Introduction

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Zinc ion is a critical component of many proteins and plays a key role in biological processes such as the regulation of DNA binding, the activation of transcription factors, or the regulation of apoptosis (Coleman, 1992; Vallee and Falchuk 1993; Reyes, 1996). The inhibitory effect of zinc on apoptosis has been attributed to its inhibitory effect on calcium- and/or magnesium-dependent endonuclease that might be involved in DNA fragmentation (Cohen and Duke, 1984; Giannakis et al., 1991; Gaido and Cidlowski, 1991).

However, the observation that cadmium, a divalent ion, induced apoptosis in intact renal cells and that it was inhibitable by zinc at micromolar concentrations raised some questions. First, given that the target for the antiapoptotic action of zinc was presumed to be the nuclear endonuclease, which requires the divalent calcium and/or magnesium for its activation, how do cadmium and zinc regulate the cell destiny? Cadmium induces apoptosis (Azzouzi et al., 1994; Ishido et al., 1995, 1998; Xu et al., 1996; Yan et al., 1997), whereas zinc prevents it (Ishido et al., 1995), although both cadmium and zinc could inhibit the activity of the endonuclease of isolated nuclei (Lohmann and Beyersmann, 1994). Second, micromolar concentrations of zinc could inhibit cadmium-induced apoptosis (Ishido et al., 1995), whereas a millimolar concentration of zinc was required for the inhibition of the purified calcium-dependent endonuclease (Reyes, 1996). These observations suggest the existence of another mechanism for the protective effect of zinc on programmed cell death. Therefore, in this study, we tested the hypothesis that zinc has mitogenic activity, which contributes to its inhibitory effect on apoptotic cell death.

	Materials and Methods

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Cell Culture and Transfection. LLC-PK₁ cells (CRL1392; American Type Culture Collection, Manassas, VA) were grown in Dulbecco's modified Eagle's Medium (Sigma Chemical Co., St. Louis, MO) supplemented with 10% fetal bovine serum (FBS; Lot no. 30A0444S; Gibco BRL, Rockville, MD), penicillin (100 U/ml), and streptomycin (100 µg/ml) in a humidified atmosphere of 95% air:5% CO₂ at 37°C. The cells were subcultured (1:4) 2 to 3 times per week. Cell viability was determined by means of the crystal violet staining method.

In Situ Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick-End Labeling (TUNEL). LLC-PK₁ cells were starved in serum-free Dulbecco's modified Eagle's medium for 16 h before the addition of the metal. The cells were exposed to nothing or to the metals for 13 h at 37°C. Then, the cells were fixed in 4% paraformaldehyde, washed twice with PBS, and permeabilized in 0.5% Triton X-100 for 5 min on ice. In situ TUNEL labeling was done with fluorescein dUTP (Boehringer-Mannheim, Mannheim, Germany) in the presence of terminal deoxynucleotidyl transferase for 1 h at 37°C. After labeling, the cells were washed with PBS twice and then directly surveyed under a fluorescence microscope.

DNA Fragmentation Analysis. DNA fragmentation analysis was carried out as described (Wylle, 1980). The cells were subcultured at 70% confluency and maintained in serum-free medium for 16 h. For examination of the effects of dialyzed FBS on the cadmium-induced DNA fragmentation, the cells were driven by dialyzed FBS (0-20%) for 4 h before the addition of cadmium. Then, the cells were exposed to cadmium for 13 h at 37°C. Treated cells (4 × 10⁶) were washed twice with PBS and lysed in 5 mM Tris buffer (pH 7.4) containing 0.5% Triton X-100 and 20 mM EDTA at 4°C for 20 min. After centrifugation at 28,000g for 20 min, DNA fragments were extracted with phenol chloroform and precipitated in ethanol. The sample was treated with 20 µg/ml RNase A (Sigma Chemical Co.) and electrophoresed on a 1.2% agarose gel.

Bromodeoxyuridine (BrdU) Incorporation. LLC-PK₁ cells were grown in an 8-well chamber slide (Nunc, Naperville, IL) and were starved in a serum-free medium for 16 h before the addition of the metal or dialyzed FBS (Lot no. 1000418; Gibco BRL). The cells were exposed to nothing, to the metal, or to dialyzed FBS for the indicated periods of time at 37°C. Then, 10 µM BrdU was added for an additional 3 h at 37°C. For visualization of BrdU incorporation, the cells were fixed with 70% ethanol in 50 mM glycine (pH 2.0) for 20 min at 20°C. Fixed cells were then treated with nuclease. Incorporated BrdU was detected with monoclonal anti-BrdU antibody and anti-mouse IgG antibody conjugated to fluorescence (Boehringer-Mannheim). For quantification of relative BrdU incorporation, LLC-PK₁ cells were grown in a 96-well plate (Costar Co., Cambridge, MA) and treated with the metals or dialysed FBS. The treated cells were fixed with 70% ethanol in 0.5 M HCl for 30 min at 20°C. After treatment with nuclease, the cells were incubated with anti-BrdU antibody conjugated to peroxidase (Boehringer-Mannheim). Bound enzyme was detected with the substrate ABTS (2,2'-azino-di-[3-ethylbenzthiazolinesulfonate]; Boehringer-Mannheim) and quantified by measuring absorbance at 405 nm with an enzyme-linked immunosorbent assay (ELISA) plate reader (Bio-Rad, Hercules, CA).

Cell Lysates and Western Blot Analysis. Cells were harvested and homogenized in 20 mM HEPES (pH 7.8) containing 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride (Wako Co., Osaka, Japan), 5 µg/ml pepstatin (Peptide Institute Inc., Osaka, Japan), and 5 µg/ml leupeptin (Peptide Institute Inc.). Protein concentrations were measured with a bicinchoninic acid kit (Pierce Chemical Co., Rockford, IL) with bovine serum albumin as a standard. Five to 20 µg of proteins were subjected to 15% polyacrylamide gels containing 0.1% SDS under reducing conditions (Laemmli, 1970). Proteins in an SDS gel were electrophoretically transferred at 2 mA/cm² for 30 min onto an Immobilon membrane (Millipore Corp., Osaka, Japan) in an Atto semidry horizontal electrophoretic transfer unit (Atto, Tokyo, Japan). The transferred membrane was incubated with monoclonal antibodies at a concentration of 0.5 µg/ml for 1 h at room temperature. The monoclonal antibodies used were raised against synthetic peptides corresponding to amino acids 41-54 of the human Bcl-2 protein. After incubation with monoclonal antibodies, the sheets were washed and the antibodies were detected with horseradish peroxidase-conjugated anti-mouse IgG with an enhanced chemiluminescence Western blotting detection kit, according to the instructions of the manufacturer (Amersham Pharmacia Biotech, Uppsala, Sweden). Gels were calibrated with prestained molecular markers (Bio-Rad).

	Results

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Previously, we demonstrated that in quiescent renal cells, 10 µM cadmium elicited DNA fragmentation and that a 5 M excess of zinc (50 µM) could antagonize the apoptotic action of cadmium (Ishido et al., 1995). To explore a more potent protection by zinc at lower concentrations, we first examined whether a lower concentration than 10 µM cadmium induced apoptosis. Figure 1 shows that the addition of 1 µM cadmium to porcine renal cultured LLC-PK₁ cells caused apoptosis (Fig. 1B), as assessed by in situ TUNEL labeling. A 5 M excess of zinc (5 µM) could block the metal-induced DNA fragmentation in situ (Fig. 1C). Thus, these data show the inhibitory effect of zinc at a lower µM concentration.

View larger version (48K): [in this window] [in a new window]   Fig. 1.   Inhibition of cadmium-induced apoptosis by lower concentration of zinc, as revealed by in situ TUNEL labeling. LLC-PK1 cells were starved in serum-free medium 16 h before the addition of the metal. The cells were exposed to nothing (A), to 1 µM cadmium alone (B), and to 1 µM cadmium plus 5 µM zinc (C) for 13 h at 37°C. In situ TUNEL labeling was done with fluorescein dUTP. Original magnification, 200×.

We then evaluated whether zinc has proliferative activity. Figure 2A shows the visualization of BrdU incorporation by fluorescent microscopy. Serum-starved LLC-PK₁ cells showed little pulsing of BrdU incorporation (Fig. 2A, f). However, 5 µM zinc alone facilitated the DNA synthesis in quiescent cells (Fig. 2A, b) as efficiently as 1% dialysed FBS (Fig. 2A, a), whereas 1 µM cadmium alone did not (Fig. 2A, e). In the presence of 1 µM cadmium, 5 µM zinc could still promote DNA synthesis (Fig. 2A, c), although the population of labeled cells was less than that of the cells that were treated with 5 µM zinc alone. A lower concentration of zinc (0.1 µM) than of cadmium (1 µM) failed to facilitate DNA synthesis (Fig. 2A, d). These data show that zinc has mitogenic activity.

View larger version (75K): [in this window] [in a new window]   Fig. 2.   A, visualization of stimulation of BrdU incorporation by zinc. LLC-PK1 cells were starved in serum-free medium 16 h before the addition of the metal or dialysed FBS. The cells were exposed to 1% dialysed FBS (a), 5 µM zinc alone (b), 5 µM zinc plus 1 µM cadmium (c), 0.1 µM zinc plus 1 µM cadmium (d), 1 µM cadmium alone (e), and nothing (f) for 13 h at 37°C. They then were pulse-labeled with 10 µM BrdU. Original magnification, 200×. B, quantification of relative BrdU incorporation by ELISA. Starved LLC-PK1 cells were exposed to 1% dialysed FBS (), 5 µM zinc alone (), 1% dialysed FBS plus 1 µM cadmium (), 5 µM zinc plus 1 µM cadmium (), and to nothing () for the indicated time at 37°C. After further incubation with 10 µM BrdU for 3 h, the relative BrdU incorporation was quantified with an ELISA kit. The results are presented as a fold of the value obtained in the corresponding cells stimulated by the metal and/or FBS for 30 min (mean ± S.E.)

Stimulation of BrdU incorporation by zinc was then quantified by ELISA. Figure 2B shows the time course of BrdU incorporation. Stimulation of the cells with dialyzed FBS (1%) and zinc (5 µM) facilitated DNA synthesis to a greater extent than that seen in unstimulated control cells. A molar excess of zinc (5 µM) could still promote BrdU incorporation in the presence of cadmium (1 µM). However, 0.1 µM zinc failed to promote BrdU incorporation, and therefore cadmium (1 µM) induced apoptosis. Thus, these data confirm the results shown in Fig. 2A, suggesting that the ability of zinc to facilitate DNA synthesis might contribute to its protective effect on apoptosis.

To confirm this, we next used dialysed FBS as a mitogen to examine whether cadmium-induced apoptotic cell death was inhibited by mitogenic factors in the serum instead of by zinc. By inductively coupled plasma atomic emission spectrometry, FBS (Lot no. 30A0444S; Gibco BRL) used for culturing the cells contained about 20 µM zinc. The dialysed FBS (Lot no. 1000418; Gibco BRL) used for this experiment also contained about 20 µM zinc, indicating that most of the zinc in FBS is bound to various proteins such as albumin and ₂-macroglobulin, as reported previously (Reyes, 1996). Figure 3 shows the effect of dialysed FBS on DNA fragmentation triggered by cadmium. Ten percent of dialysed FBS completely inhibited 1 µM cadmium-induced DNA fragmentation. One percent of dialysed FBS also was sufficient to inhibit it in correlation with the extent of the cell growth by 1% dialysed FBS in the presence of 1 µM cadmium (Fig. 2B). DNA fragmentation was not seen in the absence of cadmium (Fig. 3, lanes 8-12). Thus, cadmium-induced nephroptosis was inhibitable by zinc and by any growth factors in the serum, both of which induce cell growth, indicating that the inhibition of programmed cell death by zinc occurs via its activity in promoting the entry of cells into the S phase of DNA synthesis of the cell cycle.

View larger version (55K): [in this window] [in a new window]   Fig. 3.   Inhibition by dialysed FBS of cadmium-induced DNA fragmentation. LLC-PK1 cells (4 × 106) were exposed to 1 µM cadmium (lanes 2-6) or to nothing (lanes 8-12) in the presence of various concentrations of dialysed FBS at 37°C for 13 h: 0% (lanes 2 and 8), 0.1% (lanes 3 and 9), 1% (lanes 4 and 10), 10% (lanes 5 and 11), and 20% (lanes 6 and 12). Size markers are shown in lanes 1 and 7. DNA fragmentation was assessed in Materials and Methods.

To examine whether a death antagonist, Bcl-2, is involved in zinc-mediated cell survival, we carried out Western blot analysis. Figure 4 shows the time course of the induction of endogenous Bcl-2 protein by zinc. Bcl-2 proteins were markedly increased by 5 µM zinc as early as 3 h after exposure compared with the basal level of the proteins (Fig. 4, lanes 1 and 3). Expression levels of Bcl-2 proteins were much more stimulated by a 10-fold higher concentration of zinc (50 µM; Fig. 4, lane 7).

View larger version (33K): [in this window] [in a new window]   Fig. 4.   Western blot analysis showing the induction by zinc of endogenous Bcl-2. LLC-PK1 cells were exposed to zinc (5 or 50 µM) for the indicated periods. Cell lysates (20 µg) were subjected to a 15% SDS-gel, transferred to Immobilon membranes, and immunoblotted with monoclonal antibodies against Bcl-2. The positions of size markers (kDa) are shown on the left margin.

To examine the effect of the level of Bcl-2 proteins on zinc-stimulated DNA synthesis, the cell lines that overexpress Bcl-2 proteins were established. The human bcl-2 cDNA was transfected into the cultured LLC-PK₁ cells. Figure 5A shows the level of Bcl-2 proteins of the control cell lines (Mock) and the transfectant by Western blotting. A clone TI 14 overexpressed the protein at a rate about 20-fold higher than that of the control cells. Figure 5B showed the time course of zinc-stimulated BrdU incorporation in both TI 14 cells and control cells (Mock). Zinc (50 µM) was added to both cell cultures for the indicated periods of time. There was no significant difference between the two kinetics. Thus, overexpression of Bcl-2 proteins did not promote zinc-mediated BrdU incorporation, suggesting that increments of Bcl-2 proteins during their antiapoptotic action were independent of zinc-promoted DNA synthesis.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Overexpression of Bcl-2 proteins does not facilitate zinc-stimulated BrdU incorporation. A, LLC-PK1 cells were transfected with vector alone (lane 1) and with human bcl-2 expression vector (lane 2). Cell lysates (5 cg) from control (Mock) and bcl-2 transfectants (clone TI 14) were subjected to a 15% SDS-gel electrophoresis, transferred to Immobilon membranes, and immunoblotted with monoclonal antibodies against Bcl-2 proteins. The positions of size markers (kDa) are shown on the left margin. B, quantification of relative BrdU incorporation by ELISA. Control cells (Mock; ) and TI 14 transfectants () were exposed to 50 µM zinc for the indicated time for 37°C. After further incubation with 10 µM BrdU for 3 h, the relative BrdU incorporation was quantified. The results are presented as a fold of the value obtained in the corresponding cells stimulated by the metal for 30 min (mean ± S.E.).

	Discussion

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In retrospect, the observation of growth retardation occurring in zinc deficiency focused attention on the role of zinc in cell divisions, development, and differentiation, and led to the discovery of multiple types of zinc metalloenzymes. Zinc serves catalytic, regulatory, and structural roles in the proteins. It also was shown that zinc participates in DNA replication and transcription. More recently, it has been suggested that zinc plays a role in the regulation of the programmed cell death.

Although molecular entities containing zinc were identified, the molecular machinery of the cellular action of zinc, particularly of the antiapoptotic action of the metal, is unknown. In the present study, we demonstrate that zinc facilitates DNA synthesis during the inhibition of apoptosis in addition to the stimulation of the level of Bcl-2 proteins by quiescent renal cells. FBS-driven DNA synthesis also inhibited cadmium-induced apoptosis. It is unknown whether the inhibitory mechanisms of both FBS and zinc are the same. A recent example of apoptosis of nonproliferative cells was shown in adipocytes in which TNF- inhibited cell growth and induced apoptosis (Porras et al., 1997).

Because overexpression of Bcl-2 proteins by transfection did not facilitate zinc-mediated DNA synthesis, Bcl-2 exerts antiapoptotic action rather than accelerating the rate of cell proliferation. This was consistent with a previous report by Vaux et al. (1988).

A number of peptide factors protect the cell from apoptosis, including the neurotrophins, cytokines, and growth factors such as insulin-like growth factor-1(IGF-1) and platelet-derived growth factor (D'Mello et al.,1993; Harrington et al., 1994). These factors drove the cell to proliferate with various degrees of cell growth, suggesting that the induction of proliferation affords protection from apoptosis. One example of this is the cell fate of the fibroblasts that overexpressed c-myc proteins (Rat-1/myc cells; Evan et al., 1992). In the presence of 10% FBS, Rat-1/myc cells grew well, but in the low serum (0.05%), the cells underwent apoptosis that was inhibitable by IGF-1 and platelet-derived growth factor (Harrington et al., 1994). However, further precise analyses, such as site-directed mutagenesis of growth factor receptors, would be required to define the specific cell survival signal pathway because a receptor is coupled to multiple signal transduction pathways for many cellular functions.

The characterization of signal transduction pathways activated by these peptide factors has led to the identification of critical mediators of cell survival. So far, the best characterized mediators of cell survival are the bcl-2 family (Vaux et al., 1988; Tsujimoto, 1989) and phosphoinositide 3-kinase (PI3-kinase) and its downstream target, Akt (Yao and Cooper, 1995; Dudek et al., 1997; Marte and Downward, 1997). IGF-1 and insulin suppressed apoptosis via PI3-kinase and Akt pathways without affecting the expression levels of Bcl-2 (Jung et al., 1996). In contrast, zinc increased the level of Bcl-2. But zinc did not seem to activate PI3-kinase and Akt in renal cultured cells (data not shown). It was observed that cultured cerebellar granule neurons die by apoptosis when switched from a medium containing an elevated concentration (25 mM) of potassium (K⁺) to one with a lower concentration (5 mM) of K⁺, and that an elevated concentration of K⁺, the signaling pathway of which was PI3-kinase-independent, maintained cell survival (D'Mello et al., 1997). These observations suggest the existence of another survival signaling pathway, and it is unknown whether the inhibition of apoptosis by both ions is mediated through a common mechanism. Identification of putative growth factors in serum that contribute to the inhibition of cadmium-induced apoptosis will help to reveal the molecular mechanism of the antiapoptotic action of zinc.

Although it has been reported that caspase-3 was identified as a novel target of zinc inhibition in apoptosis (Perry et al., 1997), it still has been a subject of controversy (Takahashi et al., 1996). The caspase cascade was not involved in cadmium-induced apoptotic machinery because inhibitors for caspase protease failed to block the death-signaling by the metal (data not shown). The elucidation of a still unidentified pathway of zinc-mediated cell survival would lead to a better understanding of the regulation of cell destiny by the metal ions.