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Experimental Therapeutics, Preclinical Pharmacology |
Oncology Research Institute, Cancer Center, Greenville Hospital System [W. Y. C., N-y. C., R. S., T. E. W.], and Department of Microbiology and Molecular Medicine, Clemson University [W. Y. C., P. R., T. E. W.], Greenville, South Carolina 29605
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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hPRL3 is a neuroendocrine polypeptide hormone discovered nearly 60 years ago. It is primarily produced by the lactotrophs in the anterior pituitary gland of all vertebrates. The biological activities of PRL are mediated by specific membrane receptors, i.e., PRLRs (3) . On the basis of several conserved features (a single transmembrane domain and conserved amino acid sequences in the extracellular domain), PRLRs together with GH receptor, have been categorized into the cytokine receptor superfamily (3) . The best-characterized action of PRL is on the mammary gland. In this organ, PRL plays a decisive role in the stimulation of DNA synthesis, epithelial cell proliferation, and the promotion of milk production (4) . The generation of PRL (4) and PRLR (5) gene knock-out mice have unambiguously demonstrated that PRL and PRLR are the key regulators in mammary development.
Several lines of evidence strongly link hPRL to breast cancer development: (a) it has been reported that female hGH transgenic mice have a high incidence of breast cancer in contrast to sporadic cases found in bovine GH transgenics (6) . The high incidence of breast cancer in hGH transgenic mice is believed to be attributable to the lactogenic activity of hGH, which is a unique feature of primate GHs. A recent report of breast cancer development in hPRL transgenic mice further confirmed the role of hPRL in the stimulation of breast cancer (7) ; and (b) the finding of hPRL mRNA in mammary tissues (8, 9, 10) and the detection of biologically active hPRL in human breast cancer cells (11) suggest that hPRL is produced locally as an autocrine/paracrine growth factor within the mammary glands. This extrapituitary production of hPRL might not cause detectable systemic change of hPRL in serum yet could exert significant local stimulatory effects (12) . In support of this concept, it has also been reported that the expression levels of PRLRs are significantly higher in human breast cancer cells or in surgically removed breast cancer tissues than in normal breast epithelial tissues (13, 14, 15) . The high levels of PRLRs in malignant breast tissue make these cells highly sensitive to stimulation by hPRL (15) .
In our previous studies, we demonstrated that the third 
-helix of GH is important for its growth-promoting activities (16, 17, 18, 19, 20, 21)
. We further demonstrated that Gly 119 of bGH (18)
or Gly 120 of hGH (19)
plays a critical role in the action of GH in stimulating growth enhancement. The mechanism of these GH antagonists was further studied by other groups (22
, 23)
. It is generally accepted that GH transduces its signal via a sequential receptor binding mechanism to form a one hormone-two receptor complex (22
, 23)
. Receptor dimerization is thought to be a key step for GH signal transduction. Any amino acid substitution (other than Ala), especially one with a bulky side chain such as Arg at position 120 of hGH, will prevent receptor dimerization, resulting in a GH antagonist (16, 17, 18, 19, 20, 21)
. As a member of the GH family, hPRL is believed to share a signal transduction mechanism similar to GH (24, 25, 26, 27)
. It is, therefore, reasonable to predict that if a key amino acid within the third -helix of hPRL is substituted, it may be possible to produce a hPRL-specific antagonist in much the same manner that hGH antagonists have been produced.
In this paper, we report that by adopting a strategy similar to that which we used in designing the GH antagonist, we have developed a hPRL antagonist in which a Gly residue at position 129 was substituted with Arg (hPRL-G129R). We have demonstrated the following three hPRL-related findings: (a) single amino acid substitution mutation at position 129 of hPRL (hPRL-G129R) resulted in a hPRL antagonist, confirmed by cell proliferation assays; (b) when hPRL-G129R was applied together with 4-OH-tamoxifen, an additive inhibitory effect was observed; and (c) the inhibitory effect of hPRL-G129R on human breast cancer cells is through the induction of apoptosis. We believe that development of the hPRL-G129R, a hPRL antagonist, might open a new avenue in the design of adjuvant therapy to improve the treatment of breast cancer.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Rational Design of hPRL-G129R
We have compared the amino acid sequences of all known PRLs in the third -helical region and aligned them with GH sequences (Table 1)
. It is clear that Gly 129 of hPRL is invariable among PRLs and corresponds to hGH 120, suggesting a potentially important role in its function. We, therefore, decided to make a single amino acid substitution mutation at Gly 129 of hPRL (hPRL-G129R). We have used a similar approach to that which we have used successfully previously in the discovery of hGH antagonists in the hope of producing a hPRLR-specific antagonist (Fig. 1)
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Human Breast Cancer Cell Lines
The human breast cancer cell lines used in this study are MDA-MB-134, T-47D, BT-474, and MCF-7 from the American Type Culture Collection. These human breast cancer cell lines have been characterized as ER-positive and PRLR-positive cell lines (28)
. T-47D and BT-474 cells were grown in RPMI 1640 (phenol red free to avoid its potential estrogen-like activities) supplemented with 10% FBS (Life Technologies, Inc.) and American Type Culture Collection recommended supplements. MCF-7 cells were grown in DMEM (phenol red free), supplemented with 10% FBS. The cells were grown at 37°C in a humid atmosphere in the presence of 5% CO2. The MDA-MB-134 cells were grown in Leibovitz’s L-15 medium supplemented with 20% FBS and grown in a CO2-free atmosphere.
Expression and Production of hPRL and hPRL-G129R Proteins
Mouse L-cell transfection and stable cell selection were performed as described previously with minor modification (29)
. Briefly, cells were plated in a six-well plate and cultured until the culture was 50% confluent. On the day of transfection, cells were washed once with serum-free medium and cultured in 1 ml of serum-free medium containing 1 µg of pcDNA3-hPRL or pcDNA3-hPRL-G129R and 10 µl of LipofectAmine (Life Technologies, Inc.) for 5 h. Two ml of growth medium were added to the DNA/LipofectAmine solution, and incubation continued. After 18–24 h of incubation, fresh growth medium was used to replace the medium containing DNA/LipofectAmine mixture. At 72 h after transfection, cells were diluted 1:10 and passed into the selective medium (400 µg/ml G418) to select for neo gene expression. Individual colonies were isolated and expanded. The expression levels of the individual cell lines were determined by using an IRMA kit from Diagnostic Products Corp. (Los Angeles, CA). The cell lines with high expression levels were expanded.
Conditioned medium containing hPRL and hPRL-G129R was prepared as follows. Stable cells were plated in T-150 culture flasks at 85–90% confluence. The growth medium were then replaced with 50 ml of RPMI 1640 containing 0.5% dextran-coated charcoal-FBS and collected every other day for three times. The collected media were then pooled and filtered through a 0.22 µm filter units to remove cell debris and stored at -20°C until use. The concentration of hPRL or hPRL-G129R was determined by hPRL IRMA. Each batch product was further verified using a Western blot analysis protocol (30) . We have used this protocol in hGH analogue studies, including hGH antagonists, for in vitro studies (19) .
Radioreceptor Binding Assay
hPRLR binding assays were performed as described previously (19
, 31)
. Briefly, T-47D cells were grown in six-well tissue culture plates until 90% confluent (
105 cells/well). Monolayers of cells were starved in serum-free RPMI 1640 medium for 2 h. The cells were then incubated at room temperature in serum-free RPMI 1640 containing 8 x 104 cpm 125I-labeled hPRL (specific activity, 30 µCi/µg; NEN DuPont, Boston, MA) with or without various concentrations of hPRL (from NIH as standard) and hPRL-G129R. Cells were then washed three times in serum-free RPMI 1640 and solubilized in 0.5 ml of 0.1 N NaOH/1% SDS, and the bound radioactivity was determined by a gamma counter (model 4/600plus; ICN Biomedical, Costa Mesa, CA). EC50s of hPRL and hPRL-G129R were then determined and expressed as mean ± SD. Comparison was made by Student’s t test.
Human Breast Cancer Cell Proliferation Assays
hPRL-G129R Conditioned Media.
The assay conditions were modified from that described by Ginsburg and Vonderharr (11)
. T-47D cells were trypsinized and passed into 96-well plates in RPMI 1640 containing 0.5% FBS that was treated with charcoal/dextran-treated FBS (Hyclone, Logan, UT) in a volume of 100 µl/well. The optimal cell number/well for each cell line was predetermined after titration assay. We have found that 15,000 cell/well are optimal for T-47D cells. The cells were allowed to settle and adhere overnight (12–18 h), and subsequently various concentrations of either hPRL, hRPL-G129R, E2, or 4-OH-tamoxifen in a total volume of 100 µl of culture media were added. Purified hPRL (kindly provided by Dr. Parlow, National Hormone and Pituitary Program, NIH, Bethesda, MD) was used as a positive control for hPRL produced from stable L cells. Cells were incubated for an additional 96 h at 37°C in a humidified 5% CO2 incubator. After incubation, MTS-PMS solution (Cell Titer 96 Aqueous kit; Promega Corp.) was added to each well, following the manufacturer’s instructions. Plates were read at 490 nm using a Bio-Rad benchmark microplate reader. The experiments were carried out in triplicates and repeated three to six times for each cell line.
Coculture Experiments.
This design of the cell proliferation assay is to take advantage of stable mouse L cell lines we have established that produce hPRL and hPRL-G129R. Increasing numbers of L cells (or L-hPRL or L-hPRL-G129R cells) in a range of 4,500–27,000 cells/well were cocultured with fixed number of T-47D (9,000/well) in 96-well plates. At the same time, a correspondent set of L cells (or L-hPRL or L-hPRL-G129R cells) was cultured in the same plate (without coculture with T-47D) as background controls. The total volume of the coculture was 200 µl. The concentrations of hPRL or hPRL-G129R at the end of 72-h coculture were measured at 20–200 ng/ml, which is within the physiological range and is similar to that of the conditioned media experiments. After incubation, MTS-PMS solution was added to each well at 24, 48, or 72 h (best response was observed at 72 h and reported in this paper). Plates were then read at 490 nm using a Bio-Rad benchmark microplate reader. The absorbance (A) of T-47D cells was calculated as total A (A of T-47D plus L, L-hPRL or L-hPRL-G129R cells, respectively) minus the background As (L, L-hPRL, or L-hPRL-G129R cells alone).
TUNEL Assay
This assay (Fluorescein Apoptosis detection system; Promega Corp.) works by labeling the nicks of the fragmented DNA at the 3-OH ends. The fluorescein-labeled dUTP is incorporated at the 3-OH ends by terminal deoxynucleotidyl transferase. Four human breast cancer cell lines were used in this study. Before the assay, the breast cancer cells were switched to 10% charcoal/dextran-treated FBS (CCS) for a week. Subsequently, the cells were plated onto an eight-chambered slide system (Lab TekII) at a confluence of 60–70% per chamber. The next day, the breast cancer cells were treated with various concentrations of hPRL-G129R in conditioned medium (0.5% CCS) or 4-OH-Tamoxifen (in 0.5% CCS containing growth medium). To demonstrate the specificity of the antagonist, hPRL-G129R was also either mixed with PRL or with polyclonal anti-hPRL antibodies (kindly provided by Dr. Parlow, National Institute of Diabetes and Digestive and Kidney Diseases) before being applied to breast cancer cells. In the case of anti-hPRL antibody experiments, 125 ng/ml of hPRL-G129R were preincubated with anti-hPRL antibodies for 6 h at 4°C before adding to the cells. After the assigned period of treatment, the chambers were dismantled, and the assay was performed as per the manufacturer’s instructions. The slides were examined under a FITC filter using an Olympus IX 70 microscope system.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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]CTC). hPRL-G129R cDNA was also generated by PCR and sequenced.
Expression of hPRL and hPRL-G129R
Mouse L cell were stably transfected with either hPRL or hPRL-G129R cDNAs, and neo-resistant clones were selected and expanded. Conditioned media were collected and tested for expression by use of an IRMA kit. We have generated hPRL and hPRL-G129R stable mouse L-cell lines that produced hPRL and hPRL-G129R in a quantity of 1 mg/l every 24 h/million cells (Fig. 2)
.
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). These results were similar to our previous studies regarding bGH antagonist (bGH-G119R; Ref. 16
) and hGH antagonist (hGH-G120R; Ref. 19
).
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5
6
. hPRL stimulated T-47D proliferation in a dose-dependent manner. The maximum stimulation of hPRL (250 ng/ml) was 20% over basal levels after a single dose/4-day incubation. However, when hPRL and E2 were applied simultaneously, an additive effect was observed. The maximum response of hPRL (100 ng/ml) in the presence of 10 nM of E2 was more than tripled as compared with hPRL alone (Fig. 4)
.
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, 
). It is noteworthy to point out that the inhibitory effect of hPRL-G129R (150 ng/ml) was more potent than the maximal 500 nM dose of 4-OH-Tamoxifen in our assay system (Fig. 5)
. The maximum inhibition of a single dose of 4-OH-Tamoxifen (500 nM) is 85% of control, whereas the maximum inhibition by a single dose of hPRL-G129R resulted in 75% of control. More importantly, when hPRL-G129R was applied together with 4-OH-Tamoxifen, the inhibitory effects were doubled as compared with either the maximum dose of hPRL-G129R or 4-OH-Tamoxifen (Fig. 5)
. For example, 100 nM of 4-OH-Tamoxifen resulted in an 85% inhibition; yet in the presence of 150 ng/ml of hPRL-G129R, the inhibitory effect resulted in 58% of control. hPRL-G129R was also able to competitively inhibit hPRL-induced cell proliferation. At a 1:1 molar ratio, hPRL-G129R was able to stop the stimulatory effect of hPRL, and at 2:1 molar ratio, it inhibits cell proliferation (Fig. 6)
.
Coculture Experiments.
We found that stable mouse L-cell lines grow at a similar rate as do regular L cells, regardless of producing either hPRL or hPRL-G129R (data not shown) because of the fact that mouse L cells possess nondetectable levels of PRLR (20)
. We believe that the coculture experimental set-up sustained the presence of biologically active hPRL-G129R, resulting in a maximal response in these breast tumor cells.
T-47D cells, after coculture with L-PRL or L-PRL-G129R cells, demonstrated dose-dependent growth stimulation (with L-PRL) or inhibition (with L-PRL-G129R; Fig. 7
). The responses were rather dramatic as compared with conditioned media experiments. We nearly achieved complete inhibition of cell proliferation.
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, and the apoptosis is obvious, even at physiological concentration (50 ng/ml; Fig. 8C
). To demonstrate the specificity of hPRL-G129R to the hPRLR, hPRL or an anti-hPRL antiserum to reverse the apoptosis process and hPRL-G129R were simultaneously used to treat the cells (Fig. 8, G–I)
. As shown in Fig. 8H,
hPRL is able to competitively reverse the DNA fragmentation induced by hPRL-G129R at a ratio of 4:1 (500 ng/ml of hPRL versus 125 ng/ml of hPRL-G129R). The same results were obtained using BT-474 cells (data not shown). The DNA fragmentation in breast cancer cells is apparent even after 2 h of exposure to hPRL-G129R at a concentration of 50 ng/ml (Fig. 9, A–D)
. We also confirmed that hPRL-G129R could induced apoptosis by DNA fragmentation in four hPRLR-positive breast cancer cell lines after 24 h of treatment. (Fig. 10)
. To demonstrate the specificity of hPRL-G129R, an anti-hPRL antibody titration experiment was also included (Fig. 8I)
. It was shown that the anti-hPRL antibody could completely block the apoptotic effects of hPRL-G129R in T-47D cells after 6 h of preincubation.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recently, several lines of evidence strongly suggest that hPRL acts as an autocrine/paracrine growth factor contributing to breast cancer development (11 , 34 , 35) . More importantly, it has recently been reported that sex steroid hormones and PRL interact synergistically to control cancerous growth within the mammary gland (28) . ER and PRLR were found being coexpressed and cross-regulated in mammary tumor cell lines as well as in primary breast cancers (28) . These findings further suggest that the use of antiestrogen therapy in breast cancer may be attacking only half of the synergistic equation, which leaves an opportunity for further improvement of the ultimate therapeutic approach to breast cancer (28) . In support to this notion, a combined regimen using an antiestrogen (Tamoxifen), an anti-GH secretion drug (octreotide), and an anti-PRL secretion drug (CV 205-502) has been reported to have significantly better clinical results in metastatic breast cancer patients as compared with tamoxifen therapy alone (36) . Although this regimen does not block the autocrine/paracrine action of PRL on breast cancer, inhibition of circulating PRL from the pituitary did seem to have an additive benefit in the treatment of advanced breast cancer. This raises exciting prospects for even better results with complete PRL blockade with an antagonist that acts at the receptor level.
In this study, we report the design and production a hPRL antagonist, hPRL-G129R. We first demonstrated that hPRL and E2 exhibited an additive stimulatory effect in human breast cancer cell proliferation (Fig. 4)
. We believe that the synergistic effects between hPRL and estrogen reflect the real physiological status because the breast tissue is constantly exposed to both newly synthesized estrogen and hPRL. These results also indicate the possibility of developing new therapeutic regimens, targeting possible tumor stimuli other than the ER. The potential for additive and therefore improved benefits is significant. We further demonstrated that hPRL-G129R possessed an inhibitory effect on T-47D cell proliferation (Fig. 5)
. More importantly, when anti-estrogen (4-OH-Tamoxifen) and anti-PRL (hPRL-G129R) agents were applied simultaneously, as we had anticipated, an additive effect was observed. The inhibitory effect on cell proliferation was more than doubled (Fig. 5)
. We reason that the direct inhibitory effects of hPRL-G129R on T-47D cell proliferation are by competitive inhibition of the hPRL produced by T-47D cells (11)
. The hPRLR-specific antagonistic effects of hPRL-G129R were further substantiated by an assay that uses combinations of hPRL and hPRL-G129R. It is encouraging to note that even at the ratio of 1:1, hPRL-G129R could stop the T-47D cell proliferation induced by hPRL (Fig. 6)
.
We speculated that if we could sustain the effects of hPRL-G129R by providing a continuous fresh supply of antagonist, we might obtain even better results than by a single application and prolonged incubation. To address this question, we designed the coculture experiments. When stable L cells that produce hPRL-G129R were cocultured with T-47D cells, much more dramatic inhibitory effects were observed (Fig. 7)
. The actual concentration of hPRL-G129R at the end of the experiment is approximately the same as the beginning high dose in the conditioned media experiment; yet apparently because these antagonists are produced continuously, the effects are more dramatic.
Apoptosis (programmed cell death) is one of the central physiological mechanisms that regulates the timely and orderly death of cells (37)
. The biochemical hallmark of apoptosis is internucleosomal DNA cleavage (38, 39, 40)
, and it can be detected by the TUNEL assay or by conventional gel electrophoresis (41)
. In this report, we have presented data to demonstrate that the hPRLR antagonist, hPRL-G129R, is able to induce apoptosis by DNA fragmentation in multiple human breast cancer cell lines. The hPRL-G129R induces apoptosis in a dose-dependent manner after 24-h treatment (Fig. 8)
. The DNA fragmentation in breast cancer cells is apparent even after 2 h of exposure to hPRL-G129R at a concentration of 50 ng/ml (Fig. 9)
. We further demonstrated the specificity of hPRL-G129R by using either hPRL or an anti-hPRL antiserum to reverse the apoptosis process (Fig. 8)
. The mitogen rescue effect of hPRL is yet another indication that hPRL-G129R induces apoptosis (39)
. To our surprise, 4-OH-Tamoxifen did not induce apoptosis in the cell lines we tested at concentrations as high as 1 µM, as assayed by the same protocol (data not shown), suggesting that a different mechanism might be involved. It also explains the additive inhibitory effects on cell proliferation when two agents (hPRL-G129R and 4-OH-Tamoxifen) were applied together (Fig. 5)
.
The mechanism of induction of apoptosis by this hPRLR antagonist needs further experimental elucidation. The mammary gland is one of the few organs that undergoes most of its development in the mature organism. More importantly, the mammary gland undergoes sequential waves of apoptosis during development and involution beginning with each pregnancy and ending with each weaning. We speculate that PRL might serve as one of the major controlling factors that decides whether the breast cells should go into proliferation/differentiation (by producing more PRL) or apoptosis (deprived of PRL) under physiological conditions. In the case of breast cancer, the cancer cells are adapted to using PRL as a major growth factor by producing PRL on their own (as an autocrine/paracrine growth factor), therefore maintaining their proliferative status. Hence, it is conceivable that when we effectively deprived the mitogenic signal of PRL in breast cancer cells by competitive binding of hPRL-G129R to the hPRLR, apoptosis is induced. Whatever the mechanism of hPRL-G129R-induced apoptosis of breast cancer cells, it is clear that the hPRLR antagonist hPRL-G129R has a strong potential to be used as another line of endocrine therapy along with Tamoxifen or by itself in the treatment of breast cancer.
In summary, the appalling death rate from breast cancer is still a major health care problem in the United States. History and biology have taught us that instead of finding a single magic "bullet" for breast cancer or for any tumor, we are more likely to improve the outcome of patients with oncogenic disease if we consider the heterogeneity of the disease and explore alternative and/or combination treatment regimens. We have reported in this paper a new agent to inhibit breast cancer development, hPRL-G129R, which acts as a hPRL antagonist. These results provided strong evidence of the involvement of hPRL in human breast cancer cell proliferation and also offer a novel approach for the treatment of breast cancer. It is our belief that the development of the hPRL antagonist will have a significant impact on effective human breast cancer therapy.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported in part by the Endowment Fund of the Greenville Hospital System and the Greenville Hospital System/Clemson University Biomedical Cooperative and a Grant BC980253 from the United States Army Medical Research Command. 
2 To whom requests for reprints should be addressed, at Oncology Research Institute, Greenville Hospital System, 900 West Faris Road, Greenville, SC 29605. Phone: (864) 455-1457; Fax: (864) 455-1567; E-mail: wchen{at}ghs.org
3 The abbreviations used are: hPRL, human prolactin; PRLR, PRL receptor; GH, growth hormone; hGH, human GH; bGH, bovine GH; RT-PCR, reverse transcription-PCR; ER, estrogen receptor; FBS, fetal bovine serum; IRMA, immunoradiometric assay; E2, estradiol; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling.
Received 3/18/99; revised 7/19/99; accepted 8/17/99.
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