% FBS. At the indicated times (Lanes 1–3), cells were harvested. Clarified lysates were prepared, and equalprotein was resolved by SDS-PAGE. The endogenous cyclin A, cyclin E, cyclin D1, and RB proteins were detected by immunoblotting with the respectiveantibodies. B, LNCaP cells were transfected in CDT with the expression plasmids indicated. Cells were harvested 48 h after transfection, and clarified lysateswere prepared. Total equal protein was resolved by SDS-PAGE, and the endogenous Ras, ERK, and phospho-ERK proteins were detected by immuno-blotting with the respective antibodies (two different antibodies were used to recognize phospho-ERK, each recognizing a different epitope). C, LNCaP cellswere cotransfected with a GFP expression plasmid (0.12 mg) and the indicated plasmids (5 mg). After transfection, cells were either replaced in 10% FBSor changed to 10% CDT as indicated. BrdUrd was added 48 h after transfection for a total labeling of ;14 h. Cells were stained with anti-BrdUrd antibody,and the percentage of GFP-positive cells exhibiting BrdUrd incorporation was determined by indirect immunofluorescence. The average and deviationvalues shown are from two to three independent experiments with at least 150 GFP-positive cells counted per experiment. Representative immunofluo-rescence from the experiment is shown; arrows, GFP-positive (transfected) cells; bars, SD. D, LNCaP cells were transfected either in CDT (Lanes 1 and 2)or FBS (Lanes 3 and 4) with either vector (Lanes 1 and 3) or V12Ras (Lanes 2 and 4) expression plasmid. Cells were harvested 48 h after transfection, andequal total protein was resolved by SDS-PAGE. Cyclin D1, Cyclin E, and Cyclin A proteins were then detected by immunoblotting. E, Rat-1 cells werecotransfected with a GFP expression plasmid and the indicated plasmids. After transfection, cells were either replaced in 10% FBS or changed to 0.1%FBS as indicated. BrdUrd was added 24 h after transfection for a total labeling of ;14 h. Cells were stained with anti-BrdUrd antibody, and the percentageof GFP-positive cells exhibiting BrdUrd incorporation was determined by indirect immunofluorescence. The average and deviation values shown are fromtwo to three independent experiments with at least 150 GFP-positive cells counted per experiment; bars, SD.


the presence of V12Ras (Fig. 4D), further substantiating theidea that RB inactivation is required for androgen-indepen-dent growth, and that V12Ras is incapable of fulfilling thisrequirement in the absence of androgen (Fig. 4D). Collec-tively, these results suggest that additional mechanisms be-sides the accumulation of D-type cyclins are at play in an-drogen-dependent proliferation. To test this hypothesis,LNCaP cells were cotransfected with CDK4/cyclin D1 andmonitored for CDK4-associated kinase activity, RB phos-phorylation, and BrdUrd incorporation (Fig. 5). Initially, CDK4complexes were immunoprecipitated from transfected cellsand tested in in vitro kinase assays. As shown in Fig. 5A,endogenous CDK4 activity was high in FBS (Fig. 5, Lane 2),and this activity was further enhanced by coexpression ofCDK4/cyclin D1 (Fig. 5, Lane 3). These observations weresupported by in vivo RB kinase assays, wherein cells weretransfected with the LP fragment of RB (Fig. 5B; Refs. 25, 31,32). As has been reported previously, endogenous CDK4activity was insufficient to drive LP phosphorylation (Fig. 5B,Lane 1; Ref. 25), whereas cotransfection of CDK4/cyclin D1effectively drove LP phosphorylation in the presence of FBS(Fig. 5B, Lane 2). Therefore, ectopic CDK4/cyclin D1 wasshown to harbor RB kinase activity by both in vivo and in vitrokinase assays. LNCaP cells cultured in CDT also demon-strated enhanced in vitro RB kinase activity upon transfec-tion with CDK4 and cyclin D1 (Fig. 5A, compare Lanes 4 and

5). However, transfection of CDK4/cyclin D1 was insufficientto effectively drive in vivo phosphorylation of LP (Fig. 5B,middle panel, Lanes 3 and 4). Therefore, ectopic CDK4/cyclinD1 kinase activity is not sufficient to drive RB inactivation.Consistent with this observation, ectopic CDK4/cyclin D1expression did not restore cyclin A expression in CDT (Fig.5B, bottom panel). Moreover, ectopic CDK4/cyclin D1 wasinsufficient to drive cell cycle progression in the absence ofandrogen (Fig. 5C).

CDK4/Cyclin D and CDK2/Cyclin Complexes Are BothTargets of Androgen. It is known that complete RB phos-phorylation/inactivation requires both CDK4 and CDK2 ac-tivities (18, 21, 22). This leaves the possibility that androgenalso regulates CDK2/cyclin activity, which is required to in-activate RB. To determine this, we initially analyzed the effectof androgen on CDK inhibitory proteins, which can lead tothe attenuation of CDK2 activity either directly (p27Kip1,p21Cip1) or indirectly (p16ink4a; Ref. 33). As shown in Fig.6A, no changes in p16ink4a or p27Kip1 were observed in thepresence or absence of androgen; accordingly, no changeswere observed upon cotransfection of CDK4/cyclin D1 (com-pare Lanes 1–4). As we have reported previously (25), cellscultured in the absence of androgen exhibit low levels ofp21Cip1, as compared with cells cultured in the presence ofandrogen (compare Lanes 1 and 2 with Lanes 3 and 4). Nochanges in p21Cip1 levels were observed upon cotransfec-

Fig. 4. V12Ras inhibits the proliferation of LNCaPcells through the p53-signaling axis. A, LNCaP cellswere transfected either in CDT (Lanes 1 and 2) orFBS (Lanes 3 and 4) with either vector (Lanes 1 and3) or V12Ras (Lanes 2 and 4) expression plasmid.Cells were harvested 48 h after transfection, andequal total protein was resolved by SDS-PAGE. Thep53 and p21Cip1 proteins were then detected byimmunoblotting. B, LNCaP cells cultured in FBSwere cotransfected with a GFP expression plasmid(0.12 mg), and the indicated expression plasmids (5mg of vector, V12Ras, HPV-E6, or 2.5 mg of E6 andV12Ras together). BrdUrd was added 48 h aftertransfection for a total labeling of ;14 h. Cells werestained with anti-BrdUrd antibody, and the percent-age of GFP-positive cells exhibiting BrdUrd incorpo-ration was determined by indirect immunofluores-cence. The average and deviation values shown arefrom two to three independent experiments with atleast 150 GFP-positive cells counted per experi-ment; bars, SD. C and D, LNCaP cells cultured inCDT were cotransfected with a GFP expressionplasmid (0.12 mg), and the indicated expressionplasmids (5 mg of vector, E1A, HPV-E6, or 2.5 mg ofeach plasmid combined). BrdUrd was added 48 hafter transfection for a total labeling of ;14 h. Cellswere stained with anti-BrdUrd antibody, and the per-centage of GFP-positive cells exhibiting BrdUrd in-corporation was determined by indirect immunoflu-orescence. The average and deviation values shownare from two to three independent experiments withat least 150 GFP-positive cells counted per experi-ment; bars, SD.


tion with CDK4/cyclin D1. Collectively, these data reaffirmthat androgen deprivation does not result in increased ex-pression of these CDK inhibitory proteins, and in fact lack ofandrogen actually causes a decrease in p21Cip1. Moreover,restoration of CDK4 activity does not affect the CDK inhibitorprofile.

We also examined the effect of CDK4/cyclin D1 transfec-tion on CDK2 activity using in vitro kinase assays. As shownin Fig. 6B, LNCaP cells cultured in the presence of androgenharbored significant CDK2-associated kinase activity, as de-termined by in vitro kinase assays against an RB substrate,and ectopic expression of CDK4/cyclin D1 did not enhancethe endogenous CDK2 activity (top panel, Lanes 1–3). Con-sistent with previous reports (25), CDK2-associated kinaseactivity is attenuated in the absence of androgen, and CDK2activity was not restored by the ectopic expression of CDK4/cyclin D1 (Fig. 6B, top panel, Lanes 4 and 5). A trivial expla-nation for this would be that lack of cyclin A is responsible forthe attenuation in CDK2 kinase activity. Therefore, we ana-lyzed the kinase activity specifically associated with cyclin E(Fig. 6B, bottom panel). To do this, cyclin E complexes wereimmunoprecipitated and used in in vitro kinase assaysagainst the RB substrate. Cyclin E kinase activity was notinfluenced by ectopic expression of CDK4/cyclin D1, andcyclin E-associated activity was attenuated in the absence ofandrogen (Fig. 6B, bottom panel, compare Lanes 2 and 3 andLanes 4 and 5).

To investigate the activity of CDK2 for in vivo RB phos-phorylation, ectopically expressed cyclin E or cyclin A wasmonitored for its ability to phosphorylate the LP fragment ofRB. Both cyclin E and cyclin A overproduction promoted RBphosphorylation in the presence of androgen; however, inthe absence of androgen, RB phosphorylation was inhibited(Fig. 6C). Because CDK4/cyclin D activity is required to primethe phosphorylation of RB for subsequent hyperphosphor-ylation by CDK2/cyclin complexes, we reasoned that theinhibition of cyclin D expression and CDK4 activity mayunderlie the inhibition of RB phosphorylation when cyclin Eand cyclin A are overexpressed. To test this idea, CDK4,cyclin D1, cyclin E, and cyclin A were coexpressed with theLP fragment, and the status of LP phosphorylation was mon-itored in the presence or absence of androgen. As shown inFig. 6D, the LP fragment of RB was efficiently phosphor-ylated by this combination of CDK/cyclins in both the pres-ence and absence of androgen (compare Lanes 2 and 4),thereby indicating that these conditions overcome both theCDK4 and CDK2 inhibition observed in androgen-deprivedLNCaP cells. Because RB is a critical determinant of andro-gen-dependent cell cycle progression, we determinedwhether the cells cotransfected with CDK4, cyclin D1, cyclinE, and cyclin A were able to progress through the cell cyclein the absence of androgen. As shown in Fig. 6E, vector-transduced cells failed to incorporated BrdUrd, whereascells transfected with E1A expression plasmid incorporatedBrdUrd in the absence of androgen. Ectopic coexpression ofCDK4, cyclin D1, cyclin E, and cyclin A also promoted cellcycle progression in the absence of androgen. Together,these findings indicate that the control of RB phosphoryla-tion occurs at the level of both CDK4/cyclin D and CDK2/

Fig. 5. Ectopic expression of CDK4/cyclin D is not sufficient to promoteRB inactivation and androgen independent proliferation. A, LNCaP cellswere either mock-transfected (Lanes 2 and 4) or cotransfected with ex-pression plasmids for CDK4 and cyclin D1 (Lanes 1, 3, and 5) in either FBS(Lanes 1–3) or CDT (Lanes 4 and 5). Cells were harvested 48 h aftertransfection, lysed, and subjected to immunoprecipitation using antibod-ies direct against either E1A (Lane 1) or CDK4 (Lanes 2–5). Immunopre-cipitated complexes were used for in vitro CDK4 kinase assays, using theC-pocket of RB as a substrate. Phospho-RB was subjected to 12%SDS-PAGE and visualized by autoradiography. B, LNCaP cells were co-transfected either in FBS (Lanes 1 and 2) or CDT (Lanes 3 and 4) with eitheran expression plasmid encoding the LP fragment of RB, and vector (Lanes1 and 3) or cyclin D1 and CDK4 (Lanes 2 and 4) expression plasmids. Cellswere harvested 48 h after transfection, and equal total protein was re-solved by SDS-PAGE. Cyclin D1, LP, and cyclin A proteins were thendetected by immunoblotting. pLP, underphosphorylated LP; ppLP, hy-perphosphorylated LP. C, LNCaP cells cultured in CDT were cotrans-fected with a GFP expression plasmid (0.12 mg) and the indicated expres-sion plasmids (5 mg of vector, E1A, or 2.5 mg of cyclin D with either 2.5 mgof vector or 2.5 mg of CDK4). BrdUrd was added 48 h after transfection fora total labeling of ;14 h. Cells were stained with anti-BrdUrd antibody,and the percentage of GFP-positive cells exhibiting BrdUrd incorporationwas determined by indirect immunofluorescence. The average and devi-ation values shown are from two to three independent experiments with atleast 150 GFP-positive cells counted per experiment. Bars, SD.


cyclin kinase activities, and combined deregulation of bothcomponents leads to androgen-independent cell cycle pro-gression (Fig. 6E).

DiscussionHere we show that androgen acts through both CDK4/cyclinD and CDK2/cyclin activities to promote RB inactivation andcell cycle progression. We also show that this process isderegulated in androgen-independent tumor cells, whereassignaling capabilities from RB to its downstream effectorsremain intact. Thus, this report delineates mechanisms usedby androgen to initiate cell cycle progression and under-scores the impact of deregulated RB inactivation in prostatictumor development.

Initially, we demonstrated that in androgen-dependent pros-tate cancer cells (LNCaP), androgen withdrawal led to a ces-sation of cell cycle progression that correlated with a reductionin cyclin D1 expression, RB phosphorylation, and cyclin A ex-

pression. In contrast, in androgen independent cell lines (JCA-1and TSU-PR1), androgen withdrawal had no influence on thecyclin D/RB pathway (Fig. 1). In fact, these androgen-indepen-dent cell lines no longer required expression of the androgenreceptor to simulate cyclin D1 expression. Thus, mutations inthese cells must have rendered them capable of progressingthrough the cell cycle independent of androgen action. To de-termine whether these lesions act upstream or downstream ofRB, we expressed either p16ink4a or PSM-RB in the androgen-independent cell lines (Fig. 2). These reagents led to the ces-sation of cell cycle progression and reduced the levels of cyclinA protein, similar to that observed in LNCaP cells upon andro-gen withdrawal. These findings indicate that the lesions thatlead to cell cycle dysregulation in androgen-independent pros-tate cancer act upstream of RB. It is known that Ras activitystimulates cyclin D1 expression, thus activating CDK4/cyclinD1 complexes and initiating RB phosphorylation/inactivation (1,2, 11, 12, 26, 34). We hypothesized that androgen acts through

Fig. 6. Androgen action requires both CDK4 andCDK2 activities. A, LNCaP cells were either mock-transfected (Lanes 1 and 3) or cotransfected withexpression plasmids for CDK4 and cyclin D1(Lanes 2 and 4) in either FBS (Lanes 1 and 2) orCDT (Lanes 3 and 4). Cells were harvested 48 hafter transfection, lysed, subjected to SDS-PAGE,and immunoblotted using antibodies direct againsteither p16ink4a (top panel), p21Cip1 (middle pan-el), or p27kip1 (bottom panel). B, lysates from cellsprepared as described in A were used for immu-noprecipitation using antibodies against either E1A(Lane 1), CDK2 (top panel), or cyclin E (bottompanel). Immunoprecipitated complexes were usedfor in vitro kinase assays, using the C-pocket of RBas a substrate. Phospho-RB was subjected to12% SDS-PAGE and visualized by autoradiogra-phy. C, LNCaP cells were cultured in FBS (Lanes1–3) or CDT (Lanes 4–6) and cotransfected with anexpression plasmid encoding LP and either cyclinA (Lanes 1 and 4), vector (Lanes 2 and 5), or cyclinE (Lanes 3 and 6) plasmids. Cells were harvested48 h after transfection, and equal total protein wasresolved by SDS-PAGE. pLP, underphosphor-ylated LP; ppLP, hyperphosphorylated LP. D,LNCaP cells were cultured in FBS (Lanes 1 and 2)or CDT (Lanes 3 and 4) and cotransfected withexpression plasmids encoding LP and either vec-tor (Lanes 1 and 3) or the combination of cdk4,cyclin D1, cyclin E, and cyclin A (Lanes 2 and 4)plasmids. Cells were harvested 48 h after trans-fection, and equal total protein was resolved bySDS-PAGE. pLP, underphosphorylated LP; ppLP,hyperphosphorylated LP. E, LNCaP cells culturedin CDT were cotransfected with a GFP expressionplasmid and the indicated expression plasmids.BrdUrd was added 48 h after transfection for atotal labeling of ;14 h. Cells were stained withanti-BrdUrd antibody, and the percentage of GFP-positive cells exhibiting BrdUrd incorporation wasdetermined by indirect immunofluorescence. Theaverage and deviation values shown are from twoto three independent experiments with at least 150GFP-positive cells counted per experiment; bars,SD. F, model: androgen acts through CDK4/cyclinD and CDK2/cyclin complexes. The data pre-sented suggest that androgen stimulates both cy-clin D1 expression/activity and CDK2/cyclin E-associated activity. Both events are required tocomplete RB phosphorylation and inactivation,thus relieving RB-mediated repression of cyclin Aand initiating the G1-to-S transition.


Ras signaling to stimulate RB inactivation and cell cycle pro-gression in androgen-dependent cells. Surprisingly, althoughV12Ras stimulated the expression of cyclin D1, it did not pro-mote RB phosphorylation or androgen-independent prolifera-tion, whereas E1A rendered LNCaP cells androgen independ-ent (Fig. 3). Interestingly, V12Ras exerted a negative effect onLNCaP proliferation that was p53 dependent. Although HPV-E6overcame the growth-inhibitory action of V12Ras in LNCaPcells, it was incapable of cooperating with Ras to induce an-drogen independence (Fig. 4). Similarly, although ectopic ex-pression of cyclin D1/CDK4 promoted RB phosphorylation inthe presence of androgen, it was incapable of inactivating RB ordriving cell cycle progression in the absence of androgen (Fig.5). Thus, cyclin D expression as stimulated by V12Ras or via itsectopic expression is not sufficient for androgen-independentproliferation, indicating that androgen acts through an addi-tional signaling pathway. We show that CDK2 and cyclin E-associated activity is also regulated by androgen, and thatcombined action of CDK4/cyclin D1 and CDK2/cyclin com-plexes are sufficient to inactivate RB and drive androgen-inde-pendent cell cycle progression (Fig. 6). Thus, the data pre-sented in this report demonstrate that androgen signals to bothCDK4 and CDK2 to inactivate RB and suggest that this require-ment is subverted in androgen-independent prostate cancers.

Ras in Androgen-dependent Proliferation. Prostatic ep-ithelia and early-stage prostate cancer cells require androgenfor proliferation, as in the absence of androgen, prostate tumorscease proliferation and undergo a degree of apoptosis (24). Atpresent, the relative contributions of proliferation and apoptosisto the therapeutic treatment of prostate cancer remain contro-versial (7, 8, 24). However, it is clear that if the prostatic cancercells fail to proliferate, the cancer will be controlled. How an-drogen acts as a prostatic growth factor is largely not under-stood. Androgen, like many other growth factors, stimulatesproliferation through its cognate receptor. The androgen recep-tor acts as a DNA-binding transcriptional modulator, whichpresumably changes the pattern of gene expression to stimu-late proliferation (24). Although a number of androgen-respon-sive genes have been cloned (e.g., prostate-specific antigen),the role of these proximal targets in prostate proliferation is notwell defined (24). However, it is apparent that these early eventsare integrated to stimulate the activation of cyclin D1 expres-sion and associated kinase activity, which subsequently ini-tiates phosphorylation/inactivation of RB (25). The importanceof these events in cellular proliferation is underscored by theaction of p16ink4a or PSM-RB, as both of these reagents areeffective at inhibiting the proliferation of all prostate cancer celllines tested (16, 17). Furthermore, because androgen with-drawal attenuates RB phosphorylation and cyclin A expressionin androgen-dependent cells, p16ink4a or PSM- RB simulatedthese same responses in androgen-independent cells.

Therefore, we sought to delineate the signaling pathwaybetween androgen and RB. Ras is known to mediate signalsthat stimulate both the expression of cyclin D1 and thephosphorylation of RB. In LNCaP cells, we show that V12Rasor V12Ras 1 HPV-E6 stimulates cyclin D1 expression in theabsence of androgen. However, restored cyclin D1 did notlead to RB inactivation or cell cycle progression in the ab-sence of androgen. This contrasts with what is observed in

immortalized fibroblastic cells, wherein V12Ras stimulatesthe expression of cyclin D1 and drives RB phosphorylationand cell cycle progression under conditions of serum star-vation or anchorage deprivation (35, 36).

The role of Ras in the androgen dependence of prostatecancer is polemical. Activating Ras mutations do occur in pros-tate cancer, although the overall frequency of these mutationsis quite low (37–41). In contrast, increased loss of RB correlateswith the development of androgen-independent cancer afterandrogen blockade therapy, and mutation of RB is observed atrelatively high frequency in prostate cancers (42–44). In culture,loss of RB signaling through the use of viral oncoproteins suchas E1A or T-Ag is sufficient to enable LNCaP cells to progressthrough the cell cycle in the absence of androgen (25). Althoughothers have tested the influence of Ras on androgen depen-dence in LNCaP cells, the results are controversial (45, 46). Ourresults clearly show that in bulk-transfected LNCaP cells (whichhave not been selected), V12Ras does not induce androgen-independent proliferation, although Ras activity was apparent(Fig. 3). Importantly, under virtually identical conditions, V12Rasinduced serum-independent cell cycle progression in Rat-1cells (not shown). Thus, Ras is specifically defective in promo-ting androgen-independent growth. Furthermore, we actuallyfound that V12Ras induces cell cycle inhibition in LNCaP cells,which is mediated by a p53-dependent pathway. Importantly,even when we disrupt this antiproliferative signaling from Rasusing the HPV-E6 protein, a condition which transforms primaryfibroblasts, we were unable to achieve androgen-independentcell cycle progression. Together, these data may explain whyRas mutations are rare events in prostate tumorigenesis (37).Complementary to these findings, Ravi et al. (46) used a Raf/estrogen receptor-inducible system to demonstrate that overtactivation of Raf induces cell cycle arrest in LNCaP cells. Bycontrast, Voeller et al. (45) showed that cadmium-inducible Rasin LNCaP cells leads to a modest proliferative advantage in theabsence of androgen. However, the extent of proliferation inthis system was quite reduced in comparison with androgentreatment and was clone dependent. Because V12Ras inducescell cycle arrest in cells containing functional p53 signalingpathways, the data of Voeller et al. (45) could reflect the loss ofp53 during the selection of clones. However, our results withV12Ras and E6 suggest that additional events must beinvolved.

Androgen Regulation of CDK4 and CDK2 Activities.The results presented here suggest that in prostatic epithelia,androgen-mediated proliferation likely involves two sets of sig-nals; one that may be mediated by Ras, which stimulates theexpression and activity of cyclin D1, and another signal that isrequired for complete RB inactivation and the expression ofcyclin A. Although CDK4/cyclin D1 in vitro kinase activity wasrestored in the absence of androgen by transfection of CDK4and cyclin D1 expression plasmids, ectopic CDK4 demon-strated little RB kinase activity in vivo and did not relieve RB-mediated repression of cyclin A (Fig. 5). Moreover, ectopicCDK4/cyclin D1 did not promote androgen-independent pro-liferation. These data are also consistent with the failure ofV12Ras and E6 to promote cell cycle progression while restor-ing cyclin D1 expression. We show that androgen withdrawalalso inhibits CDK2 and cyclin E-associated kinase activity. The


ectopic overexpression of cyclin E or cyclin A drove RB phos-phorylation in the presence of androgen but failed to do so inthe absence of androgen. These findings agree with the hy-pothesis that CDK4/cyclin D-mediated phosphorylation wasalso important for priming RB for subsequent inactivation of RBby CDK2-mediated phosphorylation. The combined impor-tance of these two signaling pathways is exemplified by theobservation that ectopic expression of CDK4, cyclin D1, cyclinE, and cyclin A was sufficient to drive RB phosphorylation in theabsence of androgen and render LNCaP cells androgen inde-pendent for cell cycle progression (Fig. 6). Together, these dataindicate that androgen acts through both CDK4/cyclin D andCDK2/cyclin complexes to promote cell cycle progression.

How androgen regulates the activity of CDK/cyclin com-plexes is still a subject of study. We demonstrate that cyclinD1 protein is diminished in the absence of androgen. Be-cause cyclin D1 is regulated both at the level of transcriptionand protein stability (1, 2), it is probable that androgen reg-ulates cyclin D1 at one of these levels. We have observedthat androgen stimulates cyclin D1 promoter activity in re-porter assays (data not shown). Similarly, Ras stimulatescyclin D1 promoter activity, suggesting that the androgenreceptor may signal through Ras to stimulate cyclin D1 tran-scription and result in protein accumulation (11, 34, 35).

Although ectopic CDK4/cyclin D1 expression is capable ofphosphorylating RB in vitro, this complex lacks the ability tofully phosphorylate and inactivate RB in vivo. This findingsuggested that the CDK2/cyclin complexes that mediate RBhyperphosphorylation (47, 48) must be attenuated by andro-gen withdrawal independently of CDK4/cyclin D1 activity.Consistent with this idea, we find that CDK2 and cyclinE-associated activity (but not cyclin E expression) are down-regulated via androgen withdrawal, even when CDK4/cyclinD1 activity is restored. CDK2/cyclin complexes are regulatedby a myriad of mechanisms. Because cyclin A expression isinhibited, this would partially explain the decrease in CDK2activity but fails to explain the specific deficiency in cyclinE-associated kinase activity. It is unlikely that accumulationof the CDK2-inhibitory molecules (i.e., p21Cip1 and p27Kip1)or CDK-inhibitor shuffling (as mediated by p16ink4a induc-tion) accounts for the attenuation of cyclin E-associatedkinase activity, because none of these inhibitory moleculeswere induced by androgen withdrawal. Thus, androgen reg-ulates cyclin E-associated kinase activity by as of yet unde-fined mechanisms.

Acquisition of Androgen Independence. The progres-sion of prostatic cancer from androgen dependence to andro-gen independence represents a catastrophic consequence forpatients suffering from the disease (24). At present, endocrinetherapy represents the only viable treatment for those withearly-stage prostate cancer, and no effective therapy exists forthose individuals with androgen-independent prostate cancer(7, 8). As with the mechanism through which androgen exerts itsmitogenic effect, little is known regarding how prostate cellscan bypass this requirement (24). In most established andro-gen-independent tumor cell lines, androgen receptor expres-sion is actually lost (24). For TSU-Pr1, this has been attributedto methylation of the androgen receptor promoter (49). JCA-1cells express prostate-specific antigen (a target of the androgen

receptor; Ref. 50); therefore, we suspected that JCA-1 cellsmay express the androgen receptor. However, we failed todetect the expression of AR protein in either cell line. Becauseno androgen receptor protein is detectable, both cell lines musthave activated mechanisms for the continued proliferation in itsabsence.

In cancer, a variety of mechanisms are used to lead to de-regulate proliferation. These mechanisms can be loosely di-vided into those that act upstream of RB (e.g., deregulatedmitogenic signaling or cyclin D1 amplification) and those thatact downstream of RB (e.g., viral oncoproteins of DNA tumorviruses or deregulated cdk2 activity). Our finding that the TSU-PR1 and JCA-1 cell lines are arrested by PSM-RB andp16ink4a indicates that deregulation of the cell cycle acts up-stream of RB. Although we focused on oncogenic Ras, we alsotested the action of activated b-catenin and c-myc. Neither ofthese proteins stimulated androgen-independent cell cycle pro-gression in LNCaP cells (data not shown), although b-cateninand c-myc are known to stimulate cyclin D1 expression (51).Furthermore, ectopic expression of CDK4/cyclin D1 also failedto inactivate RB or stimulate cell cycle progression in CDT. Infact, the only proteins capable of converting LNCaP cells toandrogen independence were proteins that lead to RB inacti-vation in the absence of androgen.

In summary, we show that the signal from androgen to RB ismisregulated in androgen-independent tumor cells, whereasthe ability of RB to signal to its downstream effectors is pre-cisely maintained. These data suggest that reactivation of RB inandrogen-independent tumors should be a goal of anticancertherapeutics. In focusing on the signal from androgen to RB, wealso demonstrate that this signal has at least two components:one, which may be Ras mediated, leading to the stimulation ofcyclin D expression and CDK4 activity; and the other, which isRas independent and completes RB inactivation through theaction of CDK2/cyclin complexes. These findings underscorethe importance of RB as an integrating target for cell cyclecontrol and tumor progression in prostate cancer. Clearly, thepathways regulating RB are tightly controlled, and the deregu-lated phosphorylation of RB that occurs in androgen-indepen-dent tumors requires the dysregulation of multiple signalingpathways.

Materials and MethodsCell Culture. The human prostatic adenocarcinoma cell line, LNCaP, wasobtained from the American Type Culture Collection. The TSUPr-1 androgen-independent cell line was kindly provided by Dr. John Isaacs (Johns Hopkins),and the JCA-1 cell lines was obtained from Dr. J. W. Chiao (New YorkMedical College). Cells were passaged fewer than 10 times in the experi-ments described. For regular passage, cells were grown in Improved MEM(Biofluids) containing 10% heat-inactivated FBS (Hyclone) supplementedwith 100 units/ml penicillin-streptomycin and 2 mM L-glutamine at 37°C in ahumidified atmosphere of 5% CO2. For growth in an androgen-depletedmedia, cells were propagated in Improved MEM containing 10% CDT FBS(Hyclone). Rat-1 fibroblastic cells were kindly provided by Dr. D. Green (LaJolla Institute for Allergy and Immunology, La Jolla, CA). Cells were main-tained in DMEM supplemented with 10% FBS, 100 units/ml penicillin-strep-tomycin, and 2 mM L-glutamine at 37°C in a humidified atmosphere of 5%CO2. Transfected JCA-1 and TSUPr-1 cells were selected 24 h after trans-fection with 2 mg/ml puromycin (Sigma) for 48 h of selection.

Plasmids. The pH2B-GFP plasmid, encoding histone H2B fused tothe GFP, was obtained from Dr. Geoff Wahl (Salk Institute). The pPSM-RBand p16ink4a plasmids have been described previously (15). Dr. Gilbert


Morris (Tulane University) supplied the E1A expression plasmid, Drs. KenjiFukasawa and Tony Capobianco (University of Cincinnati) provided theV12Ras plasmid, and the RSV-cyclin D1 construct was kindly provided byDr. C. Sherr (St. Jude Children’s Research Hospital, Memphis, TN). Thepuromycin resistance plasmid, pBABE-PURO, was kindly provided by Dr.H. Land (Imperial Cancer Research Fund, London, United Kingdom). Dr.Jean Wang (University of California, San Diego, CA) provided the WTLPexpression plasmid, and the CDK4 expression plasmid was obtained fromDr. Webster K. Cavenee (Ludwig Institute, La Jolla, CA). Dr. Karen Vous-den (National Cancer Institute, Frederick, MD) kindly provided the HPV-E6expression plasmid.

Transfections/BrdUrd Incorporation. Approximately 2.5 3 105

LNCaP cells were seeded onto poly-L-lysine-coated coverslips in eachwell of a six-well dish. After 48 h, wells were washed one time withserum-free Improved MEM. Lipofectin substrate was then applied andallowed to incubate for 6 h, after which the serum-containing mediumindicated was added back. To monitor protein expression, cells wereharvested 48 h after transfection. To monitor S-phase progression, co-transfection with pH2B-GFP (0.12 mg) and a secondary effector expres-sion plasmid (5 mg) were carried out. Cells were labeled 48 h aftertransfection with Cell Proliferation Labeling Reagent (Amersham Pharma-cia Biotech), according to the manufacturer’s recommended protocol.Pulse labeling was continued for 14 h, at which time cells were fixed andprocessed for immunofluorescence. Rat-1 cells were seeded at a densityof 2 3 104 cells onto coverslips in each well of a six-well dish. Twenty-fourh later, the cells were transfected with 2 mg of total DNA (as indicated) withFuGENE 6 transfection reagent, according to the manufacturer’s protocol(Roche Diagnostics). JCA-1 and TSUPr-1 cells were seeded on coverslipsat a density of 1 3 105 cells/well of a six-well dish. Twenty-four h later, thecells were transfected with 4 mg of total plasmid DNA (as indicated) usingthe BBS/calcium phosphate method described previously (52). Forty-eight h after transfection, Cell Proliferation Labeling Reagent (Amersham)was added according to the manufacturer’s protocol. Sixteen h later, cellswere fixed with formaldehyde and processed for indirect immunofluores-cence to detect BrdUrd incorporation as described previously (53). Foreach experiment, at least 150 transfected (GFP-positive) and untrans-fected (GFP-negative) cells were counted. Data shown reflect the averageof at least 2–3 independent experiments. Images were captured using aNikon Axiophot at 340 and a SpotCam digital camera.

Quiescence. Twelve h after transfection, Rat-1 cells were washed andplaced in DMEM supplemented with 0.1% FBS, 100 units/ml penicillin-streptomycin, and 2 mM L-glutamine for 48 h prior to BrdUrd labeling.

Immunoblotting. Immunoblotting was carried out by use of standardprocedures as described previously (25). Blots were probed for the fol-lowing proteins with polyclonal antibodies: RB (851; gift of Dr. Jean Wang,University of California at San Diego, San Diego, CA); Cyclin D (sc-182 andsc-753; Santa Cruz Biotechnology); cyclin A (sc-751; Santa Cruz Biotech-nology); cyclin E (sc-198; Santa Cruz Biotechnology); Ras (sc-519; SantaCruz Biotechnology); ERK (sc-93-G; Santa Cruz Biotechnology); phos-pho-ERK#2 (9101S; New England BioLabs); androgen receptor (sc-816;Santa Cruz Biotechnology); p21(sc-757; Santa Cruz Biotechnology) andwith monoclonal antibodies: phosphoERK#1 (sc-7383; Santa Cruz Bio-technology); HA-tag (MMS-101R; Covance); p53 (OP-43; Calbiochem).Goat antimouse horseradish peroxidase, goat antirabbit horseradish per-oxidase, protein A-horseradish peroxidase (Bio-Rad), or antigoat (sc-2020; Santa Cruz Biotechnology) were used for antibody visualization viaenhanced chemiluminescence (Amersham Pharmacia Biotech).

Immunoprecipitations and CDK Kinase Assays. For in vitro kinaseassays, 2–4 3 106 cells from conditions indicated were harvested bytrypsinization and washed in PBS. To analyze CDK2 activity, cells werelysed and clarified as described previously (25). After quantitation, 160 mgof lysate were incubated for 2 h (4°C with rotation) with either anti-CDK2antiserum (Santa Cruz Biotechnology), anti-cyclin E antiserum (Santa CruzBiotechnology), or anti-E1A antiserum (Santa Cruz Biotechnology) as anegative control. Immunoprecipitations and kinase reactions were per-formed as described previously (25). CDK4 kinase assays were performedusing the technique of LaBaer et al. (54).

Time Course. LNCaP cells were seeded in medium containing 10%CDT at a density of 1.1 3 106 cells/6-cm dish. After 96 h, medium wasreplaced by 10% FBS. Cells were harvested at the times indicated in theexperiment.

AcknowledgmentsWe thank Dr. Webster K. Cavenee, Dr. Kenji Fukasawa, Dr. Zvjezdana Sever-Chroneos, and Dr. Larry Sherman for critical reading of the manuscript. Wealso thank Drs. Webster K. Cavenee, Kenji Fukasawa, Peter J. Stambrook,Karen Vousden, and Geoffrey Wahl for a generous supply of reagents.

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Differential Requirements for Ras and the Retinoblastoma ... · Differential Requirements for Ras and the Retinoblastoma Tumor Suppressor Protein in the Androgen Dependence of Prostatic