The Human Prostatic Carcinoma Cell Line Lncap Expresses Biologically Active, Specific Receptors for La,25-Dihydroxyvitamin Ãœ31

Home , ID3 (gene)

(CANCER RESEARCH 52. 515-520, February I, 1992| The Human Prostatic Carcinoma Cell Line LNCaP Expresses Biologically Active, Specific Receptors for la,25-Dihydroxyvitamin Ãœ31

Gary J. Miller,2 Gary E. Stapleton, Janet A. Ferrara, M. Scott Lucia, Stephen Pfister, Tammy E. Hedlund, and Prakash Upadhya University of Colorado Health Sciences Center, Department of Pathology; Denver, Colorado 80262

ABSTRACT cause the promiscuity which has been observed. Careful exam ination, however, has failed to identify specific receptors for The LNCaP prostatic carcinoma cell line was examined for the other sex steroids (e.g., estrogen or progestins) in LNCaP cells presence of specific receptors for la,25-dihydroxyvitamin D3 [lo,25- (<)I I )2I),]. Whole cell binding studies identified approximately 2500 high- (3,10,15). affinity (A',, = 1.4 x Id"'') binding sites per cell. Competition studies In addition to sex steroids, the superfamily of nuclear recep revealed that these receptors are specific for the la,25(OH)2 metabolite. tors contains receptors for vitamin A, vitamin D3, and thyroid Binding studies using the synthetic androgen R1881 indicate that separate hormone (17). Once felt to be limited to tissues involved in androgen and vitamin I>i receptor antibody (9A7-y) but not with a monoclonal antibody la,25(OH)2D3 have been detected in diverse normal cells and to the androgen receptor (AN1-15). The receptor/ligand complex Ã©lÃ»tes tissues including breast, kidney, and testis (18). la,25(OH)2D3 from native DNA cellulose at 0.2 M KC1. Northern blot analysis identified also appears to affect various neoplastic cells (19-21). Of special an iiiKN A of approximately 4.6 kilobases which hybridized with a specific interest are the findings that breast carcinomas (22, 23) and vitamin D3 receptor complementary DNA probe (hVDR). In the absence of androgens, la,25(OH)2D3 stimulated growth and prostate-specific leukemias (24) contain specific receptors for la,25(OH)2D3 antigen production by LNCaP cells in a dose-dependent fashion. Dose- which modify their growth and/or differentiation. response curves indicated that at physiological concentrations (Id '' M) In a preliminary report, we described the presence of high- 1<*,25(OH)2D3 was mitogenic, whereas at higher concentrations (Id"" M) affinity binding sites for [1H]la,25(OH)2D3 in human prostatic it promotes differentiation. These studies suggest that la,25(OH)2D3 carcinoma cells, including the LNCaP cell line (25). We now could play an important role in the natural history of and response to describe the further characterization of these receptors and hormone therapy by prostatic cancer. present additional evidence that la,25(OH)2D., can alter the growth and differentiation of human prostatic cancer cells. INTRODUCTION MATERIALS AND METHODS Established in 1980 by Horoszewicz et al. (1), the LNCaP cell line has become recognized as a valuable tool for the study Materials. Nonlabeled 25(OH)D3, la,25(OH)2D3, and 24,25- of prostatic cancer biology. These cells were originally obtained (OH)2D3 were the generous gift of Dr. M. Uskokovic (Hoffmann-La Roche, Nutley, NJ). [26,27-mefAy/-3H]la,25(OH)2D3 (specific activity, from an aspirate of a subcutaneous supraclavicular lymph node 180 Ci/mmol), R1881, and [\7a-methyl->H]R.lSS\ (specific activity, metastasis in a patient who had developed refractory disease following androgen deprivation therapy (1, 2). As such, LNCaP 81.8 Ci/mmol) were obtained from New England Nuclear (Boston, cells provide a model in which androgen-refractory mechanisms MA). Tandem-R kits for the radioimmunoassay of PSA were obtained can be studied. This cell line is known to contain high-affinity from Hybritech, Inc. (San Diego, CA). Cell Culture. LNCaP cells were obtained from Dr. Horoszewicz androgen receptors (2) that regulate its growth (2-4), expres (Roswell Park Memorial Institute) at the 14th passage. Initial cultures sion of prostate-specific acid phosphatase (2, 5), and expression were karyotyped and confirmed to be LNCaP by the presence of of prostate-specific antigen (6, 7) in vitro. The androgen effect characteristic Ipâ€”and 15q+ marker chromosomes (1, 25). The cells on LNCaP growth has been shown to be mediated in part via were maintained in RPMI 1640 containing 10% fetal bovine serum the expression of the epidermal growth factor receptor (8-11) (HyClone Laboratories, Logan, UT), 100 units/ml penicillin, and 100 and possibly transforming growth factor a (12). Mg/ml streptomycin (Gibco Laboratories, Grand Island, NY). The The androgen receptors of LNCaP cells are known to have nasopharyngeal carcinoma cell line COLO 2650 was obtained from Dr. unusually high affinity for progestins (13). These same recep George E. Moore (26). T47D breast carcinoma cells were obtained tors also bind estrogens and glucocorticoids to a lesser degree, from Dr. Dean Edwards (University of Colorado Health Sciences Center, Denver, CO) and were maintained as described (27). regulating the growth of LNCaP cells in proportion to their Growth dose-response curves were performed by plating 30,000 binding affinities (10, 14). It has even been shown that the cells/well into 12-well culture plates (Costar, San Diego, CA) in RPMI androgen antagonist 3-hydroxyflutamide can compete for the 1640 containing 5% CSS. After 72 h the medium was changed to RPMI androgen receptor and stimulate the growth of LNCaP cells 1640 with 5% CSS which contained the compounds of interest in (15). The presence of a mutation in the hormone-binding do ethanol (the final ethanol concentration was 0.1% or less). Controls main of the LNCaP androgen receptor (16) is now believed .to received only ethanol. The medium was subsequently changed every 2- 3 days throughout the experiment. At day 6, the cells were harvested Received 9/10/91; accepted 11/14/91. by adding 0.5 ml trypsin-EDTA (Gibco Laboratories). After 1 min 0.5 The costs of publication of this article were defrayed in part by the payment ml of RPMI 1640 with 5% CSS was added, and a small aliquot was 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. then counted using a hemacytometer. 1This work was supported in part by a grant from the Cancer League of Colorado (G. J. M.). 3The abbreviations used are: 1Â«,25(OH)2D3. lÂ«,25-dihydroxyvitamin D3; 2To whom requests for reprints should be addressed, at Department of PSA, prostate-specific antigen; CSS. charcoal-stripped, sulfatase-treated fetal Pathology (B216), University of Colorado Health Sciences Center. 4200 East bovine serum; R1881. nonlabeled methyltrienolone; TEDZ, 10 mvi Tris, I mivi Ninth Avenue, Denver, CO 80262. EDTA, 5 mvi dithiothreitol, 0.3 mvi ZnCI2. 515

Ligand-Binding Assays. Whole cell ligand-binding assays (25) were turer's instructions. Following removal of media from the flasks, the used throughout. Late log phase cells were removed from culture dishes cells were harvested by trypsinization and counted. PSA was then by brief trypsinization and were washed once in RPMI/1% fetal calf expressed as ng/cell. serum. Viable cells (1 x IO6) were aliquoted to replicate disposable Detection of Vitamin D Receptor mRN A. The plasmid hVDR (a microfuge tubes. Media containing 1% serum and radiolabeled ligand generous gift from Dr. J. W. Pike, Baylor College of Medicine, Hous were added. For competition studies either a 200-fold molar excess of ton, TX) containing complementary DNA sequences encoding the homologous or heterologous nonlabeled ligand was added. The cells human la,25(OH)2D3 receptor (33) was propagated in Escherichia coli were incubated at 37Â°Cfor l h with periodic gentle vortexing at 15- DHL Plasmid DNA was purified on a cesium chloride gradient and min intervals. The tubes were then placed on ice and allowed to cool digested with EcoRl, which released a 2-kilobase insert containing the for 15 min. The cells were washed once with media containing 1% receptor sequence. The insert was isolated and radiolabeled by nick serum and once with phosphate-buffered saline by centrifugation. Fol translation with [a-32P]dATP and [a-32P]dCTP. lowing resuspension in 50 Â»/Iofphosphate-buffered saline, the cells Total cellular RNA was extracted from 10" cells using a guanidinium were lysed by adding 200 ^1 of 0.02 M Tris-HCI, pH 7.8, and 0.25 M thiocyanate method (34). Messenger RNA was purified using the sucrose (Tris/sucrose) containing 0.5% Triton X-100. The cells were PolyATract System (Promega, Madison, WI) and was quantitated by vortexed vigorously to facilitate rupture of the cell membrane. Nuclei absorbance at 260 rtM. Northern analysis was performed as described were recovered by centrifugation, washed once with Tris/sucrose, and (35). Briefly, polyadenylated mRNA (9.4 ^g) was subjected to electro- pelleted by centrifugation as above. The pellets were resuspended in phoresis on a denaturing formaldehyde-agarose gel at 110 mA and then 100 /progesterone receptor antibody (generously provided by Dr. Dean P. were obtained. It is apparent that neither 25(OH)D3 nor Edwards, University of Colorado Health Sciences Center). 24,25(OH)2D3 competes effectively for la,25(OH)2D3 binding. DNA Affinity Chromatograph)'. Cytosol (0.5 ml) was incubated with Only at concentrations of heterologous metabolites greater than 2 mM [3H]la,25(OH)2Dj on ice for 3 h in the dark as previously 1000-fold was significant inhibition obtained. described (28). TEDZ (2.5 ml) was then added to the labeled superna The results of sucrose gradient analysis are seen in Fig. 3. tant to reduce the concentration of KCI to 50 mM. The diluted super Using 14C-labeled bovine serum albumin (4.3S) and l4C-labeled natant was loaded onto a 0.5 x 10 cm column of native DNA cellulose (Lot AG5581104; Pharmacia, Piscataway, NJ) previously equilibrated IgG (6.8S) as sedimentation markers, the la,25(OH)2 receptor with 50 mM KCI in TEDZ. Binding of the receptor/ligand complex of LNCaP cells was found to have a sedimentation coefficient was allowed to proceed at 4Â°Cfor 1 h. The unbound labeled ligand was of approximately 3.5S, similar to those of other human cell washed from the column with 50 ml of 50 mM KCI in TEDZ. The lines (26). The specificity of this finding was established using bound complex was eluted from the column using a linear gradient of two disparate approaches. First, competition with a 200-fold KCI (50-400 mM) in TEDZ. One-mi fractions were collected and excess of nonlabeled la,25(OH)2D3 eliminated the receptor/ analyzed by scintillation counting. ligand complex peak as seen in Fig. 3/Ã•.However, a 200-fold PSA Radioimmunoassay. For the radioimmunoassay of prostate- specific antigen (32), 3 x IO5LNCaP cells were plated into T-25 flasks excess of nonlabeled R1881 failed to compete for the binding of [3H]la,25(OH)2D3. Second, gradient shift assays (Fig. 35) with RPMI/5% CSS as described above. After 3 days, test substances revealed that in the presence of the anti-vitamin D receptor were added in ethanol. Controls received ethanol only. The media from these cultures were analyzed for the presence of PSA by solid-phase antibody 9A?7, the sedimentation coefficient of the receptor/ radioimmunoassay using the Tandem-R Kit according to the manufac ligand complex increased to approximately 7.0S as compared 516

by DNA affinity chromatography is seen in Fig. 4. After elution of free radioactivity by 50 mM KC1, a linear gradient was used to elute bound receptor/ligand complex. A distinct peak was eluted from native DNA at approximately 0.2 M KC1. This is similar to the affinity observed for normal human vitamin D receptors as reported by others (38). Northern analysis of polyadenylated RNAs obtained from LNCaP, T47D, and COLO 2650 are seen in Fig. 5. Both LNCaP and T47D cells contain mRNA that hybridizes with the hVDR probe. In addition to the predominant 4.6-kilobase

0.0 0.5 1.0 1.5 [3H]1a,25(OH)2 D3 (nanomoles)

5 10 15 20 25 FRACTION NUMBER

10 [ H]R1881 (nanomoles) Fig. l. A, binding of [3H]l a,25(OH)2D3 with (D) and without (â€¢)excessR1881. Specific binding data, corrected for nonspecific binding, are plotted. Points, mean of triplicate determinations from three separate experiments; bars, SE. B, binding of [3H]R1881 with (â€¢)and without (O) excess la,25(OH)2D3. Bars, Â±SEfrom triplicate determinations.

0 5 10 15 20 FRACTION NUMBER Fig. 3. A, sucrose density gradient profiles of [3H]lo,25(OH)2D3-receptor complexes from LNCaP cell cytosols, alone (â€¢),with excess lo,25(OH)2D3 (D). or with excess R1881 (A). 4.3S, position of MC-labeled bovine serum albumin; 6.SS, position of '4C-labeled IgG analyzed in parallel gradients. B, gradient shift profiles of [3H]la,25(OH)2D3-receptor complexes from LNCaP cell cytosols alone (â€¢)orwith 9A7-y (O) or AN1-15 (A) monoclonal antibodies.

3000 T

10'

MOLAR EXCESS OF COMPETITORS Fig. 2. Competition of [3H]la,25(OH)2D3 binding by la,25(OH)2D3 (A), 25(OH)D3 (â€¢),and24,25(OH)2D3 (O). Bars, Â±SEfrom triplicate determinations. The control consisted of a parallel set of samples to which nonlabeled metabolite was not added. to the '4C-labeled protein markers. This indicates that binding of the monoclonal antibody to the receptor/ligand complex has occurred. Controls with monoclonal antibodies to either the 10 20 30 40 50 60 70 80 90 FRACTION NUMBER androgen receptor or the progesterone receptor (not shown), Fig. 4. Native DNA cellulose chromatography of [3H]ltÂ»,25(OH)2D3-receptor however, failed to alter the sedimentation coefficient. complexes (O) from LNCaP cytosols. Elution was performed with a linear 50- Characterization of the LNCaP cell la,25(OH)2D3 receptor 400 mM KC1 gradient ( ) in TEDZ 517

in PSA production per cell are seen. At higher concentrations, kb a biphasic response similar to that seen with respect to growth is obtained. The amount of PSA released into the medium, however, remains higher than that produced by cells in the presence of CSS alone. The release of PSA is time dependent, as is seen in Fig. 8. At 24 and 48 h following addition of -44.8 la,25(OH)2D.,, neither the PSA per cell nor the total PSA detected (not shown) is significantly increased when compared to CSS alone. However, at 72 h, PSA is released into the medium at concentrations well over those of the untreated control cells.

DISCUSSION

Adenocarcinoma of the prostate is now recognized to be the most common malignancy of adult males in the United States. In 1991, it is estimated that this disease will be detected in 122,000 men and will cause the deaths of 32,000 (39). Over B 30% of cases of prostatic carcinoma diagnosed in the United States are detected after the disease has invaded locally or metastasized (40). For these patients, the androgen withdrawal therapy that they receive is mainly palliative. Initially, approx imately 75% respond favorably to androgen withdrawal (41). Eventually, however, the majority go on to clinical Stage D3,

Fig. 5. Northern blot analysis of polyadenylated RNA. Lane I, LNCaP; Lane 100 2. T47D; Lane 3, COLO 2650. The same nitrocellulose filter was hybridized to the hVDR vitamin D3 receptor complementary DNA probe (A), stripped, and rehybridized to the pHRp II 5.5 RNA polymerase II complementary DNA probe 80 (B), kb, kilobases. l.S and 4.8, position of the small and large ribosomal RNA subunits, respectively, on a parallel blot. O 60 ce 40 -- LiJ mRNA species T47D cells also contain a minor variant of O 20 -- approximately 2.4 kilobases (33). The nasopharyngeal carci noma cell line COLO 2650 does not contain significant o O -- amounts of receptor mRNA, which is consistent with the ob servation that these cells fail to bind [3H]1Â«,25(OH)2D.,(26). -20 -- LNCaP cells clearly contain mRNA of a size similar to that 10 " 10 ' seen in T47D cells, which is consistent with the binding results O 10 10 10 described above. The lesser amount of message present in CONCENTRATION OF METABOLITE LNCaP cells cannot be explained purely on the basis of sample Fig. 6. Dose response of LNCaP growth (cell number) to increasing concen loading, since the amounts of Pol\\ mRNA are similar in T47D trations of 1Â«,25(OH)2D3(A), 24,25(OH)2D3 (O), or 25(OH)D3 (â€¢)added to the culture medium for 6 days. Points, mean of triplicate cultures compared to CSS and LNCaP cells. controls that received vehicle only; bars, SE. We have previously shown that la,25(OH)2Dj is a mitogen for LNCaP cells for about 7 days in culture (25). The dose dependence of this growth stimulation is shown in Fig. 6. Under (n 250 -r the conditions used in the present study cell counts of control (n O cultures remained unchanged during the course of the experi 200 -- o: ments. The data for experimental conditions are expressed as Lu the percentage change from the controls, indicating an increase 150 -- or decrease in absolute cell number. As the concentration of added la,25(OH)2D3 was increased from 10"" M an increase

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1992 American Association for Cancer Research. VITAMIN Dj RECEPTORS IN LNCaP CELLS growth of advanced-stage disease. Although it is well recognized that hypervitaminosis D causes electrolyte disturbances due to 20 -- its calcium-mobilizing effects, nonhypercalcemic analogues that could be used in this application have been described. For o example, 22-oxa-lÂ«,25(OH)2D, is about 10 times more potent than la,25(OH)2D3 in suppressing proliferation and inducing differentiation of WEH1-3 mouse myelomonocytic leukemia 10 cells while displaying little or no hypercalcÃ©mieactivity (44). We are currently examining the effects of such analogues on to _ 0. 5 prostatic carcinoma cells. : ninin124 Finally, contrary to the vitamin D deficiency hypothesis it is possible that la,25(OH)2D3 promotes the growth of advanced- 72HOURS stage prostatic cancer cells. LNCaP cells were isolated from such a patient and can, therefore, be considered to be a model of "androgen-refractory" prostatic carcinoma. It is important Fig. 8. Time dependence of PSA Stimulation by 1Â«,25(OH)2D.,in LNCaP cells. D, CSS control; B, CSS with 10"' M lrt,25(OH)2D3 added to the culture to recognize that this is not inconsistent with the fact that medium. Columns, mean of triplicate determinations; bars, SE. LNCaP cells grow in response to androgens in vitro. Androgen- refractory behavior in vivo implies only that tumor cells have in which the malignant cells grow even when total androgen become capable of growing without androgens, not that they ablation has been attained. have become androgen insensitive. It is likely that the progres In spite of its prevalence, the specific causes of this disease sion to androgen-refractory behavior occurs because the cells remain somewhat obscure. Various risk factors including age, in question have become capable of growing in response to an race, and diet have been identified and are believed to contribute additional growth factor that substitutes for androgens. Since to either the initiation or the progression of this disease (42). la,25(OH)2D1 acts as a mitogen for LNCaP cells in vitro, it is Still, the mechanisms through which such factors act remain possible that vitamin D3 or even other ubiquitous steroids could the subject of speculation. Recently, Schwartz and Hulka (43) play such a role once therapeutic selection pressures have been have proposed that age, race, and diet may be interrelated applied. Direct inhibition of androgen-refractory growth would through their ability to decrease circulating vitamin D> Specif require the use of vitamin D3 antagonists. Although such a ically, they have suggested that vitamin Dj maintains the dif synthetic analogue has been described (45), none are available ferentiated phenotype of prostatic epithelium and that in defi at present for testing. Ultimately, the relative effectiveness of ciency states, carcinomas can progress to clinically manifest agonists or antagonists on the malignant behavior of prostatic disease. They note, however, that vitamin D3 receptors have carcinoma will need to be explored in vivo where endocrine not been investigated in human prostatic carcinoma cells. regulation of mineral homeostasis is operative. We have previously reported that LNCaP cell nuclei bind In summary, we have shown that a human prostatic carci pH]lo,25(OH)iD3, and these cells are stimulated to grow by noma cell line, LNCaP, contains characteristic specific high- this metabolite in the absence of androgens (25). The data affinity receptors for la,25(OH)2D,. Furthermore, we have presented above extend our earlier findings and show that shown that these receptors are biologically active, since LNCaP cells contain specific, high-affinity receptors for la,25(OH)2Dj stimulates both growth and PSA production in la,25(OH)2D.i that are similar in many respects (including a dose-dependent fashion. The possibility that a nonandrogen sedimentation coefficient, immunological cross-reactivity, receptor such as this can regulate the growth and differentiation DNA affinity, and message size) to the vitamin D, receptors of prostatic carcinoma deserves additional attention, since this that have been reported to exist in several different benign and offers alternative approaches to disease prevention and inter malignant cells (18-20, 26). LNCaP vitamin D., receptors are vention in failed metastatic disease. specific for the la,25(OH)2D, metabolite of vitamin D3 and are present in numbers per cell similar to those of other benign and ACKNOWLEDGMENTS malignant cells (19, 26). We have previously shown (25) that the nuclei of DU 145 cells and PC-3 cells also contain binding We would like to thank Nancy Hart and Clairene Mraz for their sites for [3H]lo,25(OH)2D3, suggesting that vitamin D, recep skillful assistance in preparing the manuscript for publication. tors may be present in many, if not all, prostatic carcinoma cells. We are currently investigating this possibility by exam REFERENCES ining additional established cell lines. 1. Horoszewicz, J. S., Leong, S. S., Ming-Chu, T., Wajsman, Z. L., Friedman, Our finding that the production of PSA is stimulated by M., Papsidero, L.. Kim, U., Chai, L. S., Kakati. S., Arya, S. K., and Sandberg, la,25(OH)2Di| is consistent with the hypothesis of Schwartz A. A. The LNCaP cell lineâ€”a new model for studies on human prostatic and Hulka in that physiological concentrations of vitamin D3 carcinoma. Prog. Clin. Biol. Res., 37: 115-132, 1980. 2. Horoszewicz, J. S., Leong, S. S., Kawinski, E., Karr, J. P., Rosenthal, H., promote the differentiation of prostatic carcinoma cells. This Ming Chu, T., Mirand, E. A., and Murphy, G. P. LNCaP model of human finding is similar to the action of 1Â«,25(OH)2D, on the differ prostatic carcinoma. Cancer Res., 43: 1809-1818, 1983. entiated phenotype of HL-60 human promyelocytic leukemia 3. Berns. E. M. J. J., de Boer, W., and Mulder, E. Androgen-dcpcndent growth regulation of and release of specific protein(s) by the androgen receptor cells (24). The potential significance of this finding with respect containing human prostate tumor cell line LNCaP. Prostate, 9: 247-259, to prostatic cancer is 2-fold. First, correction of deficiency states 1986. 4. Romijn, J. C., Verkoelen, C. F., and Schroeder, F. H. Application of the with supplemental dietary vitamin D., may be capable of inter MTT assay to human prostate cancer cell lines in vitro: establishment of test fering with the progression of prostatic carcinoma in the early conditions and assessment of hormone-stimulated growth and drug-induced phases of its natural history. Second, exogenous supra-physio cytostatic and cytotoxic effects. Prostate. 12: 99-110, 1988. 5. Schulz, P., Bauer, H. W., and Fittier. F. Steroid hormone regulation of logical levels of lÂ«,25(OH)2D.i may be capable of slowing the prostatic acid phosphatase expression in cultured human prostatic carcinoma 519

cells. Biol. Chcm. Hoppe-Seyler. 366: 10.13-1039. 1985. receptors for vitamin D, in human prostatic carcinoma cells. In: J. P. Karr, 6. Hasenson. M., I mullÃ.B., Stege, R., CarlstrÃ¶m, K., and Pousette. A. PAP D. S. Coffey, R. G. Smith, and D. J. Tindall (eds.). Molecular and Cellular and PSA Â¡nprostatic carcinoma cell lines and aspiration biopsies: relation to Biology of Prostate Cancer, pp. 253-259. New York: Plenum Press, 1991. hormone sensitivity and to cytological grading. Prostate, 14: 83-90, 1989. 26. Frampton, R. J.. Suva, L. J., Eisman, J. A.. Findlay, D. M., Moore, G. E., 7. Young, C. Y-F., Montgomery, B. T.. Andrews. P. E., Qiu. S.. Bilhartz, D. Moseley, J. M., and Martin, T. J. Presence of 1,25-dihydroxyvitamin D3 l and I mil.ill. D. J. Hormonal regulation of prostate-specific antigen receptors in established human cancer cell lines in culture. Cancer Res., 42: messenger RNA in human prostaticadenocarcinomacell line LNCaP. Cancer 1116-1119.1982. Res., SÃ¬:3748-3752, 1991. 27. Keydar, I., Chen, L., Karby. S., Weiss, F. R., Delarea, J., Radu, M., Chaitcik, 8. Schuurmans, A. L. C., Bolt. J.. and Mulder. E. Androgens and transforming S., and Brenner. H. J. Establishment and characterization of a cell line of growth factor rf modulate the growth response to epidermal growth factor in human breast carcinoma origin. Eur. J. Cancer, 75:659-670, 1979. human proslatic tumor cells (LNCaP). Mol. Cell. Endocrinol., 60:101-104. 28. Pike, J. W. Interaction between 1.25-dihydroxyvitamin D3 receptors and 1988. intestinal nuclei. J. Biol. Chem., 257: 6766-6775, 1982. 9. Schuurmans, A. L. G.. Bolt. J.. and Mulder, E. Androgens stimulate both 29. Pike, J. W. Monoclonal antibodies to chick intestinal receptors for 1,25- growth rate and epidermal growth factor receptor activity of the human dihydroxyvitamin D,. ]. Biol. Chem., 259: 1167-1173, 1984. prostate tumor cell LNCaP. Prostate, 12: 55-63, 1988. 30. Pike, J. W., Kesterson, R. A., Scott, R. A., Kerner, S. A., McDonnell, D. P., 10. Schuurmans. A. L. G., Bolt, J.. Voorhorst. M. M.. Blankenstein, R. A., and andO'Malley, B. W. Vitamin D3 receptors: molecular structure of the protein Mulder. E. Regulation of growth and epidermal growth factor receptor levels and its chromosomal gene. In: A. W. Norman. K. Schaefer, H-G. Grigoleit, of LNCaP prostate tumor cells by different steroids. Int. J. Cancer, 42: 917- and D. v. Herrath (eds.), Vitamin D: Molecular, Cellular and Clinical 922, 1988. Endocrinology, pp. 213-224. Berlin: Walter de Gruyter. 1988. 11. Mulder. E., van Loon, D., de Boer. W., Schuurmans. A. L. G.. Bolt. J., 31. Estes, P. A., Suba, E. J., Lawler-Heavner. J., Elashry-Stowers, D., Wei, L. Voorhorst, M. M.. Kuipcr. G. G. J. M., and Brinkmann, A. O. Mechanism L., Toft, D. O., Sullivan, W. P., Horwitz. K. B., and Edwards. D. P. of androgen action: recent observations on the domain structure of androgen Immunologie analysis of human breast cancer progesterone receptors. 1. receptors and the induction of EGF-receptors by androgens in prostate tumor Immunoaffinity purification of transformed receptors and production of cells. J. Steroid Biochem.. 32: 151-156. 1989. monoclonal antibodies. Biochemistry, 26: 6250-6262, 1987. 12. Wilding. G., Valverius, E.. Knabbe. C.. and Gelmann, E. P. Role of trans Licdtke. R. J.. and Batjer, J. D. Measurement of prostate-specific antigen by forming growth factor-Â»in human prostate cancer cell growth. Prostate, IS: radioimmunoassay. Clin. Chem., 30: 649-652, 1984. 1-12, 1989. Baker. A. R., McDonnell. D. P., Hughes. M., Crisp, T. M., Mangelsdorf, D. 13. Veldscholte, J.. Voorhorst-Ogink, M. M., Bolt-de Vries. J., van Rooij. H. C. J., Haussler, M. R., Pike, J. W., Shine, J., and O'Malley, B. W. Cloning and J., Trapman, J., and Mulder. E. Unusual specificity of the androgen receptor expression of full-length cDNA encoding human vitamin D receptor. Proc. in the human prostate tumor cell line LNCaP: high affinity for progcstagenic Nati. Acad. Sci. USA, Â«5:3294-3298, 1988. and estrogenic steroids. Biochim. Biophys. Acta, 1052: 187-194, 1990. 34. Chomczynski, P., and Sacchi, N. Single-step method of RNA isolation by 14. Sonnenschein, C., Olea, N., Pasanen, M. E., and Soto, A. M. Negative acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem., 162: 156-159. 1987. controls of cell proliferation. Human prostate cancer cells and androgens. Cancer Res., 49: 3474-3481. 1989. 35. Sambrook, J., Fritsch, E. F., and Maniatis. T. Hybridization of radiolabeled 15. Wilding. G.. Chen. M.. and Gelmann, E. P. Aberrant response in vitro of probes to immobilized nucleic acids. In: J. Sambrook, E. F. Fritsch, and T. hormone-responsive prostate cancer cells to anti-androgens. Prostate. 14: Maniatis (eds.). Molecular Cloning. A Laboratory Manual, Ed. 2, pp. 9.47- 103-115. 1989. 9.55. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1989. 16. Veldscholte, J.. Ris-Stalpers. C., Kuiper, G. G. J. M., Jenster. G., Berrevoets, 36. Thomas, P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. Nati. Acad. Sci. USA, 77: 5201-5205, C., Claassen, E., van Rooij, H. C. J., Trapman. J.. Brinkmann. A. O., and Mulder. E. A mutation in the ligand binding domain of the androgen receptor 1980. 37. Cho, K. W. Y.. Khalili, K.. Zandomeni. R., and Weinmann. R. The gene of human LNCaP cells affects steroid binding characteristics and response to anti-androgens. Biochem. Biophys. Res. Commun.. 173: 534-540. 1990. encoding the large subunit of human RNA polymcrase II. J. Biol. Chem., 260: 15204-15210. 1985. 17. Evans. R. M. The steroid and thyroid hormone superfamily. Science (Wash ington DC). 240: 889-895. 1988. 38. Malloy. P. J., Hochberg, Z., Pike, J. W., and Feldman, D. Abnormal binding of vitamin D receptors to deoxyribonucleic acid in a kindred with vitamin 18. Haussier, M. R. Vitamin D receptors: nature and function. Annu. Rev. Nutr., D-dependent rickets, type II. J. Clin. Endocrinol. Metab., 68:263-269, 1989. 6:527-562, 1986. 39. Boring. C. C., Squires, T. S., and Tong. T. Cancer statistics, 1991. CA Cancer 19. Minghetti, P. P., and Norman, A. W. l,25(OH)2-Vitamin D, receptors: gene J. Clin., 41: 19-36, 1991. regulation and genetic circuitry. FASEB J., 2: 3043-3053. 1988. 40. Schmidt, J. D., Mettlin, C. J., Natarajan, N., Peace, B. B., Beart, R. W., Jr., 20. DeLuca. H. F.. and Ostrem, V. The relationship between the vitamin D Winchester, D. P., and Murphy, G. P. Trends in patterns of care for prostatic system and cancer. Adv. Exp. Med. Biol.. 206: 413-429, 1986. cancer, 1974-1983. Results of surveys by the American College of Surgeons. 21. Manolagas. S. C. Vitamin D and its relevance to cancer. Anticancer Res.. 7: J. Urol.. 136:416-421, 1986. 625-638. 1987. 41. Venner, P. M. Therapeutic options in treatment of advanced carcinoma of 22. Frampton, R. J.. Omond. S. A., and Eisman, J. A. Inhibition of human the prostate. Semin. Oncol., 17: 73-77. 1990. cancer cell growth by 1,25-dihydroxyvitamin D3 metabolites. Cancer Res., 42. Flanders, W. D. Review: prostate cancer epidemiology. Prostate, 5:621-629, Â«.â€¢4443-4447,1983. 1984. 23. Colston, K. W., Berger. U., and Coombes, R. C. Possible role for vitamin D 43. Schwartz, G. G., and Hulka, B. S. Is vitamin D deficiency a risk factor for in controlling breast cancer cell proliferation. Lancet, /: 188-191, 1989. prostate cancer (hypothesis). Anticancer Res., 10: 1307-1312, 1990. 24. Mangelsdorf, D. J.. Koeffler. H. P., Donaldson, C. A., Pike, J. W., and 44. Abe, J.. Morikawa, M., Miyamoto, K.. Kaiho. S-i., Fukushima, M., Miyaura, Haussler, M. R. 1,25-Dihydroxyvitamin D, induced differentiation in a C., Abe. E., Suda, T., and Nishii, Y. Synthetic analogues of vitamin D3 with human promyelocytic leukemia cell line (HL-60): receptor-mediated matu an oxygen atom in the side chain skeleton. FEBS Lett., 226: 58-62, 1987. ration to macrophage-like cells. J. Cell Biol., 9Â«:391-398. 1984. 45. Dauben, W. G., Kohler, B., and Roesle, A. Synthesis of 6-fluorovitamin D3. 25. Miller, G. J.. Slapleton, G. E., Houmiel. K. L, and Ferrara, J. A. Specific J. Org. Chem.. 50: 2007-2010. 1985.

520

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1992 American Association for Cancer Research. The Human Prostatic Carcinoma Cell Line LNCaP Expresses Biologically Active, Specific Receptors for 1 α ,25-Dihydroxyvitamin D3

Gary J. Miller, Gary E. Stapleton, Janet A. Ferrara, et al.

Cancer Res 1992;52:515-520.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/52/3/515

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/52/3/515. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.