Neurotensin Is an Autocrine Trophic Factor Stimulated by Androgen

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Proc. Nat!. Acad. Sci. USA Vol. 91, pp. 4673-4677, May 1994 Medical Sciences Neurotensin is an autocrine trophic factor stimulated by androgen withdrawal in human prostate cancer (neuroendocrine/growth factor) INDER SEHGAL*, STEPHEN POWERSt*, BRENDA HUNTLEY*, GARTH POWIs§1, MARK PITrELKOWI, AND NITA J. MAIHLE*,** Departments of *Biochemistry and Molecular Biology, tGastroenterology, §Pharmacology, and I'Dermatology, Mayo Clinic, Rochester, MN 55905 Communicated by Susan E. Leeman, January 21, 1994

ABSTRACT After therapeutic hormone deprivation, pros- docrine cells have been reported to be a constituent of most tate cancer cells often develop androgen-insensitive growth prostatic adenocarcinomas (10), with incidences of up to through mechanisms thus far undefined. Neuropeptides have 100o (9). The prevalence of neuroendocrine cells in prostate been previously implicated as growth factors in some prostate cancer has been correlated with higher-grade malignancy and cancers. Here, we demonstrate that androgen-sensitive LNCaP poor prognosis (11), and prostatic neuroendocrine tumors are human prostate cancer cells produce and secrete neurotensin typically unresponsive to hormonal therapy (9). These ob- following androgen withdrawal. We show that while LNCaP servations led us to investigate the potential for neurotensin, cells express the neurotensin receptor, only androgen-deprived a tridecapeptide, to function as an androgen-independent cells exhibit a growth response to exogenous neurotensin. We trophic factor in prostate cancer. Neurotensin is a trophic further demonstrate that androgen-stimulated cells may be factor for normal intestinal cells (12) and has been speculated refractory to exogenous neurotensin due to androgen induction to play an autocrine role in small-cell lung cancer growth (13). of a metalloprotease active toward neurotensin. Thus, prostate Although neurotensin has been detected in human prostatic cancer cells deprived of androgen develop an alternative au- cancer tissues (14), this peptide is not usually expressed in tocrine growth mechanism involving neurotensin. normal prostate (15). The potential effects of sex steroids on the production and secretion of neuroendocrine peptides, Prostate cancer is the most commonly diagnosed cancer and including neurotensin, have not been studied in human ma- the second leading cause of cancer death among men in the lignancies, and the role that neuropeptides may play in United States (1). Androgens stimulate the growth of these promoting the growth of androgen-deprived cells is un- malignancies, and hormone deprivation is the primary treat- known. As clinical data suggest a possible role for small ment for advanced prostate cancer. While androgen with- peptides in prostate cancer growth, investigation of the drawal initially reduces the growth of metastatic prostatic regulatory events associated with peptide production is tumors, this clinical response is temporary and these cancers strongly warranted. ultimately recur. At this point, hormonal deprivation therapy To characterize the role of neurotensin in the process of fails and survival is usually <1 year (2, 3). To further the androgen withdrawal and subsequent hormone-independent understanding ofprostate tumor growth and progression, the cellular proliferation in human prostate tumors, the human growth-regulatory pathways associated with the develop- prostate cell line LNCaP (16) was used. ment of androgen-independent tumor cell -growth must be defined. MATERIALS AND METHODS Androgens promote the growth of prostate cancer cells, at least in part, through growth factor-mediated autocrine and Cell Culture and Growth Assays. LNCaP cells were rou- paracrine mechanisms (4). Androgen-regulated growth fac- tinely plated,- allowed to attach for 2 days and then fed tors implicated in such growth stimulatory pathways include serum-free defined medium [phenol-red free RPMI 1640 the epidermal growth factor and fibroblast growth factor supplemented with insulin (5 ,ag/ml), holo-transferrin (10 families (5, 6). Since it has been shown that androgen p.g/ml), 30 nM sodium selenite, penicillin G (100 units/ml), withdrawal leads to reduced proliferation and programmed streptomycin sulfate (100 Hg/ml), 2 mM L-glutamine, and cell death of androgen-dependent prostate cancer cells (7), it amphotericin B (1.25 pug/ml) at pH 7.4], with or without seems plausible that androgen depletion may also inhibit cell addition of 1 nM synthetic nonmetabolizable androgen, growth by decreasing the activity of autocrine/paracrine R1881 (17) (NEN/DuPont). Cells were routinely verified free growth factor loops. The evolution of androgen-insensitive of mycoplasma. For growth assays, cell were plated at low prostate cancer may, therefore, involve the selection for, or density (500 cells per cm2) in T-25 flasks, supplemented with development of, alternative growth-regulatory pathways. In 0.5, 1.0, 2.0, 5.0, 10.0, or 25 nM neurotensin in the presence this study, we have considered the potential role of neuro- or absence of androgen, and were grown with medium endocrine peptides as potential trophic factors in the devel- changes every 2 days (to minimize the accumulation of opment of such pathways. secreted growth factors) for 12 days. Cell number determi- One member ofthe family ofneuropeptides, bombesin, has nations were made with a Coulter Counter. been shown to exhibit autocrine growth activity in human Radloimmunoassays (RIAs). LNCaP cells, plated in 100- small-cell lung cancer (8). More recently, a variety of neu- mm Petri dishes (Coming) at an initial density 5 x 103 cells ropeptides have been shown to be expressed in prostatic carcinomas; these peptides are secreted by focal areas of tPresent address: Immunologic Pharmaceutical Corp., 610 Lincoln neuroendocrine differentiated cells (9). Clusters of neuroen- Street, Waltham, MA 02154. Present address: Arizona Cancer Center, University of Arizona, 1501 North Cambell Avenue, Tucson, AZ 85724. The publication costs ofthis article were defrayed in part by page charge **To whom reprint requests should be addressed at: Department of payment. This article must therefore be hereby marked "advertisement" Biochemistry and Molecular Biology, Guggenheim 14, Mayo in accordance with 18 U.S.C. §1734 solely to indicate this fact. Clinic, Rochester, MN 55905.

4673 Downloaded by guest on October 1, 2021 4674 Medical Sciences: Sehgal et al. Proc. NatL Acad. Sci. USA 91 (1994) per cm2, were grown for various times, and RIA for neuro- their potential to inhibit the 24-hr degradation of neurotensin tensin was performed as described (18) on conditioned me- (1 ng/ml). Values are the mean ± SE of four replicates. dium and acid-extracted cell lysate. Neurotensin fragments Northern RNA Analysis. Samples of poly(A)+ RNA (6 pg) are biologically active in some neural assays; these include collected from LNCaP, DU-145, and PC-3 prostate cancer amino-terminal fragments containing the peptide's midpor- cells and NCI-HI345 small-cell lung carcinoma [positive con- tion (19, 20) and carboxyl-terminal fragments consisting of trol for neurotensin receptor (15)] were denatured and elec- amino acids 8-13 and 9-13 (21, 22). Therefore, immunoas- trophoresed in a formaldehyde/0.8% agarose gel, and the says were designed to identify whole neurotensin or poten- separated RNAs were transferred to a Zeta-Probe GT mem- tially active fragments: results were derived by using an brane (Bio-Rad) by capillary transfer (23, 24). Membranes antibody which recognized the intact midportion (data not were baked at 80'C for 2 hr, prehybridized, hybridized, and shown) and were verified by using a second antibody recog- washed as per the Zeta-Probe manual. By using degenerate nizing the intact carboxyl terminus (Affinity Research Prod- primers derived from the cloned rat neurotensin receptor ucts, Nottingham, U.K.). Data, therefore, reflect quantities (25), a 300-bp fragment of the human neurotensin receptor of either whole neurotensin or potentially active fragments. was generated by PCR of human substantia nigra cDNA The LNCaP cell-associated neurotensin immunoreactivity (construct generously provided by E. Richelson, Mayo detected by RIA was confirmed as neurotensin by HPLC Clinic, Jacksonville, FL) and was subcloned into pBluescript profiles which demonstrated coelution of the immunoreac- SK(+) (Stratagene). An antisense RNA probe for neuroten- tive material from androgen-deprived LNCaP cells with sin receptor was generated by in vitro transcription of this neurotensin-(1-13) standard. Neuromedin N, a neurotensin- plasmid subclone with phage T7 polymerase and [a-32P]UTP like peptide that is cotranscribed with neurotensin, was not (800 Ci/mmol; 1 Ci = 37 GBq). Final radionucleotide con- significantly recognized by either antibody (data not shown). centration in the hybridization solution was >1 x 107 cpm/ The limit ofdetection for whole neurotensin was 20 pg/ml for ml. Molecular sizes were determined by comparison with an both antibodies. ethidium bromide-stained RNA marker (0.24- to 9.5-kb RNA Cell Lysate Preparation. LNCaP cells from sample plates ladder; GIBCO/BRL). After hybridization, blots were were removed with acell lifter (Costar) in phosphate-buffered stripped with 15 mM NaCl/1.5 mM trisodium citrate, pH saline, washed once with phosphate-buffered saline, and 7/0.1% SDS at 900C and hybridized with a probe for glycer- pelleted by centrifugation. Peptides were extracted by the aldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. A addition of 0.5 ml of 0.1 M HCl to each pellet (18), pellets 780 bp fragment ofthe cDNA sequence encoding human fetal were heated at 1000C for 10 min and recentrifuged, and the liver GAPDH was liberated from clone pHcGAP (obtained supernatant was assayed by RIA. Levels of neurotensin from the American Type Culture Collection) by double associated with cell lysates could include newly synthesized, digestion with Pst I/Xba I, and the insert was labeled with unsecreted peptide as well as receptor-bound and receptpr- [a-32P]dATP (>3000 Ci/mmol) by random priming (random internalized neurotensin. Therefore, to determine the con- primers DNA labeling system; Bethesda Research Labora- tributions that receptor-bound and/or internalized neuroten- tories). Final radionucleotide concentration was 8 x 106 sin may make to these lysates, triplicate 100-mm dishes of cpm/ml of hybridization solution. LNCaP cells were incubated for 24 hr either at 3rC or at 40C (to prevent growth factor internalization) with a medium RESULTS change containing neurotensin (1 ng/ml). Control plates were incubated without exogenous neurotensin. Cells were col- Detection ofSeced and Intracelular NeurotenuinilnLNCaP lected, lysed with 0.1 M HC1, and immunoassayed. No Cell. RIA ofserum-free medium-conditioned by LNCaP cells significant change in the level of cell-associated neurotensin in either the presence or the absence of the androgen analog was found in androgen-deprived cells either with or without R1881 (17) demonstrated that neurotensin was detectable and exogenously added neurotensin at 37°C or 4°C (see Table 1), steadily accumulated in medium from cells conditioned with- demonstrating that receptor-bound neurotensin does not out androgen over a 12-day period, reaching 400 pg per 106 significantly increase the acid-extracted cell levels. Under cells (Fig. 1A). In contrast, neurotensin was not detectable in these same conditions, no neurotensin was detectable in the conditioned medium when LNCaP cells were grown over androgen-stimulated cells (data not shown). We therefore the same time course in the presence ofandrogen (Fig. 1A). In conclude that cell-associated levels detected by this method fact, androgen addition to steroid-deprived cells induced a reflect predominantly newly synthesized, intracellular neu- marked and rapid decline of neurotensin immunoreactivity in rotensin. the conditioned medium within 48 hr. Characterization of a Neurotensin-Degrading Activity Asso- To determine whether neurotensin levels in conditioned ciated with LNCaP Cells. LNCaP cells were grown to con- medium were related to cell-associated neurotensin levels, fluence in T-75 flasks with or without R1881, and the flasks RIAs were performed on cell lysates (Fig. 1B). In these were incubated at 37°C with 10 ml of serum-free medium lysates, the quantity of endogenous neurotensin was unef- containing neurotensin (1 ng/ml). Controls were performed fected by exogenous neurotensin added to the whole cells with flasks in the absence of LNCaP cells and were used to under conditions permissive for ligand binding and internal- assay exact amounts of added peptide. After 24 hr, samples ization (37°C), or for ligand binding alone (4C) (Table 1). were frozen immediately (-70°C). For assays done with Thus, cell surface-bound or internalized neurotensin did not cell-free conditioned medium, growth medium removed from contribute to the peptide levels assayed in cell lysates. The LNCaP cells was clarified by centrifugation at 15,000 x g and lysate levels, therefore, represented unsecreted, intracellular supplemented with neurotensin (1 ng/ml) and then incubated neurotensin. Intracellular neurotensin accumulated in pros- at 37C for 24 hr. The amount ofimmunoreactive neurotensin tate cells deprived of androgens, whereas androgen-stim- remaining after exposure to cells or cell-free medium was ulated cells demonstrated no neurotensin (Fig. 1B); thus, calculated as a fraction of the amount initially added. neurotensin secretion patterns were reflected by intracellular Secreted neurotensin-degrading activity in conditioned quantities. To determine whether neurotensin might play a medium was further characterized through inhibitors ofcom- more generalized role in other hormone-responsive malig- mon protease types. Serine and thiol protease inhibitors nancies, the estrogen-responsive breast carcinoma cell line (phenylmethylsulfonyl fluoride, 5 mM; leupeptin, 10 pg/ml; MCF-7 (26) was grown with or without addition of 1 nM aprotinin, 10 ug/nml) and metalloprotease inhibitors (EDTA, 17p-estradiol for 3 and 6 days in serum-free defined medium. 1-5 mM; 1,10-phenanthroline, 1-5 mM) were assayed for Aliquots of media and cell lysates were analyzed by RIA for Downloaded by guest on October 1, 2021 Medical Sciences: Sehgal et al. Proc. Natl. Acad. Sci. USA 91 (1994) 4675 gen. Addition of neurotensin to androgen-depleted LNCaP cells stimulated growth in a dose-responsive manner up to 10 nM neurotensin (Fig. 2). Androgen-treated LNCaP cells showed no significant response to added neurotensin. Im- munoassays of growth media verified neurotensin presence H .--. throughout the growth period in both androgen-treated and androgen-deprived cell cultures (data not shown). Because androgen-treated cells can be further stimulated by other 2 H growth factors such as epidermal growth factor and trans- forming growth factor a (data not shown and ref. 27), it is unlikely that these androgen-treated cells are maximally growth-stimulated. Therefore, the absence of a growth re-

. ,-. . sponse to neurotensin in androgen-treated cells suggests that - .\-'- kJ - - neurotensin selectively promotes the growth of androgen- ii I N C' deprived cells. S Proteolytic Regulation ofNeurotensin Expression by Andro- B gen-Treated LNCaP Cells. Neurotensin expression may be -1 controlled at several levels; however, it appears to be regu- (.) lated posttranscriptionally in the intestine (28), and neurotensin is a substrate for several proteases (endopeptidases) in other malignant tissues (29-31). Addition of R1881 to androgen-depleted LNCaP cells led to the rapid loss of secreted neurotensin (Fig. 1A), suggesting that androgens might in- duce the proteolytic degradation ofneurotensin in these cells. Androgens are also known to regulate the expression of the serine protease kallikreins PSA and hGK-1 in prostate (32, ):i. rcIi jru\\ 33), but androgen-induced proteases capable of degrading FIG. 1. (A) Neurotensin secretion in LNCaP cell conditioned neurotensin were not reported. In the present study, a media. Conditioned medium was collected from androgen-depleted substantial decrease in exogenously added neurotensin was (o) and from androgen-stimulated (o) LNCaP cells at the indicated detected in androgen-treated cells compared with androgen- times and analyzed by RIA. Complete medium changes and cell deprived cells (Table 2). No exogenously added neurotensin counts were made at each time point. At 8 days, some plates were was detectable after 24 hr ofexposure to androgen-stimulated restimulated with the androgen analog R1881 (n). Plotted points cells, but 38.4% of the initial peptide remained after incuba- show mean neurotensin immunoreactivity accumulation (pg per 106 tion with androgen-deprived cells. This differential loss of cells) from the previous time point and error bars show SE of three exogenous neurotensin suggested that an androgen-inducible experiments. Asterisk indicates a neurotensin level below the limit of detection (<20 pg/ml of medium). (B) Neurotensin RIA of 0.1 M protease activity was present. However, neurotensin in con- HC-extracted LNCaP cells. Neurotensin was assayed in cell lysates ditioned medium of androgen-stimulated cells could also be to determine intracellular peptide levels from androgen-deprived decreased by metabolism; conversely, neurotensin levels in (hatched bars) and androgen-stimulated (filled bars) cells. Neuro- medium of androgen-deprived cells could be modulated by tensin levels were below the limit of detection (asterisk) for all secretion of endogenous neurotensin. Therefore, further ex- androgen-supplemented cells. Positive results indicate that the ma- periments on androgen-induced neurotensin degradation jority of plates at a given time point contained neurotensin levels were performed with cell-free conditioned medium. Results above the limit of detection. Three to six plates were used for each again showed lower levels of immunoreactive peptide re- time point, and error bars represent SD of the mean. neurotensin. No neurotensin was detected either in conditioned media or in cell lysates at either time point (data not mm .: 1., 1! ... shown). -, Neurotensin Mitogenic Activity Toward LNCaP Cells. Be- cause androgen withdrawal may lead to the loss of some -r-T types of growth-regulatory pathways and signals, the up- regulation of a potentially mitogenic neuropeptide is intrigu- ing. To test neurotensin's ability to act as a mitogen and to determine whether this peptide could counter the effects of androgen withdrawal, LNCaP cells were supplemented with exogenous neurotensin in the presence or absence of andro- Table 1. Effect of exogenous neurotensin on cell lysate- associated peptide levels in androgen-deprived LNCaP cells Exogenous Incubation neurotensin temperature, Neurotensin, (1 ng/ml) OC pg per 106 cells

i, !i I - ; I - 37 5.0 ± 0.70 , , _.._ I.;I i Ii! + 37 4.7 ± 0.28 - 4 4.2 ± 0.84 FIG. 2. Neurotensin growth stimulation of LNCaP cells. Neuro- + 4 4.5 ± 0.48 tensin was added from fresh stock to androgen-deprived (hatched Prostate cancer cells grown without androgen were incubated for bars) and androgen-stimulated (filled bars) cells at the levels shown 24 hr at 37rC or 4C with or without neurotensin (1 ng/ml). Cells were for 12 days. Medium was changed every other day. The values lysed with 0.1 M HCl and immunoassayed for neurotensin. represent mean ± SE of four flasks. Downloaded by guest on October 1, 2021 4676 Medical Sciences: Sehgal et al. Proc. Natl. Acad Sci. USA 91 (1994) Table 2. Effect of protease inhibitors on putative androgen- A .4 regulated degradation of neurotensin Neurotensin remaining after Androgen Cells Inhibitor(s) 24 hr, % + + - Undetectable - + - 38.4 ± 2.0 B : ; + - - 26.0 ± 6.9 - - - 90.4 ± 12.0 + - Aprotinin 18.4 ± 2.4 + - Leupeptin 22.9 ± 1.8 + - PMSF 28.5 ± 4.0 + - Aprotinin, leupeptin, 21.7 ± 3.2 and PMSF + - 1 mM EDTA 27.5 ± 4.9 FIG. 3. Human neurotensin receptor mRNA expression in LNCaP + - 5 mM EDTA 99.4 ± 3.3 cells. LNCaP cells were grown for either 6 days with androgen or for + - 1 mM 1,10-phenanthroline 44.0 ± 6.1 3, 6, or 9 days in the absence of androgen. Lanes: 1, NCI-H345 + - 5 mM 1,10-phenanthroline 61.0 ± 2.0 small-cell lung carcinoma; 2, LNCaP cells plus androgen; 3-5, LNCaP for 24 hr in conditioned cells without androgen for 3, 6, and 9 days; 6 and 7, DU-145 and PC-3 Neurotensin (1 ng/ml) was incubated prostate cancer cells. Probes were specific for or without (-) androgen, after androgen-independent media ofLNCaP cells treated with (+) receptor or glyceraldehyde-3-phosphate dehydroge- were by RIA. Experiments were neurotensin (A) which peptide levels quantitated nase (B) mRNA. performed in flasks containing cells or in flasks without cells but conditioned media. Results are expressed as a containing previously a as well as autocrine stimulation of percent of initial peptide level added. Inhibitors used to define the play role in the paracrine presence ofprotease type(s) involved in the loss ofneurotensin were prostate cancer. added to cell-free media, and the percent neurotensin remaining in the presence of these inhibitors is shown. PMSF, phenylmethylsul- DISCUSSION fonyl fluoride; EDTA, ethylenediaminetetraacetic acid. These data indicate that the androgen-sensitive prostate can- maining in medium from androgen-treated cells (26% remain- cer cell line LNCaP produces and secretes neurotensin during ing) compared with androgen-deprived cells (>90%o remain- androgen deprivation as a result of both increased neuropep- ing). Endogenous neurotensin levels from androgen-deprived tide synthesis/secretion and decreased neuropeptide-directed cells were calculated into the initial peptide level for these proteolytic activity. The absence of detectable neurotensin in cells and constituted <10%o of the total neurotensin. Serine androgen-stimulated cell lysates implies that androgens pri- and thiol protease inhibitors did not alter the putative andro- marily negatively regulate neurotensin at the cellular level. gen-enhanced neurotensin proteolytic activity; however, Through this mechanism, androgen-deprivation of hormone- metalloprotease inhibitors (EDTA and 1,10-phenanthroline) responsive prostate tumor cells leads to the development of a did significantly decrease the androgen-associated peptide positive growth-regulatory loop involving neurotensin. An- degradation and completely inhibited the basal degradative drogen induces a greater proliferative response than does activity observed in conditioned medium from hormone- neurotensin, demonstrating that neurotensin will not com- deprived cells. These results implicate a metalloprotease, the growth-signaling affects of androgen. extra- pletely supplant enhanced by androgen addition, in the degradation of a G-protein- in cells. The rapid However, this autocrine pathway, involving cellular neurotensin androgen-stimulated may partial resistance to the induction of this protease would also explain the loss of linked neuropeptide, impart neurotensin from previously hormone-depleted cells (Fig. growth-inhibitory effects ofhormone deprivation as androgen- 1A). sensitive cells lose the magnitude of autocrine growth- Neurotensin Receptor Expression in LNCaP Cells. In addi- promoting pathways. Androgen-treated cells have the poten- tion to the negative androgenic regulation of intra- and tial for enhanced growth in response to other mitogens (27), extracellular neuropeptide levels, modulation of autocrine and, under conditions used in our growth studies (i.e., low- growth pathways in prostate cancer may also occur at the density growth and frequent medium changes), neurotensin receptor level (5). To determine whether androgens govern peptide assays demonstrated that the peptide was available to expression of the neurotensin receptor, the level of the hormone-stimulated cells (data not shown). These androgen- transcript encoding this receptor was examined with probes treated cultures consistently showed no response to added derived from the human neurotensin receptor sequence (34). neurotensin peptide. Thus, our data suggest that this neuro- A single 4.4-kb neurotensin receptor mRNA was detectable tensin autocrine loop exists selectively in hormone-deprived in androgen-stimulated and androgen-deprived LNCaP cells, cells and further suggest that hormone deprivation may actu- as well as in the neurotensin-responsive human small-cell ally generate alternative growth-stimulatory pathways. Neu- lung carcinoma cell line NCI-H345 (Fig. 3). Thus, LNCaP rotensin is an attractive candidate for autocrine/paracrine cells express neurotensin receptor mRNA independent of roles in prostatic malignancy, since expression of both the androgen status, in contrast to the effects ofandrogen on the peptide and the receptor has been demonstrated in several levels of neurotensin peptide, suggesting that this autocrine other human cancers (12, 15, 35-38). Autocrine neurotensin pathway is regulated through modulation of peptide levels. pathways regulated through sex steroid withdrawal may be Foci of neurotensin-producing cells in a tumor could stim- unique to prostate malignancy, however, as synthesis of this ulate cells expressing neurotensin receptor through both peptide was undetectable in estrogen-responsive breast cancer autocrine and paracrine mechanisms. We therefore examined lines with or without 173-estradiol. the expression of neurotensin receptor in two androgen- In addition to regulation at the cellular level, steroid independent prostate cell lines, DU-145 and PC-3. mRNA for hormone treatment also leads to modulation of extracellular the neurotensin receptor was detected in both DU-145 and neurotensin and perhaps other neuropeptides. The presence PC-3 cells (Fig. 3), which do not secrete neurotensin. These or absence of steroid hormone-stimulated protease activity results suggest that neuropeptides such as neurotensin may could have significant impact on the growth characteristics of Downloaded by guest on October 1, 2021 Medical Sciences: Sehgal et al. Proc. Natl. Acad. Sci. USA 91 (1994) 4677 prostate cancer cells in vivo. We suggest that initial condi- 16. Horoszewicz, J. S., Leong, S. S., Kawinski, E., Karr, J. P., tions of androgen stimulation may, by inducing neuropeptide Rosenthal, H., Chu, T. M., Mirand, E. A. & Murphy, G. P. protease activity, select for and promote growth ofandrogen- (1983) Cancer Res. 43, 1809-1818. responsive cells that are reliant on growth factors other than 17. Berns, E. M. J. J., de Boer, W. & Mulder, E. (1986) Prostate neuropeptides. Subsequent androgen deprivation would en- 9, 247-259. 18. Yaksh, T. L., Michener, S. R., Bailey, J. E., Harty, G. J., hance the bioavailability of neuropeptides and could lead to Lucus, D. L., Nelson, D. K., Roddy, D. R. & Go, V. L. W. populations of cells that are more responsive to these small (1988) Peptides 9, 357-372. peptide growth factors. 19. Hernandez, D. E., Simons, K. B., Spampinato, D., Rioux, F. The synthesis of neurotensin or other neuropeptides by & St-Pierre, S. (1985) Neuropeptides 6, 561-568. androgen-sensitive prostate cancer cells undergoing hor- 20. Hernandez, D. E., Richardson, C. M., Nemeroff, C. B., Or- mone withdrawal requires additional examination. Our stud- lando, R. 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