Increased cell growth and tumorigenicity in human prostate ... - Nature

Oncogene (1998) 16, 1913 ± 1920  1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc

Increased cell growth and tumorigenicity in human prostate LNCaP cells by overexpression to cyclin D1 Yian Chen, Luis A Martinez, Margaret LaCava, Lezlee Coghlan and Claudio J Conti The University of Texas MD Anderson Cancer Center, Science Park-Research Division, Smithville, Texas 78957, USA

Deregulated expression of cyclin D1 has been found in several types of human tumors. In order to investigate factors involved in human prostate cancer progression, we studied the eects of cyclin D1 overexpression on human prostate cancer cell proliferation and tumorigenicity by transfecting LNCaP cells with a retroviral vector containing human cyclin D1 cDNA. When compared to the parental and control-vector transfected LNCaP cells, these cyclin D1-transfected cells had more cells in Sphase and lower growth factor requirements. Furthermore, these cells grew more in androgen-free medium. We also detected higher levels of Rb phosphorylation and E2F-1 protein levels in LNCaP/cyclin D1 cells than that in the parental and vector control cells in medium with or without androgen. Cyclin D1 transfected clones formed tumors more rapidly than control and parental cells. These tumors were refractory to the androgen-ablation treatment by castration, whereas tumors from parental and vector-control LNCaP cells regressed within 4 weeks after castration. These results suggest that overexpression of cyclin D1 changes the growth properties, increases tumorigenicity and decreases the requirement for androgen stimulation in LNCaP cells both in vitro and in vivo. Keywords: prostate cancer cell lines; androgen; cyclin D1; gene transfection; cell proliferation; tumorigenicity

Introduction Cyclin D1 is a 36-Kda protein that belongs to a family of cell-cycle regulators called cyclins (Xiong et al., 1991, 1992a). It functions in the G1 phase of cell cycle and activates the kinase activities of G1 cyclindependent kinases (for review, see Sherr, 1993, 1994). The active cyclin/cdk complexes phosphorylate proteins which are important in cell-cycle control. One putative substrate of these cyclin D1-activated kinases is the retinoblastoma protein (Rb). The hypophosphorylated form of Rb prevents cells from entering the S phase. When Rb is hyperphosphorylated by kinases (e.g., cdks), it can no longer prevent the cells from entering S phase (for review, see Weinberg, 1991). The activities of cyclin/cdk can be inhibited by several negative cell-cycle regulatory proteins (for review, see Sherr and Roberts, 1995). Overexpression of cyclin D1 shortens the G1 phase, decreases cell size and increases tumorigenicity in rat Correspondence: CJ Conti This work was supported by American Cancer Society CN143 Received 30 April 1995; revised 10 November 1997; accepted 10 November 1997

embryo ®broblasts (Jiang et al., 1993a; Quelle et al., 1993; Resnitzky et al., 1994). Induction of cyclin D1 in the human breast cancer cell line, T-47D, also increases the rate of transition from G1 to S phase and the induced cyclin D1 is sucient to allow cells to complete the cell cycle even if they are arrested in early G1 phase after growth-factor deprivation (Musgrove et al., 1994). Microinjection of antibodies or antisense plasmids into cells in mid-G1 inhibits cyclin D1 and prevents the cells from entering S phase (Baldin et al., 1993; Quelle et al., 1993; Lukas et al., 1994a,b; Tam et al., 1994; Zhou et al., 1995). This indicates that cyclin D1 limits the rate of progression through G1 phase. Deregulated expression of cyclin D1 has been found in several types of tumors. In some parathyroid adenomas, cyclin D1 expression is elevated by a translocation that juxtaposes the cyclin D1 parathyroid hormone genes at 11p15 (Rosenberg et al., 1991). Ampli®cation of the human 11q13 region and elevated cyclin D1 transcript levels have been reported in a subset of head and neck squamous cell carcinomas, esophagus, breast, bladder and liver carcinomas, and B-cell lymphomas. (Lammie et al., 1991; Buckley et al., 1993; Jiang et al., 1992, 1993b; Schuuring et al., 1992; Zhang et al., 1993). Thus, cyclin D1 is probably involved in tumor development. In androgen-induced rat prostate cell proliferation, cyclin D1 is greatly induced when prostate cells pass from G0 into early G1 phase. The changes in the cyclin D1 levels correlate with the proliferation rate after androgen stimulation in castrated rats (Furuya et al., 1995; Chen et al., 1996). This suggests that cyclin D1 plays an important role in prostate cell proliferation. We also examined the expression of cyclin D1 in three human prostate cancer cell lines (PC3, DU145 and LNCaP). The growth of PC3 and DU145 cells is androgen independent, and that of LNCaP is androgen dependent. We found that cyclin D1 was constitutively expressed in PC3 and DU145 cells grown with or without serum but cyclin D1 expression was significantly lower in LNCaP cells grown without serum (Chen et al., 1995). So far it is still not clear whether this constitutive high expression of cyclin D1 in androgen-independent cells is related to their nonresponsiveness to androgen ablation in prostate cancer. To further understand the role of cyclin D1 in the progression of prostate cancer, we overexpressed cyclin D1 in the LNCaP cell line by using a retroviral vector containing human cyclin D1 cDNA (Jiang et al., 1993a). The LNCaP cell line was derived from a lymph node of a patient with metastatic stage D prostate cancer (Horoszewicz et al., 1980, 1983). LNCaP cells are well dierentiated and have the characteristics of prostate cells; they produce prostatespeci®c antigen (Montgomery et al., 1992), prostate-

Cyclin D1 overexpression in LNCaP cells Y Chen et al

speci®c membrane antigen (Israeli et al., 1994) and androgen receptor (Veldscholte et al., 1990). Although there are mutations in androgen receptor of LNCaP cells, these cells are androgen responsive in cell culture (Sonnenschein et al., 1989) and androgen dependent when they form tumors in male nude mice (Horoszewicz et al., 1980, 1983; Lim et al., 1993). LNCaP cells can become more tumorigenic when they are mixed with matrigel (Pretlow et al., 1991; Lim et al., 1993) or bone ®broblasts (Wu et al., 1994) and some tumors become androgen independent in nude mice (Wu et al., 1994). Due to these characteristics, we chose this cell line to study the relationship between cyclin D1 overexpression and androgen independence in the progression of prostate cancer cells. In doing so, we examined the eects of androgen on the expression of cyclin D1 in LNCaP cells. We investigated the eects of cyclin D1 overexpression on LNCaP cell-cycle progression, cell proliferation, androgen responsiveness and involvement of the other cell-cycle regulators in cell culture. Finally, we studied the tumorigenicity of various LNCaP derivatives and their response to androgen ablation in male nude mice.

with the cyclin D1 expression vector as LNCaP/cyclin D1 cells (LN/cyl D1-A and LN/cyl D1-B) and with the vector control as LNCaP/pl (LN/pl-A). Northern blot analysis showed that when these cells were grown in RPMI 1640 medium supplemented with 10% newborn calf serum (NCS), LNCaP/cyclin D1 cells expressed more cyclin D1 mRNA than that of the parental LNCaP and LNCaP/pl cells (Figure 2a). To further con®rm that each clone was transfected, we also performed Northern blot analysis with a neo gene fragment as a probe. Figure 2b shows that a 4.8 kb transcript was detected in LNCaP/cyclin D1 cells and a 3.7 kb transcript was detected in LNCaP/pl cells (LN/ pl-A). There were no neo transcripts detected in LNCaP cells.

LNCaP 0.3

0

DHT(nM)

1

10

30

cyclin D1 —

Results Induction of cyclin D1 mRNA expression in LNCaP cells by androgen

7S RNA —

Figure 1 Induction of cyclin D1 by DHT in LNCaP cells. LNCaP cells were starved in serum-free medium for 2 days. Then 0, 0.3, 1, 10, and 30 nM of DHT were added to the medium for 1 day. About 10 mg of total mRNA from each treatment was electrophoresed through a 1% formaldehyde-denaturing agarose gel and then transferred to a nylon membrane. The blot was hybridized with an a-32P-dCTP labeled Prad-1 probe. Rehybridization to an a-32P-dCTP labeled 7S RNA probe was used to normalize RNA loading

The human cyclin D1 expression plasmid, pMV7plCCND1, (Jiang et al., 1993a) and the control vector, pMV7pl, (Kirshmeier et al., 1988) were transferred into the GP+envAm12 amphytropic packaging cell line by electroporation. These two plasmids carried the neomycin phosphotransferase (neo) gene which was used as a selection marker. Clones of transfected GP+envAm12 cells were selected with 400 mg/ml of neomycin and expanded. We harvested the viral supernatant from transfected GP+envAM12 cells and infected LNCaP cells by a method previously described (Jiang et al., 1993a). Each colony that grew after G418 selection was picked and expanded. We designated the LNCaP cells infected

cyclin D1 —

7S RNA —

LN/cyl D1-B

LN/cyl D1-A

LN/pl-A

Overexpression of cyclin D1 in LNCaP cells by a retrovirus cyclin D1 expression vector

LNCaP

1).

LN/cyl D1-B

b LN/cyl D1-A

a LN/pl-A

Previous in vivo studies show that cyclin D1 expression increases during androgen-induced rat prostate cell proliferation (Furuya et al., 1995; Chen et al., 1996). To determine the relationship between androgen stimulation and cyclin D1 expression in LNCaP cells in vitro, we starved the cells in serum-free medium for 2 days and then stimulated them with 0.3, 1, 10, 30 nM of dihydrotestosterone (DHT) for 1 day according to the protocol from Dr LWK Chung's lab (The University of Virginia Health Science Center, Charlottesville, Virginia). Northern blot analysis showed that when LNCaP cells were cultured in serum-free medium, cyclin D1 mRNA was barely detectable. When DHT was added to the serum-free medium, induction of cyclin D1 mRNA was detected with the highest induction level at 1 and 10 nM of DHT (Figure

LNCaP

1914

— Neo

— 7S RNA

Figure 2 Overexpression of cyclin D1 mRNA in LNCaP/cyclin D1 transfected cells. About 10 mg of total RNA from LNCaP, LNCaP/pl and LNCaP/cyclin D1 cells was electrophoresed through a 1% formaldehyde denaturing agarose gel and then transferred to a nylon membrane. The blot was hybridized with an a-32P-dCTP labeled Prad-1 probe (a) or a neo probe (b). Rehybridization to an a-32P-dCTP labeled 7S RNA probe was used to normalize RNA loading


Eect of cyclin D1 overexpression on LNCaP growth in vitro LNCaP cells proliferate well in RPMI 1640 medium supplemented with 5 ± 10% serum, and respond to androgen (DHT) in the culture medium (Horoszewiez et al., 1983). To characterize the LNCaP/cyclin D1 cell growth properties in vitro, we analysed cell growth in medium supplemented with 5% and 10% NCS. The LNCaP/cyclin D1 cells and their nuclei were smaller than those of the parental and vector-control cells but were spindle shaped and loosely attached to the surface of cell-culture ¯asks like the parental cells. When the cells were grown in medium with 10% NCS, ¯ow cytometry analysis showed that more LN/cyclin D1 cells than LNCaP and LN/pl cells were in S phase during the exponential phase of cell growth (Table 1). LNCaP/cyclin D1 cells doubled faster than LNCaP and LNCaP/pl cells (average 37 h versus 51 h respectively) and had greater cell saturation densities (Table 1, Figure 3). The higher saturation densities also indicate that the LNCaP/cyclin D1 cells were smaller than the parental and control cells (Table 1). When the cells were grown in medium with 5% NCS, LNCaP/ cyclin D1 cells also grew faster than LNCaP and LNCaP/pl cells (data not shown). Thus, LNCaP/cyclin D1 cells had a growth advantage over LNCaP and LNCaP/pl cells in the RPMI medium supplemented with 10% or 5% serum. To examine the androgen dependence of LNCaP/ cyclin D1 cells, we analysed the growth rates of LNCaP/cyclin D1, LNCaP/pl and LNCaP cells cultured in RPMI phenol-red free medium supplemenTable 1

Comparison of cell growth properties and cell cycle parametersa LNCaP

Doubling time 50.5 (hours) Saturation density 156105 69.3+5 Cells in G1 (%) 22.4+3.1 Cells in S (%) 8.3+2.4 Cells in G2 (%)

LN/pl-A

LN/cyl D1-A

LN/cyl D1-B

52.0

36.4

37.5

386105 71.9+4.7 19.1+1.7 9.0+2.0

606105 59.9+4.1 27.5+3.7 12.6+1.9

846105 51.0+3.5 31.9+1.4 17.1+0.6

a

Cells were grown in medium with 10% NCS, and the cell cycle parameters were examined during exponential phase of cell growth

Figure 3 Growth of LNCaP/cyclin D1 and control cells in culture medium with 10% NCS. LNCaP, LNCaP/pl and LNCaP/ cyclin D1 cells (56105 each) were seeded in a T25 ¯ask with medium containing 10% NCS on day 0. The cells were counted 2 ± 3 days

ted with 10% charcoal-stripped FBS (cFBS) and various concentrations of androgen (0, 0.001 nM and 0.1 nM of DHT). The concentration of steroid hormones (including androgen) is extremely low in this medium because of the special charcoal treatment (Breen and Leake, 1987). When 10% cFBS plus 0 or 0.001 nM of DHT were added into the medium, LNCaP and LNCaP/pl cells grew slowly, as did the LNCaP/cyclin D1 cells, but to a less extent. When the cells were fed with the medium supplemented with 10% cFBS plus 0.1 nM of DHT, all of these cells grew faster (Figure 4). These results indicated that overexpression of cyclin D1 in LNCaP cells partially reduced the cell's requirement for androgen stimulation in vitro. To understand the mechanism of the dierent androgen requirements among the LNCaP and its derivative cell lines, we further studied the involvement of cell cycle-related proteins in the regulation of these cell proliferation in vitro. It is known that cyclin D1 can form complexes and activate cdk4 in mid to late G1 phase (Xiong et al., 1992a,b; Kato et al., 1993). Once cdk4 is activated, it can further phosphorylate downstream substrates such as the Rb protein in G1 phase (Sherr, 1993, 1994). Our in vitro kinase assay, using pRb as the substrate, showed that LNCaP and its derivative cells had high cdk4 enzyme activities in medium with 10% FBS and their activities reduced dramatically in medium with 10% cFBS. However, LNCaP/cyclin D1 cells had higher cdk4 kinase activity than LNCaP/pl cells in both types of media (Figure 5A). Immunoprecipitation of the protein lysates with cyclin D1 antibody and Western blot analysis with cyclin D1 and cdk4 antibodies showed that there were more cyclin D1 protein and cyclin D1/cdk4 complexes detected when the cells were grown in medium with 10% FBS, and were less when the cells were grown in medium with 10% cFBS. However, the levels of cyclin D1 protein and cyclin D1/cdk4 complex formed in LNCaP/cyclin D1 were always higher than that in LNCaP and LNCaP/pl cells under both conditions (Figure 5B). The reduction in the cyclin D1/cdk4 complex levels may be related to the decrease of cyclin D1, but not cdk4 protein levels, after androgen deprivation in the cell culture medium (Figure 5C). We further investigated the status of other important

Figure 4 Eect of cyclin D1 overexpression on androgen dependence of LNCaP cells. Cells (26105) were seeded in a T25 ¯ask with medium with 10% NCS. After 1 day, these cells were washed twice with PBS and switched to RPMI phenol redfree medium supplemented with 10% cFBS and 0, 0.001 or 0.1 nM of DHT. The cells were counted after 8 days

1915


cell-cycle regulators, such as cyclin E, cdk2, Rb and E2F-1 proteins (Sherr, 1993, 1994; Sherr and Roberts, 1995; Weinberg, 1991). Cyclin E form complexes with and activate cdk2 which can also phosphorylate Rb

LN/cyl D1-A

LN/pl-A

LNCaP

10% cFBS LN/cyl D1-A

LNCaP

LN/pl-A

10% FBS

pRb (α-cdk4)

A

cyclin D1 B cdk4

protein in late G1 phase. Figure 6 shows that after androgen deprivation in cell culture, the protein levels of cyclin E, cdk2 (detected by Western blot analysis) and cdk2 kinase activity (detected by in vitro kinase assay using pRb as a substrate) were signi®cantly reduced in LNCaP and its derivative cells. Rb is another very important protein which inhibits cells from going through the G1/S checkpoint (Weinberg, 1991; Hinds and Weinberg, 1994). The hypophosphorylated forms of Rb can bind and suppress the function of E2F-1, which is a transcription factor for the genes required for DNA synthesis in S phase (Nevins, 1992). Hyperphosphorylation of Rb relieves this inhibition by releasing E2F-1 (Chellappan et al., 1991; Hinds and Weinberg, 1994). Western blot analysis showed that LNCaP/cyclin D1 cells had higher levels of hyperphosphorylated Rb as well as E2F-1 protein than parental and vector-control cells, even if these levels were reduced when the cells were grown in medium without androgen (10% cFBS). These dierences were more clear with cells grown in medium without androgen (Figure 7). Cyclin D1 overexpression enhanced the tumorigenicity of LNCaP cells in male nude mice

cdk4 Figure 5 Function and expression of cyclin D1 protein in LNCaP and its derivative cells before and after androgen ablation in culture. The enzyme activity of cdk4 was analysed by a kinase assay using pRb as the substrate. Protein lysates were immunoprecipitated with anti-cdk4 andibody and (A). Protein lysates were also immunoprecipitated with anti-cyclin D1 antibody and Western blotted with anti-cyclin D1 and cdk4 antibodies (B). Cyclin D1 and cdk4 expression in the cells treated with or without androgan in cell culture medium were analysed by Western blot analysis (C)

LN/cyl D1-A

LN/pl-A

LNCaP


LN/pl-A

10% FBS

LNCaP cells have very weak tumorigenicity in nude mice. They must be injected with Matrigel (Pretlow et al., 1991; Lim et al., 1993) or bone ®broblast cells (Wu et al., 1994) to form tumors in male nude mice. The growth of these tumors is mostly androgen dependent since regression of the tumors is observed after castration (Lim et al., 1993; Wu et al., 1994). However, some tumors can lose androgen dependence and grow again after castration (Wu et al., 1994). To investigate the eects of cyclin D1 overexpression on the tumorigenicity of LNCaP cells, we injected 26106 cells/site in 0.25 ml of phosphate-buered saline (PBS) with Matrigel (1 : 1) subcutaneously into intact male nude mice. The earliest visible tumor appeared 2 weeks after injection of LN/cyl D1-A, LN/cyl D1-B and LN/ cyl D1-C cells (which also expressed a high level of cyclin D1, data not shown); 6 ± 7 weeks after injection of LNCaP cells and 6 weeks after injection of LNCaP/ pl (LN/pl-A cells) (Figure 8). By 4 ± 5 weeks after cell

10% FBS

LNCaP

LN/cyl D1-A

A cdk2

LN/pl-A

LNCaP

cyclin E


C

LN/pl-A

cyclin D1

LNCaP

1916

Rb pRb (α-cdk2)

B

Figure 6 Expression and function of cyclin E and cdk2 protein in LNCaP and its derivative cells before and after androgen ablation in culture. Expression of cyclin E and cdk2 protein in the cells treated with or without androgen in cell culture medium were examined by Western blot analysis (A). The enzyme activities of cdk2 in the cells under both conditions were analysed by kinase assay using pRb as the substrate (B)

E2F-1 Figure 7 Rb phosphorylation and E2F-1 expression levels in LNCaP and its derivative cells before and after androgen ablation in culture. Rb phosphorylation and E2F-1 expression levels in LNCaP and its derivative cells cultured in medium with and without androgen were analysed by Western blot analysis. Polyclonal anti-Rb and anti-E2F-1 antibodies were used in the analysis


injection, 58.3% of the mice injected with LN/cyl D1-B cells, 53.3% of the mice with LN/cyl D1-C cells, 28.0% of the mice with LN/pl-A cells and 23.0% of the mice with LNCaP cells had tumors (Table 2). The tumorigenicity data from LN/cyl D1-A cells was not included in Table 2 because their tumors were already over 500 mm3 by 5 ± 6 weeks after injection. If we did not castrate or sacri®ce the mice, the tumors would grow too big that mice were in poor health. Thus, the tumors from LNCaP/cyclin D1 cells grew better than the parental and control cells in vivo. We also injected LNCaP, LN/pl and LNCaP/cyclin D1 cells into female and castrated male nude mice with Matrigel or intact male nude mice without Matrigel. No tumors formed during a 12-week observation period. Therefore, without Matrigel and androgen, overexpression of cyclin D1 alone could not initiate the LNCaP tumor formation in nude mice. To examine whether overexpression of cyclin D1 in LNCaP leads to resistance to hormone-ablation in vivo, we castrated the mice when they developed tumors of about 200 ± 300 mm3 and determined tumor size weekly thereafter. Figure 9 shows that the tumors derived from LNCaP and LNCaP/pl cells gradually regressed within 4 weeks after castration. On the other hand, tumors from LNCaP/cyclin D1 cells did not regress but kept growing after castration. Consistent with other reports (Lim et al., 1993; Wu et al., 1994), some of the

tumors from LNCaP cells grew 5 weeks later after castration. Thus, cyclin D1 overexpression in LNCaP cells enhances tumorigenicity of the cells and inhibits tumor regression after castration. Discussion In this study, we investigated the eects of cyclin D1 overexpression on cell proliferation and tumorigenicity in the LNCaP cell line. Compared with parental and vector-control cells, cyclin D1-transfected clones were smaller, had a larger proportion of cells in S phase and grew faster. They were less dependent on androgen in culture, developed tumors earlier and were refractory to androgen-ablation treatment in nude mice. The pMV7plCCND1 plasmid used in this study was developed in Dr IB Weinstein's lab (Jiang et al., 1993a). Rodent embryo ®broblasts cells transfected with this plasmid overexpress cyclin D1, are smaller and have an increased saturation density. They have more cells in S phase but the same doubling time as the parental and vector-control cells. These cells form tumors in nude mice but vector-control cells do not (Jiang et al., 1993a). The results obtained in our study are consistent with those of Weinstein's group except that our LNCaP/cyclin D1 cells have a faster doubling time than the parental and vector control cells. This variation may be due to the dierences between the cell lines (normal rodent embryo ®broblasts cells versus human malignant prostate cells). The growth of LNCaP cells is androgen dependent since when cultured in an androgen-free medium (10% cFBS), their growth is greatly inhibited (Sonnenschein et al., 1989). In this study, we showed that androgen ablation had a smaller inhibitory eect on LNCaP/cyclin D1 cells than the LNCaP and LNCaP/pl cells. Thus, constitutive overexpression of cyclin D1 in LNCaP led to more cells being in S phase and decreased the dependence of the cells on physiological stimuli from androgen. In the G1 phase of cell cycle, cyclin D1 functions by activating the kinase activities of cdk4 and cdk6. The activated kinases can further phosphorylate Rb at mid to late G1 phase (Sherr, 1993, 1994). Our studies showed that when cells were grown in medium with

Figure 8 In¯uence of cyclin D1 overexpression on tumorigenicity of LNCaP cells in male nude mice. LNCaP, LNCaP/pl and LNCaP/cyclin D1 cells (26106 cells/site) were mixed in 0.25 ml of PBS with Matrigel (1 : 1) and injected subcutaneously into intact male nude mice. The mice were examined weekly after injection. The incidence of tumors from each cell line was the percentage of mice with tumors

Table 2 Eect of cyclin D1 on the tumorigenicity of LNCaP cells LNCaP Number of nude mice Tumor incidence (%)a Mean tumor size (mm3)b a

13 23.0 24

LN/pl-A 7 28.0 125

LN/cyl D1-B

LN/cyl D1-C

12

10

58.3

53.3

251

554

The tumor incidence shows the percentage of mice that developed tumors within 8 weeks after being injected with 26106 cells. bThe mean tumor size was determined 8 weeks after cell injection. The formula for calculating each tumor size was: L6W6H60.5236

Figure 9 Eects of cyclin D1 overexpression in LNCaP/cyclin D1 on the refraction of androgen ablation in male nude mice. When the tumors formed from LNCaP and its derivative cells were about 200 ± 300 mm3, the mice were castrated by bilateral orchiectomy under metofane anesthesia. The tumors were measured weekly for castration

1917


1918

10% FBS, LNCaP/cyclin D1 cells had higher cdk4 kinase activity and higher levels of Rb phosphorylation than LNCaP and LNCaP/pl cells. Thus, these results were consistent with the function of cyclin D1 previously described. E2F-1 is another important protein in cell cycle regulation because it regulates the expression of genes required for DNA synthesis in S phase, including dehydrofolate reductase, DNA polymerase a, thymidine kinase and cdc2 (reviewed by Nevins 1992). The transcription of E2F-1 is also known to be activated by itself (Johnson et al., 1994). The function of E2F-1 is regulated by the members of the Rb protein. Hypophosphorylated Rb binds to E2F1 and inhibit E2F-1 from activating gene transcription. Phosphorylation of Rb relieves this inhibition by releasing E2F-1 which leads to activation of this protein and drives cells into S phase (Chellappan et al., 1991; Hinds and Weinberg, 1994; Johnson, 1995). Our studies showed that when cells were grown in medium with 10% FBS, LNCaP/cyclin D1 cells had higher expression levels of E2F-1 than LNCaP and LNCaP/pl cells. This observation is consistent with previous reports from other labs stating that cyclin D1/ cdk4 induces E2F-1 indirectly by phosphorylation of Rb resulting in the release of E2F-1 which then activates its own transcription (Schulze et al., 1994; Oswald et al., 1994; Vairo et al., 1995; Johnson, 1995; Shao and Robbins, 1995). Although cyclin D1 is overexpressed in LNCaP/ cyclin D1 cells, cell proliferation was greatly inhibited when these cells were grown in medium supplemented with 10% cFBS, as that of the LNCaP and vectorcontrol cells, but to a less degree. The medium supplemented with 10% cFBS contains most of the growth factors and hormones except steroid hormones which includes androgen. Thus, androgen is an important mitogen for the LNCaP, vector-control as well as its cyclin D1 overexpressing cells. The kinase assay and Western blot analysis performed in this study also indicate that androgen plays a role in the regulation of the cell-cycle machinery. The expression levels of cyclin D1, cyclin E, cdk2, E2F-1, phosphorylation of Rb protein and cyclin D1/cdk4 complex formation, as well as kinase activities of cdk4 and cdk2 were reduced in all of these cells after androgen deprivation from the medium. Hence, these changes may be a result of the androgen deprivation and part of the factors leading to inhibition of cell proliferation. In LNCaP/cyclin D1 cells, there was a less reduction of cyclin D1, E2F-1 protein expression, Rb phosphorylation levels and cdk4 kinase activity. This is related to the constitutively higher expression levels of cyclin D1 which may lead to less growth inhibition of these cells in androgen deprived environment. LNCaP cells are not tumorigenic when they are injected alone into nude mice. When they are injected with Matrigel (Pretlow et al., 1991; Lim et al., 1993) or bone ®broblast cells (Wu et al., 1994), they form tumors in male mice. LNCaP cells are androgen dependent not only in vitro but also in vivo as their tumors regress after castration (Lim et al., 1993; Wu et al., 1994). In our study, LNCaP/cyclin D1 cells mixed with Matrigel formed tumors in male nude mice earlier than the parental and vector-control cells did. After castration, these tumors did not regress, whereas the tumors from parental and vector-control cells did.

Thus, overexpression of cyclin D1 enhances prostate tumor growth in male nude mice and reduces tumor dependence on androgen in vivo. Interestingly, LNCaP/ cyclin D1 cells showed higher resistance to androgen ablation in vivo than in vitro. One of the reasons may be that tumor formation and growth in the nude mice are a more complicated process than cell growth in vitro, which is a dierent growth environment for cells. The degree of tumorigenicity is not only determined by the ability of cell proliferation but also by the interaction between host and tumor cells, as well as other events, such as angiogenesis and genetic instability during tumor formation and growth. Thus, the mechanisms that lead to androgen resistance of the tumors formed from LNCaP/cyclin D1 cells are very complicated and will be further studied. We also found that cyclin D1-overexpressing LNCaP cells, just like LNCaP cells, could not form tumors in male nude mice without Matrigel in female nor in castrated male nude mice with Matrigel. These results suggest that overexpression of cyclin D1 alone is not sucient for initiating LNCaP tumor formation in nude mice. Other factors such as androgen, components in the Matrigel (including extracellular matrix proteins and growth factors such as ®broblast growth factors, tissue plasminogen activators, etc.) and other biological changes are required to initiate tumor formation. Many studies have shown that resistance to androgen ablation in advanced prostate cancer may be the result of genetic alterations and deregulation of gene expression. Several groups have reported that mutations in androgen receptors or reduction in its expression may be related to androgen resistance in advanced prostate cancer (Tilly et al., 1994; Ruizeveld de Winter et al., 1994). Alterations in tumor suppressor genes, for example, p53 mutation are also associated with hormone resistance in advanced prostate cancer (Navone et al., 1993; Voeller et al., 1994; Kallakury et al., 1994). In addition, the Rb gene is mutated in 20 ± 50% of advanced prostate cancers, while mutation is rare in early tumors (Isaacs et al., 1994). In this study, we observed that androgen has an important role in the regulation of the cell-cycle machinery including induction of cyclin D1 leading to phosphorylation of Rb and release of E2F-1 transcription factor in the LNCaP cells. Thus, changes at any points in this pathway may lead to the inactivation of Rb which represents a critical event during tumorigenesis of prostate. The results from our study suggest that overexpression of cyclin D1 in LNCaP cells cooperates with other biological alterations in the cells and facilitates the cell growth, tumor formation and resistance to androgen ablation both in vitro and in vivo.

Materials and methods Cell culture The human prostate cancer cell line LNCaP (American Type Culture Collection, Rockville, MD) was cultured in RPMI 1640 medium (GIBCO, Gaithersburg, MD) supplemented with NCS or FBS (Hyclone, Logan, Utah). The amphytropic retrovirus packaging cell line, GP+envAm12, a gift from Dr Yacob Ben-David and Dr J Filmus (Sunnybrook Health Science Center, Toronto, Canada), was cultured in Dulbecco's modi®ed Eagle's medium


(DMEM) with high glucose, and 10% NCS. Both cell lines were also supplemented with 2 mM of glutamine and 50 mg/ ml of gentamicin and incubated in a humidi®ed atmosphere with 5% CO2. To examine the dependence of LNCaP and its derivatives on androgen, we cultured the cells in RPMI 1640 phenol red-free medium (Sigma Chemical Co., St. Louis, MO) supplemented with 10% FBS or 10% cFBS with various concentrations of DHT (Sigma Chemical Co.). The dextran and activated charcoal used in the preparation of cFBS were obtained from Sigma Chemical Co. Cyclin D1 retroviral expression plasmid transfection The cyclin D1 expression plasmid pMV7plCCND1 (Jiang et al., 1993a) and the control vector pMV7pl (Kirshmeir, 1988) were gifts from Dr IB Weinstein (Columbia University, New York, NY). The plasmid pMV7plCCND1 contains a 1.1 kb human cyclin D1 cDNA and the 5' Moloney murine leukemia virus long terminal repeat (MoMuLV LTR) as the cyclin D1 promoter. A neo gene was included as a selection marker (Jiang et al., 1993a). We transfected pMV7plCCND1 or pMV7pl into GP+envAM12 cells by electroporation. Forty-eight hours later, cells were trypsinized and replated in DMEM with 10% NCS and 400 mg/ml of G418. G418resistant clones were selected and expanded. To transfect pMV7plCCND1 or pMV7pl into LNCaP cells, we harvested the viral supernatant of the G418-resistant GP+envAm12 cells and infected LNCaP cells by a method previously described (Jiang et al., 1993a). Fortyeight hours after transfection, 400 mg/ml of G418 was added to the RPMI medium with 10% NCS. Individual colonies were picked and grown in RPMI medium with 10% NCS and 100 mg/ml of G418 for further analysis. Northern blot analysis Total RNA was isolated from cells by using TRI-reagent (Molecular Research Center, Inc., Cincinnati, OH). Ten micrograms of total cellular RNA from each sample was electrophoresed through a 1.5% agarose gel containing 8% formaldehyde. After electrophoresis, the RNA was transferred to a nylone membrane by the capillary method, and then cross-linked to the membrane with ultraviolet light. Prad-1 and neo probes were labeled with a-32P-dCTP by using a random primed DNA labeling kit (Boehringer Mannheim Corporation, Indianapolis IN). The Prad-1 probe, a human cyclin D1 probe, was kindly provided by Dr A Arnold (Massachusetts General Hospital, Boston, MA). The hybridization was performed at 688C with Quickhyb solution (Strategene, La Jolla, CA). The next day, each membrane was washed twice for 15 min each time at room temperature with 26SSC and 0.1% SDS and once for 30 min at 688C with 0.16SSC and 0.1% SDS and then exposed to Kodak X-AR5 ®lm for 1 day at 7708C. Rehybridization with a-32P-dCTP-labeled 7S RNA was used to normalize the amount of RNA loaded in each line. Western blot analysis and Rb kinase assay Protein was extracted from cells and kept frozen at 7808C by methods described previously (Chen, 1995). One hundred micrograms of protein lysate from each sample was electrophoresed through a 10% SDSpolyacrylamide gel, transferred to a nitrocellulose membrane by wet transfer and blotted with various antibodies. Polyclonal anti-cyclin D, cdk4, cyclin E, cdk2, Rb and E2F-1 antibodies were from Santa Cruz

Biotechnology, Inc. (Santa Cruz, CA). A secondary antibody conjugated with horseradish peroxidase (Amersham Corp., Arlington Heights, IL) was used and the ®nal detection was performed by using an ECL kit (Amersham Corp.). For the kinase assay, we followed the protocol from Dr CJ Sherr's Lab (St. Jude Children's Research Hospital, Memphis, TN) (Matsushime et al., 1994). The frozen protein lysates were thawed at 48C and 500 mg of protein lysates from each sample were immunoprecipitated with either cdk4 or cdk2 antibodies (Santa Cruz, CA). pRb, a GST fusion protein produced in E. coli (Santa Cruz, CA), was used as substrate in the kinase assay. The phosphorylation of pRb was labeled by g-32P-ATP. For quanti®cation of cyclin D1/cdk4 complexes formed in the cells, protein lysates were immunoprecipitated with cyclin D1 antibody and Western blotted with cdk4 antibodies. In vitro cell growth analysis To characterize cell growth in vitro, we performed ¯ow cytometric analysis as well as studies of cell growth curves and cell population doubling time. For the ¯ow cytometric analysis, exponentially growing cells in medium with 10% NCS were trypsinized, washed with cold PBS and ®xed in 75% ethanol. Propidium iodide with 2 mg/ml RNase was used to stain the cells. DNA content and the percentage of cells in various phases of the cell cycle were determined by measuring ¯uorescent intensity with a Coulter Elite Flow cytometer. To examine cell growth, 56105 cells were plated in each of three 25 mm2 ¯asks in DMEM with 10%, 5% NCS or 26105 cells in RPMI 1640 phenol red-free medium supplemented with 10% cFBS with 0, 0.001, 0.1 nM of DHT. Cells were counted every 2 ± 3 days with a hematocytometer. The cells were fed twice a week. Tumorigenicity assay in nude mice Male nude mice were purchased from Frederick Cancer Research and Development Center (Frederick, MD) and kept aseptically in an athymic animal room. The pMV7pland pMV7plCCND1-transfected LNCaP cells and parental LNCaP cells were trypsinized, washed with PBS, resuspended in PBS and mixed with an equal volume of Matrigel (Collaborative Biomedical Products/Becton Dickinson, Bedford, MA). Then 26106 cells in 0.25 ml of the mixture were injected subcutaneously into nude mice by a method previously described (Lim et al., 1993). The mice underwent bilateral orchiectomy under metofane anesthesia (Pitman-Moore, Washington Crossing, NJ) when the tumors were 200 ± 300 mm3. All the mice were examined once a week, and each tumor's length (L), width (W) and thickness (H) were measured. Tumor volume was calculated by the formula L6W6H60.5236 (Janek et al., 1975). Acknowledgements We would like to thank Dr IB Weinstein for providing the pMV7plCCND and pMV7pl plasmids; Dr Yacob BenDavid and Dr J Filmus for providing GP+envAM12 cells; Dr A Arnold for providing the Prad-1 (cyclin D1) probe; Dr LWK Chung for sharing their protocol of DHT supplementation in cell culture and Dr CJ Sherr for providing their protocol for kinase assays. Dale Wiss and Robert B Whalin for their extensive assistance with the animal surgery and care; Dennis Walker for ¯ow cytometric analysis; Judy G Ing for artwork and Dr Maureen Goode for editing the paper.

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