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J. Biol. Chem., Vol. 280, Issue 29, 27076-27084, July 22, 2005

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Antisense-mediated Inhibition of the Plasma Membrane Calcium-ATPase Suppresses Proliferation of MCF-7 Cells^*

Won Jae Lee, Jodie A. Robinson, Nicola A. Holman, Martin N. McCall, Sarah J. Roberts-Thomson, and Gregory R. Monteith¶

From the School of Pharmacy, University of Queensland, Brisbane, Queensland 4072, Australia and Acyte Biotech Pty. Ltd., Sydney, New South Wales 2052, Australia

Received for publication, December 16, 2004 , and in revised form, April 15, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Alterations in Ca²⁺ signaling may contribute to tumorigenesis and the mechanism of action of some anti-cancer drugs. The plasma membrane calcium-ATPase (PMCA) is a crucial controller of intracellular Ca²⁺ signaling. Altered PMCA expression occurs in the mammary gland during lactation and in breast cancer cell lines. Despite this, the consequences of PMCA inhibition in breast cancer cell lines have not been investigated. In this work, we used Tet-off PMCA antisense-expressing MCF-7 cells to assess the effects of PMCA inhibition in a human breast cancer cell line. At a level of PMCA inhibition that did not completely prevent PMCA-mediated Ca²⁺ efflux and did not induce cell death, a dramatic inhibition of cellular proliferation was observed. Fluorescence-activated cell sorting analysis indicated that PMCA antisense involves changes in cell cycle kinetics but not cell cycle arrest. We concluded that modulation of PMCA has important effects in regulating the proliferation of human breast cancer MCF-7 cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Free intracellular Ca²⁺, acting as a second messenger, is crucial for a diverse range of biological functions, such as fertilization, neurotransmission, muscle contraction, and gene transcription (1, 2). Intracellular Ca²⁺ signaling is also a key regulator of proliferation (3), cell cycle progression (4), and apoptosis (5). Modulation of capacitative Ca²⁺ entry, a mechanism whereby the influx of extracellular Ca²⁺ is coupled to the depletion of intracellular Ca²⁺ stores within the endoplasmic reticulum, is associated with the proliferative phenotype (6). Indeed, blockers of Ca²⁺ entry may be potential anti-proliferative agents (7, 8). Inhibition of Ca²⁺/calmodulin-dependent serine/threonine protein kinase II also arrests HeLa cells in the G₂ phase of the cell cycle (9), and apoptotic resistance conferred by overexpression of the anti-apoptotic oncoprotein Bcl-2 involves modulation of Ca²⁺ handling by the endoplasmic reticulum (5) and capacitative Ca²⁺ entry (10, 11). Although the essential role of Ca²⁺ signaling in maintaining homeostatic functions is well established, less is known about the role of aberrant Ca²⁺ signaling in disease (12, 13).

The plasma membrane Ca²⁺-ATPase (PMCA),¹ or pump, belongs to the family of P-type ATPases and is a critical regulator of free intracellular Ca²⁺ (14). It actively extrudes Ca²⁺ across the plasma membrane and is important for maintaining basal cytosolic Ca²⁺ levels and lowering free intracellular Ca²⁺ after the generation of Ca²⁺ transients and other Ca²⁺ signaling events (2, 15). PMCA has important roles in shaping the dynamics of Ca²⁺ signaling, as its activation can limit Ca²⁺ influx during capacitative Ca²⁺ entry (16). Moreover, PMCA overexpression decreases both the amplitude and duration of cytosolic Ca²⁺ responses after agonist-induced stimulation (17).

There are four isoforms of PMCA (PMCA1-4) with additional isoform diversity generated by alternative splicing from the primary PMCA transcripts (14, 18). Up-regulation of PMCA2 and down-regulation of PMCA4 in the rat mammary gland during lactation may indicate that PMCA isoforms regulate differentiation and proliferation of the mammary gland (19).

There is also an increasing awareness that PMCA alterations are associated with tumorigenesis, including that of the mammary gland (20). Indeed, relative PMCA1 mRNA expression is greater in tumorigenic breast cancer MCF-7 and MDA-MB-231 cells than that in non-tumorigenic MCF-10A cells (21). PMCA1 and PMCA4 expression is also lower in simian virus 40-transformed human skin fibroblasts (22). Thus, perturbed PMCA regulation or function may be important in diseases such as cancer. Therapeutic modulation of PMCA expression and/or activity may have functional consequences in suppressing the tumorigenic phenotype. The appropriateness of PMCA as a possible drug target is also demonstrated by the prominence of other clinically used inhibitors of P-type ATPases, digoxin and omeprazole, as inhibitors of Na⁺/K⁺-ATPase and H⁺/K⁺-ATPase, respectively (23, 24).

Despite the dynamic regulation of PMCA expression in the mammary gland, the consequences of PMCA inhibition in breast cancer cell lines has not been addressed. Our aim was to investigate whether PMCA inhibition alters Ca²⁺ homeostasis, cell proliferation, and cell death in MCF-7 human breast cancer cells.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cell Culture—MCF-7 Tet-off cells (Clontech) were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 4 mM L-glutamine, 100 µg/ml G418, 100 units/ml penicillin G, and 100 µg/ml streptomycin with the addition of 110 µg/ml hygromycin B and 100 ng/ml doxycycline (DOX) as required. All cultures were maintained at 37 °C in a humidified 5% CO₂/95% air incubator.

Construction of PMCA Antisense cDNA and Cloning—A 286-base-pair cDNA fragment was amplified by reverse transcription-PCR from MCF-7 Tet-off cells and cloned into the bidirectional pBI-G response plasmid (Clontech) to generate the PMCA antisense construct. The following primers were used to amplify the cDNA fragment: forward primer, 5'-TAGGTCGACGAAATGTCTATGACAGCAT-3'; and reverse primer, 5'-TAGGTCGACGGGCCCATTATCTTCTTCATCAT-3'. These primers were based on sequence information that is conserved across all human PMCA isoforms. A SalI restriction enzyme site (italics) was introduced into each primer to facilitate cloning, and an ApaI site (underlined) was incorporated into the reverse primer to assist in directional cloning. The PCR product was digested with SalI and inserted into the SalI site of the pBI-G plasmid in the antisense orientation. Following nucleotide sequencing of the antisense construct, the cDNA fragment sequence was compared against GenBank^TM using BLAST version 2.2.8.

Generation of a Double Stable MCF-7 Tet-off PMCA Antisense-transfected Cell Line—Parental MCF-7 Tet-off cells already stably transfected with the regulatory tetracycline-controlled transactivator were co-transfected using Lipofectamine 2000 (Invitrogen) with the prepared pBI-G PMCA antisense plasmid and the pTK-Hyg hygromycin B resistance plasmid (Clontech). Hygromycin B-resistant clones were isolated (total of 43) and screened for tetracycline-regulated -galactosidase expression. -Galactosidase acted as an indirect reporter for the simultaneous expression of PMCA antisense RNA when DOX was removed from the culture medium. Screening was performed using the -galactosidase enzyme assay system with reporter lysis buffer in accordance with the manufacturer's instructions (Promega, Annandale, New South Wales, Australia). Eight colonies that showed differing degrees of tetracycline-regulated -galactosidase activity (2-10-fold increases upon DOX removal) were further screened for their effect on PMCA protein with the non-isoform-specific PMCA antibody 5F10. From these colonies, three clones were assessed in subsequent proliferation assays and from the three, one clone (A7) was selected for more detailed characterization.

Proliferation Assays—Cells were plated at 800 cells/well in 96-well plates by initially seeding them in DOX- and hygromycin B-free medium (100 µl/well). DOX was then added to or omitted from the wells at the time of plating (day 0). The cells were grown over a 2-week period with the medium renewed every 2-3 days, except on the last day of the experiment. The proliferation of viable cells was monitored daily from days 3 to 13 via a colorimetric MTS assay (25) by directly adding CellTiter 96® AQueous One Solution reagent (20 µl; Promega) to each well and incubating plates for 2 h at 37 °C in a humidified 5% CO₂/95% air environment. After 2 h, absorbances were read at 490 nm with a Bio-Rad Model 550 microplate reader.

Immunoblotting—Protein was isolated from cells plated into 10-cm diameter dishes at 1 x 10⁵ cells/dish. The cells were seeded in DOX- and hygromycin B-free medium and then were adjusted at the time of plating to include hygromycin B, either with or without DOX (100 ng/ml). On days 3, 7, and 13, the cells were trypsinized and stored as cell pellets at -80 °C until further processing for total protein isolates. Total protein was analyzed by immunoblotting, as described previously (26). For each sample, 40 µg of total protein was loaded onto 7.5% gels. Antibodies were used at the following dilutions and incubated for 1 h at room temperature: 1:2000 for 5F10 (Affinity BioReagents, Golden, CO), 1:1000 for JA9 (kind gift from Dr. John T. Penniston, Department Biochemistry and Molecular Biology, Mayo Foundation, Rochester, MN), 1:1000 for NR1 (Affinity BioReagents), 1:1000 for NR2 (Affinity BioReagents), 1:1000 for IID8 (Affinity BioReagents), and 1:10,000 for anti--actin (AC-15, Sigma). A goat anti-mouse or goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody (Bio-Rad) was diluted 1:2000 or 1:1000, respectively, and used as required with incubations for 1 h at room temperature. Proteins were visualized via enhanced chemiluminescence (ECL^TM, Amersham Biosciences). Densitometry measurements of immunoblot bands were analyzed using MetaMorph® version 4.01 imaging software (Universal Imaging Corporation, Downingtown, PA). For each immunoblot, the optical density values for bands representing total PMCA, PMCA4, PMCA1, PMCA2, and SERCA2 were normalized to the corresponding value for the -actin band in each lane.

Fluorescence-activated Cell Sorting (FACS) Analysis of DNA Content—Cells were plated at 5 x 10⁵ cells/dish in 10-cm diameter dishes, in DOX- and hygromycin B-free medium and then grown in either the presence or absence of DOX. On days 7 and 13, the cells were harvested from the dishes by collecting trypsinized cells together with floating cells in the medium. For each condition, a volume of the cell suspension corresponding to 2 x 10⁶ cells was centrifuged, and the resultant cell pellet was resuspended in ice-cold phosphate-buffered saline (0.5 ml). The cells were fixed in ice-cold 70% ethanol and stained with propidium iodide. FACS analysis was performed using a BD Biosciences FACS-Calibur^TM flow cytometer. DNA content histograms and cell cycle phase distributions were modeled from at least 10,000 single events by excluding the cell aggregates based on scatter plots of the fluorescence pulse area versus fluorescence pulse width using ModFit LT^TM version 2.0 (Mac) software (Verity Software House, Topsham, ME).

5-Bromo-2'-deoxyuridine (BrdUrd) Pulse-Chase Analysis of Cell Cycle Progression—Cells that had been growing in the presence or absence of DOX-containing medium for 6 days were plated at 1 x 10⁶ cells/dish in 10-cm diameter dishes. After a further 24 h in the appropriate medium (±DOX), the cells were pulsed with either Dulbecco's phosphate-buffered saline or 5-bromo-2'-deoxyuridine 10 µM (BrdUrd) for 1 h, followed by one wash with Dulbecco's phosphate-buffered saline. After washing, the cells were then maintained in the appropriate medium (±DOX) until being trypsinized, centrifuged at 300 x g for 5 min, and fixed at 0, 3, 6, 12, and 24 h post-BrdUrd pulse. Collected cells were fixed and stained with the BD Biosciences fluorescein isothiocyanate BrdUrd flow kit, according to the manufacturer's instructions. Briefly, paraformaldehyde-fixed and saponin-permeabilized cells were incubated for 1 h at 37 °C with DNase (300 µg/ml in Dulbecco's phosphate-buffered saline). BrdUrd and total DNA were then stained with a fluorescein isothiocyanate-conjugated anti-BrdUrd antibody and 7-amino-actinomycin D (7-AAD), respectively. Data were acquired using a BD Biosciences FACSCalibur^TM flow cytometer with BD Biosciences CellQuest^TM version 3.3 software. For each sample, data were displayed using WinMDI version 2.8 (Joseph Trotter, facs.scripps.edu/software.html) by first gating at least 10,000 events to select a population of single cells based on density plots of the 7-AAD fluorescence pulse area (FL3-A) versus the 7-AAD fluorescence pulse width (FL3-W). Using this subpopulation of cells, density plots of fluorescein isothiocyanate-conjugated anti-BrdUrd antibody fluorescence pulse height (FL1-H) versus 7-AAD fluorescence pulse height (FL3-H) were generated. Negative BrdUrd controls, where cells were pulsed with Dulbecco's phosphate-buffered saline, were used to set the positions of quadrant markers.

Assessment of Cell Morphology—Double stable MCF-7 Tet-off PMCA antisense-transfected cells were plated onto 25-mm diameter coverslips placed in a 6-well plate at 1.8 x 10⁴ cells/well and grown either in the presence or absence of DOX. On day 13 of the culture, phase contrast images at 200x magnification were taken from five random fields using a Nikon Eclipse TE300 microscope (Nikon Instech, Kawasaki, Japan) and MetaFluor® version 4.01 imaging software (Universal Imaging Corporation, Downingtown, PA).

Assessment of Calcium Efflux—The efflux of free intracellular Ca²⁺ was assessed using a fluorescence microplate reader to monitor the rate of decline in free intracellular Ca²⁺ concentration ([Ca²⁺]_i) after the generation of ionomycin-induced Ca²⁺ transients that ranged in magnitude with varying concentrations of the Ca²⁺ ionophore. Subconfluent double stable MCF-7 Tet-off PMCA antisense cells grown in DOX were subcultured at a split ratio of 1:10 into new T-75 cm² flasks containing fresh medium, either with or without DOX (100 ng/ml), and grown for 6 days. After 6 days, the cells were plated into polystyrene 96-well plates at 4 x 10⁴ cells/well in growth medium (100 µl/well) with or without DOX, as previously treated. The cells were allowed to adhere for 24 h before loading the cells with the non-ratiometric Ca²⁺ indicator fluo-4 AM (8 µM; Molecular Probes, Eugene, OR) in the loading buffer. The loading buffer (pH 7.3) was composed of 5.9 mM KCl, 1.4 mM MgCl₂, 10 mM HEPES, 1.2 mM NaH₂PO₄, 5 mM NaHCO₃, 140 mM NaCl, 11.5 mM glucose, 1.8 mM CaCl₂, and 3 mg/ml bovine serum albumin. The cells were loaded at 37 °C for 30 min and washed once with loading buffer (no indicator) and once with physiological salt solution (PSS). PSS (pH 7.3) contained 5.9 mM KCl, 1.4 mM MgCl₂, 10 mM HEPES, 1.2 mM NaH₂PO₄, 5 mM NaHCO₃, 140 mM NaCl, 11.5 mM glucose, and 1.8 mM CaCl₂. The cells were then incubated for 15 min in PSS at room temperature (21 ± 2 °C) to minimize indicator leakage and sequestration into intracellular organelles. The PSS was removed, and the cells were washed once with loading buffer and then twice with PSS in which NaCl was replaced with an equimolar amount of N-methyl-D-glucamine and CaCl₂ was excluded (Na⁺/Ca²⁺-free PSS). Cells loaded with fluo-4 were stimulated with 50-420 nM ionomycin in Na⁺/Ca²⁺-free PSS containing 100 µM BAPTA (Molecular Probes) and 5 µM cyclopiazonic acid to obtain various levels of peak [Ca²⁺]_i and establish a Ca²⁺ efflux profile curve. A calcium chelator, BAPTA was included to block Ca²⁺ influx (27), and the addition of cyclopiazonic acid prevented Ca²⁺ sequestration into the endoplasmic reticulum by SERCA; this concentration was selected based on previous studies (28) and after cyclopiazonic acid dose response curves were generated in MCF-7 cells (data not shown). Na⁺/Ca²⁺-free PSS was used throughout the experiment to prevent the Na⁺/Ca²⁺ exchanger from contributing to Ca²⁺ efflux (27, 29). Hence the rate of decline in [Ca²⁺]_i was predominately a measure of PMCA-mediated free intracellular Ca²⁺ efflux. Experiments were performed using a NOVOstar® fluorescence microplate reader (BMG LabTechnologies, Offenburg, Germany). Fluo-4 was excited at 485 nm, and fluorescence emission was detected at 520 nm. Fluo-4 fluorescence data points were acquired every 1.5 s. All experiments were performed at 31 ± 1 °C. Relative changes in [Ca²⁺]_i using fluo-4 were determined by calculations of F/F, where F/F = (F_t - F₀)/F₀. In this equation, F_t equals the fluorescence reading at each time point (t), and F₀ represents initial fluorescence at t = 0 (30). For each well, where peak [Ca²⁺]_i was obtained within 90.5 s following the addition of ionomycin, the fluorescence data corresponding to the 60 s after and including the peak F/F was fitted to a monoexponential curve as a measure of the rate of PMCA-mediated free intracellular Ca²⁺ efflux (29).

Statistical Analyses—All data points are presented as means ± S.D., unless otherwise stated, for the number of replicates (n) indicated. Meaningful comparisons between groups were analyzed for statistical significance using a two-sided Student's t test with significance set at p < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
PMCA Antisense Induction Suppresses Proliferation of MCF-7 Tet-off Breast Cancer Cells—To generate a cell line where PMCA antisense could be tightly controlled and equivalent levels of expression could be obtained, we generated a MCF-7 Tet-off PMCA antisense cell line, where the removal of DOX induced PMCA antisense expression (see "Experimental Procedures"). Based upon initial screening for tetracycline-regulated -galactosidase expression as a marker for the simultaneous expression of PMCA antisense and the sensitivity of PMCA expression to doxycycline removal, proliferation assays were conducted for three MCF-7 Tet-off PMCA antisense-transfected clones. All three clones exhibited inhibition of proliferation upon antisense induction, and one clone (A7) was selected for more detailed characterization. The effect of antisense expression on proliferation is shown in Fig. 1A for A7. Controls included identical experiments using parental MCF-7 Tet-off cells (Fig. 1B) and a MCF-7 Tet-off cell line with regulated expression of both -galactosidase and a protein unrelated to PMCA (Fig. 1C). The induction of PMCA antisense (DOX absence) profoundly suppressed the proliferation of MCF-7 (A7) cells over a period of 13 days (Fig. 1A). The removal of DOX did not affect the proliferation of the controls (Fig. 1, B and C).

Effect of PMCA Antisense Induction on PMCA Protein Expression—To evaluate whether tetracycline-regulated PMCA antisense inhibits PMCA protein expression, we used immunoblotting (Fig. 2) to probe for total PMCA protein using the non-isoform-specific 5F10 antibody and isoform-specific antibodies to PMCA4, PMCA1, and PMCA2 (31). Immunoblots were repeated twice for two independent sets of protein isolated from MCF-7 (A7) cells. A representative immunoblot is shown for total PMCA protein (Fig. 2A). As shown by densitometry measurements, a significant (p = 0.005) decrease in total PMCA protein expression was observed on day 13 in cells expressing PMCA antisense (DOX absence) compared with no antisense-expressing cells (DOX presence) (Fig. 2B). Relative PMCA4 levels increased with days in culture, implicating a potential role for PMCA4 in proliferation, and this increase was inhibited in cells expressing antisense (Fig. 2, C and D). By day 13 of growth, PMCA4 protein expression was significantly (p = 0.007) lower in PMCA antisense-induced MCF-7 (A7) cells, in contrast to the parallel non-induced control (Fig. 2D). We also showed for the first time, PMCA1 and PMCA2 protein in MCF-7 cells (Fig. 2, E and G). PMCA1 was only modestly increased in relative expression with days in culture, which was not significantly attenuated by PMCA antisense (Fig. 2, E and F). Consistent with its likely role in specific cellular functions, such as lactation (19, 32), PMCA2 levels did not alter with days in culture and proliferation (Fig. 2, G and H).

View larger version (13K): [in this window] [in a new window]   FIG. 1. Effect of PMCA antisense induction on the proliferation of MCF-7 cells as monitored by MTS assays. Cells were cultured in either the presence (+) or absence (-) of doxycycline (DOX; 100 ng/ml) for up to 13 days. A, MCF-7 Tet-off PMCA antisense cells (clone A7). B, parental MCF-7 Tet-off cells. C, MCF-7 Tet-off cells with regulated expression of an unrelated protein. The data points in Fig. 1 represent means of four replicate wells ± S.D. and are representative of two independent experiments.

View larger version (62K): [in this window] [in a new window]   FIG. 2. Protein expression of total PMCA and specific PMCA isoforms in MCF-7 Tet-off PMCA antisense cells (clone A7). Cells were cultured in either the presence (+) or absence (-) of doxycycline (DOX; 100 ng/ml) and harvested after 3, 7, and 13 days of growth. A, a representative immunoblot probed for total PMCA protein with the 5F10 antibody. B, the corresponding (to A) densitometry measurements of bands representing total PMCA normalized to the -actin band in each lane. C, a representative immunoblot probed for PMCA4 protein with the JA9 antibody. D, the corresponding (to C) densitometry measurements of bands representing PMCA4 normalized to the -actin band in each lane. E, a representative immunoblot probed for PMCA1 protein with the NR1 antibody. F, the corresponding (to E) densitometry measurements of bands representing PMCA1 normalized to the -actin band in each lane. G, a representative immunoblot probed for PMCA2 protein with the NR2 antibody. H, the corresponding (to G) densitometry measurements of bands representing PMCA2 normalized to the -actin band in each lane. The bars in B, D, F, and H represent means ± S.E. for n = 4 replicates. The asterisk denotes a statistically significant difference (p < 0.05).

View larger version (29K): [in this window] [in a new window]   FIG. 3. Expression of sarco(endo)plasmic reticulum calcium-ATPase 2 (SERCA2) protein in MCF-7 Tet-off PMCA antisense cells (clone A7). Cells were cultured in either the presence (+) or absence (-) of doxycycline (DOX; 100 ng/ml) and harvested after 3, 7, and 13 days of growth. A, a representative immunoblot probed for SERCA2 protein with the IID8 antibody. B, the corresponding densitometry measurements of bands representing SERCA2 normalized to the -actin band in each lane. The bars in B represent means ± S.E. for n = 4 replicates. The asterisk denotes a statistically significant difference (p < 0.05).

PMCA Antisense Induction Alters Cell Cycle Phase Distribution—We next investigated the effect of PMCA antisense on cell cycle phase distribution by performing FACS analysis of DNA content using asynchronous cultures of MCF-7 (A7) cells, which were grown in either the presence or absence of DOX for 7 and 13 days. Fig. 4 shows (for each growth condition) percentages of the total number of cells modeled that were present in G₀/G₁, S, or G₂/M phases of the cell cycle. There were no significant differences in the percentage of cells in G₀/G₁ phase between MCF-7 (A7) cells grown in either the presence or absence of DOX for both 7 and 13 days (Fig. 4A). However, PMCA antisense induction significantly (p = 0.005, day 7; p = 0.006, day 13) decreased the percentage of cells present in S phase (Fig. 4B). PMCA antisense induction also significantly (p = 0.008, day 7; p = 0.013, day 13) increased the percentage of cells in G₂/M phase (Fig. 4C). FACS analysis revealed the absence of a noticeable sub-G₀/G₁ peak, at either day 7 or 13, in a set of propidium iodide-stained PMCA antisense-induced MCF-7 (A7) cells (data not shown).

To further assess the mechanism by which PMCA antisense induction inhibited the proliferation of MCF-7 (A7) cells, we conducted BrdUrd pulse-chase experiments. Results (Fig. 5) are representative of two independent experiments. PMCA antisense induction (DOX absence) in MCF-7 (A7) cells resulted in fewer BrdUrd-positive cells at 0 h after the BrdUrd pulse (12-12.9%) compared with the controls (DOX presence; 26-26.8%). This is consistent with the significant reduction of S phase cells, as previously shown in Fig. 4B. At 6 h post-pulse, cells were progressing into G₂/M in both control and antisense-induced groups. At 12 h, the emergence of a BrdUrd-positive peak with a G₀/G₁ DNA content in both PMCA antisense-induced (DOX absence) and -non-induced controls (DOX presence) indicated that PMCA antisense-induced cells were still actively transiting S and G₂/M phases (Fig. 5A). Thus PMCA antisense expression in MCF-7 (A7) cells did not result in S or G₂/M phase arrest. At 24 h post-pulse, PMCA antisense induction resulted in fewer BrdUrd-positive cells with a G₀/G₁ DNA content (11.3-12.4%) compared with controls (26.2-27.5%), indicating a slower transition through G₂/M phase. No apparent differences in S and G₂/M phase progression were found for parental MCF-7 Tet-off cells at the 0, 6, and 12 h time points (data not shown) or the 24 h time point (Fig. 5B).

Induction of PMCA Antisense Inhibits PMCA-mediated Efflux of Free Intracellular Calcium—The effect of PMCA antisense on the primary functional role of PMCA was assessed by comparing the rate of PMCA-mediated efflux of free intracellular Ca²⁺ between MCF-7 (A7) cells that were cultured either with or without DOX for 7 days (Fig. 6). Data were obtained by stimulating Ca²⁺ transients with ionomycin (50-420 nM) and then monitoring PMCA-mediated declines in [Ca²⁺]_i between pairs of wells containing either PMCA antisense-induced or -non-induced MCF-7 (A7) cells. Ionomycin was selected to reduce the activation of other pathways known to alter PMCA activity (28, 34). Ca²⁺ transients induced by 300 nM ionomycin and subsequent declines in DF/F, as a measure of relative change in [Ca²⁺]_i, show that, at this level of PMCA antisense induction, cells could still recover to basal levels of [Ca²⁺]_i after Ca²⁺ ionophore-induced increases (Fig. 6A). However, cells expressing PMCA antisense (DOX absence) had a slower rate of PMCA-mediated free intracellular Ca²⁺ efflux. This is illustrated by comparisons of the rate of PMCA-mediated decline in [Ca²⁺]_i between antisense-expressing cells and cells with no antisense induction at similar levels of peak relative [Ca²⁺]_i (Fig. 6B). To assess the effects of PMCA inhibition on the calcium homeostasis changes induced by physiological stimuli, the peak responses to the purinergic receptor activator ATP (100 mM) were assessed. PMCA inhibition increased peak responses to this stimulus (Fig. 6C).

PMCA Antisense Induction Alters the Morphology of MCF-7 Tet-off Cells—Phase contrast images of MCF-7 (A7) cells plated at the same cell density and then grown for 13 days, either in the presence or absence of DOX, were taken from five random fields (Fig. 7). PMCA antisense-induced cells (DOX absence) were subconfluent (Fig. 7B) compared with antisense-non-induced controls (DOX presence) (Fig. 7A). Moreover, a proportion of MCF-7 (A7) cells with PMCA antisense induction exhibited a large, rounded, and flat cell morphology (Fig. 7B), similar to that we have previously observed for the non-tumorigenic, "normal" breast cell line MCF-10A (data not shown).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Intracellular Ca²⁺ signaling is essential for a diverse array of cellular functions and regulates processes that control both life and death. PMCA is not merely a mechanism for the maintenance of a low resting [Ca²⁺]_i. PMCA also plays a role in shaping the complex dynamics of Ca²⁺ signals (35). The versatility of Ca²⁺ signaling in terms of spatial and temporal regulation is believed to form the basis of how fluctuations in [Ca²⁺]_i are able to encode a diverse array of specific messages controlling many cellular functions. For instance, changes in the amplitude and duration of Ca²⁺ signals can mediate the specificity and efficiency in activating different transcription factors (36), and PMCA is likely to play a key role in these events. We sought to investigate the effects of inhibiting PMCA, a critical regulator of intracellular Ca²⁺ signaling (14), in MCF-7 human breast cancer cells using a tetracycline-regulated antisense approach. PMCA antisense induction, at levels that do not completely block recovery of [Ca²⁺]_i after stimulation and do not induce cell death, suppresses cell proliferation, affects cell cycle progression, and alters cell morphology in MCF-7 cells.

View larger version (14K): [in this window] [in a new window]   FIG. 4. Effect of PMCA antisense induction on cell cycle phase distribution as determined by FACS analysis of MCF-7 Tet-off PMCA antisense cells (clone A7). Cells were cultured in either the presence (+) or absence (-) of doxycycline (DOX; 100 ng/ml) and harvested after 7 and 13 days of growth. The panels represent results for the percentage of the total number of cells analyzed that were present in G0/G1 phase (A), S phase (B), and G2/M phase (C) of the cell cycle. The bars are indicative of means ± S.D. for three independent experiments. The asterisk denotes a statistically significant difference (p < 0.05).

We used a tetracycline-regulated antisense approach to assess the functional consequences of PMCA inhibition in the MCF-7 human breast cancer cell line. This approach was necessary because of the current lack of a highly specific pharmacological inhibitor of PMCA. Other commonly used pharmacological agents include orthovanadate ([VO₃ (OH)]^2-), which is a general inhibitor of all P-type ATPases (14), and lanthanum (La³⁺), which has been used to inhibit PMCA-mediated Ca²⁺ efflux (37); but at high concentrations of lanthanum, there may also be inhibition of Ca²⁺ efflux mediated by the Na⁺/Ca²⁺ exchanger (34). Stable antisense approaches have been used previously by others to study the functional role of PMCA. For instance, inhibition of PMCA1 expression in PC6 pheochromocytoma cells via stable antisense hinders neuronal differentiation and neurite extension in response to nerve growth factor (38). A tetracycline-regulated approach to antisense expression possesses advantages over the use of antisense oligonucleotides or transient siRNA transfection. It bypasses the issues surrounding inefficient and variable uptake (39), which may cause either partial or complete inhibition of expression in some cells. A transient transfection would make it difficult to confirm whether decreased proliferation is because of partial target inhibition or complete inhibition in some cells. Moreover, the stable expression of antisense under the control of a tetracycline-regulated promoter also allows for the long term inhibition of protein expression (40), which is of particular relevance for proteins with relatively long half-lives (41). Indeed, the half-life of PMCA has been reported to be 12 days (42). Moreover, the ability to induce antisense expression only during experiments and not during colony selection or culture maintenance reduces the likelihood of very long term adaptive changes to signaling pathways.

Our findings show that the induction of PMCA antisense in the MCF-7 breast cancer cell line profoundly suppresses cell proliferation (Fig. 1A), being neither a nonspecific effect of DOX removal from the culture medium (Fig. 1B) nor a random effect of expression from a tetracycline-regulated promoter (Fig. 1C). Immunoblot densitometry measurements indicated that PMCA antisense induction inhibited total PMCA protein expression (Fig. 2, A and B). Our studies also demonstrated for the first time PMCA1, PMCA4, and PMCA2 protein expression in MCF-7 cells. This places the well characterized MCF-7 cell line as an outstanding tool to explore the role of individual PMCA isoforms in specific signaling pathways and functions. The relatively long half-life of PMCA (42) may account for a significant difference in the PMCA protein level being evident only after extended days of culture and suggests that the inhibition of new PMCA protein synthesis regulates the observed effect on cell proliferation. Even when PMCA-mediated free intracellular Ca²⁺ efflux is inhibited to levels that allow recovery of [Ca²⁺]_i (Fig. 6A), we saw dramatically reduced cell proliferation, identifying PMCA as a potential drug target in breast cancer. Indeed, the non-estrogen-mediated effects of tamoxifen have been likened to the augmentation of Ca²⁺ responses (43), an effect which would also arise from the inhibition of PMCA.

View larger version (22K): [in this window] [in a new window]   FIG. 5. Effect of PMCA antisense induction on cell cycle phase progression as determined by dual 5-bromo-2'-deoxyuridine (BrdUrd)- and 7-amino-actinomycin D (7-AAD)-stained FACS analysis of MCF-7 Tet-off PMCA antisense cells (clone A7). Cells were cultured in either the presence (+) or absence (-) of doxycycline (DOX; 100 ng/ml) for 7 days, pulsed with BrdUrd 10 µM for 1 h, and then harvested 0, 3, 6, 12, and 24 h post-BrdUrd pulse. A, results are shown for MCF-7 Tet-off PMCA antisense cells (clone A7) at 0, 6, 12, and 24 h after the BrdUrd pulse. B, results are shown for parental MCF-7 Tet-off cells at 24 h after the BrdUrd pulse. The y-axis (anti-BrdUrd fluorescein isothiocyanate (FITC)) indicates whether cells are synthesizing DNA or have undergone DNA synthesis with the incorporation of BrdUrd. The x-axis (7-AAD) indicates whether cells have a G0/G1 or G2/M DNA content.

View larger version (12K): [in this window] [in a new window]   FIG. 6. PMCA-mediated efflux of free intracellular calcium and peak ATP responses as determined by relative changes in fluo-4 fluorescence (F/F). MCF-7 Tet-off PMCA antisense cells (clone A7) were cultured for 7 days, either in the presence (+) or absence (-) of doxycycline (DOX; 100 ng/ml) loaded with the intracellular Ca2+ indicator fluo-4 and then PMCA-mediated free intracellular Ca2+ efflux and peak ATP responses were assessed. A, example traces of Ca2+ transients induced by 300 nM ionomycin are shown for a pair of wells housing cells that were grown either in the presence of DOX (+DOX; no antisense) or absence of DOX (-DOX; antisense). The rate of decline in F/F, after reaching peak F/F, is a relative measure of PMCA-mediated free intracellular Ca2+ efflux. B, rates of PMCA-mediated decline in F/F were plotted against peak F/F values for valid comparisons of PMCA-mediated Ca2+ efflux rates in antisense-induced and -non-induced cells. Data points in B are means ± S.E. Numbers of replicates for each ionomycin concentration used are as follows: 6 at 50 nM, 14-18 at 100 nM, 23-26 at 175 nM, 34 at 300 nM, and 35-38 at 420 nM. C, peak F/F values induced by ATP at 100 µM are shown for cells that were grown either in the presence of doxycycline (+DOX; no antisense) or absence of doxycycline (-DOX; antisense). Bars represent mean ± S.E. (n = 6).

View larger version (75K): [in this window] [in a new window]   FIG. 7. Assessment of cell morphology as seen by phase contrast microscopy after the induction of PMCA antisense. MCF-7 Tet-off PMCA antisense cells (clone A7) were plated onto coverslips placed in a 6-well plate at 1.8 x 104 cells/well and then cultured in either the presence (+) or absence (-) of doxycycline (DOX; 100 ng/ml). After 13 days of growth, images of cells at 200x magnification were taken from five random fields. A, the panels on the left are images of cells grown in the presence of doxycycline (+DOX; no antisense). B, the panels on the right are images of cells grown in the absence of doxycycline (-DOX; antisense).

To evaluate whether the induction of PMCA antisense affected PMCA function and not only protein expression, we assessed the relative rates of PMCA-mediated intracellular Ca²⁺ efflux in PMCA antisense-expressing and -non-expressing MCF-7 cells (Fig. 6B). The suppression of cell proliferation by PMCA antisense induction correlated with the inhibition of PMCA-mediated intracellular Ca²⁺ efflux. Although PMCA antisense altered PMCA-mediated intracellular Ca²⁺ efflux, this degree of antisense induction did not affect the apparent ability of the cell to eventually recover basal intracellular Ca²⁺ levels after ionomycin-induced increases in [Ca²⁺]_i (Fig. 6A). This may explain why suppression of cell proliferation was observed upon PMCA antisense induction, without the concomitant presence of noticeable cell death. Indeed ATP-induced calcium signaling remained during partial PMCA inhibition, although as predicted, when calcium efflux is reduced, peak calcium responses were augmented (Fig. 6C). Collectively, our results suggest that moderate changes in PMCA-mediated Ca²⁺ efflux can lead to profound cellular consequences, despite the absence of major deleterious effects on global intracellular Ca²⁺ homeostasis. It also supports the possibility of how alterations in PMCA function may affect the kinetics of Ca²⁺ signals in specifically regulating various cellular processes such as proliferation (3). The complex dynamic aspects of intracellular Ca²⁺ signaling in regulating transcription factors has been an area of active research (1, 2) and represents a link by which PMCA-mediated modulation of Ca²⁺ signaling could control gene transcription and cell phenotype. Indeed, frequency modulation of Ca²⁺ signals can regulate the activity of nuclear factor B (NFB) (45), a key transcription factor that is important in the control of proliferation in lymphocytes and is also abnormally expressed in breast cancer (46).

There is a growing body of evidence suggesting PMCA plays important functional roles in the breast. Our data support this notion, and although inhibition of PMCA-mediated Ca²⁺ efflux may limit breast cancer cell line proliferation alone, compromised PMCA function may be able to augment cytotoxic Ca²⁺ responses together with other existing anti-tumor agents like tamoxifen (43). This and the consequences of inhibiting specific PMCA isoforms in MCF-7 cells warrant further investigation.

	FOOTNOTES

 
^* This work was supported by the National Health and Medical Research Council (NHMRC) of Australia (102426), the Queensland Cancer Fund (Q34-02), and by a Dora Lush (Biomedical) Postgraduate Research Scholarship (awarded to W. J. L.) by the NHMRC.

^¶ To whom correspondence should be addressed: School of Pharmacy, University of Queensland, Steele Bldg., Brisbane, Queensland 4072, Australia. Tel.: 61-7-3365-7442; Fax: 61-7-3365-1688; E-mail: G.Monteith{at}pharmacy.uq.edu.au.

¹ The abbreviations used are: PMCA, plasma membrane Ca²⁺-ATPase; DOX, doxycycline; FACS, fluorescence-activated cell sorting; PBS, phosphate-buffered saline; BrdUrd, 5-bromo-2'-deoxyuridine; 7-AAD, 7-amino-actinomycin D; PSS, physiological salt solution; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrapotassium salt; SERCA, sarco(endo)plasmic reticulum Ca²⁺-ATPase; MTS, (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt.

	ACKNOWLEDGMENTS

 
We thank Grace Chojnowski at the Queensland Institute of Medical Research, Herston, Australia for technical assistance with FACS analysis. We also thank Dr. Ibtissam Abdul-Jabbar of the Centre for Cancer Research and Immunology for help with FACS analysis of BrdUrd and 7-AAD-stained cells.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. Berridge, M. J., Lipp, P., and Bootman, M. D. (2000) Nat. Rev. Mol. Cell Biol. 1, 11-21[CrossRef][Medline] [Order article via Infotrieve]
2. Carafoli, E., Santella, L., Branca, D., and Brini, M. (2001) Crit. Rev. Biochem. Mol. Biol. 36, 107-260[CrossRef][Medline] [Order article via Infotrieve]
3. Lipskaia, L., and Lompre, A. M. (2004) Biol. Cell 96, 55-68[CrossRef][Medline] [Order article via Infotrieve]
4. Kahl, C. R., and Means, A. R. (2003) Endocr. Rev. 24, 719-736[Abstract/Free Full Text]
5. Orrenius, S., Zhivotovsky, B., and Nicotera, P. (2003) Nat. Rev. Mol. Cell Biol. 4, 552-565[CrossRef][Medline] [Order article via Infotrieve]
6. Ichikawa, J., Furuya, K., Miyata, S., Nakashima, T., and Kiyohara, T. (2000) Cell Biochem. Funct. 18, 215-225[CrossRef][Medline] [Order article via Infotrieve]
7. Nie, L., Oishi, Y., Doi, I., Shibata, H., and Kojima, I. (1997) Cell Calcium 22, 75-82[CrossRef][Medline] [Order article via Infotrieve]
8. Haverstick, D. M., Heady, T. N., MacDonald, T. L., and Gray, L. S. (2000) Cancer Res. 60, 1002-1008[Abstract/Free Full Text]
9. Patel, R., Holt, M., Philipova, R., Moss, S., Schulman, H., Hidaka, H., and Whitaker, M. (1999) J. Biol. Chem. 274, 7958-7968[Abstract/Free Full Text]
10. Williams, S. S., French, J. N., Gilbert, M., Rangaswami, A. A., Walleczek, J., and Knox, S. J. (2000) Cancer Res. 60, 4358-4361[Abstract/Free Full Text]
11. Vanden Abeele, F., Skryma, R., Shuba, Y., Van Coppenolle, F., Slomianny, C., Roudbaraki, M., Mauroy, B., Wuytack, F., and Prevarskaya, N. (2002) Cancer Cell 1, 169-179[CrossRef][Medline] [Order article via Infotrieve]
12. Berridge, M. J., Bootman, M. D., and Roderick, H. L. (2003) Nat. Rev. Mol. Cell Biol. 4, 517-529[CrossRef][Medline] [Order article via Infotrieve]
13. Rizzuto, R., and Pozzan, T. (2003) Nat. Genet. 34, 135-141[CrossRef][Medline] [Order article via Infotrieve]
14. Carafoli, E. (1994) FASEB J. 8, 993-1002[Abstract]
15. Monteith, G. R., and Roufogalis, B. D. (1995) Cell Calcium 18, 459-470[CrossRef][Medline] [Order article via Infotrieve]
16. Klishin, A., Sedova, M., and Blatter, L. A. (1998) Am. J. Physiol. 274, C1117-C1128[Medline] [Order article via Infotrieve]
17. Brini, M., Bano, D., Manni, S., Rizzuto, R., and Carafoli, E. (2000) EMBO J. 19, 4926-4935[CrossRef][Medline] [Order article via Infotrieve]
18. Strehler, E. E., and Zacharias, D. A. (2001) Physiol. Rev. 81, 21-50[Abstract/Free Full Text]
19. Reinhardt, T. A., Filoteo, A. G., Penniston, J. T., and Horst, R. L. (2000) Am. J. Physiol. 279, C1595-C1602
20. Kamrava, M. R., Michener, C. M., and Kohn, E. C. (2002) Pharmaceutical News 9, 435-442
21. Lee, W. J., Roberts-Thomson, S. J., Holman, N. A., May, F. J., Lehrbach, G. M., and Monteith, G. R. (2002) Cell. Signal. 14, 1015-1022[CrossRef][Medline] [Order article via Infotrieve]
22. Reisner, P. D., Brandt, P. C., and Vanaman, T. C. (1997) Cell Calcium 21, 53-62[CrossRef][Medline] [Order article via Infotrieve]
23. Chene, P. (2002) Nat. Rev. Drug. Discov. 1, 665-673[CrossRef][Medline] [Order article via Infotrieve]
24. Johnson, P. H., Walker, R. P., Jones, S. W., Stephens, K., Meurer, J., Zajchowski, D. A., Luke, M. M., Eeckman, F., Tan, Y., Wong, L., Parry, G., Morgan, T. K., Jr., McCarrick, M. A., and Monforte, J. (2002) Mol. Cancer Ther. 1, 1293-1304[Abstract/Free Full Text]
25. Goodwin, C. J., Holt, S. J., Downes, S., and Marshall, N. J. (1995) J. Immunol. Methods 179, 95-103[CrossRef][Medline] [Order article via Infotrieve]
26. Smith, S. A., Monteith, G. R., Holman, N. A., Robinson, J. A., May, F. J., and Roberts-Thomson, S. J. (2003) J. Neurosci. Res. 72, 747-755[CrossRef][Medline] [Order article via Infotrieve]
27. Rosado, J. A., and Sage, S. O. (2000) J. Biol. Chem. 275, 19529-19535[Abstract/Free Full Text]
28. Usachev, Y. M., DeMarco, S. J., Campbell, C., Strehler, E. E., and Thayer, S. A. (2002) Neuron 33, 113-122[CrossRef][Medline] [Order article via Infotrieve]
29. Sedova, M., and Blatter, L. A. (1999) Cell Calcium 25, 333-343[CrossRef][Medline] [Order article via Infotrieve]
30. Laskey, A. D., Roth, B. J., Simpson, P. B., and Russell, J. T. (1998) Cell Calcium 23, 423-432[CrossRef][Medline] [Order article via Infotrieve]
31. Filoteo, A. G., Elwess, N. L., Enyedi, A., Caride, A., Aung, H. H., and Penniston, J. T. (1997) J. Biol. Chem. 272, 23741-23747[Abstract/Free Full Text]
32. Reinhardt, T. A., Lippolis, J. D., Shull, G. E., and Horst, R. L. (2004) J. Biol. Chem. 279, 42369-42373[Abstract/Free Full Text]
33. Kuo, T. H., Liu, B. F., Yu, Y., Wuytack, F., Raeymaekers, L., and Tsang, W. (1997) Cell Calcium 21, 399-408[CrossRef][Medline] [Order article via Infotrieve]
34. Furukawa, K. I., Tawada, Y., and Shigekawa, M. (1988) J. Biol. Chem. 263, 8058-8065[Abstract/Free Full Text]
35. Bautista, D. M., Hoth, M., and Lewis, R. S. (2002) J. Physiol. (Lond.) 541, 877-894[Abstract/Free Full Text]
36. Dolmetsch, R. E., Lewis, R. S., Goodnow, C. C., and Healy, J. I. (1997) Nature 386, 855-858[CrossRef][Medline] [Order article via Infotrieve]
37. Shimizu, H., Borin, M. L., and Blaustein, M. P. (1997) Cell Calcium 21, 31-41[CrossRef][Medline] [Order article via Infotrieve]
38. Brandt, P. C., Sisken, J. E., Neve, R. L., and Vanaman, T. C. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 13843-13848[Abstract/Free Full Text]
39. White, P. J., Fogarty, R. D., McKean, S. C., Venables, D. J., Werther, G. A., and Wraight, C. J. (1999) J. Investig. Dermatol. 112, 699-705[CrossRef][Medline] [Order article via Infotrieve]
40. Sazani, P., Vacek, M. M., and Kole, R. (2002) Curr. Opin. Biotechnol. 13, 468-472[CrossRef][Medline] [Order article via Infotrieve]
41. Slodzinski, M. K., and Blaustein, M. P. (1998) Am. J. Physiol. 275, C459-C467[Medline] [Order article via Infotrieve]
42. Ferrington, D. A., Chen, X., Krainev, A. G., Michaelis, E. K., and Bigelow, D. J. (1997) Biochem. Biophys. Res. Commun. 237, 163-165[CrossRef][Medline] [Order article via Infotrieve]
43. Zhang, W., Couldwell, W. T., Song, H., Takano, T., Lin, J. H., and Nedergaard, M. (2000) Cancer Res. 60, 5395-5400[Abstract/Free Full Text]
44. Munster, P. N., Srethapakdi, M., Moasser, M. M., and Rosen, N. (2001) Cancer Res. 61, 2945-2952[Abstract/Free Full Text]
45. Hu, Q., Natarajan, V., and Ziegelstein, R. C. (2002) Biochem. Biophys. Res. Commun. 292, 325-332[CrossRef][Medline] [Order article via Infotrieve]
46. Sovak, M. A., Bellas, R. E., Kim, D. W., Zanieski, G. J., Rogers, A. E., Traish, A. M., and Sonenshein, G. E. (1997) J. Clin. Investig. 100, 2952-2960[Medline] [Order article via Infotrieve]

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