Intracrine PTHrP Protects against Serum Starvation- Induced ...

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Endocrinology 143(2):596 – 606 Copyright © 2002 by The Endocrine Society

Intracrine PTHrP Protects against Serum StarvationInduced Apoptosis and Regulates the Cell Cycle in MCF-7 Breast Cancer Cells VERONICA A. TOVAR SEPULVEDA, XIAOLI SHEN,

AND

MIRIAM FALZON

Department of Pharmacology and Toxicology and Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77555 PTHrP is secreted by breast cancer cells in vivo and in vitro. In the breast cancer cell line MCF-7, PTHrP overexpression is associated with increased mitogenesis. We used this cell line to study the mechanism for the proliferative effects of PTHrP. Clonal MCF-7 lines were established overexpressing wildtype PTHrP or PTHrP mutated in the nuclear localization signal (NLS). Mutation of the NLS negated the proliferative effects and nuclear trafficking of PTHrP, indicating that increased mitogenesis is mediated via an intracrine pathway. Cells overexpressing wild-type PTHrP were enriched in G2 ⴙ M stage of the cell cycle compared with cells overexpressing NLS-mutated PTHrP, indicating an intracrine role for PTHrP

in cell cycle regulation. Wild-type PTHrP also protected MCF-7 cells from serum starvation-induced apoptosis. Cells overexpressing wild-type PTHrP showed significantly greater cell survival than cells overexpressing NLS-mutated PTHrP. The ratios of the apoptosis-regulating proteins Bcl-2 to Bax and Bcl-xL to Bax were higher in cells overexpressing wild-type, but not NLS-mutated, PTHrP compared with control cells. These findings suggest that the proliferative effects of PTHrP in breast cancer cells are mediated through regulation of the cell cycle and apoptosis, and that controlling PTHrP production in breast cancer may be therapeutically useful. (Endocrinology 143: 596 – 606, 2002)

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THrP WAS INITIALLY identified through its role in humoral hypercalcemia of malignancy, one of the most frequent paraneoplastic syndromes and a very common complication in breast cancer patients (1– 6). More recently, the protein was found to be widely distributed in most fetal and adult tissues (7–9). However, PTHrP is not normally present in the circulation (10), suggesting that it may act in an autocrine/paracrine manner. The mature peptide can undergo extensive posttranslational processing to generate secretory forms of PTHrP representing N-terminal, midregion, and C-terminal portions of the molecule (11, 12). Each region exhibits unique biological activities and presumably acts through its own cognate receptor (13, 14). However, only the PTH/PTHrP (PTH 1) receptor that binds PTH, PTHrP, and their N-terminal analogs has been cloned to date (15, 16). Physiological roles attributed to PTHrP include regulation of cell growth and differentiation, smooth muscle relaxation, promotion of transplacental calcium transport, and cartilage development (9, 17–21). The peptide is secreted in a regulated or constitutive fashion depending on the cell type (12). In addition to effects that are mediated via signal transduction cascades initiated at membrane receptors, PTHrP can also function in an intracrine manner after translocation to the nucleus or nucleolus (22–24). The PTHrP molecule contains a midregion nuclear localization sequence (NLS) comprising multibasic clusters in the 88 –106 region, which is similar to nuclear localization signals found in viral and mammalian transcription factors (22–25). Therefore, PTHrP

may function locally in an autocrine/paracrine or intracrine fashion. PTHrP is closely linked to normal mammary gland function (26), with enhanced expression during lactation when the mammary gland is in a proliferative state (27). Studies in transgenic mice have shown the involvement of the protein in branching morphogenesis of the mammary gland, indicating active participation in normal mammary development (28). Conversely, the absence of PTHrP in knockout mice leads to mammary epithelial degeneration (29). Most breast cancer cells secrete higher levels of PTHrP than do normal breast cells (1, 30). In fact, it has been proposed that PTHrP production by breast cancer cells may be one of the key elements instrumental in supporting carcinogenesis (31, 32). Therefore, the effects of PTHrP in breast cancer cells have both biological and clinical significance. PTHrP exerts a mitogenic effect in the breast cancer cell line MCF-7 (33). Here, we report that the increase in cell number is mediated via an intracrine pathway through both cell cycle regulation and inhibition of apoptosis through the Bcl-2 family of proteins. These findings suggest that endogenously produced PTHrP may enhance breast cancer growth via these pathways. Materials and Methods Materials Synthetic human (h) PTHrP-(1–34), hPTHrP-(1– 86), and hPTHrP(107–139) were purchased from Bachem (Torrance, CA). Recombinant hPTHrP-(1–139) was prepared using the IMPACT (intein-mediated purification with an affinity chitin-binding tag) method (New England Biolabs, Inc., Beverly, MA) (34). Peptide stocks (10⫺4 m) were prepared in 10 mm acetic acid. FBS and NuSerum were obtained from Atlanta Biologicals (Norcross, GA) and Collaborative Research (Bedford, MA),

Abbreviations: 7-AAD, 7-Aminoactinomycin D; FITC, fluorescein isothiocyanate; hPTH, human PTH; NCS, newborn calf serum; NLS, nuclear localization signal; PerCP, peridinin chlorophyll protein.

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respectively. Tissue culture supplies were purchased from Life Technologies, Inc. (Gaithersburg, MD). FuGENE 6 and annexin V were obtained from Roche Molecular Biochemicals (Indianapolis, IN). 7Aminoactinomycin D (7-AAD) and valinomycin were obtained from Sigma (St. Louis, MO). Anti-Bax (mouse IgG2b, clone B-9) antibody, anti-Bcl-xL (rabbit IgG, clone H-62) antibody, and isotype normal antirabbit IgG antibody were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Fluorescein isothiocyanate (FITC)-labeled anti-Bcl-2 (hamster IgG, clone 6C8) antibody, FITC-labeled hamster IgG antibody, biotin-labeled antirabbit IgG, and peridinin chlorophyll protein (PerCP)labeled streptavidin were obtained from PharMingen (San Diego, CA). Phycoerythrin-labeled goat antimouse IgG antibody, FITC-labeled goat antimouse IgG, and isotype mouse IgG2b antibody were obtained from Caltag Laboratories, Inc. (Burlingame, CA). Anti-PTHrP mouse monoclonal antibody and the FITC-labeled goat antimouse antibody (Alexa 488) were purchased from Oncogene Research Products (Cambridge, MA) and Molecular Probes, Inc. (Eugene, OR), respectively.

Plasmid constructs The PTHrP construct expressing wild-type PTHrP was constructed by cloning PTHrP cDNA coding for amino acids ⫺5 to ⫹139 (a gift from Dr. W. I. Wood, Genentech, Inc., South San Francisco, CA) in the sense orientation into the expression vector pcDNA3.1⫹ (Invitrogen, San Diego, CA; ⫹ refers to the orientation of the multiple cloning site within the vector, relative to the direction of transcription from the T7 promoter). This construct was used to prepare PTHrP cDNAs mutated over the NLS using a Transformer Site-Directed Mutagenesis Kit (CLONTECH Laboratories, Inc., Palo Alto, CA). The cDNAs encoded either a single deletion (elimination of residues 88 –91 or 102–106) or a double deletion (elimination of residues 88 –91 and 102–106). Mutations were confirmed by DNA sequencing. These constructs as well as the empty vector control pcDNA 3.1⫹ were transfected into MCF-7 cells using FuGENE 6 transfection reagent (Roche Molecular Biochemicals). The DNA template used to prepare the probe for Northern blot analysis was a 231-bp cloned DNA fragment spanning exons 3 and 4 of the PTHrP gene (35, 36).

Cell culture and stable transfection MCF-7 cells were obtained from American Type Culture Collection (Manassas, VA) and grown at 37 C in a humidified 95% O2-5% CO2 atmosphere in DMEM supplemented with 10% FBS and l-glutamine. MCF-7 cells were stably transfected using FuGENE 6 transfection reagent, according to the manufacturer’s specifications (Roche Molecular Biochemicals). Two days after transfection, 600 ␮g/ml G418 (Geneticin, Life Technologies, Inc.) were added, and resistant clones were selected. Single clones of stably transfected cells, isolated by limiting dilution in 96-well plates, were transferred to individual flasks and cultured in medium containing 150 ␮g/ml G418. Individual clones were tested for PTHrP production using an immunoradiometric assay (described below), and transfected mRNAs were detected by Northern blot analysis (also described below). To measure the effects of added peptides on cell cycle regulation and apoptosis, MCF-7 cells were plated in 100-mm dishes in the presence of 10% FBS. To measure apoptosis, cells were transferred to 0.1% NuSerum after 24 h. When they had reached about 30% confluence, the cells were treated with 10⫺8 or 10⫺7 m of the indicated hPTHrP peptides for 3 or 5 d. Control cells received an equivalent volume of 10 mm acetic acid as a vehicle control. When the cells were treated for 5 d, the growth medium was replaced with fresh peptide-containing medium after 3 d. The cells were then analyzed by FACS for their cell cycle or annexin V profile.

Northern blot analysis Total RNA was isolated using RNA STAT-60 (Tel-Test B, Friendswood, TX). RNA gel electrophoresis was performed under standard conditions (37), using 25 ␮g total RNA. The RNA was then blotted onto nitrocellulose (Schleicher & Schuell, Inc., Keene, NH) by capillary action. Probes for hybridization were prepared by the asymmetric PCR (38). The PCR template was a 231-bp cloned DNA fragment spanning exons 3 and 4 of the hPTHrP gene (35, 36). To detect RNA produced by the transfected sense PTHrP, an antisense probe was prepared using the down-

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stream primer from exon 4 of the human PTHrP gene (5⬘-GTTAGGGGACACCTCCGAGGT-3⬘). The blots were prehybridized for 30 min and hybridized for 2 h in Expresshyb (CLONTECH Laboratories, Inc.) at 65 C. After hybridization, the blots were washed twice in 2⫻ SSC/0.05% SDS for 15 min at room temperature and then twice in 0.1⫻ SSC/0.1% SDS at 50 C for 30 min. The washed membranes were exposed to Kodak X-Omat film (Eastman Kodak Co., Rochester, NY) at ⫺70 C with intensifying screens. The ethidium bromide-stained RNA on the nitrocellulose membrane was photographed immediately after transfer. 18S ribosomal RNA signal was used to provide a reference to normalize for equal RNA loading and transfer. The intensities of the bands representing PTHrP and 18S ribosomal RNA were evaluated using the Sigmagel program (Jandel Scientific, San Rafael, CA).

Immunoassay for secreted PTHrP The amount of PTHrP secreted into the culture medium was measured using an immunoradiometric sandwich assay (Nichols Institute Diagnostics, San Juan Capistrano, CA), as previously described (35). Single clones of transfected and control (untransfected) cells were plated in 24-well dishes (5 ⫻ 104 cells/well), and the medium was replaced after 24 h. Conditioned medium was collected after a further 4 d and frozen at ⫺80 C for future use; cell number was determined using a Coulter counter (Hialeah, FL). Unconditioned medium (never exposed to cells) served as a negative control. The assay was carried out according to the manufacturer’s specifications. The detection limit of the assay is 0.7 pmol/liter (39).

Determination of cell number To measure the effects of overexpression of wild-type and NLSmutated PTHrP on cell number, cells were plated into 24-well dishes at 104 cells/well in medium containing 10% FBS (d 0). Cells were then trypsinized and counted on d 1, 2, 4, and 7.

Thymidine incorporation assay For these experiments, cells were plated in 12-well plates at 2 ⫻ 104 cells/well in medium containing 10% FBS. After 3 d, when cells had reached about 70% confluence, they were pulse-treated with [3H]thymidine (0.2 ␮Ci/ml) for 3 h. To determine [3H]thymidine incorporation, the cell monolayer was washed twice with PBS, and nucleic acids in the cell fraction were precipitated with trichloroacetic acid and solubilized with 1 m sodium hydroxide for scintillation counting and protein determination. The protein concentration was measured using the Bio-Rad Laboratories, Inc. assay (Hercules, CA). The results were expressed as counts per ␮g protein.

PTHrP immunofluorescence labeling Cells grown on Lab-Tek chamber slides (Nalge Nunc International, Naperville, IL) were washed with cold PBS, fixed in acetone for 10 min at ⫺20 C, and washed again with PBS. Acetone permeabilizes the cells so they can stain intracellularly. The cells were then incubated in 50 mm ammonium chloride in PBS for 15 min at room temperature and washed with PBS. After blocking nonspecific protein-binding sites by preincubation with 5% milk (Novagen, Madison, WI) for 1 h at room temperature and washing with PBS, the cells were incubated with primary antibody (anti-PTHrP mouse monoclonal IgG, Oncogene Research Products) for 60 min at room temperature. Control cells received no primary antibody. After washing with cold PBS, the cells were treated with goat antimouse secondary antibody (Alexa 488, Molecular Probes) for 1 h at room temperature. The cells were washed again in PBS, mounted using a Prolong Antifade Kit (Molecular Probes), and analyzed using a fluorescence microscope (Olympus Corp., Melville, NY) with a wide-band green filter. The percentage of cells whose nuclei stained positively were counted in a blinded fashion by two observers using computerized histomorphometry (Optimus Corp., Bothell, WA).

Flow cytometry Cell cycle analysis of MCF-7 cells overexpressing wild-type and NLSmutated PTHrP was performed as follows. Cells were plated and grown

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in 100-mm culture dishes in medium containing 10% FBS. When the cells reached about 75% confluence, they were trypsinized and collected by centrifugation. The cell pellets were then suspended in 200 ␮l PBS, and 5 ml ice-cold 75% ethanol were added dropwise to the cell suspension with agitation at 4 C. The cells were collected by centrifugation, and the cell pellets were washed twice with PBS and resuspended in PBS containing 50 ␮g/ml propidium iodide (Sigma) and 50 ␮g/ml deoxyribonuclease-free ribonuclease A (Sigma). The cell suspension was incubated on ice for 1 h in the dark, then analyzed with a FACScan flow cytometer (Becton Dickinson and Co., Franklin Lakes, NJ). Early stage apoptosis was measured using annexin V-Fluos (Roche Molecular Biochemicals) in conjunction with 7-AAD (Sigma) to distinguish among early apoptosis, late apoptosis, and necrosis (40, 41). Cells were plated in T-75 flasks in medium containing 10% FBS and were transferred to medium containing 0.1% NuSerum after 24 h. When the cells had reached about 50% confluence, they were synchronized by treating with aphidicolin (10 ␮g/ml) for 16 h, then cultured in the absence of aphidicolin for a further 24 h. The cells were then trypsinized and counted, a fraction of the cells (0.2 ⫻ 106) was collected by centrifugation, and the pellet was washed twice with PBS. After a further centrifugation, the cell pellet was resuspended in 100 ␮l labeling solution containing 2 ␮l annexin V-FITC labeling reagent in 100 ␮l HEPES buffer [10 mm HEPES/NaOH (pH 7.4), 140 mm NaCl, and 5 mm CaCl2]. For the negative control, the cell pellet was resuspended in HEPES buffer in the absence of annexin V. After a 15-min incubation on ice, the cell suspension was collected by centrifugation and washed twice with PBS containing 1 mm CaCl2. The cell pellet was then resuspended in PBS and 1 mm CaCl2 containing 20 ␮g/ml 7-AAD. Apoptosis was measured by two-color flow cytometry on a FACScan flow cytometer (Becton Dickinson and Co.). Viable cells were electronically gated on forward and side scatter parameters. In addition, viable cells were gated on their ability to exclude the dye 7-AAD (40, 41). The expression of Bcl-2, Bcl-xL, and Bax was measured after aphidicolin synchronization (described above) by staining intracellularly with antibodies to Bcl-2, Bcl-xL, and Bax antibody (42). Briefly, 0.2 ⫻ 106 cells were incubated in 100 ␮l 0.2% saponin buffer [0.2% saponin in PBS containing 2% newborn calf serum (NCS), 5% nonfat dry milk, and 0.02% sodium azide] on ice for 30 min. Primary antibody was then added, and the cells were incubated on ice for a further 30 min. The cells were then washed twice with 1 ml 0.1% saponin buffer (0.1% saponin in PBS containing 2% NCS and 0.02% sodium azide), incubated on ice with secondary antibody for 30 min, and washed twice with 0.1% saponin buffer and once with PBS containing 2% NCS and 0.02% sodium azide. For calculation of the Bcl-xL/Bax ratio, cells were further incubated on ice with PerCP-labeled streptavidin for 30 min, then washed twice with 0.1% saponin buffer. The samples were analyzed on a FACScan flow cytometer (Becton Dickinson and Co.). Viable cells were electronically gated on forward and side scatter parameters. Acquired data were analyzed with CellQuest software (Becton Dickinson and Co.). The ratio of Bcl-2/Bax or Bcl-xL/Bax was calculated as follows: % Bcl-2- or BclxL-positive cells/% Bax-positive cells. Levels of Bcl-2 and Bax were calculated in the same cells by staining with FITC-labeled anti-Bcl-2 (or hamster IgG as isotype control) and anti-Bax (or mouse IgG2b as isotype control) as first antibodies and phycoerythrin-labeled goat antimouse IgG as second antibody. The levels of Bcl-xL and Bax were calculated in the same cells by staining with anti-Bcl-xL (or normal rabbit IgG as isotype control) and anti-Bax (or mouse IgG2b as isotype control) as first antibodies, FITC-labeled goat antimouse IgG and biotin-labeled goat antirabbit Ig as secondary antibodies, and PerCP-labeled streptavidin to bind to biotin.

Nuclear staining assay Cells were grown on Lab-Tek chamber slides (Nalge Nunc International) and at 50% confluence were transferred to medium containing 0.1% NuSerum for 24 h, then treated with the potassium ionophore valinomycin (40 ␮m) for 4 h (43). Control cells were kept in 10% FBS. Apoptosis was induced, and cells were then stained with the DNAbinding fluorescent dye Hoechst 33342 (10 ␮m in PBS) for 10 min. After washing with PBS, the cells were analyzed under a fluorescence microscope (Olympus Corp.) with excitation at UV 360 nm (44).


Statistics Numerical data are presented as the mean ⫾ sem. The data were analyzed by ANOVA, followed by a Bonferroni posttest to determine the statistical significance of differences. P ⬍ 0.05 was considered significant. All statistical analysis were performed using Instat Software (GraphPad Software, Inc., San Diego, CA).

Results Establishment and characterization of cell lines overexpressing NLS-mutated PTHrP

We have previously shown that transfection of MCF-7 cells with a sense PTHrP construct increases the proliferation of these cells (33). To determine whether the growth effects of PTHrP in MCF-7 cells require an intact NLS, cells were transfected with PTHrP cDNA constructs mutated at the NLS. Transfected wild-type and NLS-mutated sense PTHrP mRNAs were detected by Northern blot analysis. Transfected sense PTHrP mRNA was only detected in MCF-7 cells transfected with the sense wild-type and NLS mutant PTHrP expression constructs (⬃1.1-kb transcript; Fig. 1), but not in untransfected (Fig. 1) or empty vector-transfected (data not shown) cells. Detection of the endogenous 1.5-kb transcript required much longer exposure of the autoradiograms (33). PTHrP secretion was measured by immunoassay. The amount of PTHrP secreted by control cells was 1.3 ⫾ 0.04 pm/105 cells. PTHrP secretion from the empty vector-transfected cells was not significantly different from that from untransfected cells (Fig. 2). Transfection with the wild-type PTHrP constructs produced a significant increase in PTHrP production compared with empty vector-transfected and untransfected cells (Fig. 2). Similarly, each of the clones overexpressing NLS-mutated PTHrP (⌬88 –91, ⌬102–106, and ⌬88 –91 ⫹ ⌬102–106) secreted significantly higher PTHrP levels (Fig. 2). Secretion from these NLS-mutated PTHrP transfectants was also higher than that from the wild-type PTHrP transfectants. During the process of cloning, secretion from a minimum of four clones for wild-type PTHrP and each of the deletions was tested. Clones expressing NLSmutated PTHrP consistently showed higher levels of PTHrP secretion compared with clones expressing wild-type

FIG. 1. Characterization of MCF-7 cells overexpressing wild-type or NLS-mutated PTHrP. Northern blot analysis of total RNA was carried out to detect transfected transcript from untransfected (parent) cells (P) and from cells transfected with a vector expressing wild-type sense PTHrP mRNA (SN) or with vectors expressing NLS-mutated PTHrP mRNA (⌬1, ⌬88 –91; ⌬2, ⌬102–106; ⌬⌬, ⌬88 –91 ⫹ ⌬102–106). The probe was prepared by asymmetric PCR of a cDNA fragment spanning exons 3 and 4 of the human PTHrP gene, as described in Materials and Methods. The positions of the 5S, 18S, and 28S ribosomal bands are indicated. Top panel, PTHrP mRNA; bottom panel, 18S RNA.



firmed using the [3H]thymidine incorporation assay. After 3 d in culture, [3H]thymidine incorporation of wild-type sense PTHrP transfectants was approximately 1.7-fold that of empty vector transfectants (Fig. 4). This value matches the growth rate obtained by direct cell counting, where, after 4 d in culture, the number of wild-type PTHrP-overexpressing cells was approximately twice that of the empty vector controls (Fig. 3). The [3H]thymidine incorporation rate of the NLS-mutated PTHrP transfectants tended to be lower, but was not significantly different from that of empty vector transfectants (Fig. 4). We cannot at present explain the difference between the growth rate of empty vector-transfected and NLS-mutated PTHrP as measured by direct cell counting (⬃2.5-fold after 4 d in culture; Fig. 3) and by [3H]thymidine FIG. 2. PTHrP secretion by wild-type and NLS mutated PTHrP transfectants. Conditioned medium from MCF-7 cells transfected with a vector expressing wild-type or mutant NLS PTHrP was collected after 4 d in culture; secreted PTHrP was measured by the immunoradiometric assay. P, Untransfected (parent) cells; V, empty vector-transfected cells; SN, cells expressing wild-type PTHrP; ⌬1, ⌬2, and ⌬⌬, cells expressing PTHrP ⌬88 –91, ⌬102–106, and ⌬88 –91 ⫹ ⌬102–106, respectively. Each bar, representing an independent clone, is the mean ⫾ SEM of four wells obtained after subtracting the background value, represented by unconditioned medium (not exposed to cells). Asterisks denote significant differences from untransfected cells (P ⬍ 0.001).

PTHrP. Thus, clones overexpressing wild-type PTHrP secreted between 30 and 50 pm PTHrP/105 cells, whereas clones overexpressing NLS-mutated PTHrP secreted between 80 and 125 pm/105 cells. The empty vector transfectants secreted less than 1.5 pm PTHrP/105 cells. It may be possible that secretion of wild-type PTHrP on the same scale as that for NLS-mutated PTHrP is detrimental to cell viability, or that secretion levels are affected by the inability of NLS-mutated PTHrP to enter the nucleus. Two clones for wild-type PTHrP and each of the NLS mutants (⌬88 –91, ⌬102–106, and ⌬88 –91 ⫹ ⌬102–106) were chosen for further experiments; secretion from these clones is shown in Fig. 2. These clonal lines were used to examine whether the mutated NLS sequence influenced the ability of PTHrP to promote cell growth.

FIG. 3. Proliferation of MCF-7 cells overexpressing wild-type or NLS mutant PTHrP. Cells were plated in 10% FBS at a density of 104 cells/well in 24-well dishes. At the indicated time intervals, cells were trypsinized, and cell numbers were determined using a Coulter counter. Each point is the mean ⫾ SEM of three independent experiments for each of two individual clones (four wells per experiment). Where no error bar is shown, the SEM is smaller than the point. F, Empty vector; E, sense; , ⌬88 –91; f, ⌬102–106; ƒ, ⌬88 –91 ⫹ ⌬102– 106. *, Significantly different from control (empty vector transfectant) at P ⬍ 0.001.

Mutant NLS negates the proliferative effects of PTHrP overexpression

The growth rates of cells stably transfected with cDNAs expressing wild-type or NLS-mutated PTHrP are shown in Fig. 3. The growth rate of MCF-7 clones overexpressing wildtype PTHrP was more rapid than that of empty vector-transfected cells, such that after 4 and 7 d in culture, the cell number of the PTHrP-overexpressing clones was approximately 2 and 3 times that of the empty vector-transfected clones, respectively. On the other hand, the growth rate of the clones overexpressing NLS-mutated PTHrP was about 0.4 times that of empty vector-transfected clones (Fig. 3). NLS deletions still permit the secretion of large concentrations of N-terminally intact PTHrP, which may act to inhibit MCF-7 cell proliferation through an autocrine/paracrine pathway (33), but cannot increase cell proliferation through the intracrine pathway. The differences in the rate of proliferation of clones transfected with wild-type or NLS-mutated PTHrP were con-

FIG. 4. Thymidine incorporation by MCF-7 cells overexpressing wildtype or NLS-mutated PTHrP. Cells were plated in 12-well dishes at 2 ⫻ 104 cells/well in medium containing 10% FBS. After 3 d, cells were pulse-treated with [3H]thymidine for 3 h. Thymidine incorporation was determined as described in Materials and Methods. Each bar is the mean ⫾ SEM of six independent experiments (three experiments for each of two independent clones). P, Parent; V, empty vector; SN, sense; ⌬1, ⌬88 –91; ⌬2, ⌬102–106; ⌬⌬, ⌬88 –91 ⫹ ⌬102–106. *, Significantly different from control (empty vector transfectant) at P ⬍ 0.001.

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incorporation (Fig. 4). The small difference in the [3H]thymidine incorporation rate over 3 d of culture may account for a relatively larger difference in cell number. Mutant NLS inhibits nuclear localization

Immunoflurescence was used to detect the presence of PTHrP in transfected and control MCF-7 cells. Wild-type PTHrP showed strong nuclear and cytoplasmic staining (Fig. 5A). Cytoplasmic staining was present in all cells, whereas nuclear staining was present in approximately 70% of nuclei. Cells transfected with NLS-mutated PTHrP ⌬88 –91 and ⌬88 –91 ⫹ ⌬102–106 still showed strong cytoplasmic staining in all cells, but in this case nuclear staining was present in less than 10% of nuclei (Fig. 5A). The ⌬102–106 mutant showed approximately 35% nuclear staining (Fig. 5A). Cytoplasmic staining was present in all cells. Control (empty vector-transfected) cells showed significantly less intense cytoplasmic staining, and nuclear staining was present in about 25% of nuclei (Fig. 5A). Cells stained in the absence of primary antibody showed no cytoplasmic or nuclear staining (Fig. 5A). Data for nuclear staining in the different clones are summarized in Fig. 5B. Wild-type, but not NLS-mutated, PTHrP transfectants are enriched in G2 ⫹ M

To determine whether the proliferative effects of PTHrP overexpression are accompanied by an effect on the cell cycle profile, this parameter was measured in asynchronously cycling cells grown in 10% FBS. For this purpose, MCF-7 clones overexpressing wild-type or NLS-mutated PTHrP, and control (empty vector-transfected) cells were stained with propidium iodide and analyzed by flow cytometry to determine


the distribution of cells in the various stages of the cell cycle. All clones exhibited a major diploid peak (G0/G1). As shown in Fig. 6, A and B, cells overexpressing wild-type PTHrP were enriched in the G2 ⫹ M phase of the cell cycle (32% in G2 ⫹ M), whereas empty vector-transfected cells were largely in G1 (only 4% in G2 ⫹ M). Parental MCF-7 cells were also largely in G1 (only 3.7% in G2 ⫹ M; Fig. 6B). Cells transfected with the NLS mutants ⌬88 –91, ⌬102–106, and ⌬88 –91 ⫹ ⌬102–106) showed the same profile as empty vector-transfected cells (4.2– 6.7% in G2 ⫹ M; Fig. 6, A, B). We also determined the cell cycle profile of parental MCF-7 cells cultured in 10% FBS in the presence of hPTHrP(1–34), -(1–139), or -(107–139) for 3 or 5 d. Under these conditions, there was no difference in the cell cycle profile between treated and untreated cells (data not shown). As PTHrP decreases MCF-7 cell proliferation when acting through the autocrine/paracrine pathway (33), we expected that treatment with exogenous PTHrP analogs would result in fewer cells in G2 ⫹ M. The lack of any significant effect probably can be ascribed to the fact that a very low percentage of cells (3.7%) are normally in G2 ⫹ M in parental MCF-7 cells (data not shown). Wild-type, but not NLS-mutated, PTHrP inhibits apoptosis

Because growth factors and cytokines are known to play important roles in maintaining cell health and survival, serum deprivation has become a widely used strategy to induce apoptosis in a variety of cells (45– 47). In the early stages of apoptosis, phosphatidylserine migrates from the inner part of the plasma membrane to the outer layer, and thus becomes exposed on the external surface of the cell (48). Annexin V is a Ca2⫹-dependent phospholipid-binding pro-

FIG. 5. Immunofluorescence detection of PTHrP in MCF-7 cells overexpressing wild-type or NLS-mutated PTHrP. SN, Wild-type PTHrP transfectants; ⌬1, ⌬88 –91; ⌬2, ⌬102–106; ⌬⌬, ⌬88 –91 ⫹ ⌬102–106; V, empty vector transfectants; C, negative control (absence of primary antibody). A, Cells overexpressing wild-type PTHrP show abundant nuclear and cytoplasmic staining. Cells overexpressing NLS-mutated PTHrP show abundant cytoplasmic, but significantly less nuclear, staining. Cells transfected with empty vector show reduced cytoplasmic and nuclear staining. Magnification, ⫻100. B, The percentage of nuclei that contain PTHrP in the various MCF-7 clones. Each bar represents the mean of 8 slides (4 slides for each of 2 individual clones), and 100 –200 cells were counted per slide in a blinded fashion by 2 observers. Data are presented as the mean ⫾ SEM. *, P ⬍ 0.01.



tein with high affinity for phosphatidylserine. Apoptosis in serum-depleted (0.1% NuSerum) transfected, clonal MCF-7 cells was evaluated using annexin V as a marker of early stage apoptotic cells. This methodology was chosen over DNA fragmentation techniques because a number of reports have concluded that apoptosis in MCF-7 cells is not associated with DNA degradation (49 –51). Annexin V labeling was measured by FACS analysis. When coupled with 7-AAD, this method differentiates between apoptotic and necrotic cells (40, 41). After serum starvation, cells overexpressing wild-type PTHrP showed significantly less apoptosis than control cells (Fig. 7). The level of apoptosis in cells expressing NLS-mutated PTHrP was not significantly different from that in control cells (Fig. 7). Apoptosis in parental MCF-7 cells cultured in serumdepleted medium (0.1% NuSerum for 24, 48, or 72 h) was also measured by FACS analysis in the presence of hPTHrP-(1– 34), -(1–139), or -(107–139). None of the analogs, tested at a dose of 10⫺7 m for each of the three time points, influenced the extent of apoptosis, as determined by FACS analysis (data not shown). Wild-type and NLS-mutated PTHrP-overexpressing cells express different levels of the Bcl-2 family of proteins

The Bcl-2 family of proteins includes central regulators of the apoptotic pathway regardless of the stimulus for apo-

FIG. 6. Cell cycle analysis of wild-type or NLS-mutated PTHrP overexpressing and empty vector-transfected MCF-7 cells. Cells were plated in 10% FBS and at 75% confluence were collected and analyzed by flow cytometry as described in Materials and Methods. SN, Wildtype PTHrP transfectants; V, empty vector transfectants; ⌬1, ⌬88 –91; ⌬2, ⌬102–106; ⌬⌬, ⌬88 –91 ⫹ ⌬102–106. A, Cell cycle profile. B, Percentage of cells in G2 ⫹ M. Each bar is the mean ⫾ SEM of six independent experiments (three experiments for each of two individual clones). *, Significantly different from control (empty vector transfectant) at P ⬍ 0.001.

FIG. 7. Annexin V staining of MCF-7 cells overexpressing wild-type or NLS-mutated PTHrP. Cells were plated in 10% FBS, then transferred to 0.1% NuSerum after 24 h. At 50% confluence, they were synchronized with aphidicolin and analyzed by FACS analysis as described in Materials and Methods. Viable cells were gated based on their forward and side light scatter characteristics. Additional gating of viable cells was based on their ability to exclude 7-AAD. A, Annexin V vs. 7-AAD staining of cells. Percentages refer to the number of annexin V-positive cells in the lower right quadrant. B, Percentage of annexin V-stained cells (from values in the lower right quadrant). SN, Sense; V, empty vector; ⌬1, ⌬88 –91; ⌬2, ⌬102–106; ⌬⌬, ⌬88 –91 ⫹ ⌬102–106. Each bar is the mean ⫾ SEM of six independent experiments (three experiments for each of two individual clones). *, Significantly different from all other values at P ⬍ 0.001.

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ptosis. Certain family members (e.g. Bcl-2 and Bcl-xL) act as potent suppressors of apoptosis, whereas others (e.g. Bax and Bak) have opposing functions and promote cell death. The ratio of anti- to proapoptotic molecules determines the response to a death signal (52–54). We performed FACS analysis to determine whether PTHrP alters the expression of these apoptosis-related proteins. Figures 8 and 9 show an increase in Bcl-2/Bax and Bcl-xL/Bax ratios in wild-type PTHrP-overexpressing cells compared with those in NLSmutated PTHrP-overexpressing and vector control cells. Wild-type, but not NLS-mutated, PTHrP inhibits nuclear degradation

We compared the nuclear morphology of MCF-7 cells overexpressing wild-type or NLS-mutated PTHrP after treatment with valinomycin, a potassium ionophore that gives rise to atypical cell death, with chromatin condensation appearing with (43) or without (55) DNA fragmentation. This treatment was chosen because, compared with serum starvation alone, it produced very dramatic differences in terms of nuclear vulnerability between wild-type PTHrP-overexpressing vs. control (empty vector-transfected) and NLSmutated PTHrP-overexpressing cells. Cells cultured in 10%

FIG. 9. Determination of the Bcl-xL/Bax ratio in MCF-7 cells overexpressing wild-type or NLS-mutated PTHrP. Cells were plated in 10% FBS, then transferred to 0.1% NuSerum after 24 h. At 50% confluence, they were synchronized with aphidicolin and analyzed for intracellular Bcl-xL and Bax by FACS analysis as described in Materials and Methods. SN, Sense; V, empty vector; ⌬1, ⌬88 –91; ⌬2, ⌬102–106; ⌬⌬, ⌬88 –91 ⫹ ⌬102–106. A, Dot plot of Bcl-xL vs. Bax staining of cells. B, Bcl-xL/Bax ratio, calculated as described in Materials and Methods. Each bar is the mean ⫾ SEM of six independent experiments (three experiments for each of two individual clones). Where no error bar is shown, the SEM is smaller than the bar line. *, Significantly different from all other values at P ⬍ 0.01.

FIG. 8. Determination of the Bcl-2/Bax ratio in MCF-7 cells overexpressing wild-type or NLS-mutated PTHrP. Cells were plated in 10% FBS, then transferred to 0.1% NuSerum after 24 h. At 50% confluence, they were synchronized with aphidicolin and analyzed for intracellular Bcl-2 and Bax by FACS analysis as described in Materials and Methods. SN, Sense; V, empty vector; ⌬1, ⌬88 –91; ⌬2, ⌬102–106; ⌬⌬, ⌬88 –91 ⫹ ⌬102–106. A, Dot plot of Bcl-2 vs. Bax staining of cells. B, Bcl-2/Bax ratio, calculated as described in Materials and Methods. Each bar is the mean ⫾ SEM of six independent experiments (three experiments for each of two individual clones). *, Significantly different from all other values at P ⬍ 0.01.

serum in the absence of valinomycin show predominantly round or oval nuclei (healthy nuclei; Fig. 10A). The predominant effect of valinomycin treatment in MCF-7 cells overexpressing wild-type PTHrP was nuclear condensation (micronuclei; Fig. 10, A and B). There were no degraded nuclei. In contrast, valinomycin treatment of empty vector-transfected cells predominantly caused nuclear degradation (Fig. 10, A and B). Nuclei from cells overexpressing NLS-mutated PTHrP (⌬88 –91 and ⌬88 –91 ⫹ ⌬102–106) were either condensed or degraded, with very few healthy nuclei (Fig. 10, A and B). There were approximately 30% healthy nuclei in valinomycin-treated cells overexpressing the ⌬102–106 PTHrP mutant, with about 60% micronuclei and the rest degraded nuclei (Fig. 10, A and B). Discussion

Determining the factors involved in promoting breast cancer cell growth is crucial for understanding the pathogenesis of the disease. This study has examined the mechanism by which PTHrP enhances growth of the breast cancer cell line MCF-7. Our main findings are that overexpression of PTHrP in clonal MCF-7 cells stably transfected with PTHrP cDNA


FIG. 10. DNA staining with Hoechst 33342 of MCF-7 cells overexpressing wild-type or NLS-mutated PTHrP. U, Control cells (10% serum); all other cells were treated with valinomycin (40 ␮M) for 4 h in the presence of 0.1% NuSerum. SN, Wild-type PTHrP transfectants; V, empty vector transfectants; ⌬1, ⌬88 –91; ⌬2, ⌬102–106; ⌬⌬, ⌬88 –91 ⫹ ⌬102–106 NLS-mutated PTHrP transfectants. A, Arrows point to condensed nuclei (micronuclei). Magnification, ⫻100. B, The percentage of micronuclei and degraded nuclei in the various MCF-7 clones. Each bar represents the mean ⫾ SEM of 8 slides (4 slides for each of 2 individual clones), and 50 –100 cells were counted per slide in a blinded fashion by 2 observers. *, Significantly different from respective vector control at P ⬍ 0.01.

protects against apoptosis induced by serum starvation. PTHrP overexpression also increased [3H]thymidine incorporation and the percentage of cells in the G2 ⫹ M phase of the cell cycle, indicating that the peptide increases the mitotic rate in these cells. Taken together, these effects on apoptosis and cell cycle regulation account for the observed increased growth rate of PTHrP-overexpressing MCF-7 cells (33). Regulation of cell proliferation by PTHrP can be mediated via autocrine/paracrine or intracrine pathways. We have previously shown that exogenously added N-terminal or full-length PTHrP exerts an antimitogenic effect in MCF-7 cells, and that this effect is mediated via the cell surface PTH/PTHrP receptor, which is present and functional in these cells (33). These same exogenously added analogs had no effect on apoptosis, suggesting that the antiapoptotic effects are mediated solely via an intracrine pathway. It is noteworthy, however, that nuclear targeting of PTHrP can be accomplished via protein that is endo-


cytosed from the cell exterior, even in cells that do not express the PTH/PTHrP receptor (56 –58), providing nuclear targeting of exogenously added PTHrP. However, MCF-7 cells may lack the necessary cellular component(s) to endocytose and translocate PTHrP to the nucleus. In these cells, endogenous PTHrP may normally translocate to the nucleus without ever leaving the cytosol. Such a mechanism has been proposed by workers who propose that alternative translation start sites may bypass the PTHrP N-terminal signal sequence and result in a peptide that cannot enter the endoplasmic reticular space to be secreted, but, having the NLS sequence, can travel directly to the nucleus (56). In addition, we have not tested all available PTHrP analogs [e.g. PTHrP-(38 – 64) and -(87–106)]. The basic question of whether the antiapoptotic effects of PTHrP observed in this study involved a nuclear action was addressed by overexpressing PTHrP with a mutated NLS, an approach often used to answer this question (47, 56 – 60). The results, obtained with MCF-7 cells overexpressing mutant PTHrP, showed unequivocally that deleting either multibasic cluster (⌬88 –91 or ⌬102–106) or both (⌬88 –91 ⫹ ⌬102– 106) in the NLS of PTHrP negated its ability to protect against apoptosis. Mutant PTHrP was also ineffective in preventing against valinomycin-induced nuclear degradation. These findings indicate that an intact NLS is required for the PTHrP-mediated protective effects against apoptosis and suggest that this action requires that PTHrP enter the nucleus. The protective effects of PTHrP on apoptosis and survival have been noted in a number of other systems, including chondrocytes (23), cerebellar granule cells (61), and prostate cancer cells (62). The mechanisms underlying the ability of PTHrP to protect against apoptosis may be mediated via the Bcl-2 family of proteins. The proapoptotic protein Bax binds to the antiapoptotic Bcl-2 and Bcl-xL through interaction of their BH3 domains, forming heterodimers (63, 64). The balance of homodimers of Bcl-2 and Bcl-xL to Bcl-2/Bax and Bcl-xL/Bax heterodimers influences cell fate (53, 54, 65). When Bax is expressed preferentially, Bax homodimers predominate, promoting apoptosis. When Bcl is expressed preferentially, Bcl homodimers form, and apoptosis is inhibited. Therefore, the ratio of expression of Bcl-2 and Bcl-xL to Bax influences cell survival. Overexpression of PTHrP increases the Bcl-2/Bax and Bcl-xL/Bax ratios, suggesting that the protective effect of wild-type PTHrP overexpression against apoptosis may be mediated via this pathway. Whether additional pathways are involved in these PTHrP effects are at present unknown and are currently being addressed. PTHrP overexpression has also been found to modulate Bax and Bcl-xL levels in IEC-6 cells, a nontumor rat intestinal crypt cell line (47). Interestingly, in this cell line, PTHrP overexpression enhanced apoptosis via an intracrine pathway, and Bcl-xL expression was decreased whereas Bax expression was increased (47). Overall, the peptide still increased the proliferation of IEC-6 cells. It is at present not understood how the peptide can exert opposing effects in different cell lines. However, both proliferative and antiproliferative effects have pre-

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viously been attributed to PTHrP, depending on the cell type (33, 56, 59, 66, 67). Deletion of either one or both of these multibasic clusters within the NLS also abolished the proliferative effects of PTHrP in MCF-7 cells. Similar results have been reported in a number of studies, including normal rat small intestinal crypt cell-derived IEC-6 cells (59), vascular smooth muscle cells (56), and COS-1 cells (23). Mutation of the NLS also abolished the effect of PTHrP on the cell cycle, thus supporting an intracrine role for the peptide in cell cycle regulation. Decreased proliferation of the NLS mutants was reflected by a decrease in nuclear localization, as determined by immunofluorescence. Thus, significantly more nuclei stained positively for PTHrP in cells transfected with the wild-type peptide than in cells transfected with NLS mutants or empty vector. Interestingly, more nuclei stained positively for PTHrP in the ⌬102–106 mutant compared with the ⌬88 –91 and the ⌬88 –91 ⫹ ⌬102–106 mutants. The single NLS-mutated PTHrP clones target two different regions of PTHrP. Amino acids 88 –91 are recognized by ␤-importin (58), and therefore the presence of this sequence in the ⌬102– 106 mutant may still permit some trafficking of PTHrP to the nucleus. However, the increased nuclear presence of PTHrP in the ⌬102–106 mutant did not appear to influence the growth rate (as measured by direct cell counts, [3H]thymidine incorporation, and the cell cycle profile) or extent of apoptosis, compared with the ⌬88 –91 and ⌬88 –91 ⫹ ⌬102– 106 mutants. Taken together, these data demonstrate a critical role for nuclear targeting in the antiapoptotic and cell cycle regulatory effects of PTHrP. PTHrP plays an important role in breast cancer. Breast cancer cells secrete high levels of PTHrP, accounting for the high prevalence of humoral hypercalcemia of malignancy in breast cancer patients (68), and there is a positive correlation between PTHrP expression in breast cancer and skeletal metastasis (69 –71). In a mouse model of bone metastases, neutralizing antibodies to PTHrP-(1–34) inhibited the development of breast carcinoma metastases to bone of the human breast carcinoma cells MDA-MB-231, which produce moderate amounts of PTHrP (72). Conversely, in the same mouse model, overexpression of PTHrP by MCF-7 cells, which normally express low levels of PTHrP and do not cause osteolytic metastases, induced marked bone destruction associated with increased osteoclast formation compared with controls (73). These data suggest that tumor production of PTHrP is important for the establishment and progression of osteolytic bone metastases. However, Henderson et al. (74) report that patients presenting with PTHrP-positive breast cancers have a more favorable outcome and have fewer metastases to bone and other sites. This study used an antiPTHrP-(1–14) antibody to detect PTHrP by immunocytochemistry (74). This antibody may not recognize all forms of PTHrP generated by posttranslational processing. In addition, the intracrine form(s) of PTHrP has not all been identified and again may not be recognized by this antibody. It is possible that breast cancer growth and bone metastasis are mediated by specific forms of PTHrP not recognized by anti-PTHrP-(1–14) antibody. Therefore, these findings do not exclude a central role for PTHrP in breast cancer growth and metastasis. The PTHrP-mediated modulation of cell prolif-


eration through apoptosis and cell cycle regulatory pathways in breast cancer is of both physiological and clinical relevance. We believe that ours is the first report demonstrating the mechanisms responsible for imparting a growth advantage to PTHrP-expressing breast cancer cells. In summary, our studies show that overexpression of PTHrP in clones of MCF-7 cells stably transfected with PTHrP cDNA significantly increases the proliferation of these cells through both antiapoptotic and cell cycle regulatory effects. The presence of an intact NLS within PTHrP is required for this action. These findings suggest that PTHrP, produced in abundance by breast cancer cells, may increase the proliferation of these cells in an intracrine manner, thereby establishing a positive regulatory loop between protein production and breast tumor burden. Acknowledgments We thank Dr. Rolf Konig for advice with FACS analysis, and Drs. David Konkel, Patricia K. Seitz, Mary L. Thomas, and Cheryl S. Watson for critical reading of the manuscript. Received July 19, 2001. Accepted October 15, 2001. Address all correspondence and requests for reprints to: Dr. Miriam Falzon, Department of Pharmacology and Toxicology and Sealy Center for Molecular Science, University of Texas Medical Branch, 10th and Market Streets, Galveston, Texas 77555. E-mail: [email protected].

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