The Journal of Nutrition Vol. 128 No. 10 October 1998, pp. 1600-1605

Zinc Deprivation of Murine 3T3 Cells by Use of Diethylenetrinitrilopentaacetate Impairs DNA Synthesis upon Stimulation with Insulin-Like Growth Factor-I (IGF-I)^1,2

Ruth S. MacDonald³, Lavonna C. Wollard-Biddle, Jimmy D. Browning, William H. Thornton Jr., and Boyd L. O'Dell

Nutritional Sciences Program and Department of Biochemistry, University of Missouri, Columbia, MO 65211

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Growth failure in zinc-deficient animals is associated with decreased DNA synthesis; zinc deprivation of 3T3 cells, by use of diethylenetrinitrilopentaacetate (DTPA), impairs thymidine incorporation when the cells are stimulated with fetal bovine serum (FBS). The purpose of this study was to determine the step of cell cycle progression that is affected by zinc deprivation. Swiss murine 3T3 cells were cultured for 3 d in complete media and then for 2 d in low serum media. Cells were then placed in serum-free media and stimulated in sequence with platelet-derived growth factor (PDGF; 3 h), epidermal growth factor (EGF; 0.5 h) and insulin-like growth factor-I (IGF-I; 16 h). The combination of growth factors stimulated thymidine incorporation to the same extent as 10% FBS, and DTPA or EDTA (0.6 mmol/L) inhibited thymidine incorporation. Inhibition was prevented by addition of zinc, but not calcium, iron or cadmium (0.4 mmol/L). When DTPA was present during all stages with no addition of zinc, or zinc added during the competency-priming (PDGF and EGF) step, the IGF-I step, or both steps, the zinc effect occurred at the IGF-I step. Zinc addition 4 h before the measurement of thymidine incorporation had no ameliorative effect, but the presence of zinc during the prior 12 h increased incorporation. Thus zinc exerts its major effect on DNA synthesis during the IGF-I stimulatory phase of the cell cycle. The total zinc concentration of 3T3 cells treated with DTPA for 16 h was not different from that of untreated cells; hence only a small compartment of the cell is affected by DTPA.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Impaired growth is a cardinal sign of zinc deficiency in animals, and growth is largely dependent upon cell division. The latter requires DNA synthesis, a process that is more susceptible to lack of zinc than is protein synthesis (Williams and Chesters 1970). For this reason, the role of zinc in nucleic acid metabolism has received considerable research attention, but the basic role of zinc in DNA synthesis remains unresolved.

Zinc deficiency in rats, whether induced by nutritional deprivation (Eckhert and Hurley 1977, Williams and Chesters 1970) or by infusion of a chelator such as EDTA (Fujioka and Lieberman 1964), decreases DNA synthesis as measured by thymidine incorporation. Chelators also decrease DNA synthesis in cultured cells, and the detrimental effect is prevented specifically by zinc (Rubin 1972). Chesters et al. (1989) studied DNA synthesis in 3T3 fibroblasts that were maintained for 36 h in media that had a low fetal bovine serum (FBS)⁴ concentration (2%), then stimulated with 12% FBS in the presence or absence of diethylenetrinitrilopentaacetate (DTPA). DTPA dramatically decreased thymidine incorporation, an effect that was specifically prevented by zinc supplementation. In an extension of this work, Chesters and Boyne (1991) showed that both mRNA and protein synthesis were required for completion of this zinc-dependent process.

Initiation of cell division in quiescent (G_o) 3T3 cells requires several growth factors that are present in FBS, including platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and insulin-like growth factor-I (IGF-I). The mitotic process is regulated largely by events that occur during the G₁ phase of the cell cycle (Campisi and Pardee 1984). Growth-arrested (G_o) cells must be made competent and primed by stimulation with PDGF and EGF, peptides that are required to initiate subsequent response to IGF-I (Yoshinouchi and Baserga 1993). Activation of the IGF-I receptor is considered to be the primary restriction point of the cell cycle (Pardee et al. 1986).

Chesters and Boyne (1991) speculated that zinc deprivation affects the cell cycle by reducing the concentration of a protein necessary for progression of cells into the S phase. It is postulated here that the same endpoint, failure of DNA synthesis and mitosis, would result from lack of growth factor stimulation, i.e., failure of signal transduction. The purpose of this project was to test this hypothesis by determining the effect of zinc deprivation of 3T3 cells during stimulation by PDGF, EGF and IGF-I. The 3T3 cells were chosen for this study because of their longstanding (Rubin 1972) and extensive (Chesters et al. 1989) use in the investigation of the role of zinc in DNA synthesis. Conditions similar to those of Chesters et al. (1989) were used because this study is largely an extension of earlier work. Cells were grown in the presence of DTPA with and without the addition of zinc, and DNA synthesis was determined by measurement of thymidine incorporation into DNA. A combination of the three growth factors stimulated thymidine incorporation to the same extent as FBS, and deprivation of zinc at the IGF-I step was critical. In spite of the detrimental effect of DTPA on thymidine incorporation, it did not decrease the measurable zinc concentration in the cells.

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

Materials.  Cell culture media and plasticware were obtained from Collaborative Biomedical (Bedford, MA). PDGF and EGF were purchased from Sigma Chemical (St. Louis, MO) and IGF-I from Amgen Biologicals (Thousand Oaks, CA). ³H-Thymidine (3.15 TBq/mmol) was purchased from Amersham Corporation (Arlington Heights, IL). DTPA, receptor-grade bovine serum albumin (BSA) and FBS were from Sigma Chemical. All other chemicals were of reagent grade and came from Sigma Chemical or Fisher Scientific (St. Louis, MO).

Cells and cell culture.  3T3 Swiss murine fibroblasts were obtained from ATCC (Rockville, MD). They were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mmol/L glutamine and 10% FBS. Cells were cultured under 95% CO₂/5% O₂ at 37°C, passaged weekly and maintained at subconfluent density. For experimental use, 15,000 cells per well were plated in 24-well plates with DMEM (1.5 mL) containing 10% FBS. Cells for zinc analysis were plated in 35-mm dishes. After 3 d, the medium was replaced with DMEM containing 2% FBS and incubation continued for 2 d. In some experiments, the cells were stimulated by the addition of 10% FBS to the DMEM; in others, they were stimulated with combinations of PDGF, EGF and IGF-I. The growth factors (GF) were added to the medium with sufficient BSA to provide a final concentration of 0.1% BSA. Initially, the cells were incubated sequentially with the individual GF as follows: PDGF (0.1 nmol/L) for 3 h, EGF (100 nmol/L) for 0.5 h and IGF-I (10 nmol/L) for 16 h. At each stage, the medium was removed and replaced by the appropriately supplemented medium. In some experiments, GF were added collectively, after it was observed that similar results were obtained. To determine the effect of zinc deprivation, DTPA (0.6 mmol/L) was added with or without zinc sulfate (0.4 mmol/L) at each stage of stimulation, according to an experimental protocol described in the Results section. By analysis, the zinc concentration of the DMEM with 2% FBS was 1.1 µmol/L and DMEM with 10% FBS was 5.0 µmol/L.

Thymidine incorporation.  After stimulation by FBS or GF, thymidine incorporation into DNA was determined by adding ³H-thymidine to the medium [1.33 kBq (0.036 µCi) per well]. The cell monolayers were incubated at 37°C for 4 h, washed twice with PBS, and the cells solubilized in 1 mol/L NaOH. Aliquots of the solution were added to scintillation solution (Ultima Gold, Packard Instrument, Downers Grove, IL) and counted in a scintillation counter (Packard). Protein was determined by the Bio-Rad micromethod (Bio-Rad Laboratories, Hercules, CA), using BSA as the standard. To verify that the thymidine counts solubilized by NaOH after the 4-h treatment were incorporated into DNA, comparable cells were treated with trichloroacetic acid (TCA) according to the procedure of Blakesley et al. (1996). Briefly, the monolayers were washed twice with PBS and once with 0.3 mol/L TCA. They were allowed to stand at room temperature for 10 min, then washed twice with 95% alcohol. The washed cells were then solubilized with 1 mol/L NaOH and counted.

Cell number and determination of zinc concentration.  For determination of the number of cells per well, monolayers were washed with PBS, trypsinized and the collected cells stored in RPMI buffer containing 1.3 mol/L dimethyl sulfoxide (Vindelov et al. 1983) at 80° C. The suspensions were thawed, diluted and cell number determined by use of a Coulter Counter (Hiahleah, FL).

The zinc concentration of cells comparable to those used for thymidine incorporation after FBS stimulation was determined. Monolayers were washed four times with zinc-free PBS, dissolved in 0.2% Triton X-100, then diluted with nitric acid to provide final concentrations of 0.016 mol/L HNO₃ and 0.03% Triton X-100. Triplicate aliquots of the solution were injected into a furnace atomic absorption spectrophotometer (SpectrAA-40 "Zeeman," Varian Techtron, Mulgrave, Australia) with autosampler. Zinc was measured using peak area calibration and a standard additions method, with additions of 5, 10 and 15 pg of zinc per sample. The furnace program specified ashing and atomizing temperatures of 300 and 1900°C, respectively.

Statistical treatment.  The data were analyzed with the use of the GLM procedure of SAS (SAS Institute, Cary, NC). All analyses were performed on log-transformed data to correct for nonhomogeneity of variance. The data in Figure 2 were analyzed as a split-plot design, with DTPA treatment as the main plot and DNA isolation method (NaOH or TCA) as the subplot. Data in Table 1 and Figures 3 and 5 were analyzed by one-way ANOVA, and those in Figures 4 and 6 as 2 × 2 factorial designs. Main effects were considered significant at P < 0.05, and interactions at P < 0.10. Group mean comparisons were made using the LSMEANS component of GLM, with probabilities <0.05 considered significant.

View larger version (46K): [in this window] [in a new window]   Fig 2. Comparative thymidine incorporation into DNA as measured by direct NaOH solubilization and by prior trichloroacetic acid (TCA) treatment of cells stimulated with 10% fetal bovine serum (FBS), 10% FBS with 0.6 mmol/L diethylenetrinitrilopentaacetate (DTPA), or 10% FBS with DTPA plus 0.4 mmol/L zinc. Cells were pretreated by culturing for 2 d in 2% FBS. Means (n = 8) are represented by bars and SEM by bar extensions. Significant differences (P < 0.05) within the isolation methods are indicated by different letters. Split-plot analysis revealed significant main (DTPA treatment, P = 0.0006) and subplot (NaOH vs. TCA, P < 0.0001) effects, as well as a significant interaction (P < 0.0001).

View larger version (47K): [in this window] [in a new window]   Fig 3. Specificity of zinc in reversing the effect of two chelators (Chel), diethylenetrinitrilopentaacetate (DTPA) and EDTA, (0.6 mmol/L each) on thymidine incorporation into 3T3 cells. Cells were cultured in 2% fetal bovine serum (FBS) for 2 d, then stimulated by growth factor (GF) [platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) added together followed by insulin-like growth factor (IGF)]. All cations (zinc, calcium, ferric iron, and cadmium) were added with the chelator at 0.4 mmol/L. Significantly different (P < 0.05) means within treatments for a given chelator are indicated by different letters. Other designations as in Figure 2; n = 4 for DTPA and 8 for EDTA.

View larger version (42K): [in this window] [in a new window]   Fig 5. Effect of time of free zinc deprivation on thymidine incorporation by 3T3 cells stimulated with insulin-like growth factor (IGF-I) in the presence of diethylenetrinitrilopentaacetate (DTPA). Cells were made competent and primed with platelet-derived growth factor (PDGF) and epidermal growth factor (EGF), then treated with IGF-I and DTPA (0.6 mmol/L) for a period of 20 h. Zinc (0.4 mmol/L) was added for various periods, ranging from 0 to 20 h; n = 8 in duplicate trials. Bars with different letters are significantly different, P < 0.05.

View larger version (33K): [in this window] [in a new window]   Fig 4. Effect of zinc on thymidine incorporation when added at different stages of growth factor (GF) stimulation in the presence of diethylenetrinitrilopentaacetate (DTPA). Cells were stimulated with all GF in sequence and DTPA (0.6 mmol/L) was present at all steps. Zinc (0.4 mmol/L) was added with specific GF [platelet-derived growth factor (PDGF) + epidermal growth factor (EGF); insulin-like growth factor (IGF-I)], in a factorial design: no zinc, zinc during PDGF-EGF (PE) step, zinc during IGF-I step and zinc during both steps; n = 9. Factorial analysis revealed significant effects of zinc addition during the IGF-I step (P < 0.0001) and a significant PE step × IGF-I step interaction (P = 0.0632). Bars with different letters are significantly different (P < 0.05).

View larger version (39K): [in this window] [in a new window]   Fig 6. Degree of quiescence in 3T3 cells treated with 2% fetal bovine serum (FBS) for 2 d as indicated by their response to insulin-like growth factor-I (IGF-I) with and without priming with platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) (PE) in a factorial design. After pretreatment by culturing for 2 d in 2% FBS, the cells were placed in serum-free medium (SFM) or SFM supplemented with PDGF and EGF, IGF-I or a combination of PDGF, EGF and IGF-I. Factorial analysis showed a significant effect of PE (P < 0.0001) and IGF-I (P < 0.0001) and a significant interaction (P = 0.0058). Bars with different letters are significantly different.

	RESULTS

Abstract Introduction Methods Results Discussion References

Before proceeding with investigation of the mechanism by which DTPA impairs DNA synthesis, it was important to establish the characteristics of the cell culture system. We first determined whether DTPA affected cell appearance or growth rate. The addition of 0.6 mmol/L DTPA to culture media that contained 10% FBS had little or no effect on cell morphology, but tended to decrease cell density (Fig. 1). In view of the reported dramatic effect of DTPA in reducing thymidine incorporation by 3T3 cells and its specific reversal by zinc (Chesters et al. 1989), the zinc concentration in the cells was determined. As shown by the results in Table 1, addition of 0.6 mmol/L DTPA to the medium had no effect on the total measurable zinc concentration of cells placed in that culture for 16 h. However, cells grown in media containing added DTPA (0.6 mmol/L) and zinc (0.4 mmol/L) had a zinc concentration significantly higher than those grown in 10% FBS.

View larger version (153K): [in this window] [in a new window]   Fig 1. Photomicrographs of 3T3 cells cultured in 10% fetal bovine serum (FBS; upper panel) or in 10% FBS supplemented with 0.6 mmol/L diethylenetrinitrilopentaacetate (FBS + DTPA; lower panel). The cells were pretreated by culturing in 2% FBS for 2 d before stimulation with FBS or FBS plus DTPA for 24 h. Bar = 50 µm.

To determine the point in cell cycle progression at which DTPA inhibits growth factor stimulation, it was necessary to establish that the sequential addition of the three growth factors (PDGF, EGF and IGF-I) to the serum-free DMEM stimulates thymidine incorporation to the same extent as 10% FBS. In a series of seven trials involving 28 cultures, it was found that the combination of GF stimulated the incorporation of thymidine to the same extent as 10% FBS (12.0 ± 1.6 vs. 12.9 ± 1.4 Bq/µg protein). In these and subsequent trials, thymidine incorporation after a 4-h exposure was measured by solubilizing the cells with NaOH. To verify that this procedure measured incorporation into DNA, it was compared with the TCA method; the results are presented in Figure 2. Thymidine incorporation as measured by NaOH solubilization was slightly higher (12%) than that measured by TCA, but the measured effects imposed by chelator treatment were the same. However, as expected, in other trials that involved 1-h exposure to thymidine, NaOH gave significantly higher values than TCA (data not shown). To confirm that thymidine incorporation into DNA as measured by this method relates directly to mitotic rate, cells in similar 24-well plates were counted over a 2-d period. The doubling time was ~16 h for control cells cultured in 10% FBS, over a density range of 1.3-9.3 × 10⁴ cells/cm². After 2 d, wells containing DTPA + Zn had 82% of the number in control wells; those containing DTPA alone had 10% less than the starting number.

The dramatic effect of DTPA on thymidine incorporation was confirmed in a factorial experiment in which cells were stimulated with 10% FBS or GF in the presence of DTPA or DTPA + Zn. As a percentage of controls (10% FBS), the 10% FBS-stimulated cultures containing DTPA + Zn incorporated 94.8 ± 4.9% as much thymidine per unit of protein; this compares with 1.9 ± 0.3% for similar cultures containing DTPA alone. For GF-stimulated cells, the comparable values were 97.9 ± 5.3 versus 5.3 ± 1.6%, respectively. The effect of zinc was highly significant (n = 16, P < 0.05 for each treatment).

A degree of zinc specificity in reversing the effect of two chelators, DTPA and EDTA, in the GF-stimulated system is shown in Figure 3. Addition of DTPA plus zinc supported thymidine incorporation to the same extent as observed in control cells (GF-stimulated cells without the chelator), but the addition of Ca²⁺, Fe³⁺ or Cd²⁺ at the same concentration, 0.4 mmol/L, had no beneficial effect. The results obtained with EDTA (0.6 mmol/L) were analogous to those obtained with DTPA.

To determine at which stage of cell cycle progression DTPA exerts its inhibiting effect on thymidine incorporation, cells were treated with DTPA at all stages of GF stimulation and were supplemented with zinc at specific stages of stimulation. All cells were stimulated with PDGF, EGF and IGF-I, but zinc was added in a factorial design, i.e., not at all, at the PDGF-EGF priming step, at the IGF-I step, or at both steps. The results of this experiment are presented in Figure 4. As shown, addition of zinc during the priming-competency step (PDGF-EGF) had little effect. Addition of zinc during the IGF-I step alone significantly increased thymidine incorporation compared with addition of zinc during the priming steps. Zinc addition during all stages of stimulation (PDGF, EGF and IGF-I) also improved thymidine incorporation significantly but to a lesser extent than when zinc was present during the IGF-I step only. The latter result suggests that zinc had a negative regulatory role during the priming-competency step, probably during EGF stimulation. Data not presented in Figure 4 show that the addition of zinc during stimulation with EGF and IGF-I also supported less thymidine incorporation than when it was present with IGF-I only (7.2 ± 0.6 vs. 11.3 ± 2.0 Bq/µg protein, P < 0.09).

Because zinc in the presence of DTPA had its major effect at the stage of IGF-I stimulation, it was important to establish the period of time during which zinc is essential for IGF-I stimulation. To investigate this question, cells were stimulated with IGF-I in the presence of DTPA; zinc was added at intervals from 0 to 20 h before determination of thymidine incorporation. The results, summarized in Figure 5, show the effect of various periods of zinc exposure before thymidine incorporation was measured. Four hours of zinc treatment was no better than none, but 8 h provided significant improvement. There was a linear response to zinc treatment from 8 to 16 h but no difference between 16 and 20 h. Thus, zinc had no effect during the first 4 h of IGF-I stimulation but had a progressively increasing effect over the next 12 h.

The interpretation of the results presented in Figure 5 depends in part on whether all cells were synchronized and quiescent before stimulation with IGF-I. To address this question, cells were cultured in 2% FBS as usual, transferred to serum-free medium and stimulated with PDGF, EGF, or IGF-I or combinations of these GF. Thymidine incorporation was measured, and the results are summarized in Figure 6. A combination of PDGF and EGF had little stimulatory effect, and the combination of PDGF, EGF and IGF-I gave near maximal response. IGF-I alone stimulated thymidine incorporation, ~40% of maximum (all GF), showing that PDGF and EGF were not needed by all cells to be made competent and primed.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

DTPA is an extremely potent chelator of zinc, with a K_d of ~2 × 10¹⁹ mol/L (Martell and Smith 1975), but it is not specific for zinc. All biologically important cations have comparably low dissociation constants. Both DTPA and EDTA are membrane impermeant (Cleton et al. 1963), and this property undoubtedly has an important bearing on their mode of action. As observed here, when DTPA was present in the medium at a concentration of 0.6 mmol/L for 16 h, it had no effect on the measurable zinc concentration of 3T3 cells. This fact suggests that DPTA exerts its effect on thymidine incorporation by reducing the zinc concentration of a small compartment of the cell, such as the plasma membrane, resulting in a change too small to be detected by total zinc analysis. It is of considerable interest that addition of the combination of DTPA (0.6 mmol/L) and zinc (0.4 mmol/L) to the medium increased the zinc concentration of the cells significantly. It is not clear how the DTPA-zinc complex increases the cell zinc concentration. Even with added zinc, the calculated concentration of free zinc in the medium is extremely low. The calculated free zinc concentration of a solution containing 0.6 mmol/L DTPA and 0.4 mmol/L total zinc at pH 7.0 is 1.6 × 10¹³ mol/L (estimation calculated using the software program MaxWinc v1.7, http://www-leland.stanford.edu/sim;cpatton). This leaves essentially all of the zinc in the form of a Zn-DTPA complex. The cells may absorb zinc directly from the bound complex, or they may strongly adsorb the complex. In any case, the higher level of total zinc had no detectable effect on cell performance compared with the basal level without DTPA. There is increasing evidence that the first limiting functional defect in zinc-deficient animals occurs at the cell plasma membrane (Bettger and O'Dell 1993). Erythrocytes from zinc-deficient rats and pigs are more fragile to osmotic stress; their plasma membranes contain a lower zinc concentration than normal, whereas the cytoplasm zinc concentration is unchanged (Bettger and Taylor 1986, Johanning and O'Dell 1989, Johanning et al. 1990, O'Dell et al. 1987). In view of these findings, the impaired thymidine incorporation by 3T3 cells treated with DTPA, which occurs without a detectable change in total cell zinc concentration, may be the result of a decreased concentration of zinc in the plasma membrane.

Cells stimulated with the growth factors PDGF, EGF and IGF-I incorporated thymidine into DNA to the same extent as did those stimulated with 10% FBS. Furthermore, the addition of DTPA to the medium of GF-stimulated cells had a similar inhibitory effect. Of the cations tested, only zinc prevented the effect of DTPA. EDTA had a similar inhibitory effect. The equivalency of stimulation by GF and FBS allowed determination of the stage of cell cycle progression at which zinc plays its critical role. Zinc had its major effect during the IGF-I stimulation phase of the cycle (Fig. 4). The addition of zinc during both phases of GF stimulation did not result in greater thymidine incorporation than did its addition during the IGF-I stage. The periods of GF stimulation, 16 h for IGF-I, 3 h for PDGF and 0.5 h for EGF, are those commonly used (Kojima et al. 1988 and 1990), but the different times of exposure to DTPA without zinc could have a bearing on the results. Conceivably, the 16-h exposure to IGF-I without zinc could disrupt cell metabolism to a greater extent than the 3- or 0.5-h exposures. However, the results suggest that the presence of zinc during the EGF stage had a negative rather than a positive regulatory effect and this occurred during a 0.5-h exposure to DTPA without zinc. It is of considerable interest and importance that zinc exerts its major effect at the IGF-I step, the chief stimulation before the restriction point (Pardee et al. 1986).

As shown in Figure 5, addition of zinc with DTPA during the first 4 h of IGF-I stimulation had no effect on subsequent thymidine incorporation, but it was critical during the next 12 h. This observation is similar to that of Chesters et al. (1989) who found that the addition of zinc to 3T3 cells stimulated with FBS was critical during the period 8 h after stimulation to 3 h before the start of the S phase. In their experiments, the measure of cell entry into the S phase was the proportion of cell nuclei labeled after continuous exposure to radioactive thymidine, whereas here it was the rate of thymidine incorporated during a 4-h period after GF stimulation. In subsequent work, Chesters and Boyne (1991) confirmed that zinc is required during the second half of the G₁ transition phase and proposed that zinc plays a critical role in the accumulation and maintenance of a protein required for cell cycle progression into the S phase. This a reasonable hypothesis, granted that the cells are quiescent and synchronized before stimulation and zinc treatment, but in our experience, not all cells were quiescent after exposure to 2% FBS for 48 h. Under our conditions, the results could be explained by the fact that there is a proportion of continuously cycling cells that required zinc ~4 h after IGF-I stimulation in order to enter the S phase. The number of cells entering the S phase continued to increase as they progressed through the cycle, and thus the total thymidine incorporated increased.

As suggested by Chesters and Boyne (1991), zinc deficiency induced by DTPA could impair DNA synthesis by direct interference with the production of a protein (Pardee et al. 1986) essential for progression of 3T3 cells into the S phase of the cell cycle. Considering the fact that in our hands the cells were not synchronized, the results could be explained as well by failure of IGF-I signal transduction. Transduction of the IGF-I signal requires calcium uptake (Kojima et al. 1988), and zinc deficiency has been shown to impair calcium uptake by platelets (O'Dell and Emery 1991) and by brain synaptic membranes (Browning and O'Dell 1994 and 1995). Based on these observations, it is postulated that failure of IGF-I signal transduction, as a result of a faulty calcium channel, is the basis of the impaired DNA synthesis observed in 3T3 cells treated with DTPA.

	FOOTNOTES

Manuscript received 3 April 1998. Initial reviews completed 30 April 1998. Revision accepted 26 May 1998.

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