International Urology and Nephrology 27 (1). pp. 67-80 (1995)

Effects of Sex Steroids on Cell Growth and C-myc Oncogene Expression in LN-CaP and DU-145 Prostatic

Carcinoma Cell Lines*

F. K. ASADI, R. SHARIFI

Department of Urology, University of Illinois Medical Center at Chicago, Chicago, Illinois

(Accepted September 6, 1994)

The prostate gland is dependent on androgens for the maintenance of its nor­mal growth and functional integrity. Initially, growth of the majority of prostate tu­mours can be manipulated by endocrine therapy. In the present study, we evaluated the effects of sex steroids on the cell growth and expression of the C-myc oncogene in two human prostatic adenocarcinoma cell lines. We found that dihydrotestos­terone increases the proliferation rate of prostatic cells, and amplification of C-myc oncogene is hormone-dependent. We also demonstrated a positive correlation be­tween the number of cells positive for C-myc oncogene and oncoprotein in hor­mone-treated prostate cell lines.

Introduction

Carcinoma of the prostate is the most common non-skin cancer in men, and the second leading cause of male cancer death in the United States [1]. The prostate gland is dependent on androgens for the maintenance of its normal growth and functional integrity. In addition, prostatic tumours are initially an­drogen responsive, and decrease in size in response to androgen ablation or anti-androgen therapy. The extent and duration of the response are variable, and ultimately, prostate cancer reverts to an androgen-independent state. The molecular mechanism of this loss of growth control by androgens is poorly un­derstood [2]. Oncogenic products have been demonstrated to have sequence homologies similar to proteins that are important mediators of hormone action on target cells. It is therefore likely that growth factors within the prostate may be related to oncogene products. Both human and animal systems have suggest­ed a role for oncogenes in prostatic carcinogenesis [3-12]. Elevated expression of the C-myc oncogene is often associated with cell proliferation, and the p62 C-myc oncogene product has been correlated closely with tumour grade [13]. Expression of the C-myc oncogene generally appears higher in prostatic cancer tissue than in benign hyperplasia [14]. While Rijnders and colleagues [11] dem­onstrated that the expression of C-myc oncogenes was independent of hor-

* Supported by Internal Grant from Section of Urology at West Side Veterans Administration Hospital.

VSP. Utrecht Akademiai Kiad6. Budapest

68 Asadi, Shariji: Sex-steroid modulation

monal treatment, a recent paper indicates that oestradiol produces an increase' in the level ofC-myc mRNA [15]. Griffiths and associates [16] have found tran­scripts of C-myc and p53 in benign adenoma and/or carcinoma tissue. By use of a recombinant retroviral vector, the C-myc oncogene is recently introduced singly or in combination with the ras oncogene into the foetal urogenital sinus, resulting in distinct phenotypes of prostatic pathology [17]. These observa­tions prompted us to explore whether expression of C-myc oncogene was under hormonal control. A combination of immunohistochemistry, in situ hybridiza­tion, and Southern hybridization has been used to study the expression of the C-myc oncogene within two human prostatic cell lines. We found that C-myc oncogene and oncoprotein are present in LN-CaP and DU-145 prostatic carci­noma cell lines and in both prostatic carcinomatous tissues. With regard to hormonal effects, we showed that dihydrotestosterone (DHT) and estrogen in­crease expression of C-myc oncogene and oncoprotein; however, DHT appears to be more effective in eliciting C-myc expression.

Materials and methods

Cell culture

Two human adenocarcinoma cell lines, DU-145 and LN-CaP were ob­tained from the American Type Culture Collection (Rockville, MD). Both cell lines were cultured at 37°C in Roswell Park Memorial Institute (RPMI)-1640 medium, with added glutamine, supplemented with 10% (V N) dextran-coated charcoal stripped foetal bovine serum (FBS). The control cells received no hor­mones, but were maintained in RPMI-1640 supplemented with 10% stripped serum. The method used for the preparation of stripped serum was a modified procedure originally described by Drovin [18]. The treated cells in a period of one week received either 10-3 5a.-dihydrotestosterone or 200 pg/ml 1713-estra­diol purchased from Sigma Chemical Co. (St. Louis, MO, 63178). The control cells for hormone experiments received also 0.2% solution of hormone solvent mixture (ethanol: propylene glycol 9: 1).

In vitro proliferation assay

A suspension of 1 x 103 cells/ml was seeded in tissue culture 6-well plates and incubated overnight in a CO2 incubator. The following day, 100 III of 5-bromo-2' -deoxyuridine (Amersharn Corp., Arlington Heights, IL) was added to each well. After one-hour incubation, cells were gently washed with phos­phate buffer saline (PBS), fixed in 5% acetic acid in ethanol, washed in 0.1% Tween 20 in PBS, and incubated with 50 III of anti-5-bromo-2' -deoxyuridine monoclonal antibody for 30 minutes. This was followed by incubating with the secondary peroxidase rabbit anti-mouse antibody for 25 minutes at room temperature. Slides were then treated with the peroxidase substrate solution (O.l% DAB in Tris-HCl, pH 7.2, containing 0.005% hydrogen peroxide) for

International Urology and Nephrology 27, 1995

Asadi, Sharifi: Sex-steroid modulation 69

5 minutes. Five slides of each specimen were selected at random and counted. The percentage of positive cells was determined by counting a total of 250 and positive cells tallied.

In situ hybridization

Slides of cell cultures were immersed in 3% peroxide for five minutes, followed by rinsing in deionized distilled water. Slides were treated with 1 mM lavamisole (Sigma Chemical Co., St. Louis, MO) and rehydrated by se­quential double washes in 95%, 80%, 45%, and 0% ethanol in water. To each slide 100 III of hybridization mixture was added. Hybridization mixtures were made from 50 III deionized formamide (Fisher Scientific, Fair Lawn, NJ), 10 III of 50% dextran sulfate (Sigma Chemical Co., St. Louis, MO), 10 11120 X SSPE (3.6 M sodium chloride, 0.2 M sodium phosphate, pH 7.4, 0.02 M EDTA, pH 7.4), 20 III of 5 pg/1l1 stock DNA probe (ENZO Biochem, New York, NY), and 10 III deionized water. For denaturation, slides were placed in a 14 cm glass Petri dish, and incubated in 0.2 N HCl solution at room tempera­ture for 20 minutes. After denaturation, slides were washed in 0.05% Triton X-I00 and immersed in 50% formamide in 0.1 X SSC (1 M NaCl, 1 mM Na3 citrate - H20, pH 7.0) at 37°C for 10 minutes. After a formaldehyde bath, slides were washed in 0.05% Triton X-lOO, then 200 III of streptavidin and biotinylated horseradish peroxidase detection complex (ENZO Biochem, New York, NY) were added and incubation continued at room temperature for 20 minutes. Finally, slides were rinsed in wash buffer (1 X PBX, 5 mM EDTA), 400 III of substrate mixture (25 III of 1% H20 2, 20 III of aminoethylcarbazole, 1 1110.5 M sodium acetate, pH 4.9) was added to the specimen and incubated at room temperature for 10-15 minutes. The slides were then rinsed with wash buffer and cover slips were mounted on the slide and viewed by light micro­scopy at x400 magnification.

Southern experiment

Total DNA was purified and analyzed by the Southern procedure [19]. Digestion of DNA by restriction endonuclease was accomplished as recom­mended by the supplier (Boehringer Mannheim, Indianapolis, IN). DNA frag­ments were fractionated on 0.8% agarose gels, transferred to nylon membrane, hybridized to the nick-translated C-myc probe, and visualized by autoradiogra­phy. The blots were hybridized and washed at 42°C in 0.3 M NaCl.

Statistical analysis

Analysis of variance employing the BMDP Statistical package was used to analyze the cell growth data (available on the main frames of UIC comput­er). The program BMDP8V was applied to differentiate between fixed and random factors.

International Urology and Nephrology 27, 1995

70 Asadi, Sharifi: Sex-steroid modulation

Results

Serum modulation of cell growth

Human prostate cells utilized in vitro require serum supplement for growth. The results (Fig. 1) show that both cell lines can grow in the presence of either intact or dextran coated charcoal stripped serum. However, the dif­ference between the growth rates in both media was more pronounced with LN-CaP cells compared to that with DU-145 cells.

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Fig. 1. Growth curves of human prostatic adenocarcinoma cell lines DU-145 and LN-CaP maintained in RPM! containing either intact or stripped serum

International Urology and Nephrology 27, 1995

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Asadi. Sharifi: Sex-steroid modulation 71

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Fig. 5

Figs 2-5. Growth curves of human prostatic adenocarcinoma cell lines DU-145 (Fig. 2 and Fig. 4) and LN-CaP (Fig. 3 and Fig. 5) maintained on media containing stripped serum supplemented

with different concentrations ofDHT (Fig. 2 and Fig. 3) and l7P-EST (Fig. 4 and Fig. 5)

72 Asadi, Sharifi: Sex-steroid modulation

Steroid modulation of cell growth

The growth rate ofOU-145 and LN-CaP cells treated with different con­centrations ofDHT (10-3 M -10-7 M) and 17 M-EST (40 pg/ml to 200 pg/ml) are shown in Figs 2-5. Concentrations of 10-3 M and 10-4 M of DHT had the most stimulatory effect on the proliferation of DU-145 cells. LN-CaP cells showed the highest growth rate when treated with the highest concentration of DHT (10-3 M). On the other hand, there was no evident effect of EST concen­trations on the growth ofOU-145 and LN-CaP cells and the log total cell counts were very close to that of the control cells.

Proliforation assay

Proliferation assay showed that the percentages of proliferating cells on day 7 were comparable between the two cell lines when untreated (18-20%) or increased by treatment with OHT (26-27%). The increase in per cent prolif­eration was statistically significant with DHT treatment for both cell lines (Table 1). Estrogen treatment, however, differentially decreased the percentage of proliferating cells (Table 1), as well as the total number of cells (data are not shown). Both the decrease from the control values as well as the difference be­tween cell lines were statistically significant (P<O.OOOI).

Table 1 Percentage of cells positive for proliferations

DU-145

LN-CaP

Untreated controls

20.5± 1.4

l7.7±1.2

Number of proliferating cells (±SEM) (N=3)

Testosterone treated

26.3±1.3

26.6±1.3

Estradiol treated

13.7±0.4

6.9±O.9

Two-way analysis of variance establishes that there are significant differences between cell lines, among treatments, as well as in inter­group effect. A Duncan multiple range test shows significant (95% confidence) differences between all groups except for the two groups within each set of continuously underlined groups.

Pattern of oncogene and oncoprotein staining

In situ DNA-DNA hybridization revealed that both cell lines showed am­plified C-myc oncogene (Fig. 6). Double immuno-enzyme staining of prostate cell lines for either oncoprotein and proliferation or oncoprotein and oncogene suggests that C-myc expression is related in some way to the cellular prolifera-

International Urology and Nephrology 27, 1995

Asadi. Sharifi: Sex-steroid modulation

Fig. 6. Photomicrograph ofLN-CaP cells treated with DHT for seven days and assayed by in situ hybridization using

a peroxidase labelled C -myc oncogene probe. Arrows show cells positive for C-myc (x450)

1 • Fig. 7. Photomicrograph ofLN-CaP cells treated with DHT for seven days.

Cells producing oncoprotein (arrows) (blue reaction product from alkaline phosphatase-labelled anti-C-myc oncoprotein) are also positive for C-myc oncogene (brown reaction product of peroxidase-labelled C-myc

oncogene probe) (x450)

International Urology and Nephrology 27. 1995

73

74 Asadi, Sharifi: Sex-steroid modulation

tion of prostate cells (Fig. 7). We showed that DHT and EST increased the per­centage of positive LN-CaP cells for C-myc oncogene from 19.3±O.2 'to 39.2±1.0 and to 26.02±2.0, respectively, and for DU-145 cells from 15.6±1.7 to 29.O±O.7 and to 21.5±1.9, respectively (Table 2). On the other hand, DHT in­creased the percentage of cells expressing C-myc oncoprotein from 6.2±O.9 to 14.6±O.7 for DU-145 and from 9.6±10 to 12.4±1.11 for LN-CaP (Table 3). Estradiol decreases the percentage of cells expressing C-myc oncoprotein from 9.6±1.1 to 6.1±1.1 for LN-CaP cells (Table 3). The results of the regression

Table 2 Percentage of cells positive for C-myc oncogene.

N=3 for control groups, N=6 for testosterone (DHT) and estrogen (EST) treatment groups

Control DHT EST

LN-CaP 19.3±O.2 30.2±l.O 26.2±2.0

DU-145 15.6±1.7 29.0±O.7 2 l.5± 1.9

DHT and EST cause an increase in C-myc oncogene expression in both cell types. The only difference between the two cell types is that in the DU-145 cells EST was less effective in increasing C-myc oncogene ex­pression.

Table 3 Percentage of cells positive for C-myc protein.

N=3 for control groups, N=6 for testosterone (DHT) and estrogen (EST) treatment groups

Control DHT EST

LN-CaP 9.6±1.0 12.4 ±l.l 6.1±1.l

DU-145 6.2±O.9 14.65±O.7 6.8±0.4

ANOV A shows a significant hormone treatment effect with a signifi­cant interaction with cell type. A subsequent Duncan multiple range test shows that DHT causes an increase in DU-145 cells and EST causes a decrease in LN-CaP cells.

analysis done on the oncogene/oncoprotein data showed a negative correlation between cells positive for oncogene and oncoprotein in DU-145 and LN-CaP control cells. However, DHT and EST treated DU-145 cells and DHT treated LN -CaP cells showed a positive linear correlation between presence of onco­gene and oncoprotein. Estradiol treated LN-CaP cells did not show any such correlation (Fig. 8a, b).

International Urology and Nephrology 27, 1995

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Asadi. Sharifi: Sex-steroid modulation

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75

76 Asadi. Sharifi: Sex-steroid modulation

Southern blotting experiment

To correlate the results from in situ hybridization to the level of C-myc oncogene, DNA from two human prostate adenocarcinoma cell lines, DU-145 and LN-CaP were subjected to Southern blot hybridization (Fig. 9). Lanes B and E represent the DNA from control cultures which received no hormones, but were maintained on RPMI-1640-GA supplemented with 10% stripped serum, and received only 0.2% hormone solvent mixture. DNA from cells treated with either 10--3 M DHT or 200 pg/ml EST over one week are shown in Lanes C, D, F, and G. A 2.0 Kb band was detected in all groups which repre­sents DNA regions containing sequences homologous to C-myc oncogene. However, treatment of DU-145 cells with 10-3 M DHT for seven days has shown an amplification ofC-myc related sequences (Fig. 9, Lane C) compared to a faint band at the 2.0 Kb in DU-145 control group (Fig. 9, Lane B). An intense band corresponding to 2.0 Kb was detected in LN-CaP cells treated with 10--3 M for seven days (Fig. 9, Lane F). The intensity of the 2.0 Kb band decreased in both cell lines when treated with 200 pg/ml EST for seven days (Fig. 9, Lanes D and G).

Fig. 9. Southern DNAIDNA hybridization of DNA from human prostatic adenocarcinoma cell lines, DU-145 and LN-CaP, which were maintained on media containing 10% stripped serum and supplemented with 10-3 M DHT or 200 pg/l.L1 of EST for seven days. DNA (15 Ilg/lane) was digested with Hind III, separated on 0.8 agarose gel and transferred to a nylon membrane and labelled with 32p nick-translated probes specific for C-myc oncogene. Lambda phage digested with Hind III was used as marker DNA; sizes of the fragments (in Kb) are indicated on the left. Lane A. Lambda phage digested with Hind III; Lane B. DU-145 (control); Lane C. DU-145 (DHT-treated); Lane D. DU-145 (EST-treated); Lane E. LN-CaP (control); Lane F. LN-CaP

(DHT-treated); Lane G. LN-CaP (EST-treated)

international Urology and Nephrology 27. 1995

Asadi, Sharifi: Sex-steroid modulation 77

Discussion

The prostate gland requires a permanent androgen stimulus for its differ­entiation and function. Prostatic tumour cells, however, can overcome androgen dependence and escape hormone ablation therapy. The C-myc oncogene is a highly conserved DNA sequence and is a cellular homologue of C-myc viral oncogenes. It encodes a nuclear protein that has been linked with cell prolifera­tion and DNA synthesis. Expression of C-myc has been reported in prostatic carcinoma cell lines such as PC3, PC133, and PC 135 [11]. They have been re­ported in hyperplastic and carcinomatous prostatic tissue as well [14]. Hie­rowski et al. [20] have reported the potential dependence of human prostatic growth factor on stimulating thymidine incorporation into DNA. They have shown that stimulation was significantly enhanced by DHT and EST in the an­drogen sensitive LN-CaP cell lines, which express high level of androgen re­ceptor [21]. We have used a fast growing colony from this LN-CaP cell line, designated LN-CaP-FGC, and have observed that growth of these cells is also stimulated by androgen (DHT), whereas estradiol was suppressive. It appears that the DU-145 cell line is apparently not hormone sensitive since it grows equally well in either 10% intact or stripped serum. The cell line DU-I45 does not express androgen receptor, although the androgen receptor gene is present [22]. The presence of a wide range of concentrations of DHT and estradiol was found to be without significant effect on the growth ofDU-I45 cells. How­ever, these hormones show a significant effect on the growth ofLN-CaP cells. The stimulatory effect of DHT or inhibitory effect of 17P-EST on the mitotic index (PDT) of the LN -CaP cell line might indicate its degree of androgen sensitivity. We obtained similar results as those reported by Horoszewicz et al. [23], indicating that the LN-CaP cell line is responsive to hormonal treatment. We were also able to confirm the results reported by Tilley et al. [22] indicating that the DU-I45 cell line is not dependent on hormones for its survival.

Recent studies indicate that the C-myc gene is expressed throughout the cell cycle. Our data demonstrate the expression of the C-myc oncogene in both human prostatic adenocarcinoma cell lines as well as benign and malignant prostatic tumours. However, the level of expression of the C-myc oncogene is significantly higher in adenocarcinoma of the prostate than in benign prostatic hyperplasia (Figs 10 and II). Although p62 oncoprotein expression correlated with histologic differentiation and thus indirectly with survival, the prognostic value of p62 quantitation in prostate cancer will have to be determined in future studies. Elevated expression of the C-myc oncogene is often associated with cell proliferation. In our study, the expression of C-myc oncoprotein in prolifer­ating cells confirms the relationship between cell proliferation and C-myc ex­pression. Whether oncogene activation occurs at a late stage in prostate cancer or whether it occurs only as an apparently random, infrequent event, awaits the results of further studies.

To assess co-expression of C-myc oncogene and oncoprotein we studied prostate cell lines by in situ hybridization using a peroxidase-labelled C-myc

International Urology and Nephrology 27, 1995

78 Asadi, Sharifi: Sex-steroid modulation

Fig. 10. Photomicrograph of prostate tissues showing strong positive brown reaction product from peroxidase-labelled C-myc oncogene probe in a mod­erately to well differentiated gland (large arrow). It also shows a blue reac­tion product from alkaline phosphatase-labelled anti-C-myc oncoprotein (small arrow). Note the negative reactions for both C-myc oncogene and

C-myc oncoprotein in poorly differentiated glands (x4S0)

Fig. 11. Photomicrograph of prostatic tissue. Note the strong positive reaction for C-myc oncoprotein in the moderately differentiated glands (thick arrow) and the weak staining reaction in the well differentiated glands (thin arrow).

Normal glands are completely negative (arrow heads) (x4S0)

International Urology and Nephrology 27, 1995

Asadi, Sharifi: Sex-steroid modulation 79

probe and by immunocytochemical staining with an alkaline phosphatasc­labelled, mouse anti-p62 monoclonal antibody, simultaneously. Dihydrotestos­terone and estradiol increased the percentage of cells positive for C-myc onco­gene in both cell lines. On the other hand, the percentage of cells positive for oncoprotein increased with DHT treatment in DU-145 cells and decreased with EST treatment in LN-CaP cells. Regression analysis showed a negative correlation between the number of cells positive for oncogene and for onco­protein in DU-145 and LN-CaP control cells. DHT- and EST-treated DU-145 cells and DHT -treated LN-CaP cells, however, showed a positive association between the presence of oncogene and oncoprotein. EST -treated LN -CaP cells did not show any such association. This suggests that translation may be linked to other intracellular controlling factors.

Southern blotting using a 32P-C-myc probe also showed a more intense band of 2.0 Kb corresponding to C-myc sequences in cells treated with ste­roids when compared with untreated control cells. The intensity of the DNA band from DHT -treated cells, however, was more prominent than the band from EST-treated cells. The difference between the in situ hybridization experi­ments and the Southern blotting experiments can be reconciled on the basis that in situ hybridization only reflected individual cell differences in a semi­quantitative manner.

The data discussed in these studies strongly implicate a link between hor­monal treatment and C-myc expression in human prostatic adenocarcinomas. The prognostic significance of elevated levels of C-myc expression in hor­mone-treated human prostatic adenocarcinoma warrants further investigation. In view of the claims that over-expression of C-myc and other oncogenes has prognostic significance, it is of great interest to identify the type of tumours that express these genes.

Acknowledgements

We are grateful to Drs. Joe Monaligod, Herbert Yamashiroya, and Paul Weiz-Caring­ton for advice and discussion during the course of this work. We also thank B. Branham for help in preparing the manuscript.

References

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adenocarcinoma. Prostate, 10, 281 (1987). 4. Konishi, N., Enomoto, T., Buzard, G., Ohshima, M., Ward, J. M., Rice, J. M.: Ki-ras

activation and ras 21 expression in latent prostatic carcinomas in Japanese men. Cancer, 69,2293 (1992).

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International Urology and Nephrology 27, 1995