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[Cancer Research 59, 3821-3830, August 1, 1999]
© 1999 American Association for Cancer Research
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[Cancer Research 59, 3821-3830, August 1, 1999]
© 1999 American Association for Cancer Research

Tumor Biology

Acquisition of Neuroendocrine Characteristics by Prostate Tumor Cells Is Reversible

Implications for Prostate Cancer Progression1

Michael E. Cox, Paul D. Deeble, Saquib Lakhani and Sarah J. Parsons2

Cancer Center and Department of Microbiology, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neuroendocrine (NE) cells occur as scattered foci within prostatic adenocarcinoma, similar to their distribution within ductal epithelial cells of the normal prostate. However, the density of NE cells is often greater in prostate carcinomas than in normal tissue, and the frequency of NE cells correlates with tumor grade, loss of androgen sensitivity, autocrine/paracrine activity, and poor prognosis. Although NE cells are nonmitotic, proliferating cells are found in direct proximity to them, suggesting that NE cells provide paracrine stimuli for surrounding carcinoma cells. In vitro, differentiation of the LNCaP and PC3M prostatic tumor cell lines to a NE phenotype can be induced by dibutyryl cyclic AMP (cAMP), suggesting that physiological agents that increase intracellular concentrations of cAMP might regulate NE differentiation in vivo. Indeed, we demonstrate in this report that LNCaP cells acquire NE characteristics in response to treatment with physiological and pharmacological agents that elevate intracellular cAMP, agents such as epinephrine, isoproterenol, forskolin, and dibutyryl cAMP. The androgen-independent LNCaP-derived cell line C4-2 also responded to these agents, indicating that cells representing later stages of tumor progression are also capable of differentiation. The NE phenotype in this study was monitored by the appearance of dense core granules in the cytoplasm, the extension of neuron-like processes, loss of mitogenic activity, and expression of the NE markers neuron-specific enolase, parathyroid hormone-related peptide, neurotensin, serotonin, and chromogranin A. However, contrary to previous reports, we observed rapid loss of the NE phenotype in both LNCaP and C4-2 cells upon withdrawal of inducing agents. Withdrawal also resulted in a rapid, dramatic increase in tyrosine kinase and mitogen-activated protein kinase activities, suggesting that activation of these intracellular signaling enzymes may be important for reentry into the cell cycle. Together, these results indicate that chronic cAMP-mediated signaling is required to block proliferation of prostate tumor cells and to induce NE differentiation.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The need for novel treatments for advanced prostate cancer has prompted the study of unique features of prostate physiology with the goal of uncovering potentially new therapeutic targets. One such potential target is the NE3 cell of the prostate. In addition to basal and exocrine cells, the prostate gland possesses a third population of highly specialized epithelial cells known variably as endocrine-paracrine cells, amine precursor uptake and decarboxylation cells, or NE cells (1) . NE cells comprise a minor fraction of the total epithelial population, but they are distributed throughout the prostate as morphologically heterogeneous cells with irregular neurite-like processes extending between epithelial cells and often into the lumen. NE cells are identified by the presence of neurosecretory granules and by their ability to express a wide variety of neuronal markers, such as chromogranins (2) , NSE, and a number of potentially mitogenic hormones, including PTHrP, NT, serotonin, Bomb, calcitonin, and thyroid-stimulating hormone (3, 4, 5, 6) .

Although little is known regarding their functional role, NE cells are present in the developing prostate, where, by analogy with respiratory, gastrointestinal, and pancreatic systems, they are thought to play a paracrine role during tissue growth and differentiation. NE cells persist in the fully developed prostate, where it is conjectured that they participate in the regulation of secretory processes of the mature gland. However, unlike NE cells of the adrenal and pituitary glands, which are derived from the neural crest, prostatic NE cells appear to arise from an epithelial stem cell within the prostate (7 , 8) . This conclusion is based on the findings that some NE cells, like other prostate epithelial cells, have been shown to express prostate-specific antigen (9 , 10) , basal cell-specific cytokeratins (10) , and androgen receptor (11 , 12) .

NE cells are also found in primary prostatic malignancies and in metastatic adenocarcinomas (13) and have been suggested to arise as part of the malignancy, rather than having been trapped by the tumor (14) . Cancers composed purely of malignant NE cells (small cell carcinomas) and NE-containing carcinoid tumors are rare and highly aggressive diseases (15 , 16) . Prostatic adenocarcinomas that contain foci of apparently nonproliferating NE cells are more typically seen (1) . Although there are reports both supporting (17, 18, 19, 20) and refuting (21 , 22) the prognostic value of the extent of NE differentiation in conventional prostatic adenocarcinoma, strong links between NE status and long-term, disease-specific survival have been suggested (23) .

Tumor cell populations have been reported to become enriched for NE cells after long-term antiandrogen therapy (2) , and although the NE cells appear to be nonmitotic in these tissue samples, proliferating carcinoma cells have been found in close proximity to them (24 , 25) . These observations suggest that NE cells provide paracrine stimuli for proliferation of the surrounding carcinoma cells and that NE differentiation is associated with progression of prostate cancer toward an androgen-independent state, a condition for which there is currently no successful therapy. Therefore, determining what factors regulate NE differentiation from epithelially derived prostatic tumor cells, assessing the potential paracrine activity of NE cells, and elucidating the significance (if any) of NE cells in the development of androgen independence are important areas of investigation. Knowledge of the mechanisms underlying the development and function of prostatic NE cells will provide the information needed to determine whether NE cells are potential targets for novel therapeutic approaches.

The choice of the prostate tumor cell lines used for our studies was based on a previous report that showed that LNCaP cells acquire a NE phenotype in response to treatment with dbcAMP and the phosphodiesterase inhibitor IBMX (26) . LNCaP is an androgen-responsive cell line originating from a lymph node metastasis of a prostate cancer patient (27) . C4-2 is an androgen-independent cell line that was derived from a tumor formed in nude mice after coinjection of LNCaP cells with bone fibroblasts (28) . In our studies, we found that both LNCaP and C4-2 cells acquire NE characteristics not only in response to treatment with pharmacological agents, such as dbcAMP/IBMX and Fsk/IBMX, but also in response to the physiologically relevant ß-adrenergic receptor agonists Epi and Isop. However, in contrast to the results reported previously, we found that the acquisition of these NE characteristics is fully reversible. These findings suggest that the phenotype of cells within tumors (proliferating carcinoid cells or postmitotic NE cells) is dynamic and will be determined in part by the balance of differentiative and mitogenic factors in the local environment. Prolonged exposure of susceptible tumor cells to differentiating agents is proposed to increase the probability of conversion to a NE cell, whereas immersion in paracrine factors is postulated to promote growth and progression to metastatic, androgen-independent disease.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Treatments.
LNCaP and C4-2 cell lines were obtained from Dr. L. W. Chung (University of Virginia, Charlottesville, VA) and maintained in T-Media with 5% FBS (Life Technologies, Inc., Gaithersburg, MD) at 37°C in a humidified, 5% CO2 environment (28) . Fsk, Epi, Isop, Bomb, IBMX, dbcAMP, and dHT were purchased from Sigma (St. Louis, MO).

cAMP Assays.
Assays for intracellular cAMP levels were performed as described previously (29) by the acetylated radioimmunoassay developed by Harper and Brooker (30) . Assays were carried out by the University of Virginia Diabetes Core Laboratory. Cells were cultured in serum-free, phenol red-free RPMI 1640 for 20–24 h before stimulation with the indicated agents at 37°C in 5% CO2 for the times noted. Reactions were terminated by aspirating the media and extracting the cells with 1 ml of 0.1 N HCl for 1 h at room temperature.

ELISAs.
Conditioned culture media from LNCaP, C4-2, PC3, and PC3M prostate cancer cell lines and C3H10t1/2 murine fibroblasts were prepared by plating 2 x 105 cells/well in 24-well culture dishes. Cells were allowed to adhere overnight and treated with the appropriate agents as indicated in 0.5 ml of culture media for 72 h. Media from the cells were collected, cleared by centrifugation (14,000 x g, 10 min) and stored at -70°C before analysis.

For detection of ChrA, conditioned culture media from LNCaP, C4-2, PC3, and C3H10t1/2 cells were incubated in Immulon 4HBX flat-bottomed microtiter plates (Dynatech Laboratories, Chantilly, VA) overnight at 4°C to allow adherence of neuropeptides to the wells. Unoccupied protein binding sites were blocked by incubation with 200 µl of 5% goat serum (Life Technologies, Inc., Grand Island, NY) and 0.2% BSA in TBST [50 mM Tris-HCl, 150 mM NaCl, and 0.5% Tween 20 (pH 7.4)] for 1.5 h. This and all subsequent steps were performed at room temperature. Each well was then incubated with 200 µl of rabbit anti-ChrA (DAKO, Carpinteria, CA) diluted 1:500 in blocking solution for 2 h, washed three times (5 min each) with TBST, incubated with 200 µl of goat antirabbit horseradish peroxidase (Amersham Life Sciences, Arlington Heights, IL) diluted 1:1000 in blocking buffer for 2 h, and washed three times (5 min each) in TBST. For immunodetection, 200 µl of Sigma Fast o-Phenylenediamine dihydrochloride peroxidase substrate (0.4 mg/ml) were added to each well for 30 min, and plates were read at 450 nm in a spectrophotometer plate reader (Molecular Devices, Sunnyvale, CA). Using a ChrA (286–301) peptide (Peninsula Laboratories, Inc., Belmont, CA) as a standard for the assay, ChrA concentrations were determined for each experimental condition, as indicated.

Detection of the neurosecretory peptides PTHrP and NT was performed using EIAs for the respective peptides (Peninsula Laboratories, Inc.) as recommended by the manufacturer. All treatments were assessed in three to four independent experiments, each of which was performed in duplicate.

Transmission Electron Microscopy.
Cells were prepared for transmission electron microscopy following the indicated treatments by fixation in 4.0% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) warmed to 37°C and then held overnight at 4°C. Fixed cells were scraped from the flasks and transferred to 1.5-ml microcentrifuge tubes. The cell pellets were post-fixed for 1 h in 2% osmium tetroxide, dehydrated in acetone, and embedded in epoxy resin. After polymerization, ultrathin sections (70–80-nm thick) were prepared and stained with uranyl acetate (50% in acetone) and lead citrate (31) and examined using a JEOL 100-CX transmission electron microscope.

Immunocytochemistry.
For immunocytochemical analysis, LNCaP and C4-2 cells were plated onto glass coverslips and treated as described. Cells were fixed in 4% paraformaldehyde and 4% sucrose in PBS [50 mM sodium phosphate and 150 mM NaCl (pH 7.4)] at room temperature for 10 min, permeabilized with 0.1% Triton X-100 in PBS for 5 min, and washed three times (10 min each) with PBS. Fixed and permeabilized cells were then blocked in 20% heat-inactivated goat serum in PBS for 1 h at 37°C before primary antibody incubation at 37°C for 1 h. Anti-serotonin antibody (ICN Pharmaceuticals, Inc., Costa Mesa, CA) was used at a 1:50 dilution, and control serum from an unimmunized rabbit was used at a 1:20 dilution, all in PBS containing 5% heat-inactivated goat serum. Cells were then washed three times (10 min each) with PBS, incubated at 37°C for 1 h with Texas Red-conjugated goat antirabbit IgG (Jackson Immunoresearch Laboratories, West Grove, PA) at 1 µg/ml in PBS containing 5% heat-inactivated goat serum, washed, mounted on slides, and imaged by photomicroscopy using phase-contrast and immunofluorescence optics (Leica, Rijswijk, the Netherlands).

Cell Counts and [3H]Thymidine Labeling.
Cell growth was assessed by total cell counts and by [3H]thymidine incorporation. LNCaP and C4-2 cells were plated at 2 x 105 cells/well in 6-well culture dishes, allowed to adhere overnight, and treated with the appropriate agents, as indicated. Cells were refed media containing the respective treatments every third day. Cells were harvested at the indicated times by trypsinization; the contents of each well were counted using a hemocytometer, and the results are expressed as the mean cell number/well ± SE. [3H]Thymidine labeling was performed as described previously (32) , using LNCaP and C4-2 cells plated and treated as described above by adding 10 µCi of [3H]thymidine (20 Ci/mmol; DuPont New England Nuclear, Boston, MA) to the wells for 20 h before harvesting the cells for mitotic activity analysis. After trypsinization and counting, the cells were pelleted by centrifugation (500 x g, 5 min), resuspended in 1.0 ml of 10% cold trichloroacetic acid, and incubated on ice for 20 min. The resulting precipitate was pelleted (10,000 x g, 10 min) and solubilized in 0.2 ml of 0.4 N sodium hydroxide, and the acid-insoluble radioactivity was measured by liquid scintillation counting. The mitotic index was calculated as the mean acid-insoluble 3H cpm/cell ± SE for three independent experiments, each of which was performed in triplicate and normalized to the mitotic index of the respective untreated cultures.

Immunoblotting.
After treatment, cells were washed with PBS and lysed in HO buffer [50 mM HEPES, 100 mM NaCl, 1% NP40, 2 mM EDTA, 1 µg/ml leupeptin, 2 µg/ml aprotinin, 0.5 mM sodium vanadate, 40 mM p-nitrophenyl phosphate, and 2 µM microcystin (pH 7.2)] on ice. Lysates were clarified by centrifugation at 10,000 x g for 10 min at 4°C and processed for SDS-PAGE and electrophoretic transfer to nitrocellulose membranes (Schleicher and Schuell, Keene, NH), as described previously (33) . MAPK and NSE were immunoblotted with the protein G-purified anti-MAPK monoclonal antibody 1B3B9 (34) and anti-NSE antibody (ICN Pharmaceuticals, Inc.), respectively. Primary antibodies were visualized by the binding of horseradish peroxidase-conjugated protein A (Amersham Life Science, Inc.) and enhanced chemiluminescence (DuPont New England Nuclear). Detection of tyrosine-phosphorylated proteins in immunoblots of whole cell lysates was visualized by enhanced chemiluminescence using the horseradish peroxidase-conjugated anti-phosphotyrosine antibody RC20 (Upstate Biologicals, Inc., Lake Placid, NY). Relative band intensities were determined by densitometry using ImageQuant software.

Kinase Assays.
MAPK activity was assessed by immunocomplex kinase assay using TR10 rabbit polyclonal anti-MAPK antiserum (34) and the substrate MBP, as described previously (35) . The amount of MAPK in the immunocomplex was determined by blotting the immunoprecipitates with 1B3B9 antibody as described previously (34) .


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Physiological and Pharmacological Factors That Increase Intracellular Levels of cAMP Induce Development of a NE Morphology in LNCaP Cells.
The ability of dbcAMP/IBMX to induce NE differentiation of LNCaP cells (26) suggested that an increased accumulation of intracellular cAMP might underlie the conversion of prostatic tumor cells to NE cells during disease progression. A variety of physiological factors are known to function through cell surface receptors to induce such accumulations, with the archetypic example being the ß-adrenergic receptor complex and its agonists, Epi and Isop. Pharmacological factors, such as Fsk, which bypass the receptor and chronically activate intracellular downstream components of the ß-adrenergic pathway, are also known to induce increased levels of cAMP in a variety of cell types. To determine whether activators upstream of dbcAMP could induce NE differentiation and to verify that the morphological changes observed in response to dbcAMP/IBMX treatment were due to the effects of increased cAMP levels and not to nonspecific effects of the dibutyryl form of cAMP, LNCaP cells were stimulated with Fsk (an activator of adenylyl cyclase; Ref. 36 ) and IBMX (Fsk/IBMX) or with Epi for 3 days, and the morphology of the cells was examined by phase-contrast microscopy. Cells treated with Fsk/IBMX (Fig. 1C)Citation or Epi (Fig. 1E)Citation developed compact, rounded cell bodies and extended numerous long, fine, branched processes with defined growth cones, whereas untreated LNCaP cells exhibited a fusiform morphology, tapering into unbranched processes typically less than one cell body in length (Fig. 1A)Citation . The morphology of Fsk/IBMX- or Fsk-treated cells was indistinguishable from the neuritic phenotype induced by dbcAMP/IBMX (Fig. 1BCitation ; Ref. 26 ). Morphological differentiation also occurred in response to treatment with the ß-adrenergic receptor agonist Epi, but not in response to treatment with the mitogens Bomb or dHT (Fig. 2)Citation . The formation of neuritic processes was visible within the first hour of treatment, continued to develop, and persisted as long as the cells were maintained under differentiating conditions. NE differentiation occurred even in the presence of 10% FBS and on tissue culture plastic, glass coverslips, or coverslips coated with laminin, fibronectin, or type II collagen (data not shown). These results indicate that physiological and pharmacological agents known to elevate intracellular cAMP levels in other cell types are capable of inducing LNCaP cells to acquire a neuritic morphology typical of NE cells.



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  		Fig. 1. Physiological and pharmacological cAMP-elevating agents induce reversible morphological changes in LNCaP cells. Cells were seeded at 1 x 104 cells/cm2 on glass coverslips in T-media + 5% FBS and left untreated (A) or treated for 3 days with 0.1 mM dbcAMP/100 mM IBMX (B), 10 µM Fsk/100 mM IBMX (C), or 10 µM Epi (E) or cultured for 3 days with Fsk/IBMX or Epi, followed by withdrawal from treatment for 1 day (D and F, respectively). Photomicrographs were taken at x40 magnification.

 


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  		Fig. 2. Specific induction of the NE morphological phenotype in LNCaP cells by agents that are known to elevate intracellular levels of cAMP. LNCaP cells were cultured for 3 days in serum-free, phenol red-free RPMI 1640 alone (S-F) or in the presence of 0.1 nM dHT, 10 µM Fsk, 10 µM Epi, or 1 µM Bomb for 3 days. NE morphology was determined by scoring the length of a cell’s longest neuritic process from the edge of the cell body linearly to its most distal tip. Process length was determined for at least 50 cells in three random fields, and the results are expressed as the mean ± SE of three independent experiments. *, P < 0.005 as compared to the S-F group by Student’s t test.

 
To determine whether elevations in cAMP levels accompanied morphological differentiation, LNCaP cells were incubated with various agents over a time course of 0–30 min, and intracellular levels of cAMP were determined as described in "Materials and Methods." Of the agents tested in this study, only those that stimulated an increase in intracellular cAMP levels induced morphological differentiation (Fig. 3)Citation . Whereas Fsk increased intracellular cAMP to levels 40-fold greater than those in unstimulated cells, Epi and Isop treatment increased cAMP levels 5-fold higher than Fsk; however, all three agents induced strikingly similar morphological changes. Additionally, treatment of LNCaP cells with Fsk, Isop, or Epi for 2 or 5 min resulted in an indistinguishable increase in cAMP-dependent protein kinase kinase activity as measured in the immune complex using the peptides kemptide or malantide as substrates (data not shown). These results suggest that a modest threshold level of cAMP is required for differentiation of LNCaP cells.



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  		Fig. 3. Intracellular concentrations of cAMP induced by various agents in LNCaP cells. Cells (5 x 105) were cultured in serum-free, phenol red-free RPMI 1640 and treated with 10 µM Fsk, 10 µM Epi, 10 µM Isop, 0.1 nM dHT, or 1 µM Bomb for 0, 0.5, 2.5, 10, and 30 min. HCl lysates were prepared from triplicate wells of cells for each treatment and time point and subjected to cAMP analysis by ELISA (Diabetes Core Facility, University of Virginia). Results are expressed as the mean cAMP concentration (pmol/105 cells) ± SE.

 
Morphological Differentiation Is Accompanied by the Appearance of Dense Core Secretory Granules.
A hallmark of NE cells is the presence of dense core secretory granules in the cytoplasm. These granules contain secretory products that are released into the extracellular milieu through a regulated mechanism (37) . The presence of these granules denotes a dedicated secretory cell and can be used as a distinguishing characteristic. To determine whether gross morphological alterations induced in response to differentiating agents were accompanied by the appearance of dense core secretory granules, LNCaP cells were treated with Fsk/IBMX or Epi for 6 days, and the differentiated cells were examined by transmission electron microscopy for the presence of neurosecretory granules. Fsk and Epi treatment resulted in the appearance of pleomorphic dense core granules and lamellar bodies in the cytoplasm of LNCaP cells (Fig. 4, B and C)Citation which was not observed in untreated cells (Fig. 4A)Citation . The morphology of the granules detected in Fsk/IBMX- and Epi-treated LNCaP cells is consistent with that observed previously in normal prostatic NE cells (38) and in LNCaP cells induced to undergo NE differentiation (26) .



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  		Fig. 4. Ultrastructural characterization of LNCaP cells induced to undergo NE differentiation. Cells were cultured for 7 days in T-media + 5% FBS (untreated cells; A) or in the presence of 10 µM Fsk (B) or 10 µM Epi (C) and subjected to analysis by transmission electron microscopy (x15,000 magnification).

 
Production and Secretion of Neuropeptides by LNCaP Cells.
In addition to morphological characteristics, prostatic NE cells are defined by the expression of a number of neurosecretory products. ChrA is considered to be the most reliable indicator of prostatic NE differentiation. Consistent with previously published observations (26 , 39) , we found that both LNCaP and C4-2 cells constitutively secrete elevated levels of ChrA and that these levels are not appreciably altered by agents that increase intracellular cAMP (26) . When expressed as the amount of ChrA secreted into conditioned media normalized to the cell number as measured by the cell lysate protein level, C4-2 cells secreted twice as much ChrA (69 pg/mg) as LNCaP cells (36 pg/mg) and approximately four times the amount detected in the media from PC3 (17 pg/mg), PC3M (17 pg/mg), and murine fibroblasts (20 pg/mg) that were used as a negative control. These latter levels represent the minimum detection limits of the linear range for the ELISA used. These observations suggest that LNCaP and C4-2 cells are predisposed to a NE phenotype in that they already exhibit the ability to secrete ChrA before an exogenous stimulation to differentiate.

Additional neurosecretory factors produced by prostatic NE cells that have been postulated to contribute to prostate cancer progression are PTHrP (40) and NT (41) . To determine whether production of these factors was a characteristic of LNCaP cells stimulated to acquire a NE phenotype, cells were treated with Fsk for 3 days, and the media from these cells were assessed for the presence of PTHrP or NT by EIAs (Fig. 5)Citation . Under these conditions, PTHrP accumulated in the culture media to a concentration of ~200 pg/ml, and NT accumulated to ~90 pg/ml. These concentrations were within the linear range for detection of these factors by the EIAs (50–1000 pg/ml). The concentration of PTHrP and NT in conditioned media from untreated cells or in T-media itself was below the level of detection of these assays. The level of these neurosecretory factors in conditioned media from Fsk-stimulated LNCaP cells is in good agreement with the level of PTHrP detected in LNCaP-conditioned media under serum-free conditions (6) and the levels of NT detected under androgen-depleted conditions (42) . These results indicate that acquisition of a NE morphology in LNCaP cells treated with agents that increase intracellular cAMP levels is correlated with the production of neurosecretory factors, indicative of a NE phenotype.



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  		Fig. 5. Secretion of PTHrP and NT into the media of Fsk-stimulated LNCaP cells. Cells were cultured in T-media and either left untreated (No Trt.) or treated with 10 µM Fsk and 500 µM IBMX (Fsk/IBMX) for 3 days. PTHrP and NT levels were measured in conditioned media or in T-media alone using an ELISA for the respective peptides (Peninsula Laboratories) and expressed as the mean of quadruplicate assays ± SE.

 
Androgen-independent C4-2 Tumor Cells Also Undergo NE Differentiation in Response to cAMP-elevating Agents.
The C4-2 cell line is an androgen-unresponsive derivative of LNCaP cells that exhibits a more malignant phenotype in the nude mouse than LNCaP cells. It is therefore frequently considered to represent a later stage in tumor development than LNCaP. To determine whether acquisition of an androgen-unresponsive phenotype affects the capacity of cells to acquire cAMP-mediated NE characteristics, C4-2 cells were analyzed morphologically for their responsiveness to agents that induce NE phenotypic changes in LNCaP cells. When treated with dbcAMP/IBMX, C4-2 cells responded in a manner nearly identical to that of LNCaP cells, exhibiting contracted cell bodies and the extension of neuritic-like processes (Fig. 6, D and H)Citation . C4-2 cells also acquire a neuritic morphology in response to Fsk and Epi (data not shown). These results demonstrate that androgen-unresponsive as well as androgen-responsive prostate tumor cells can acquire a NE morphology.



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  		Fig. 6. Serotonin expression in C4-2 cells is regulated concomitantly with NE differentiation. Cells were cultured in T-media + 5% FBS and left untreated (A and B), treated with 0.1 mM dbcAMP/100 mM IBMX for 6 days (C, D, G, and H), or treated for 6 days, washed, and refed with media without dbcAMP/IBMX for 2 days (E and F). Formalin-fixed C4-2 cells were subjected to indirect immunofluoresence microscopy using either antiserotonin antiserum (A, C, and E) or serum from unimmunized rabbits (G) and phase-contrast photomicroscopy (x25 magnification; B, D, F, and H) of the corresponding fields, as described in "Materials and Methods." A, C, E, and G were subjected to equal exposure times (2 s) to allow comparison of antiserotonin fluorescence intensity.

 
Reversibility of cAMP-mediated NE Differentiation of LNCaP and C4-2 Cells.
Neuroendocrine cells of the normal prostate and other tissues are terminally differentiated and postmitotic. To determine whether prostate tumor cells become terminally differentiated upon treatment with cAMP-elevating agents, LNCaP or C4-2 cells were subjected to the withdrawal of differentiating agents after being induced to undergo NE differentiation. Withdrawal of Fsk or Epi from LNCaP cells (Fig. 1, D and F)Citation or withdrawal of dbcAMP from C4-2 cells (Fig. 6F)Citation induced the loss of neuritic processes and the reacquisition of a morphology typical of untreated cells. Loss of the neuritic phenotype was initiated almost immediately upon withdrawal and was characterized by dramatic ruffling of the plasma membrane and either retraction or shedding of the neuritic processes such that by 10 h after withdrawal, cells reattained their predominantly fusiform morphology.

Another characteristic ascribed to prostatic NE cells is the loss of mitogenic activity. To determine whether acquisition and loss of the NE phenotype correlated with changes in mitotic activity, cell number and [3H]thymidine incorporation were measured for LNCaP and C4-2 cells after treatment with and withdrawal from dbcAMP, Fsk, or Epi. LNCaP and C4-2 cells treated with dbcAMP/IBMX failed to increase in cell number, whereas untreated cells exhibited the expected positive growth in serum-containing media (Fig. 7, A and B)Citation . Similarly, LNCaP cells treated with either Epi (Fig. 7C)Citation or Fsk/IBMX (Fig. 7D)Citation failed to increase in cell number. Cells treated with dbcAMP/IBMX, Fsk/IBMX, or Epi exhibited a rapid loss of mitotic activity, as measured by [3H]thymidine incorporation, in comparison to that of cells growing in T-media (Table 1)Citation . The mitotic index of cells cultured in the presence of differentiating agents was indistinguishable from that of cells maintained in serum-free, phenol red-free RPMI 1640 for 48 h (data not shown). Cells from which these agents were withdrawn and replaced with T-media reacquired a mitotic index comparable to that of untreated cells within 1 day, whereas cells remaining in the presence of differentiating agents continued to exhibit no growth and depressed mitotic indexes (Table 1)Citation . These results correlate with the morphological analysis and suggest that induction of increased intracellular cAMP levels in LNCaP and derivative cell lines results in the rapid acquisition of NE characteristics that are lost upon withdrawal from treatment, as measured by the loss of the NE phenotype and reentry into the cell cycle.



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  		Fig. 7. Inhibition of LNCaP and C4-2 cell proliferation by cAMP-elevating agents. Cells were plated in T-media + 5% FBS at 2 x 105 cells/35-mm tissue culture well and left untreated (•) or treated with 0.1 mM dbcAMP/100 mM IBMX ({circ}; LNCaP cells, A; C4-2, B), 10 µM Epi ({square}; LNCaP cells, C), or 10 µM Fsk/100 mM IBMX ({triangleup}; LNCaP cells, D) for up to 7 days. Cells were refed every third day with T-media + 5% FBS and the appropriate agents. At the indicated times, cells were trypsinized and counted manually. Results are expressed as the mean number of cells x 105/well ± SE for three independent experiments, each of which was performed in triplicate.

 

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  		Table 1 Mitotic index of LNCaP and C4-2 cells upon treatment and withdrawal of agents that induce NE differentiation

 
Induction of NE Differentiation of LNCaP Induces Reversible Changes in the Expression of NE Markers.
LNCaP and C4-2 cells were also tested for the reversibility of increased expression of factors characterized as markers of NE cells. By immunoblot analysis, NSE levels were increased 6–10-fold in both cell lines after differentiation by dbcAMP or Epi, whereas lysates from either untreated or Epi-treated murine fibroblasts exhibited no detectable NSE (Fig. 8A)Citation . The accompanying MAPK immunoblot of the same filters serves as a protein loading control. As was the case for the other indicators of NE differentiation, withdrawal of dbcAMP or Fsk treatment for 48 h resulted in a return to near basal levels of expression of NSE (Fig. 8B)Citation . The induction of NSE expression in LNCaP cells by Epi, Fsk, or dbcAMP treatment was comparable to the levels detected in equivalent extracts from murine brain or bovine chromaffin cell lysates.



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  		Fig. 8. NSE expression is regulated concomitantly with NE differentiation. Immunoblots were prepared from 50 µg of whole cell lysate from LNCaP cells (A, Lanes 1–3; B, Lanes 1–5), C4-2 cells (A, Lanes 4–6), C3H10t1/2 murine fibroblasts (10t1/2; A, Lanes 7 and 8; B, Lane 6), rat brain (Br; B, Lane 7), and bovine chromaffin cells (CC; B, Lane 8) using anti-NSE antiserum (top panels) or anti-MAPK serum (bottom panels). Cells were either left untreated (A, Lanes 1, 4, and 7; B, Lanes 1 and 6–8); treated for 3 days with 10 µM Epi (A, Lanes 2, 5, and 8), 0.1 mM dbcAMP/100 mM IBMX (dbcAMP; A, Lanes 3 and 6; B, Lane 2), or 10 µM Fsk (B, Lane 4); or withdrawn from dbcAMP or Fsk treatment for 2 days (B, Lanes 3 and 5, respectively).

 
Serotonin expression was also reversibly increased upon NE differentiation. As measured by indirect immunofluorescence, a relatively low basal level of expression was detected in C4-2 cells as compared to cells stained with normal rabbit serum (Fig. 6, A and G)Citation . After 6 days of induction with dbcAMP/IBMX, the level of serotonin was dramatically elevated (Fig. 6C)Citation , and withdrawal of dbcAMP/IBMX resulted in a decrease in serotonin levels, approaching that observed in unstimulated cells (Fig. 6E)Citation . Similar results were obtained for LNCaP cells. The changes in NSE and serotonin levels seen in both LNCaP and C4-2 cells upon treatment and withdrawal of agents that induce the acquisition of NE characteristics correlated with changes in the morphology and mitotic activity of these cells, further supporting the hypothesis that reversible interconversion can occur between the nonmitotic NE phenotype and the mitotic carcinoid phenotype.

Loss of the NE Phenotype Is Accompanied by Changes in Tyrosine Kinase and MAPK Signaling.
Induction of proliferative activity from quiescence is often accompanied by increased intracellular kinase signaling events. To investigate whether such changes occurred in LNCaP cells upon withdrawal of treatments that induce and maintain NE differentiation, MAPK and tyrosine kinase activities were assessed in cells at various times after withdrawal from dbcAMP/IBMX treatment. Tyrosine kinase activity was measured by antiphosphotyrosine immunoblotting of whole cell lysates (Fig. 9A)Citation . Tyrosine phosphorylation of a number of proteins was increased upon the withdrawal of differentiating agents. At 2.5 min after withdrawal, tyrosine phosphorylation of a 35–40 kDa protein was detected. The tyrosine phosphorylation of this protein persisted through 120 min in T-media, with some variation in phosphorylation levels over the time course. At 30 min after withdrawal, the tyrosine phosphorylation of additional proteins became apparent, including those with molecular masses of 100–150 kDa, a broad spectrum centered at 55 kDa, and two proteins with molecular masses of 44 and 42 kDa. The phosphorylation of these proteins remained elevated throughout the 120-min time course.



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  		Fig. 9. Withdrawal from NE differentiation is accompanied by increased protein tyrosine phosphorylation and MAPK activation. A, whole cell lysates were prepared from LNCaP cells cultured in T-media for 6 days in the absence (Lane 1) or presence of dbcAMP/IBMX (Lane 2) or after withdrawal of dbcAMP/IBMX for the indicated times (Lanes 3–10). Immunoblot analysis was performed with antiphosphotyrosine (top panel) or MAPK (bottom panel) antibodies. B, MAPK activity was also assessed by immune complex kinase assays using MBP as a substrate, as described in "Materials and Methods." After SDS-PAGE and electrophoretic transfer of phosphorylated proteins to nitrocellulose membranes, 32P-labeled MBP was visualized by autoradiography (top panel) and normalized for the amount of immunoprecipitated MAPK by immunoblotting with MAPK antibodies (bottom panel). Results are representative of three independent experiments.

 
The coincident tyrosine phosphorylation of the 44- and 42-kDa proteins with reentry of the LNCaP cells into the cell cycle suggested that these proteins may be the MAPK family members ERK1 and ERK2, respectively. To assess whether tyrosine phosphorylation of these proteins upon withdrawal of NE induction represented activation of the MAPKs, lysates were subjected to immunoprecipitation with anti-MAPK antibodies, and the resulting immunocomplexes were used to phosphorylate the exogenous MAPK substrate, MBP, in vitro (Fig. 9B)Citation . At 30 min after the withdrawal of dbcAMP/IBMX from the culture media, MAPK activity was increased 5-fold as compared to untreated or dbcAMP/IBMX-treated cells (Fig. 9BCitation , Lanes 1 and 2). This activation persisted throughout the withdrawal time course but declined to 2-fold above the level of untreated and dbcAMP/IBMX-treated cells at 120 min. The activation of tyrosine kinase and MAPK signaling pathways after the withdrawal of differentiating agents is further support for the transition of these cells to a more proliferative phenotype. These observations indicate that trans-differentiation of prostatic carcinoma cells may occur under physiologically relevant conditions and that the ability of these carcinoid cells to acquire a NE phenotype is reversible. Thus, in contrast to NE cells of the normal gland, NE cells derived from prostatic tumor cells are not terminally differentiated.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
With growing recognition of cancer as a multifaceted process, the identification and characterization of specific cellular phenotypes within a given tumor that may contribute to the malignant process offers the possibility of targeting those cells for future therapies. The presence in prostatic carcinoma of NE cells, which are hypothesized to provide proliferative stimuli to surrounding cancer cells, represents one such opportunity. This report examines the types of agents that may induce prostatic tumor cells to differentiate into NE-like cells and the propensity of different stages of cancer cells to respond to these agents. We have found that those agents that increase intracellular levels of cAMP induce LNCaP prostate tumor cells and their androgen-unresponsive derivatives (C4-2 cells) to acquire a neuritic morphology, develop secretory granules, cease growth, and increase expression of the neurosecretory products PTHrP, NT, NSE, and serotonin. In contrast to a previous report by Bang et al. (26) , we found that this process is completely reversible and that maintenance of the NE phenotype requires the continual presence of differentiating agents. These findings have important implications for our understanding of the apparently complex network of events that eventually leads to androgen-independent growth of prostate tumors and progression of the disease.

Prostatic NE cells have been demonstrated to express a variety of potentially mitogenic hormones including PTHrP (40) , NT (41) , serotonin (43) , calcitonin (44) , Bomb-like factor (45 , 46) , and thyroid-stimulating hormone-like peptide (2) . The ability to produce these factors is theorized to be responsible for the observed increased mitotic index in carcinoma cells juxtaposed to the foci of NE cells in some tumors (24) . If NE cells are able to contribute to the proliferative capacity of the surrounding tumor cells and are themselves able to reenter the cell cycle under appropriate environmental conditions, then tumors retaining such cells would gain a strong selective advantage and possibly account for the development of a loss of androgen dependence. Our demonstration that LNCaP cells are able to produce three of these mitogenic neurosecretory factors indicates that these cells acquire physiologically significant NE features in response to increased intracellular cAMP levels, supporting the use of this model system for characterizing the molecular mechanisms that control NE differentiation in prostatic carcinomas.

The ability of LNCaP-derived prostatic carcinoma cell lines to undergo trans-differentiation into a cell type with strong NE characteristics suggests the intriguing possibility that such events might underlie the apparent accumulation of NE cells in tumors during progression. Although NE cells in prostatic tumors are histologically considered nonproliferative because they do not stain positively with the human mitotic indicator antibodies Ki-67 or MIB1, the acquisition of this phenotype may represent a transient response to environmental influences. Accumulation of a constant, effective concentration of differentiating agents may not be readily achieved in vivo, giving rise to a dynamic situation in which tumor cells can interconvert between a proliferative phase and a nonmitotic, secretory phase, depending upon the balance of factors in the surrounding milieu that promote differentiation versus growth. If differentiation were terminal, and all cells in a tumor were capable of undergoing differentiation, then the possibility is raised that induction of differentiation could cause growth cessation and tumor stasis. However, differentiated cells would still be capable of secreting growth factors and stimulating any remaining tumor cells. Furthermore, not all transformed cells within a tumor may be responsive to differentiating agents, although this report demonstrates that both the androgen-responsive LNCaP cells and the androgen-unresponsive C4-2 cells are sensitive to differentiating factors. Perhaps tumor cells that represent earlier stages in neoplastic development may be less responsive to differentiation factors and more responsive to proliferative factors. The findings that advanced tumors have a higher NE status (12 , 13) and that detection of ChrA in primary tumors is a poor prognosticator for disease-specific survival (23 , 47 , 48) suggest that in those tumors that do develop and advance, a balance is achieved in which the proportion of NE-like cells to proliferating cells approaches an optimum. The presence of ChrA in LNCaP cells under culture conditions in which the cells exhibit the androgen-responsive, exocrine phenotype typical of prostatic adenocarcinomas, together with the observation that some tumor cells exhibit both exocrine and endocrine characteristics (49) , suggests that the acquisition of NE characteristics can occur gradually in response to the phenotypic potential of a cell and the influence of its environment.

The nature of the environmental conditions that promote NE trans-differentiation in vivo are not clearly understood. However, our report and the study by Bang et al. (26) indicate that factors capable of increasing adenylate cyclase activity are obvious candidates. In this regard, our demonstration that the ß-adrenergic agonists Epi and Isop are capable of inducing LNCaP NE differentiation suggests that the patient’s own stress response may contribute to the development of tumors with NE characteristics. Whereas Epi is not produced by cells of the prostate to any measurable extent, it can reach the prostate via the vasculature. Therefore, it is a reasonable physiological candidate for inducing NE differentiation. Additionally, recent reports indicating that unidentified factors in media from phytohemagglutinin-activated lymphocytes (50) , interleukin 1 (39) , interleukin 6 (51) , and long-term serum/androgen deprivation (52) can induce acquisition of NE characteristics by LNCaP cells suggest that factors present in lymph nodes and bone marrow, in conjunction with androgen ablation therapy, may also contribute to the development of tumors with NE characteristics. These findings also indicate a possible mechanism for the propensity of prostate tumors to metastasize to these locations.

The reversibility of the NE phenotype complicates our ability to test the NE cell/paracrine hypothesis both in tissue culture systems and in the animal model. However, it does provide an opportunity to examine the role of tyrosine kinase and MAPK signaling in the regulation of prostate carcinoma cells during the transition from a nonproliferative state to a proliferative state. In this system, we have shown the activation of MAPK and the tyrosine phosphorylation of as yet unidentified proteins upon the withdrawal of adenylate cyclase activators or cAMP analogues from LNCaP cells. These results suggest that activation of these intracellular signaling pathways may function in the regulation of prostatic carcinoma proliferation in vivo and that controlling these signaling events may prove to be alternative therapeutic targets for controlling tumor growth.


   ACKNOWLEDGMENTS
 
We thank Drs. L.W. Chung and R. Sikes for providing the LNCaP and C4-2 cell lines and advice on their maintenance; Drs. M. J. Weber, C. E. Myers, H. F. Frierson, and D. Theodorescu for conceptual insights; and J. A. Redick (University of Virginia Central Electron Microscope Facility) for assistance with the transmission electron micrographs.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by NIH Grants DHHS-NCI PO1 40042, DHHS-NCI R21 69848, and DHHS-NCI RO1 76649. Back

2 To whom requests for reprints should be addressed, at Cancer Center and Department of Microbiology, Box 441, University of Virginia Health Sciences Center, Charlottesville, VA 22908. Phone: (804) 924-2532; Fax: (804) 982-0689; E-mail: sap{at}virginia.edu Back

3 The abbreviations used are: NE, neuroendocrine; cAMP, cyclic AMP; dbcAMP, dibutyryl cAMP; Fsk, forskolin; Epi, epinephrine; Isop, isoproterenol; IBMX, isobutylmethylxanthine; PTHrP, parathyroid hormone-related peptide; NT, neurotensin; NSE, neuron-specific enolase; ChrA, chromogranin A; Bomb, bombesin; EIA, enzyme-linked immunoassay; MAPK, mitogen-activated protein kinase; MBP, myelin basic protein; dHT, dihydrotestosterone; FBS, fetal bovine serum. Back

Received 2/ 9/99. Accepted 6/ 2/99.


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 RESULTS
 DISCUSSION
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