Departments of 1Reproductive Biology, 3Physiology and Biophysics, and 4Oncology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106; and 2Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814
Submitted 25 May 2004 ; accepted in final form 15 July 2004
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ABSTRACT |
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cervix; epithelium; ATP; 2'-3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate
The ectocervical epithelium is maintained by a balance between proliferation of the basal layer of cells and death of cells in upper layers. Cells in the basal layer can either replicate or cease proliferation and exit from the mitotic cycle, stratify, and undergo terminal differentiation into cornified envelopes (superficial cells) (14). Our current state of knowledge suggests three levels of growth control of cervical epithelial cells: proliferation, controlled by mitogenic signals [e.g., estrogen and epidermal growth factor (EGF)] vs. growth inhibitory factors [e.g., transforming growth factor (TGF)-] (21); terminal differentiation, controlled mainly by estrogen (57); and senescence of cells evading growth control and terminal differentiation due to the erosion of telomeres (32). Preliminary data from our lab (13) suggest that growth of human cervical epithelial cells is also regulated by apoptosis, but the mechanisms of apoptosis-induced growth control of the normal cells are unknown. The objective of the present study was to better understand the phenomenon and the mechanisms involved.
Apoptosis is a regulated homeostatic process, orchestrated by the host's genome, of selective cell deletion without stimulating inflammatory response (7, 8, 61). Under certain conditions such as deficiency of necessary growth factors or hormones, apoptosis can lead to premature death of cells, loss of tissue, and aging (50). Dysregulation of apoptotic cell death has been implicated in the neoplastic transformation and in states of disease (7, 8, 44, 61). Many studies have examined apoptosis of cervical cancer cells in response to therapies, but only a few looked into regulation of apoptosis in normal human cervical cells. Ter-Harmsel et al. (55) reported higher expression of markers of proliferation and apoptosis protection in premalignant lesions compared with normal cervical tissues. Lee et al. (27) reported that tumor progression in the cervical epithelium was associated with an increase in both cell proliferation and cell apoptosis. In contrast, Mozzetti et al. (34) did not find a significant difference in the expression of apoptosis-related markers between normal and cancer cervical cells.
A number of studies used cervical cells immortalized with human papillomavirus (HPV) to determine the effects of drugs on apoptosis. Rorke and Jacobberger (45) reported that TGF-1 enhanced apoptosis in HPV type 16-immortalized human ectocervical epithelial cells. Brown et al. (4) found that HPV-16 E6 sensitized cells to atractyloside-induced apoptosis. Rorke et al. (46) reported that HPV-immortalized cervical cells are less sensitive to toxicant damage, including apoptosis induction, than normal cervical epithelial cells. More recently, Thomas et al. (56) and others have shown that in HPV-infected cells the HPV E6 protein complexes with the tumor suppressor p53 and targets it for rapid proteasome-mediated degradation, abrogating p53's growth-arrest and apoptosis-inducing activities. Because of the mechanism by which HPV infection alters regulation of apoptosis, it remains difficult to extrapolate these results to the normal cervix.
A number of studies also looked into apoptosis of vaginal-cervical cells in rodents. Perfettini et al. (40) reported that infection of female mice with Chlamydia trachomatis enhances secretion of tumor necrosis factor (TNF)- and augments apoptosis of endocervical cells. The authors also showed that infection of human HeLa cells with C. trachomatis increases secretion of TNF-
and results in apoptosis. Sato et al. (47) reported that in the middle and basal layers of the rat vaginal epithelium the apoptotic index was high at metestrus and negatively correlated with the mitotic rate in that tissue. Rao et al. (42) confirmed those results and suggested that the apoptosis is part of the terminal differentiation of the epithelium. The authors also found a few scattered cells throughout the thickness of the vaginal epithelium undergoing apoptotic death involving DNA fragmentation. Unaltered levels of Bcl-2 message on estradiol administration prompted them to question the role of Bcl-2 in preventing death of the vaginal-cervical epithelium by apoptosis.
The above studies suggest that apoptosis plays a role in the regulation of proliferation and extinction of cervical cells. However, our present state of knowledge is incomplete with regard to phenomena and mechanisms in the normal human cervix. These data could be important for the understanding of cervical cell biology and tumorigenesis. In preliminary experiments we observed (13) that normal human cervical epithelial cells undergo apoptosis spontaneously and that the effect could be blocked by incubating cells in low extracellular calcium. The main objective of the present study was to understand the regulation and mechanisms of this effect. Because the only agent that could stimulate prolonged increases in cytosolic calcium in human cervical epithelial cells is ATP (Gorodeski GI, unpublished observations), we hypothesized that ATP is the mediator of the baseline apoptosis. The experiments utilized cultures of normal human ectocervical epithelial (hECE) cells, an experimental system previously characterized (14, 16) and found to be adequate and useful for apoptosis research. The results show that ATP, acting via the P2X7 receptor mechanism, stimulates apoptosis that involves predominantly the calcium-dependent caspase-9-mediated mitochondrial pathway.
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METHODS |
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Preliminary experiments were conducted on cells plated on culture plates, and definitive experiments were repeated on cells plated on filters. The latter method improves culturing conditions and promotes epithelial cell polarization and differentiation (16). Anocell-10 filters (Sigma) are ceramic-base filters with a pore size of 0.02-µm width and 50-µm depth. Filters were coated on their upper (luminal) surface with 35 µg/cm2 collagen type IV and incubated at 37°C overnight. The remaining collagen solution was aspirated, and the filter was dried at 37°C. Before plating, both sides of the filters were rinsed three times with Hanks' balanced salt solution. Cells were plated on the upper surface of the filter at 3 x 105 cells/cm2. Plated at this high density, the cultures became confluent within 12 h after plating.
In some experiments, treatments with apoptosis-inducing drugs were carried out on cells incubated in their respective culture medium. In other experiments, cells were shifted to modified Ringer solution composed of (in mM) 120 NaCl, 1.2 CaCl2, 1.2 MgSO4, 5 KCl, 10 NaHCO3, 10 HEPES, and 5 glucose with 0.1% BSA, pH 7.2. Levels of extracellular Ca2+ were controlled by adding EGTA. All treatments involved adding drugs to both the luminal and subluminal solutions.
Western blot analysis.
The postnuclear supernatant of cells was solubilized in lysis buffer [50 mM Tris·HCl, pH 6.8, 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and 5 mM EDTA, pH 8.0] containing 50 µg/ml PMSF, 10 µg/ml benzamidine, 10 µg/ml bacitracin, 10 µg/ml of leupeptin, and 2 µg/ml aprotinin. Aliquots normalized to 15 µg of protein (45 µl; determined by Bio-Rad protein assay solution, Hercules, CA) were loaded on 10% polyacrylamide-SDS gel, and vertical electrophoresis was conducted at 50 mA for 1.5 h. Gels were transferred onto Immobilon membrane (Millipore, Bedford, MA) at 200 V for 1.5 h; membranes were blocked in 5% milk and exposed to the primary antibody at 4°C overnight. Membranes were washed three times in PBS and fluorescent stained for 1 min with an enhanced chemiluminescence (ECL) kit of peroxidase-conjugated secondary antibody from Amersham (Piscataway, NJ).
DNA fragmentation assay. The DNA fragmentation assay was modified from Lizard et al. (30). Cells attached on plates were harvested and combined with floaters recovered from the medium. Cells were washed in ice-cold PBS lacking Ca2+ and Mg2+ and resuspended in the same medium, and cellular DNA was extracted with a DNA extraction kit (Stratagene, La Jolla, CA). In some experiments the latter step was done by using lysis buffer composed of (in mM) 10 EDTA, 400 NaCl, 35 SDS, and 10 Tris·HCl, pH 8.2, with 1 mg/ml proteinase K. Cells were spun briefly at 180 g, and the pellet was resuspended in the lysis buffer, transferred to an Eppendorf tube, and incubated overnight at 37°C. The tubes were spun for 5 min at Eppendorf high speed, and the supernatant containing the DNA was precipitated in 2 volumes of 100% ethanol and incubated overnight at 20°C. Tubes were spun for 5 min at Eppendorf high speed, and the pellet containing the DNA was saved. For assays, the DNA-containing pellets were resuspended in 100 µl of TE buffer (composed of 10 mM Tris·HCl and 0.2 mM Na-EDTA, pH 7.5) and equal amounts of DNA (determined by spectrofluorometry) were separated on 1.8% agarose gel electrophoresis for 15 h at 20 V. Gels were prepared in Tris-buffered ethanolamine buffer (9 mM Tris-borate, pH 8, 2 mM EDTA) plus 0.1 µg/ml ethidium bromide and photographed under ultraviolet light.
DNA solubilization assay. Twenty-four hours before the end of the experiment cells were labeled with 3H-labeled thymidine (specific activity 100 Ci/mmol; 5 µCi per 1 x 106 cells) for 16 h. The medium was removed, and cells were washed three times with fresh medium lacking [3H]thymidine and incubated in the same medium for an additional 6 h. At the end of incubation the "supernatant" was stored and cells were lysed in 0.5 ml of lysis buffer (10 mM Tris·HCl, pH 7.5, + 1 mM EDTA-0.2% Triton X-100) for 1 h at 4°C. The intact chromatin was separated from the fragmented DNA by 5-min centrifugation at 4°C in an Eppendorf microcentrifuge at 12,000 g. The supernatant (referred to as "lysate") was stored, and the pellet was resuspended in 0.5 ml of 1% SDS. The radioactivity contained in the supernatants, lysates, and pellets was counted in a scintillation counter, and DNA fragmentation was calculated as [% solubilized DNA] = [(cpm supernatant + cpm lysate)/(cpm supernatant + cpm lysate + cpm pellet)] x 100.
In situ detection of DNA fragmentation. In situ detection of DNA fragmentation was done by terminal deoxynucleotidyl transferase (TdT)-mediated nick-end labeling (TUNEL) with an ApoAlert DNA fragmentation assay kit from ClonTech (Palo Alto, CA); procedures were carried out at room temperature according to the manufacturer's instructions. Cells on filters were fixed with 4% paraformaldehyde for 15 min, washed with PBS, and treated with 20 µg/ml proteinase K for 5 min. Cells were fixed again with 4% paraformaldehyde for 5 min, reincubated for 10 min with PBS, and equilibrated with TdT equilibration buffer for 10 min. Cells were then incubated with TdT and fluorescein-dUTP for 60 min at 37°C in the dark, and the reaction was terminated by immersing the cells in 0.3 M NaCl plus 30 mM sodium citrate solution for 15 min. After two PBS washes (5 min each) cells were costained with propidium iodide and mounted in antifade solution.
ATP assays. Cells were grown on plates, and for experiments cells were maintained in a volume of 300 µl of either regular medium or modified Ringer solution. Aliquots of 50 µl were collected into a polypropylene tube 20 min after stabilization and left at 4°C for 2 h. Two hundred microliters (3.33 mg/ml) of luciferase-luciferin was added to each tube, and ATP in the medium was measured by a chemiluminescence method linked to firefly luciferase-luciferin (Sigma) as described previously (54) with minor modifications. Bubbled Ringer solution was used as blank to determine background ATP, and ATP in the medium was determined from a standard curve of samples with known ATP concentrations. The limit of detection for ATP was 0.25 nM.
Antibodies.
Mouse monoclonal anti-human caspase-3, -8, and -9 antibodies were from Chemicon (Temecula, CA). Mouse monoclonal anti-human -actin antibody was from Zymed Laboratory (San Francisco, CA). The antibodies were used according to the manufacturers' instructions.
Statistical analysis of the data. Data are presented as means ± SD, and significance of differences among means was estimated by performing Student's t-test. Trends were calculated with GB-STAT V5.3 (1995; Dynamic Microsystems, Silver Spring, MD) and analyzed by ANOVA.
Chemicals and supplies.
ATP, ADP, AMP, adenosine, adenine, UTP, GTP, CTP, ITP, XTP, TTP, adenosine 5'-O-(3-thiotriphosphate) (ATPS),
,
-methyleneadenosine 5'-triphosphate (AMP-PCP),
,
-methyleneadenosine 5'-triphosphate (AMP-CPP), adenosine 5'-(
,
-imido)triphosphate (AMP-PNP), N6-([6-aminohexyl]carbamoylmethyl)adenosine 5'-triphosphate (A-8889),8-azidoadenosine 5'-triphosphate (A-2392), 2'-3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP), adenosine 5'-O-(2-thiodiphosphate) (ADP
S), ADPase, and ATPase (apyrase) were from Sigma. 2-Methylthio-ATP (2-MeS-ATP) was obtained from Research Biochemicals (Natick, MA). Nucleotides were dissolved in water or dimethyl sulfoxide as appropriate. Leu-Glu-His-Asp-O-methyl-fluoromethylketone (LEHD-FMK), Asp-Glu-Val-Asp-O-methyl-fluoromethylketone (DEVD-FMK), Ile-Glu-Thr-Asp-O-methyl-fluoromethylketone (IETD-FMK), and N-benzyloxycarbonyl-Val-Ala-Asp-O-methyl-fluoromethylketone (Z-VAD-FMK) were from Calbiochem (La Jolla, CA) and used at a concentration of 50 µM. All other culture medium additives and chemicals were obtained from Sigma, unless specified otherwise.
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RESULTS |
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DNA fragmentation assay on homogenates of hECE or CaSki cells grown in medium enriched with serum did not detect significant laddering (not shown). However, DNA solubilization assays in those cells revealed values of 1.4% and 1%, respectively (Fig. 1), indicating a low baseline degree of apoptosis. When cells were shifted to serum-free medium these levels increased to 2.4% and 1.6%, respectively (Fig. 1; P < 0.05), and in some experiments laddering was observed (not shown). Shifting cells to low Ca2+ (1.2 mM 0.6 mM) resulted in low levels of DNA solubilization (0.40.8%), regardless of whether cells were incubated in serum-enriched medium or in serum-free medium (Fig. 1).
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The apoptotic effect of ATP was time and dose related. In CaSki cells DNA fragmentation was already observed 13 h after addition of ATP and reached submaximal effect 69 h after treatment (Fig. 4). Similar effects were obtained in hECE cells (not shown). In hECE cells DNA fragmentation began already with 0.11 µM ATP (Fig. 5A) and in CaSki cells with 110 µM ATP (Fig. 5B). Also determined were the effects of ATP and BzATP on DNA solubilization. In hECE cells (Fig. 6) and in CaSki cells (not shown) the dose-response curves of DNA solubilization vs. ATP or BzATP did not reach saturation even at millimolar concentrations of the nucleotides (Fig. 6). BzATP had a more efficacious and potent apoptosis-inducing effect than ATP (Fig. 6), similar to effects described in other types of cells (6, 41).
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The objective of the next set of experiments was to determine the mechanism by which P2X7 receptor activation induces apoptosis in human cervical epithelial cells. As shown in Fig. 8, the effect of BzATP could be attenuated by lowering extracellular Ca2+. In contrast, the effect of TNF- was independent of extracellular Ca2+, suggesting that TNF-
and BzATP operate via different mechanisms. To determine the degree to which BzATP and TNF-
utilize different signaling mechanisms cells were treated with both BzATP and TNF-
. Cotreatment with 10 ng/ml TNF-
had an effect on DNA solubilization partially additive to that of BzATP (Fig. 9). This was observed in the range of 050 µM BzATP, which are doses that produce submaximal effects for both BzATP (Fig. 6) and TNF-
(Table 1). In contrast, at 100 µM BzATP and TNF-
had no additional effect on DNA solubilization (Fig. 9).
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ATP is secreted from human cervical epithelial cells.
One of the questions regarding the physiological role of P2X7 receptors in the regulation of apoptosis is that of ATP activity in the extracellular fluid. ATP is present in millimolar concentrations in the cytosol of all cell types, but extracellular levels of the nucleotide are normally maintained at lower levels because of minimal permeation of ATP across lipid bilayers and because ubiquitous ecto-ATPases and nucleotidases hydrolyze extracellular nucleotides (reviewed in Ref. 6). To determine the degree to which human cervical epithelial cells secrete ATP into the extracellular fluid, aliquots of conditioned medium from hECE and CaSki cultures were collected and analyzed by an ECL method linked to firefly luciferase-luciferin. ATP values ranged from 5 nM to 1 µM, with mean ± SD of 485 ± 217 nM. ATP levels in conditioned medium of hECE cells tended to be higher than in CaSki cells (570 ± 170 vs. 350 ± 219 nM). Lowering extracellular Ca2+ from 1.2 mM to 0.6 mM tended to lower extracellular ATP (to 405 ± 125 and 175 ± 110 nM in hECE and CaSki cells, respectively), whereas shifting cells to serum-free medium tended to increase extracellular ATP (to 650 ± 170 and 575 ± 130 nM, respectively). However, none of the differences was significant. These data indicate that cultured human cervical cells secrete ATP into the extracellular fluid, and the mean steady-state ATP activity is 0.5 µM, which is within the range of values that suffice to activate the P2X7 receptor (Fig. 6).
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DISCUSSION |
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The role of calcium in apoptosis was discussed previously (39). However, the present data are novel with regard to the role of calcium as mediator of ATP-induced apoptosis. In squamous epithelial cells, including cervical cells, augmented calcium influx stimulates terminal differentiation and envelope formation (22). Because terminal differentiation and apoptosis share signaling pathways (33, 60), a number of authors have used those terms interchangeably to describe the process in which dividing basal cells withdraw from the cell cycle and progressively differentiate as they are displaced toward the epithelial surface (see, e.g., Ref. 23). It is now realized that terminal differentiation and apoptosis are distinct phenomena. Terminal differentiation follows a structured topographical pattern whereby cells in suprabasal layers begin to lose their nucleus and other organelles to become flattened squamae. These are finally shed from the surface as bags of cross-linked keratin filaments enclosed in a cornified envelope (60). Apoptosis, on the other hand, can be observed throughout the thickness of the epithelium, including in basal/parabasal layers (Refs. 38 and 60 and the present results).
In the present study we show, for the first time, that apoptosis can be induced unrelated to terminal differentiation. hECE cells cultured in low (0.6 mM) Ca2+ remain as a monolayer of proliferating cells, resembling the basal/parabasal layers of the ectocervical epithelium (16). Switching cells to normal (1.2 mM) Ca2+ induces terminal differentiation (22) and biochemical changes of apoptosis (present results). However, in cells maintained in 0.6 mM Ca2+, TNF- and BzATP induced apoptosis without stimulating terminal differentiation. Interestingly, low extracellular calcium had little effect on the TNF-
-induced apoptosis but it attenuated the BzATP effect, suggesting that the apoptotic effect of BzATP utilizes predominantly a calcium-dependent mechanism.
In human cervical epithelial cells the apoptotic effect of ATP utilizes mainly the caspase-9-mitochondrial pathway. This pathway involves disruption of the inner mitochondrial transmembrane potential by hyperoxidation and elevated cytosolic calcium, and it leads to the formation of mitochondrial permeability transition pores (25, 28). The present data are consistent with this model, because lowered extracellular calcium blocked the effect of BzATP. Human cervical epithelial cells express the P2X7 receptor, and activation of the receptor leads to prolonged and sustained calcium influx and increased cytosolic calcium (15). It is therefore suggested that the apoptosis in human cervical epithelial cells involves calcium-induced mitochondrial dysfunction.
The mechanism by which activation of the P2X7 receptor induces uncontrolled increases in cytosolic calcium is unclear, but it probably involves increased plasma membrane permeability to Ca2+. The P2X7 receptor is unique in its ability to form pores in the plasma membrane in the continued presence of the ligand (58). Plasma membrane pore formation depends on the long COOH terminus of the receptor (5), and oligomerization of neighboring molecules is believed to cause progressive dilatation of the pore to a diameter of 4 nm and an increase in the permeation path to molecules of 400- to 900-Da molecular mass (24). In its "final" size the pore is relatively permeable to Ca2+, but it remains selective to other cations and is impermeable to anions (41).
Activation of P2X7 receptors can also induce apoptosis by other mechanisms, including IL-1 (9), TNF-
-TNF-related apoptosis-inducing ligand (TRAIL) (1), and the p38, JNK/SAPK (19), and NF-
B (10) pathways. We found that the caspase-8 inhibitor IETD-FMK mildly attenuated the BzATP-induced apoptosis, suggesting involvement of the caspase-8 pathway. One explanation is that, in addition to the mitochondrial pathway, ATP activated another pathway such as the p38, JNK/SAPK, or NF-
B cascades. The TNF-
-TRAIL pathway could be one such mechanism because effects of BzATP were partially additive to those of TNF-
, suggesting some degree of sharing of signaling pathways. Another explanation is cross-interaction between the caspase-9 and caspase-8 pathways and amplification of caspase-3 activation (20). Regardless of the mechanism involved, the contribution of the caspase-8 pathway was significantly smaller compared with that of the caspase-9 pathway.
The present results provide insight into the physiological relevance of P2X7 receptor actions. In vivo, the only known ligand of the P2X7 receptor is ATP. Extracellular levels of ATP are determined by the amount of ATP released from cells and by ATP degradation. Intracellular levels of ATP range from 3 to 5 mM, and most cells constitutively secrete ATP into the extracellular fluid via specialized transporters (6). Extracellular ATP is usually hydrolyzed to ADP and AMP by surface ectonucleotidases, and AMP can be converted into adenosine by 5'-nucleotidases (62). Similar to other recent reports (17, 18, 31, 51), in the conditioned medium of cultured human cervical epithelial cells ATP steady-state activity was 0.5 µM, which is in the range that is sufficient to activate the human P2X7 receptor (present study). This finding can explain activation of the P2X7 receptor by ATP in vivo; accordingly, cervical cells self-regulate cytosolic calcium and apoptosis by secreting ATP into the extracellular fluid. A similar concept was previously suggested for the regulation of cell volume (59) and for the regulation of renal microcirculation (37). In summary, our findings suggest a novel autocrine-paracrine mechanism of P2X7 receptor control of cervical cell apoptosis. This model could be important for the understanding of cervical cell biology and cancer development.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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