Differential Requirements for Ras and the Retinoblastoma Tumor Suppressor Protein in the Androgen Dependence of Prostatic Adenocarcinoma Cells 1

Vol. 11, 361 372, July 2000 Cell Growth & Differentiation 361 Differential Requirements for Ras and the Retinoblastoma Tumor Suppressor Protein in the Androgen Dependence of Prostatic Adenocarcinoma Cells 1 Anne F. Fribourg, Karen E. Knudsen, Matthew W. Strobeck, Clint M. Lindhorst, and Erik S. Knudsen 2 Department of Cell Biology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0521 Abstract Prostate cells are dependent on androgen for proliferation, but during tumor progression prostate cancer cells achieve independence from the androgen requirement. We report that androgen withdrawal fails to inhibit cell cycle progression or influence the expression of cyclin-dependent kinase (CDK)/cyclins in androgen-independent prostate cancer cells, indicating that these cells signal for cell cycle progression in the absence of androgen. However, phosphorylation of the retinoblastoma tumor suppressor protein (RB) is still required for G 1 -S progression in androgen-independent cells, since the expression of constitutively active RB (PSM-RB) or p16ink4a caused cell cycle arrest and mimicked the effects of androgen withdrawal on downstream targets in androgen-dependent LNCaP cells. Since Ras is known to mediate mitogenic signaling to RB, we hypothesized that active V12Ras would induce androgen-independent cell cycle progression in LNCaP cells. Although V12Ras was able to stimulate ERK phosphorylation and induce cyclin D1 expression in the absence of androgen, it was not sufficient to promote androgen-independent cell cycle progression. Similarly, ectopic expression of CDK4/cyclin D1, which stimulated RB phosphorylation in the presence of androgen, was incapable of inactivating RB or driving cell cycle progression in the absence of androgen. We show that androgen regulates both CDK4/cyclin D1 and CDK2 complexes to inactivate RB and initiate cell cycle progression. Together, these data show that androgen independence is achieved via deregulation of the androgen to RB signal, and that this signal can only be Received 1/4/00; revised 5/11/00; accepted 5/11/00. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported in part by Grant R01-CA82525-01 from the National Cancer Institute-NIH and a grant from the Sidney Kimmel Cancer Foundation (to E. S. K.). K. E. K. is supported by Postdoctoral Fellowship CA82034 from the National Cancer Institute. 2 To whom requests for reprints should be addressed, at Department of Cell Biology, Neurobiology, and Anatomy, University of Cincinnati College of Medicine, P. O. Box 670521, 3125 Eden Avenue, Cincinnati, OH 45267-0521. Phone: (513) 558-6849; Fax: (513) 558-4454; E-mail: Erik.Knudsen@UC.edu. partially initiated by the Ras pathway in androgendependent cells. Introduction Tumorigenic growth is characterized by proliferation occurring under inappropriate environmental conditions (1, 2). This phenomenon is clearly observed during the progression of prostate cancer. Initially, prostatic epithelia or early-stage prostatic cancer cells are androgen-dependent and thus require androgen to proliferate (3 5). In the most commonly used therapy for prostate cancer, androgens are removed either by chemical or surgical means, leading to the cessation of proliferation (6). Despite this initial remission, the tumor cells rapidly become androgen-independent, rendering hormonal treatment ineffective (7, 8). Strikingly, there is no efficient treatment for androgen-independent tumors. Furthermore, little is known about how these recurrent tumor cells are able to proliferate in the absence of androgen. Androgen is a known growth factor for prostatic epithelial cells (9). However, the mechanism through which androgen signals to promote proliferation is poorly understood (10). In general, mitogenic factors act through the Ras/ERK 3 pathway to activate a host of CDK/cyclin complexes, which are required for cell cycle progression and cellular proliferation (1, 2, 11, 12). Early in the G 1 phase of the cell cycle, mitogenic signals activate CDK4/cyclin D complexes, which are required to initiate phosphorylation of RB (2, 13). Reagents that block RB phosphorylation (e.g., the CDK4 inhibitor p16ink4a) or constitutively active RB proteins (PSM-RB) arrest cells in G 1 (2, 14, 15). Thus, inactivation of RB is typically required for entry into S-phase, although specific cell types acquire resistance to the actions of these reagents, either through the attenuation of downstream signaling or through the expression of viral oncoproteins of DNA tumor viruses (16, 17). We and others have shown recently that RB inhibits cell cycle progression by attenuating cyclin A expression, thus diminishing CDK2 activity (16, 18 20). Because both CDK2/cyclin E and CDK2/cyclin A activities are required for entry into S-phase, inhibition of cyclin A expression is a critical component of the antiproliferative action of RB (18, 21, 22). The LNCaP (androgen-dependent) tumor cell line is a model system for the study of androgen-dependent proliferation (23, 24). Androgen deprivation causes a documented reduction in cyclin D protein levels and CDK4/cyclin D kinase activity. As a result, RB remains underphosphorylated/active, and cyclin A 3 The abbreviations used are: ERK, extracellular signal-regulated kinase; CDK, cyclin-dependent kinase; RB, retinoblastoma tumor suppressor protein; BrdUrd, bromodeoxyuridine; CDT, charcoal dextran treated; GFP, green fluorescent protein; HPV, human papillomavirus; LP, large pocket; FBS, fetal bovine serum.

362 RB Regulates the Androgen Requirement Fig. 1. Androgen-independent cells do not arrest and fail to regulate RB in the absence of androgen. A, JCA-1, LNCaP, and TSUPr-1 cells were propagated for 96 h in the presence of either 10% CDT FBS (CDT) or 10% FBS. Cells were then labeled with BrdUrd for a period of 14 h. Subsequent to the pulse, cells were fixed and processed to monitor BrdUrd incorporation via direct immunofluorescence. Data shown are the average of at least three independent experiments in which at least 150 cells/experiment were analyzed; bars, SD. Representative immunofluorescence from the experiment described are shown in the inset. B, JCA-1, TSUPr-1, and LNCaP cells were propagated for 96 h in the presence of either 10% CDT (Lanes 2, 4, and 6) or 10% FBS (Lanes 1, 3, and 5), harvested, and clarified, and protein lysates were prepared. Equal protein was resolved by SDS-PAGE, and the endogenous cyclin A, cyclin E, cyclin D1, and RB proteins were detected by immunoblotting with the respective antibodies. prb, underphosphorylated RB; pprb, hyperphosphorylated RB. C, lysates from JCA-1, TSUPr-1, and LNCaP cells as described in B were resolved by SDS-PAGE, and the androgen receptor (AR) was detected by immunoblotting. protein levels (as well as CDK2 activity) are attenuated (25). Thus, these cells arrest in G 1 when androgen is removed. Conversion to androgen independence in LNCaP cells can be achieved by ectopic expression of viral oncoproteins E1A and T-Ag, which functionally disrupt RB (25). Together, these results suggest that RB plays an important role in maintaining androgen dependence in prostatic tumor cells. Furthermore, this suggests that androgen independence occurs through signals that disrupt RB function, either through upstream or downstream signaling. In an effort to understand the molecular changes that underlie androgen independence, we evaluated the ability of androgenindependent cells to transmit signals to RB and for RB to signal to its downstream effectors. We show that androgen-independent cells remained responsive to RB activation and that RB targets were invoked in an appropriate fashion. By contrast, RB inactivation in these cells was deregulated and occurred in the absence of androgen. Thus, signals upstream of RB are misregulated in androgen-independent prostate cells. Because Ras is known to link growth factor signaling to the cyclin D/RB network, we investigated the function of activated Ras in androgen-dependent cells. Surprisingly, although Ras did induce cyclin D1 in the absence of androgen, Ras had an overall negative effect on cell cycle progression that was p53 dependent. Even when the growth-inhibitory activity of Ras was disrupted with HPV-E6 or when CDK4/cyclin D1 was ectopically expressed, androgen-independent cell cycle progression failed to occur. These data indicate that androgen must regulate more than CDK4/cyclin D to initiate cell cycle progression. In fact, we demonstrate that androgen also regulates CDK2/cyclin activity, and the collective activities of CDK4/cyclin D and CDK2/cyclin complexes are required to inactivate RB and initiate cell cycle progression in the absence of androgen. These data demonstrate that maintenance of the androgen to RB pathway requires factors distinct from Ras and involves the combined regulation of CDK4/cyclin D and CDK2/cyclin complexes. Results Androgen Signaling to RB Is Deregulated in Androgenindependent Cells. The LNCaP cell line is androgen dependent and fails to incorporate BrdUrd when cultured in CDT serum that lacks androgen (Fig. 1A). We have shown previously that cell cycle progression in LNCaP cells is re-

Cell Growth & Differentiation 363 stored by supplementing CDT with dihydrotestosterone (25). In contrast, the androgen-independent cell lines JCA-1 and TSUPr-1 continue to incorporate BrdUrd when cultured in CDT (Fig. 1A). Consistent with the BrdUrd incorporation results, whereas androgen-independent cells proliferate in the absence of androgen, androgen-dependent LNCaP fail to proliferate, as determined by growth curves (not shown). When deprived of androgen, LNCaP cells exhibited decreased levels of cyclin D1, and as a consequence RB was dephosphorylated (Fig. 1B, compare Lanes 5 and 6). Cyclin E expression remained constant in the presence or absence of androgen, whereas cyclin A protein levels were diminished in the absence of androgen (Fig. 1B, Lanes 5 and 6). These data are consistent with a model wherein androgen acts through cyclin D1 to stimulate RB phosphorylation, which then inactivates RB and derepresses cyclin A expression. Thus, we hypothesize that androgen-independent cells have either lost this signal from androgen to RB or from RB to its known downstream effectors. To distinguish between these possibilities, JCA-1 and TSUPr-1 cells were propagated in either the presence or the absence of androgen. Regardless of androgen status, these cells maintained high levels of cyclin D1 protein and demonstrated RB hyperphosphorylation, indicating that CDK4 activity was also maintained (Fig. 1B, compare Lanes 1 and 2, Lanes 3 and 4). Cyclin A protein levels remained unchanged, consistent with the phosphorylation status of RB. Cyclin E protein levels also remained constant (Fig. 1B, compare Lanes 1 and 2, Lanes 3 and 4). These data indicate that androgen-independent cells have lost the ability to regulate RB in response to androgen withdrawal. A possible explanation for this finding would be that the androgen receptor in these cell lines is constitutively active and thus functions in the absence of androgen. However, immunoblot analysis of the androgen receptor showed that JCA-1 and TSU-PR1 cells do not express detectable receptor (Fig. 1C, Lanes 1 4), whereas in LNCaP cells the receptor was readily detectable (Fig. 1C, Lanes 5 and 6). Thus, these androgen-independent cell lines have subverted the requirement for the androgen receptor to promote cell cycle progression. RB Signaling Is Retained in Androgen-independent Cells. The finding that RB phosphorylation was observed in the androgen-independent cells in the absence of androgen suggested that signals upstream of RB (i.e., cyclin D-associated kinase activity) must be deregulated for ongoing cell cycle progression. However, it is known that disruption of RB by the oncoproteins of DNA tumor viruses or deregulation of downstream targets leads to inappropriate cell cycle progression (17, 18). Therefore, we designed experiments to determine whether RB signaling is intact in androgen-independent cell lines. JCA-1 cells were cotransfected in complete serum with a GFP expression plasmid and either parental vector, p16ink4a, or PSM-RB expression plasmids, and cell cycle progression of transfected cells (GFP positive) was monitored by BrdUrd incorporation. Cells transfected with vector showed approximately the same level of BrdUrd incorporation as nontransfected cells (GFP negative) from the same coverslip (Fig. 2A). By contrast, cells transfected with p16ink4a or PSM-RB showed significant reduction in BrdUrd incorporation in comparison to vector-transfected cells or nontransfected cells from the same coverslip. Together with our previous observation that PC3 and TSU-PR1 cells also arrest in response to active RB (17), these results indicate that signaling of RB to its downstream targets is intact in androgen-independent tumors. To assess the importance of RB signaling in these cells, we compared the molecular response of RB-arrested androgenindependent cells (JCA-1 and TSU-PR1) with that observed upon androgen withdrawal in androgen-dependent cells (LNCaP). JCA-1 and TSU-PR1 cells were cotransfected in the presence of androgen with a puromycin-resistance plasmid and either parental vector, p16ink4a, or PSM-RB. Twentyfour h after transfection, cells were then selected with puromycin for 48 h, at which time the majority of untransfected cells were eliminated by puromycin (data not shown). Transfected cells were harvested and protein levels were examined by immunoblot analyses. Cyclin D1 and cyclin E protein levels remained constant in each cell line whether the cells were transfected with p16ink4a, PSM-RB, or vector (Fig. 2B, Lanes 1 6). By contrast, when the androgen-independent prostatic tumor cells were either transfected with p16ink4a or PSM-RB, cyclin A protein levels were reduced in comparison with the same cells transfected with parental vector (Fig. 2B, compare Lanes 1 and 3 with Lane 2 or Lanes 4 and 6 with Lane 5). In addition, the endogenous RB maintained high levels of phosphorylation when transfected with PSM-RB (Fig. 2B, Lanes 3 and 6), whereas after transfection with p16ink4a, only the underphosphorylated form of RB was predominant in JCA-1 and TSUPr-1 (Fig. 2B, Lanes 1 and 4). This indicates that as reported previously (16), PSM-RB does not feed back to modify the phosphorylation of endogenous RB, whereas p16ink4a blocks the CDK4 kinase activity that is required to initiate RB phosphorylation. Taken together, these data show that the signaling pathway from androgen to RB is lost in androgen-independent cells, but these cells have not lost the ability to arrest in response to RB. Moreover, active RB or p16ink4a transfected into androgen-independent cells mimics the effect of androgen withdrawal in androgen-dependent cells (i.e., down-regulation of cyclin A but not cyclin E). Thus, androgen-independent cells have achieved independence not by subverting RB activity directly or by downstream disruption of RB signaling but rather by dysregulation of upstream signaling pathways. Ras Induction of Cyclin D Is Insufficient to Promote Androgen-independent Proliferation. These data suggest that androgen acts through cyclin D1 to stimulate RB phosphorylation/inactivation, thus leading to induced cyclin A expression and cell cycle progression. Consistent with this idea, a kinetic study of androgen stimulation in LNCaP cells revealed that increased cyclin D1 expression correlates with increased RB phosphorylation that preceded the accumulation of cyclin A protein (Fig. 3A, Lanes 1 3). It is known that growth factors can act through the Ras signaling pathway to activate cyclin D1 expression (1, 2, 11, 12, 26). We therefore surmised that androgen acts through Ras to stimulate cyclin D1 expression and inactivate RB. To test whether activation of Ras is sufficient to promote proliferation in LNCaP cells, we used activated V12Ras. To confirm that the V12Ras

364 RB Regulates the Androgen Requirement Fig. 2. Androgen-independent cells arrested by PSM-RB mimic that of androgen-dependent cells deprived of androgen. A, JCA-1 cells were cotransfected with a GFP expression plasmid (0.12 g) and the indicated plasmids (4 g). BrdUrd was added 24 h after transfection for a total labeling of 14 h. Cells were stained with anti-brdurd antibody, and the percentage of GFP-positive and GFP-negative cells exhibiting BrdUrd incorporation was determined by indirect immunofluorescence. The average and deviation values shown are from two to three independent experiments with at least 150 GFP-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 g) and the indicated expression plasmids (15 g) in 10-cm dishes by the calcium phosphate method. Twenty-four h after transfection, JCA-1 and TSUPr-1 cells were selected with 2 g/ml of puromycin for 48 h. Then, the cells 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-RB proteins were detected by immunoblotting with the respective antibodies. (activated Ras) expression plasmid expressed active protein when transfected into LNCaP cells in androgen-deprived media, cells were transfected in CDT with either V12Ras expression plasmid or parental vector. Cells were harvested 48 h after transfection, and immunoblot analyses were performed. As shown in Fig. 3B, Ras protein was overexpressed when the expression plasmid was transfected into LNCaP cells 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 a major downstream target of Ras (27 29). No significant difference in ERK protein expression was observed between the two transfections, although ERK1 and ERK2 phosphorylation was dramatically induced when LNCaP cells expressed V12Ras (Fig. 3B, compare Lanes 1 and 2). Furthermore, V12Ras led to an increase in the expression of cyclin D1 in CDT cultured LNCaP cells (Fig. 3D, compare Lanes 1 4). Thus, these data confirmed that V12Ras is expressed and active when transfected into the LNCaP cells in the absence of androgen and acts to promote the accumulation of cyclin D1. To determine whether Ras signaling is sufficient to promote androgen-independent cell cycle progression, LNCaP cells cultured in CDT were cotransfected with a GFP expression plasmid and either parental vector, V12Ras, or E1A expression plasmids, and BrdUrd incorporation was monitored. As shown in Fig. 3C, cells transfected with vector alone failed to incorporate BrdUrd in the absence of androgen. Strikingly, LNCaP cells transfected with activated Ras also failed to demonstrate androgen-independent cell cycle progression. By contrast, E1A rescued cell cycle progression of LNCaP cells to approximately the same level as vectortransfected cells in the presence of androgen. Using a similar protocol, V12Ras promoted cell cycle progression in serumstarved Rat-1 cells (Fig. 3E). Thus, V12Ras signaling is not sufficient to promote cell cycle progression in LNCaP cells. Consistent with this finding, although we observed an increase in cyclin D1 expression, we did not observe an increase in cyclin A expression (Fig. 3D), nor did we observe RB phosphorylation (not shown). Cyclin E levels were unaffected by the expression of V12Ras (Fig. 3D). V12Ras Inhibits Cell Cycle Progression in LNCaP Cells via p53. Although the expression of cyclin D1 was stimulated when V12Ras was transfected into LNCaP cells cultured in CDT, neither increased RB phosphorylation nor was cyclin A expression detected. These data suggested that V12Ras was either incapable of acting to activate the cyclin D1-associated kinase activity, or alternatively that V12Ras was stimulating an antiproliferative pathway. Because it has been shown recently that V12Ras can inhibit proliferation of

Cell Growth & Differentiation 365 Fig. 3. E1A but not V12Ras is sufficient to promote androgen-independent cell cycle progression. A, LNCaP cells were seeded and grown in 10% CDT for 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 equal protein was resolved by SDS-PAGE. The endogenous cyclin A, cyclin E, cyclin D1, and RB proteins were detected by immunoblotting with the respective antibodies. B, LNCaP cells were transfected in CDT with the expression plasmids indicated. Cells were harvested 48 h after transfection, and clarified lysates were prepared. Total equal protein was resolved by SDS-PAGE, and the endogenous Ras, ERK, and phospho-erk proteins were detected by immunoblotting with the respective antibodies (two different antibodies were used to recognize phospho-erk, each recognizing a different epitope). C, LNCaP cells were cotransfected with a GFP expression plasmid (0.12 g) and the indicated plasmids (5 g). After transfection, cells were either replaced in 10% FBS or 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 deviation values shown are from two to three independent experiments with at least 150 GFP-positive cells counted per experiment. Representative immunofluorescence 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, and equal total protein was resolved by SDS-PAGE. Cyclin D1, Cyclin E, and Cyclin A proteins were then detected by immunoblotting. E, Rat-1 cells were cotransfected 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 percentage of GFP-positive cells exhibiting BrdUrd incorporation was determined by indirect immunofluorescence. The average and deviation values shown are from two to three independent experiments with at least 150 GFP-positive cells counted per experiment; bars, SD. primary fibroblasts (30), we determined whether V12Ras exerted a growth-inhibitory effect in LNCaP cells. To test this, LNCaP cells cultured in FBS were cotransfected with a GFP expression plasmid and either vector control or V12Ras expression plasmids (Fig. 4B). Cells were labeled with BrdUrd, and the incorporation of BrdUrd in GFP-positive cells was determined. Although the vector had no influence on BrdUrd incorporation, transfection of V12Ras significantly inhibited BrdUrd incorporation, thus demonstrating that V12Ras inhibits cell cycle progression in LNCaP cells. Because the growth-inhibitory activity of V12Ras is known to act through the p53/p21cip1 signaling axis (30), the ability of V12Ras to stimulate expression of p53 and p21cip1 (a p53 target) was determined. In cells transfected with V12Ras, there was a consistent increase in p53 and p21cip1 protein levels, regardless of androgen status, suggesting that the growth-inhibitory activity of V12Ras is mediated via p53 (Fig. 4A, compare Lanes 1 and 2 or Lanes 3 and 4). To test this directly, LNCaP cells cultured in FBS were cotransfected with GFP expression plasmid and either V12Ras or V12Ras and HPV-E6 expression plasmids. HPV-E6 is known to promote p53 degradation and thereby overcome the antiproliferative activity associated with p53 (Fig. 4B). Although the GFP-positive cells cotransfected with V12Ras expression plasmid arrested, coexpression of HPV-E6 partially reversed this effect of V12Ras (Fig. 4B). Cyclin D Expression Is Not Sufficient for Androgen Independence. Because the growth-inhibitory effects of V12Ras can be overcome by HPV-E6, we reasoned that this combination should be sufficient to render LNCaP cells androgen independent. LNCaP cells cultured in CDT were cotransfected with GFP expression plasmid and either Vector, E1A, or V12Ras HPV-E6 expression plasmids. Consistent with our earlier observations, expression of E1A promoted BrdUrd incorporation in CDT (Fig. 4C). Surprisingly, cotransfection of V12Ras with HPV-E6 was not sufficient to drive androgen-independent cell cycle progression (Fig. 4C). However, E1A rendered LNCaP cells androgen independent in

366 RB Regulates the Androgen Requirement Fig. 4. V12Ras inhibits the proliferation of LNCaP cells through the p53-signaling axis. A, 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, and equal total protein was resolved by SDS-PAGE. The p53 and p21cip1 proteins were then detected by immunoblotting. B, LNCaP cells cultured in FBS were cotransfected with a GFP expression plasmid (0.12 g), and the indicated expression plasmids (5 g of vector, V12Ras, HPV-E6, or 2.5 g ofe6and V12Ras together). 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 deviation values shown are from two to three independent experiments with at least 150 GFP-positive cells counted per experiment; bars, SD. C and D, LNCaP cells cultured in CDT were cotransfected with a GFP expression plasmid (0.12 g), and the indicated expression plasmids (5 g of vector, E1A, HPV-E6, or 2.5 g of each plasmid combined). 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 deviation values shown are from two to three independent experiments with at least 150 GFP-positive cells counted per experiment; bars, SD. the presence of V12Ras (Fig. 4D), further substantiating the idea that RB inactivation is required for androgen-independent growth, and that V12Ras is incapable of fulfilling this requirement in the absence of androgen (Fig. 4D). Collectively, these results suggest that additional mechanisms besides the accumulation of D-type cyclins are at play in androgen-dependent proliferation. To test this hypothesis, LNCaP cells were cotransfected with CDK4/cyclin D1 and monitored for CDK4-associated kinase activity, RB phosphorylation, and BrdUrd incorporation (Fig. 5). Initially, CDK4 complexes were immunoprecipitated from transfected cells and 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 of CDK4/cyclin D1 (Fig. 5, Lane 3). These observations were supported by in vivo RB kinase assays, wherein cells were transfected with the LP fragment of RB (Fig. 5B; Refs. 25, 31, 32). As has been reported previously, endogenous CDK4 activity was insufficient to drive LP phosphorylation (Fig. 5B, Lane 1; Ref. 25), whereas cotransfection of CDK4/cyclin D1 effectively drove LP phosphorylation in the presence of FBS (Fig. 5B, Lane 2). Therefore, ectopic CDK4/cyclin D1 was shown to harbor RB kinase activity by both in vivo and in vitro kinase assays. LNCaP cells cultured in CDT also demonstrated enhanced in vitro RB kinase activity upon transfection with CDK4 and cyclin D1 (Fig. 5A, compare Lanes 4 and 5). However, transfection of CDK4/cyclin D1 was insufficient to effectively drive in vivo phosphorylation of LP (Fig. 5B, middle panel, Lanes 3 and 4). Therefore, ectopic CDK4/cyclin D1 kinase activity is not sufficient to drive RB inactivation. Consistent with this observation, ectopic CDK4/cyclin D1 expression did not restore cyclin A expression in CDT (Fig. 5B, bottom panel). Moreover, ectopic CDK4/cyclin D1 was insufficient to drive cell cycle progression in the absence of androgen (Fig. 5C). CDK4/Cyclin D and CDK2/Cyclin Complexes Are Both Targets of Androgen. It is known that complete RB phosphorylation/inactivation requires both CDK4 and CDK2 activities (18, 21, 22). This leaves the possibility that androgen also regulates CDK2/cyclin activity, which is required to inactivate RB. To determine this, we initially analyzed the effect of androgen on CDK inhibitory proteins, which can lead to the 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 the presence or absence of androgen; accordingly, no changes were observed upon cotransfection of CDK4/cyclin D1 (compare Lanes 1 4). As we have reported previously (25), cells cultured in the absence of androgen exhibit low levels of p21cip1, as compared with cells cultured in the presence of androgen (compare Lanes 1 and 2 with Lanes 3 and 4). No changes in p21cip1 levels were observed upon cotransfec-

Cell Growth & Differentiation 367 Fig. 5. Ectopic expression of CDK4/cyclin D is not sufficient to promote RB inactivation and androgen independent proliferation. A, LNCaP cells were either mock-transfected (Lanes 2 and 4) or cotransfected with expression 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 after transfection, lysed, and subjected to immunoprecipitation using antibodies direct against either E1A (Lane 1) or CDK4 (Lanes 2 5). Immunoprecipitated complexes were used for in vitro CDK4 kinase assays, using the C-pocket of RB as a substrate. Phospho-RB was subjected to 12% SDS-PAGE and visualized by autoradiography. B, LNCaP cells were cotransfected either in FBS (Lanes 1 and 2) or CDT (Lanes 3 and 4) with either an expression plasmid encoding the LP fragment of RB, and vector (Lanes 1 and 3) or cyclin D1 and CDK4 (Lanes 2 and 4) expression plasmids. Cells were harvested 48 h after transfection, and equal total protein was resolved by SDS-PAGE. Cyclin D1, LP, and cyclin A proteins were then detected by immunoblotting. plp, underphosphorylated LP; pplp, hyperphosphorylated LP. C, LNCaP cells cultured in CDT were cotransfected with a GFP expression plasmid (0.12 g) and the indicated expression plasmids (5 g of vector, E1A, or 2.5 g of cyclin D with either 2.5 g of vector or 2.5 g of CDK4). 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 deviation values shown are from two to three independent experiments with at least 150 GFP-positive cells counted per experiment. Bars, SD. tion with CDK4/cyclin D1. Collectively, these data reaffirm that androgen deprivation does not result in increased expression of these CDK inhibitory proteins, and in fact lack of androgen actually causes a decrease in p21cip1. Moreover, restoration of CDK4 activity does not affect the CDK inhibitor profile. We also examined the effect of CDK4/cyclin D1 transfection on CDK2 activity using in vitro kinase assays. As shown in Fig. 6B, LNCaP cells cultured in the presence of androgen harbored significant CDK2-associated kinase activity, as determined by in vitro kinase assays against an RB substrate, and ectopic expression of CDK4/cyclin D1 did not enhance the endogenous CDK2 activity (top panel, Lanes 1 3). Consistent with previous reports (25), CDK2-associated kinase activity is attenuated in the absence of androgen, and CDK2 activity was not restored by the ectopic expression of CDK4/ cyclin D1 (Fig. 6B, top panel, Lanes 4 and 5). A trivial explanation for this would be that lack of cyclin A is responsible for the attenuation in CDK2 kinase activity. Therefore, we analyzed the kinase activity specifically associated with cyclin E (Fig. 6B, bottom panel). To do this, cyclin E complexes were immunoprecipitated and used in in vitro kinase assays against the RB substrate. Cyclin E kinase activity was not influenced by ectopic expression of CDK4/cyclin D1, and cyclin E-associated activity was attenuated in the absence of androgen (Fig. 6B, bottom panel, compare Lanes 2 and 3 and Lanes 4 and 5). To investigate the activity of CDK2 for in vivo RB phosphorylation, ectopically expressed cyclin E or cyclin A was monitored for its ability to phosphorylate the LP fragment of RB. Both cyclin E and cyclin A overproduction promoted RB phosphorylation in the presence of androgen; however, in the absence of androgen, RB phosphorylation was inhibited (Fig. 6C). Because CDK4/cyclin D activity is required to prime the phosphorylation of RB for subsequent hyperphosphorylation by CDK2/cyclin complexes, we reasoned that the inhibition of cyclin D expression and CDK4 activity may underlie the inhibition of RB phosphorylation when cyclin E and cyclin A are overexpressed. To test this idea, CDK4, cyclin D1, cyclin E, and cyclin A were coexpressed with the LP fragment, and the status of LP phosphorylation was monitored in the presence or absence of androgen. As shown in Fig. 6D, the LP fragment of RB was efficiently phosphorylated by this combination of CDK/cyclins in both the presence and absence of androgen (compare Lanes 2 and 4), thereby indicating that these conditions overcome both the CDK4 and CDK2 inhibition observed in androgen-deprived LNCaP cells. Because RB is a critical determinant of androgen-dependent cell cycle progression, we determined whether the cells cotransfected with CDK4, cyclin D1, cyclin E, and cyclin A were able to progress through the cell cycle in the absence of androgen. As shown in Fig. 6E, vectortransduced cells failed to incorporated BrdUrd, whereas cells transfected with E1A expression plasmid incorporated BrdUrd in the absence of androgen. Ectopic coexpression of CDK4, cyclin D1, cyclin E, and cyclin A also promoted cell cycle progression in the absence of androgen. Together, these findings indicate that the control of RB phosphorylation occurs at the level of both CDK4/cyclin D and CDK2/

368 RB Regulates the Androgen Requirement Fig. 6. Androgen action requires both CDK4 and CDK2 activities. A, LNCaP cells were either mocktransfected (Lanes 1 and 3) or cotransfected with expression plasmids for CDK4 and cyclin D1 (Lanes 2 and 4) in either FBS (Lanes 1 and 2) or CDT (Lanes 3 and 4). Cells were harvested 48 h after transfection, lysed, subjected to SDS-PAGE, and immunoblotted using antibodies direct against either p16ink4a (top panel), p21cip1 (middle panel), or p27kip1 (bottom panel). B, lysates from cells prepared as described in A were used for immunoprecipitation using antibodies against either E1A (Lane 1), CDK2 (top panel), or cyclin E (bottom panel). Immunoprecipitated complexes were used for in vitro kinase assays, using the C-pocket of RB as a substrate. Phospho-RB was subjected to 12% SDS-PAGE and visualized by autoradiography. C, LNCaP cells were cultured in FBS (Lanes 1 3) orcdt(lanes 4 6) and cotransfected with an expression plasmid encoding LP and either cyclin A(Lanes 1 and 4), vector (Lanes 2 and 5), or cyclin E(Lanes 3 and 6) plasmids. Cells were harvested 48 h after transfection, and equal total protein was resolved by SDS-PAGE. plp, underphosphorylated LP; pplp, hyperphosphorylated LP. D, LNCaP cells were cultured in FBS (Lanes 1 and 2) or CDT (Lanes 3 and 4) and cotransfected with expression plasmids encoding LP and either vector (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 transfection, and equal total protein was resolved by SDS-PAGE. plp, underphosphorylated LP; pplp, hyperphosphorylated LP. E, LNCaP cells cultured in CDT were cotransfected with a GFP expression plasmid and the indicated expression plasmids. 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 GFPpositive cells exhibiting BrdUrd incorporation was determined by indirect immunofluorescence. The average and deviation values shown are from two to three independent experiments with at least 150 GFP-positive cells counted per experiment; bars, SD. F, model: androgen acts through CDK4/cyclin D and CDK2/cyclin complexes. The data presented suggest that androgen stimulates both cyclin D1 expression/activity and CDK2/cyclin E- associated activity. Both events are required to complete RB phosphorylation and inactivation, thus relieving RB-mediated repression of cyclin A and initiating the G 1 -to-s transition. cyclin kinase activities, and combined deregulation of both components leads to androgen-independent cell cycle progression (Fig. 6E). Discussion Here we show that androgen acts through both CDK4/cyclin D and CDK2/cyclin activities to promote RB inactivation and cell cycle progression. We also show that this process is deregulated in androgen-independent tumor cells, whereas signaling capabilities from RB to its downstream effectors remain intact. Thus, this report delineates mechanisms used by androgen to initiate cell cycle progression and underscores the impact of deregulated RB inactivation in prostatic tumor development. Initially, we demonstrated that in androgen-dependent prostate cancer cells (LNCaP), androgen withdrawal led to a cessation of cell cycle progression that correlated with a reduction in cyclin D1 expression, RB phosphorylation, and cyclin A expression. In contrast, in androgen independent cell lines (JCA-1 and TSU-PR1), androgen withdrawal had no influence on the cyclin D/RB pathway (Fig. 1). In fact, these androgen-independent cell lines no longer required expression of the androgen receptor to simulate cyclin D1 expression. Thus, mutations in these cells must have rendered them capable of progressing through the cell cycle independent of androgen action. To determine whether these lesions act upstream or downstream of RB, we expressed either p16ink4a or PSM-RB in the androgenindependent cell lines (Fig. 2). These reagents led to the cessation of cell cycle progression and reduced the levels of cyclin A protein, similar to that observed in LNCaP cells upon androgen withdrawal. These findings indicate that the lesions that lead to cell cycle dysregulation in androgen-independent prostate cancer act upstream of RB. It is known that Ras activity stimulates cyclin D1 expression, thus activating CDK4/cyclin D1 complexes and initiating RB phosphorylation/inactivation (1, 2, 11, 12, 26, 34). We hypothesized that androgen acts through

Cell Growth & Differentiation 369 Ras signaling to stimulate RB inactivation and cell cycle progression in androgen-dependent cells. Surprisingly, although V12Ras stimulated the expression of cyclin D1, it did not promote RB phosphorylation or androgen-independent proliferation, whereas E1A rendered LNCaP cells androgen independent (Fig. 3). Interestingly, V12Ras exerted a negative effect on LNCaP proliferation that was p53 dependent. Although HPV-E6 overcame the growth-inhibitory action of V12Ras in LNCaP cells, it was incapable of cooperating with Ras to induce androgen independence (Fig. 4). Similarly, although ectopic expression of cyclin D1/CDK4 promoted RB phosphorylation in the presence of androgen, it was incapable of inactivating RB or driving cell cycle progression in the absence of androgen (Fig. 5). Thus, cyclin D expression as stimulated by V12Ras or via its ectopic expression is not sufficient for androgen-independent proliferation, indicating that androgen acts through an additional signaling pathway. We show that CDK2 and cyclin E- associated activity is also regulated by androgen, and that combined action of CDK4/cyclin D1 and CDK2/cyclin complexes are sufficient to inactivate RB and drive androgen-independent cell cycle progression (Fig. 6). Thus, the data presented in this report demonstrate that androgen signals to both CDK4 and CDK2 to inactivate RB and suggest that this requirement is subverted in androgen-independent prostate cancers. Ras in Androgen-dependent Proliferation. Prostatic epithelia and early-stage prostate cancer cells require androgen for proliferation, as in the absence of androgen, prostate tumors cease proliferation and undergo a degree of apoptosis (24). At present, the relative contributions of proliferation and apoptosis to the therapeutic treatment of prostate cancer remain controversial (7, 8, 24). However, it is clear that if the prostatic cancer cells fail to proliferate, the cancer will be controlled. How androgen acts as a prostatic growth factor is largely not understood. Androgen, like many other growth factors, stimulates proliferation through its cognate receptor. The androgen receptor acts as a DNA-binding transcriptional modulator, which presumably changes the pattern of gene expression to stimulate proliferation (24). Although a number of androgen-responsive genes have been cloned (e.g., prostate-specific antigen), the role of these proximal targets in prostate proliferation is not well defined (24). However, it is apparent that these early events are integrated to stimulate the activation of cyclin D1 expression and associated kinase activity, which subsequently initiates phosphorylation/inactivation of RB (25). The importance of these events in cellular proliferation is underscored by the action of p16ink4a or PSM-RB, as both of these reagents are effective at inhibiting the proliferation of all prostate cancer cell lines tested (16, 17). Furthermore, because androgen withdrawal attenuates RB phosphorylation and cyclin A expression in androgen-dependent cells, p16ink4a or PSM- RB simulated these same responses in androgen-independent cells. Therefore, we sought to delineate the signaling pathway between androgen and RB. Ras is known to mediate signals that stimulate both the expression of cyclin D1 and the phosphorylation of RB. In LNCaP cells, we show that V12Ras or V12Ras HPV-E6 stimulates cyclin D1 expression in the absence of androgen. However, restored cyclin D1 did not lead to RB inactivation or cell cycle progression in the absence of androgen. This contrasts with what is observed in immortalized fibroblastic cells, wherein V12Ras stimulates the expression of cyclin D1 and drives RB phosphorylation and cell cycle progression under conditions of serum starvation or anchorage deprivation (35, 36). The role of Ras in the androgen dependence of prostate cancer is polemical. Activating Ras mutations do occur in prostate cancer, although the overall frequency of these mutations is quite low (37 41). In contrast, increased loss of RB correlates with the development of androgen-independent cancer after androgen blockade therapy, and mutation of RB is observed at relatively high frequency in prostate cancers (42 44). In culture, loss of RB signaling through the use of viral oncoproteins such as E1A or T-Ag is sufficient to enable LNCaP cells to progress through the cell cycle in the absence of androgen (25). Although others have tested the influence of Ras on androgen dependence in LNCaP cells, the results are controversial (45, 46). Our results clearly show that in bulk-transfected LNCaP cells (which have not been selected), V12Ras does not induce androgenindependent proliferation, although Ras activity was apparent (Fig. 3). Importantly, under virtually identical conditions, V12Ras induced serum-independent cell cycle progression in Rat-1 cells (not shown). Thus, Ras is specifically defective in promoting androgen-independent growth. Furthermore, we actually found 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 Ras using the HPV-E6 protein, a condition which transforms primary fibroblasts, we were unable to achieve androgen-independent cell cycle progression. Together, these data may explain why Ras 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 overt activation of Raf induces cell cycle arrest in LNCaP cells. By contrast, Voeller et al. (45) showed that cadmium-inducible Ras in LNCaP cells leads to a modest proliferative advantage in the absence of androgen. However, the extent of proliferation in this system was quite reduced in comparison with androgen treatment and was clone dependent. Because V12Ras induces cell cycle arrest in cells containing functional p53 signaling pathways, the data of Voeller et al. (45) could reflect the loss of p53 during the selection of clones. However, our results with V12Ras and E6 suggest that additional events must be involved. Androgen Regulation of CDK4 and CDK2 Activities. The results presented here suggest that in prostatic epithelia, androgen-mediated proliferation likely involves two sets of signals; one that may be mediated by Ras, which stimulates the expression and activity of cyclin D1, and another signal that is required for complete RB inactivation and the expression of cyclin A. Although CDK4/cyclin D1 in vitro kinase activity was restored in the absence of androgen by transfection of CDK4 and cyclin D1 expression plasmids, ectopic CDK4 demonstrated little RB kinase activity in vivo and did not relieve RBmediated repression of cyclin A (Fig. 5). Moreover, ectopic CDK4/cyclin D1 did not promote androgen-independent proliferation. These data are also consistent with the failure of V12Ras and E6 to promote cell cycle progression while restoring cyclin D1 expression. We show that androgen withdrawal also inhibits CDK2 and cyclin E-associated kinase activity. The

370 RB Regulates the Androgen Requirement ectopic overexpression of cyclin E or cyclin A drove RB phosphorylation in the presence of androgen but failed to do so in the absence of androgen. These findings agree with the hypothesis that CDK4/cyclin D-mediated phosphorylation was also important for priming RB for subsequent inactivation of RB by CDK2-mediated phosphorylation. The combined importance of these two signaling pathways is exemplified by the observation that ectopic expression of CDK4, cyclin D1, cyclin E, and cyclin A was sufficient to drive RB phosphorylation in the absence of androgen and render LNCaP cells androgen independent for cell cycle progression (Fig. 6). Together, these data indicate that androgen acts through both CDK4/cyclin D and CDK2/cyclin complexes to promote cell cycle progression. How androgen regulates the activity of CDK/cyclin complexes is still a subject of study. We demonstrate that cyclin D1 protein is diminished in the absence of androgen. Because cyclin D1 is regulated both at the level of transcription and protein stability (1, 2), it is probable that androgen regulates cyclin D1 at one of these levels. We have observed that androgen stimulates cyclin D1 promoter activity in reporter assays (data not shown). Similarly, Ras stimulates cyclin D1 promoter activity, suggesting that the androgen receptor may signal through Ras to stimulate cyclin D1 transcription and result in protein accumulation (11, 34, 35). Although ectopic CDK4/cyclin D1 expression is capable of phosphorylating RB in vitro, this complex lacks the ability to fully phosphorylate and inactivate RB in vivo. This finding suggested that the CDK2/cyclin complexes that mediate RB hyperphosphorylation (47, 48) must be attenuated by androgen withdrawal independently of CDK4/cyclin D1 activity. Consistent with this idea, we find that CDK2 and cyclin E-associated activity (but not cyclin E expression) are downregulated via androgen withdrawal, even when CDK4/cyclin D1 activity is restored. CDK2/cyclin complexes are regulated by a myriad of mechanisms. Because cyclin A expression is inhibited, this would partially explain the decrease in CDK2 activity but fails to explain the specific deficiency in cyclin E-associated kinase activity. It is unlikely that accumulation of the CDK2-inhibitory molecules (i.e., p21cip1 and p27kip1) or CDK-inhibitor shuffling (as mediated by p16ink4a induction) accounts for the attenuation of cyclin E-associated kinase activity, because none of these inhibitory molecules were induced by androgen withdrawal. Thus, androgen regulates cyclin E-associated kinase activity by as of yet undefined mechanisms. Acquisition of Androgen Independence. The progression of prostatic cancer from androgen dependence to androgen independence represents a catastrophic consequence for patients suffering from the disease (24). At present, endocrine therapy represents the only viable treatment for those with early-stage prostate cancer, and no effective therapy exists for those individuals with androgen-independent prostate cancer (7, 8). As with the mechanism through which androgen exerts its mitogenic effect, little is known regarding how prostate cells can bypass this requirement (24). In most established androgen-independent tumor cell lines, androgen receptor expression is actually lost (24). For TSU-Pr1, this has been attributed to methylation of the androgen receptor promoter (49). JCA-1 cells express prostate-specific antigen (a target of the androgen receptor; Ref. 50); therefore, we suspected that JCA-1 cells may express the androgen receptor. However, we failed to detect the expression of AR protein in either cell line. Because no androgen receptor protein is detectable, both cell lines must have activated mechanisms for the continued proliferation in its absence. In cancer, a variety of mechanisms are used to lead to deregulate proliferation. These mechanisms can be loosely divided into those that act upstream of RB (e.g., deregulated mitogenic signaling or cyclin D1 amplification) and those that act downstream of RB (e.g., viral oncoproteins of DNA tumor viruses or deregulated cdk2 activity). Our finding that the TSU- PR1 and JCA-1 cell lines are arrested by PSM-RB and p16ink4a indicates that deregulation of the cell cycle acts upstream of RB. Although we focused on oncogenic Ras, we also tested the action of activated -catenin and c-myc. Neither of these proteins stimulated androgen-independent cell cycle progression in LNCaP cells (data not shown), although -catenin and c-myc are known to stimulate cyclin D1 expression (51). Furthermore, ectopic expression of CDK4/cyclin D1 also failed to inactivate RB or stimulate cell cycle progression in CDT. In fact, the only proteins capable of converting LNCaP cells to androgen independence were proteins that lead to RB inactivation in the absence of androgen. In summary, we show that the signal from androgen to RB is misregulated in androgen-independent tumor cells, whereas the ability of RB to signal to its downstream effectors is precisely maintained. These data suggest that reactivation of RB in androgen-independent tumors should be a goal of anticancer therapeutics. In focusing on the signal from androgen to RB, we also demonstrate that this signal has at least two components: one, which may be Ras mediated, leading to the stimulation of cyclin D expression and CDK4 activity; and the other, which is Ras independent and completes RB inactivation through the action of CDK2/cyclin complexes. These findings underscore the importance of RB as an integrating target for cell cycle control and tumor progression in prostate cancer. Clearly, the pathways regulating RB are tightly controlled, and the deregulated phosphorylation of RB that occurs in androgen-independent tumors requires the dysregulation of multiple signaling pathways. Materials and Methods Cell Culture. The human prostatic adenocarcinoma cell line, LNCaP, was obtained from the American Type Culture Collection. The TSUPr-1 androgenindependent 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 York Medical College). Cells were passaged fewer than 10 times in the experiments described. For regular passage, cells were grown in Improved MEM (Biofluids) containing 10% heat-inactivated FBS (Hyclone) supplemented with 100 units/ml penicillin-streptomycin and 2 mm L-glutamine at 37 C in a humidified atmosphere of 5% CO 2. For growth in an androgen-depleted media, cells were propagated in Improved MEM containing 10% CDT FBS (Hyclone). Rat-1 fibroblastic cells were kindly provided by Dr. D. Green (La Jolla Institute for Allergy and Immunology, La Jolla, CA). Cells were maintained in DMEM supplemented with 10% FBS, 100 units/ml penicillin-streptomycin, and 2 mm L-glutamine at 37 C in a humidified atmosphere of 5% CO 2. Transfected JCA-1 and TSUPr-1 cells were selected 24 h after transfection with 2 g/ml puromycin (Sigma) for 48 h of selection. Plasmids. The ph2b-gfp plasmid, encoding histone H2B fused to the GFP, was obtained from Dr. Geoff Wahl (Salk Institute). The ppsm-rb and p16ink4a plasmids have been described previously (15). Dr. Gilbert