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Endocrinology |
Division of Biomedical Sciences, University of California, Riverside, California 92521
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We conclude that PRL is used as an autocrine growth factor by human prostate cancer cells both in vitro and in vivo and that interruption of this growth loop in vivo inhibits tumor initiation and the growth of well-established tumors.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although a role for PRL in prostate physiology has been evident for some time (21, 22, 23, 24, 25, 26) , the importance of PRL in prostate disease has been underestimated because a variety of clinical studies have failed to find a clear correlation between circulating PRL levels and the presence or absence of disease (14 , 27 , 28) . There may be two very important reasons why this is so. The first is that human pituitary PRL is released in a variety of posttranslationally modified forms, which are not equally recognized in clinical radioimmunoassays and some of which have been shown to have very different biological activities (29) . Thus, total assayable plasma levels may be elevated with no concomitant increase in prostate stimulatory activity and vice versa. The second, as mentioned above, is production of PRL within the prostate itself. If extrapituitary sources of PRL, which include a large number of tissues (reviewed in Ref. 30 ), collectively contribute 10–20% of circulating PRL, then a doubling of PRL production in the prostate alone may not be sufficient to convincingly elevate circulating PRL to a degree that was significant over normal interpatient variation. However, this doubling of PRL within the prostate itself could have a most significant effect on prostate cell proliferation.
More recently, work in animal model systems and with normal human organ cultures has added substantially to the evidence that PRL is an important growth factor in the prostate. Thus, in mice overexpressing rat PRL there was massive prostate hyperplasia at 10–15 months of age (31) , and knockout of the PRL gene resulted in a prostate that was 30% smaller than normal (32) . In the Noble rat, prostate dysplasia and eventually cancer can be induced by estrogen and testosterone administration (33, 34, 35) , and this can be inhibited by reducing PRL release from the pituitary with bromocriptine (35) . In human prostate cultures, PRL has been shown to be an autocrine growth factor (20 , 36) , with the majority of PRL and PRLR expression occurring in the epithelial cells of the gland.
In regard to the posttranslationally modified forms of PRL (reviewed in Ref. 37 ), work from our laboratory has demonstrated that phosphorylated PRL can act as an antagonist to unmodified PRL in tissues where unmodified PRL promotes cell proliferation (38 , 39) . More recent work using a molecular mimic of phosphorylated PRL has extended these observations to many additional tissues (40) . The molecular mimic of phosphorylated PRL is produced by substituting an aspartate residue for the normally phosphorylated serine (39 , 41) thereby producing an S179D PRL mutant, which serves in many tissues as a PRLR antagonist. In this study, we have asked two important questions. The first is whether the PRL autocrine growth loop of normal prostate tissue is retained in cell lines representative of the androgen-dependent and androgen-insensitive stages in the evolution of the human disease, and the second is whether antagonism of such a growth loop with the molecular mimic of phosphorylated PRL could influence progression of the disease. To answer these questions we made use of the LnCAP, PC3, and DU145 cell lines.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Male nude mice were purchased from Charles River Laboratories (Wilmington, MA). They were homozygous (nu/nu) and 8–9 weeks old at the time of experiments. They were kept in sterilized laminar flow cages under 12-h light and 12-h dark standardized environmental conditions throughout the experiments. Sterile lab coats, boots, hats, and masks were required inside the animal room. Sterilized food and water were supplied ad libitum.
Alzet Minipump Implantation.
Nude mice were restrained in open-ended conical plastic bags and anesthetized in the interscapular region with lidocaine (0.7 ml of 0.2 mg/ml s.c. injection). Alzet minipumps (model 2004; Alza, Palo Alto, CA) containing WT PRL, S179D PRL, or no PRL were implanted s.c. between the scapulae after a single lateral incision. The incisions were closed by wound clips. All of the procedures were conducted in a laminar flow hood with sterile technique.
DU145 Cell Injection.
When confluent, cells were washed with PBS, and harvested with trypsin/EDTA (2.5%/1% in PBS). Cells (5 x 106) were then resuspended in MEM supplemented with penicillin/streptomycin (20 units/ml and 20 µg/ml, respectively) before injection into nude mice. More than 90% of cells in the suspension were viable as assessed by trypan blue exclusion. DU145 cells were injected s.c. into the left hind leg of the nude mice. Cells from the same flask were used to inject animals from different groups, and a record was kept of this to control for potential interflask variation.
All of the animal procedures were approved by the University of California, Riverside, Committee on Laboratory Animal Care and were in accordance with NIH guidelines.
Recombinant Protein Expression.
Recombinant WT hPRL and the molecular mimic of phosphorylated PRL (S179D hPRL) were prepared by expression in Escherichia coli (39)
. The WT and S179D hPRL were expressed at the same time and collected and refolded in parallel to ensure comparability. Both proteins were expressed at a similar level. The proteins were then tested for their relevant biological activity in an Nb2 bioassay and compared with an NIDDK standard preparation of hPRL, as described previously (39)
. Nb2 cells are T lymphoma cells originally isolated from the lymph node of an estrogen-treated rat, which proliferate in response to PRL. Proteins were quantified by Coomassie blue staining and gel densitometry using NIDDK hPRL to produce a standard curve.
To prepare the proteins used in nude mice experiments, refolded WT and S179D recombinant PRLs were sterilized by passage through a 0.45-µm filter (Gelman Science, Ann Arbor, MI), and then concentrated using a sterile Mr 10,000 molecular weight cutoff concentrator (Amicon, Beverly, MA). WT and S179D recombinant PRL were concentrated to 0.76 µg/µl and 0.71 µg/µl in saline, respectively. The filtration and concentration processes reduced the biological activity of the recombinant WT PRL to be equivalent to NIDDK standard PRL. Proteins were administered to the animals via the Alzet minipumps with delivery rates of 6 µl/24 h or 4.56 µg/24 h (WT) and 4.26 µg/24 h (S179D).
Determination of Circulating Levels of the PRLs.
Circulating levels of administered recombinant proteins were determined by immunoprecipitation of 35S-biosynthetically labeled recombinant proteins produced as described by Giovane et al. (45)
. Blood plasma samples were taken at day 5 after minipump implantation. (Previous experiments had demonstrated that circulating levels of recombinant PRLs stabilized between days 3 and 4 after minipump implantation.) On the basis of the specific activity of the radiolabeled PRLs and the knowledge that excess anti-PRL showed 85% of the counts to still be in the PRLs, we determined that administration of 4.56 and 4.26 µg/24 h resulted in circulating levels of 47 ng/ml and 54.4 ng/ml of WT and S179D PRL, respectively.
Cell Proliferation Assay.
Cells were plated at 5000 cells/well (DU145) or 2000 cells/well (LnCAP and PC3) in a 96-well plate in their growth medium containing 10% FBS. Cells were allowed to attach overnight. On day 2, the medium was changed to 5% FBS (DU145) or 10% HS (LnCAP and PC3) containing 20 ng/ml WT PRL and increasing concentrations of S179D PRL (0, 2, and 20 ng/ml and 2 and 20 µg/ml), and the cells were incubated for 3 days (DU145 and PC3), or 5 days (LnCAP) with a medium change on day 3. In additional wells, the responses to S179D PRL were titrated out with WT PRL to test the reversibility of the inhibition. The number of cells plated, the exact medium, and the incubation times were dictated by the growth characteristics of each cell line. Cell numbers were determined by MTS assay (Promega, Madison, WI). Briefly, the MTS dye, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxymethoxyphenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium, is converted to a formazan product that is soluble in tissue culture medium and measured at 492 nm. Cell number was proportional to MTS dye conversion within the range used in these experiments.
Nb2 Bioassay of DU145-conditioned Medium.
DU145 cells were cultured in growth medium with 10% FBS. When near to confluence, the cells were washed four times with PBS to be free of FBS. Cells were then incubated in medium supplemented with 10% HS. After 24 h, the conditioned medium was collected, and cell number was determined. Nb2 cells were used to assay for growth-promoting PRL in the conditioned medium. Nb2 cells were prepared in lactogen-free medium overnight before assay as described previously (39)
. Nb2 cells were plated at 5000 cells/well with serially diluted DU145-conditioned medium and incubated for 3 days. The amount of PRL being produced by DU145 cells was quantified using a concomitant Nb2 cell dose-response curve to standard NIDDK hPRL B2.
Reverse Transcription-PCR for PRL and PRLR.
Total RNA was extracted from cells using Tri-reagent (Molecular Research Center, Inc., Cincinnati, OH). Equal amounts of total RNA (1.5 µg) were subjected to the action of reverse transcriptase for 1 h at 37°C (Life Technologies, Inc.). Twenty µl of the product were diluted into 50 µl. Two µl of the product were then incubated in the PCR to amplify PRL and PRLR mRNA. The primers used were 5'GGG TTC ATT ACC AAG GCC ATC and 3'TTC AGG ATG AAC CTG GCT GAC for PRL mRNA and 5'AAT GTG GCA TCT GCA ACC GTT TTC ACT C and 3'CTC CAT GCA CTC CAG TAT CCA TGG TCT G for the PRLR mRNA. Both of the primers were designed to flank introns. Final concentrations of 0.5 µM of each primer, 2 units of Ampli-Taq Gold (Perkin-Elmer, Branchburg, NJ), and 1.5 mM and 2 mM MgCl2 for PRL and PRLR mRNA, respectively, were included in a 50 µl volume of each PCR reaction. RNA extracted from the human breast cancer cell line MCF7, well known to express PRL and PRLR (46)
, was used as a positive control, and the absence of reverse transcriptase served as a negative control. Thirty-six cycles of amplification were performed with the following profile: 1 min 30 s at 95°C, 1 min and 30 s at 63°C, 3 min at 72°C, followed by extension for 15 min at 72°C. Amplification products were resolved on a 2% agarose gel and stained with ethidium bromide.
Tumor Experiments.
In the first experiment, Alzet minipumps containing no PRL, WT PRL, or S179D PRL were implanted s.c. on day 1. On day 4, 5 x 106 DU145 cells were injected s.c. into the left hindquarter. On day 22, the animals were killed; tumors were removed and measured by calipers then fixed and processed for histopathological analysis.
In the second experiment, 5 x 106 DU145 cells were injected on day 1. On day 18, Alzet minipumps containing no PRL, WT PRL, or S179D PRL were implanted s.c. On day 42 (24 days of PRL exposure), animals were killed; tumors were removed and measured by calipers then fixed and processed for histopathological analysis.
Statistics.
The data are represented as mean ± SE. For comparisons between two groups, unpaired t tests or Mann-Whitney tests were used. For comparisons of multiple treatment groups, one-way ANOVA were used. Ps are indicated in the figure legends.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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DU145-conditioned Medium Assay.
Expression of the message does not mean expression of the protein. To test for the expression of PRL, we assayed the DU145-conditioned medium. Because of the known interaction of lactogens in FBS with the rat PRLR of Nb2 cells, DU145 cells were washed free of FBS and incubated in medium supplemented with HS before collection of conditioned medium. Using an Nb2 cell growth response curve to NIDDK standard PRL B2 as the reference, assay results showed that undiluted conditioned medium induced the same degree of Nb2 cell proliferation as 160 pg/ml of B2 PRL (Fig. 3)
. Because there were a total of 30 ml of conditioned medium at the end of the collection period, and there were 107 cells, we calculated that DU145 cells produced 0.48 ng of PRL/106 cells/24 h.
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, in the presence of 20 ng/ml priming WT PRL the addition of the molecular mimic of phosphorylated PRL (S179D PRL) resulted in inhibition of growth of DU145 cells. A dose-related growth inhibition was observed from 2 ng/ml to 2 µg/ml S179D PRL (2 µg/ml was the highest concentration tested). The antagonism of growth could be reversed by adding excess WT PRL. The addition of 200 ng/ml WT PRL reversed the antagonism of 20 ng/ml S179D PRL. The priming WT PRL had no stimulatory effect on growth, but omission of the priming WT PRL resulted in no significant inhibition of proliferation by S179D PRL.
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, both LnCAP and PC3 cells expressed PRL mRNA, and a dose-related growth inhibition by S179D PRL was observed (Figs. 5
and 6
). Addition of 200 ng/ml WT PRL reversed the antagonism of 20 ng/ml S179D PRL for LnCAP cells and 2 µg/ml and 20 µg/ml S179D PRL for PC3 cells.
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shows that the incidence of tumor formation in the no-PRL control group was 9/11 animals (82%). In the animals treated with WT PRL, the incidence was 8/10 (80%), whereas in the animals treated with the S179D mutant PRL, the incidence was markedly reduced to 3/11 (27%). Although WT PRL had no effect on the incidence of tumors, the average size of the tumors was increased from 25.80 ± 6.69 mm3 in the no-PRL-treated control animals to 66.66 ± 18.06 mm3 in the WT PRL-treated animals.
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. Compared with the no-PRL-treated animals, treatment with WT PRL shifted the distribution to the right, with more animals having larger tumors. In the S179D mutant PRL-treated group, the distribution curve shifted to the left, with animals in this group having much smaller tumors (some of them were microscopic tumors) and a significant number of animals having no tumor at all.
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except those that related to overall tumor size.
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The same number of DU145 human prostate cancer cells were injected but this time on day 1, and the animals were randomly assigned to three groups. To be comparable with the first in vivo study, tumors were allowed to grow inside the nude mice for 18 days before the Alzet minipumps containing no PRL, WT PRL, or S179D PRL were implanted.
Table 2
shows the average tumor size after exposure to the PRLs for 24 days. Tumor size was significantly reduced from 1731.35 ± 283.4 mm3 in the no-PRL control animals to 1030.98 ± 294.6 mm3 in the S179D mutant PRL-treated animals. The average tumor size in the WT PRL-treated animals was 2117.75 ± 630.32 mm3 and not significantly different from the controls.
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The distribution of the tumors among the animals in the three different groups was analyzed (Fig. 9)
. S179D PRL treatment shifted the distribution curve toward the left, graphically indicating that more animals in this group have a smaller size tumor.
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. These more advanced tumors often invaded the spinal column and femur (51.4%). Some blood pools were present in the interior, as were necrotic areas, but there were no obvious morphological differences that distinguished the three groups.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PCR analyses demonstrated the presence of both PRL and PRL receptor mRNA in DU145 cells, and Nb2 bioassay of DU145 cell-conditioned medium demonstrated that the PRL mRNA was translated and secreted into the medium as a proliferative form of the hormone, i.e., one that stimulated Nb2 cell proliferation. This PRL is therefore unmodified PRL (20 , 38) and equivalent to WT PRL. Thus, all of the essential elements of an autocrine loop were present. However, demonstration of the autocrine growth loop in DU145 cells in the in vitro assay was dependent on the presence of sufficient WT PRL in the medium to prime the system. The priming amount of WT PRL was not sufficient to increase cell proliferation over no additional PRL, and so the experiment is not analyzing simple antagonism of an added stimulator. We propose that the priming PRL up-regulates the PRLR, because this has been demonstrated in other systems (47) and also in prostate (48) , therefore increasing sensitivity to the S179D PRL. When appropriately primed, the S179D PRL showed a dose-related inhibition of cell proliferation, which was evident at 2 ng/ml, i.e., only one-tenth the amount of priming PRL. This is consistent with previous reports of the efficacy of this antagonist (39) . The necessity for priming in vitro and not in vivo (no WT PRL was administered with the S179D PRL, and mouse PRL is thought to not interact with the hPRLR) probably reflects the fact that the amount of PRL produced by the DU145 cells themselves is insufficient for priming when diluted into culture medium but is sufficient within the confines of a tumor. Alternatively, some other factor in vivo achieves the same result. Interestingly, Fuh and Wells (49) also reported use of what we refer to as priming WT PRL when analyzing inhibition of the growth of a mammary cell line by the G120R PRL antagonist in vitro. The specificity of the inhibition with S179D PRL is demonstrated by its reversal by an 11-fold excess of WT PRL.
Reversible growth inhibition of LnCAP and PC3 cells was also demonstrated. The different concentrations of S179D PRL used to achieve the maximum inhibition were different in different cells probably because of a combination of factors including the rate of PRL production, PRL degradation, PRL receptor production, and PRL receptor display and turnover. Mutation of the PRL receptor may also play a role. In this regard, we were unable to detect the PRLR in LnCAP and PC3 cells using the same primers as used for DU145 cells. However, LnCAP and PC3 cells are responsive to PRL as illustrated by Janssen et al. (12) and the results herein and, therefore, must display functional, if mutated, PRLR. Additional work will be required to determine the nature of the receptor mutation. However, it is clear that the cells are using a PRL autocrine growth loop that can be antagonized by S179D PRL.
The degree of inhibition of cell proliferation during the 3- or 5-day incubation periods was modest even at high S179D PRL concentrations, but even a modest inhibition of tumor cell growth can have a significant impact on long-term tumor development. In this regard, one can see maintenance of 
50 ng/ml in the circulation of nude mice was sufficient to have a major impact on both tumor initiation and tumor growth.
Metastatic spread from a primary tumor involves changes in the expression of cell adhesion molecules and proteases to allow release of a cell and its movement through connective tissue toward a lymphatic or blood capillary. In addition, once free, the liberated cell will initiate formation of a secondary tumor. Our first in vivo experiment demonstrated that treatment of the animals with S179D PRL markedly inhibited tumor initiation and, hence, could potentially contribute to the inhibition of metastatic spread of prostate cancer.
In this first experiment, when the tumor cells were exposed both to autocrine PRL and administered WT PRL, an increase in tumor size in response to WT PRL was observed. No statistically significant increase was seen in the second experiment when the treatment began later, although there was a trend toward larger size. This illustrates the potential under normal circumstances for pituitary PRL, in addition to autocrine PRL, to contribute to prostate cancer growth, at least when tumors are small, and explains the clinical benefit of bromocriptine in human trials (50) and animal model systems (35) .
In the second in vivo experiment, we analyzed the effect of the S179D PRL on the growth of very well-established tumors and saw significant inhibition of growth. There was a 40% reduction in size compared with the control group and a 62% reduction compared with the WT PRL-treated group. Unmodified PRL (equivalent to WT PRL) and the S179D PRL work through the same receptor but result in different intracellular signaling (51) . In other words, the S179D PRL does not merely block signal transduction from unmodified PRL. Indeed, in some cell systems, signaling from S179D induces differentiation as well as inhibition of proliferation (40 , 52) . However, no morphologically apparent signs of differentiation were seen in the S179D PRL-treated tumors.
We conclude that DU145 cells can use unmodified PRL as an autocrine growth factor when grown at sufficient density in vitro or when grown as tumors in vivo, that pituitary PRL has the potential to contribute to prostate cancer growth at least in its early stages, and that S179D PRL holds promise as a treatment for androgen-independent prostate cancer through its ability to slow tumor growth and limit metastatic spread. Furthermore, inhibition of the growth of LnCAP cells suggests the possibility that S179D PRL may also be useful during the androgen-dependent stages of the disease.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the Cancer Research Fund, under Interagency Agreement 97-12013 (University of California contract 98-00924V) with the Department of Health Services, Cancer Research Program. 
2 To whom requests for reprints should be addressed, at Phone: (909) 787-5942; Fax: (909) 787-5504; E-mail: ameae.walker{at}ucr.edu
3 The abbreviations used are: PRL, prolactin; WT, wild type; PRLR, prolactin receptor; FBS, fetal bovine serum; PBS, Dulbecco’s 0.01 M PBS; NIDDK, National Institute of Diabetes and Digestive and Kidney Diseases; hPRL, human prolactin; HS, horse serum.
Received 12/ 5/00. Accepted 6/20/01.
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