1Department of Biotechnology, College of Engineering, and 2Protein Network Research Center, Yonsei University, Seoul 120-752; 3Laboratory of Molecular Oncology, Korea Institute of Radiological and Medical Sciences, Seoul 136-706; and 4Department of Oral Biology, College of Dentistry, Yonsei University, Seoul 120-752, South Korea
Submitted 28 January 2003 ; accepted in final form 17 June 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
signal transduction; extracellular signal-regulated kinase; p21Cip/WAF1
Extracellular zinc is reported to activate signaling pathways related to cell growth regulation, such as MAPK and phosphoinositol 3-kinase pathways (17, 20, 28, 32), and the chelation of intracellular zinc does not change the activation status of MAPK by extracellular zinc (14). Moreover, cellular growth regulation by zinc is not likely to be mediated by direct interaction with intracellular metalloproteins, because symptoms of zinc deficiency precede changes in the intracellular zinc content (6, 20). Recently, Hershfinkel et al. (15) reported the presence of an extracellular zinc-sensing receptor (ZnR) in HT-29 colorectal cancer cells and suggested its involvement in extracellular receptor-mediated signaling pathway(s) for growth regulation.
In the present study, we investigated the mechanism of the differential regulation of HT-29 colorectal cancer cell growth at nontoxic micromolar concentrations of extracellular zinc. HT-29 colorectal cancer cell growth was found to be positively and negatively regulated by different extracellular zinc concentrations, which resulted from differential ERK activation. Moreover, cyclin D1 and p21Cip/WAF1 cell cycle regulators were also differentially induced, and this was also found to depend on ERK activation and to be related to the differential regulation of cell growth. No significant accumulation of intracellular zinc by the application of micromolar concentrations of extracellular zinc suggests that the effects of zinc are mediated by receptor-mediated signal transduction rather than being due to complex effects associated with intracellular zinc accumulation (18, 34).
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell counting and thymidine incorporation. HT-29 colorectal cells were grown in minimally serum-starved medium (MSSM) containing 1% FBS for 16 h. The cell culture medium was replaced with fresh 1 or 10% FBS medium containing different concentrations of ZnCl2 (0-500 µM). The attached cell numbers were counted by mounting 10 µl of the cell mixture (1:1 mixture of 0.4% trypan blue and HT-29 cell suspension) onto a Tiefe Depth Profondeur 0.0025-mm2 cell-counting plate (Superior) and observing plated cells under a microscope.
For thymidine incorporation, the HT-29 cells were plated in 12-well culture dishes at 1.3 x 105 cells/well and then grown in MSSM for 16-20 h. The medium was then replaced with fresh MSSM containing different ZnCl2 concentrations (0, 10, or 100 µM), and the cells were further incubated for 40 h. When required, PD-98059 was also added 30 min before ZnCl2 treatment. Finally, the incubations were continued in the presence of [3H]thymidine (0.5 µCi/well) for 8 h. After cells were washed twice with 5% trichloroacetic acid, they were solubilized by the addition of 0.2 N NaOH, and DNA-associated 3H activity was counted by using a liquid scintillation counter.
Western blot analysis. Cell extracts were prepared from harvested cells as previously described (28). Samples of 10-50 µg protein from the whole cell extracts were separated by SDS-PAGE, and the blots were prepared on a Protran membrane. The following antibodies were used as probes: for ERK, phosphospecific anti-ERK (New England Biolabs, Beverly, MA) and anti-ERK (Stratagene, La Jolla, CA) antibodies; for cyclin D1 and p21Cip/WAF1, anti-cyclin D1 and -p21Cip/WAF1 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). The blots were probed with horseradish peroxidase-conjugated secondary antibodies (Transduction Laboratories, San Diego, CA), and the proteins were visualized by enhanced chemiluminescence.
Measurement of intracellular zinc. The intracellular zinc concentration ([Zn2+]i) in HT-29 cells was measured by using a modification of a method described previously (34). HT-29 cells were plated onto coverslips at a density of 3 x 105 cells/coverslip into six-well plates, and the cells were grown overnight in McCoy's 5A medium containing 1 or 10% FBS and treated with ZnCl2 at 1, 100, or 500 µM. Cells were then loaded with the fluorescent indicator mag-fura-2 at 37°C in a 5% CO2 incubator by including 3 µM of the acetoxymethyl ester of mag-fura-2 (mag-fura-2 AM; Molecular Probes, Eugene, OR) for 20 min in a HCO3--buffered solution containing (in mM): 110 NaCl, 4.5 KCl, 1 NaH2PO4, 1 MgSO4, 1.5 CaCl2, 5 HEPES-Na, 5 HEPES-free acid, 25 NaHCO3, and 10 D-glucose (pH 7.4). Cells were then rinsed twice and incubated in the HCO3--buffered solution for at least 20 min before use.
[Zn2+]i was measured on the stage of an inverted microscope (Nikon, Tokyo, Japan) by spectrofluorometry (Photon Technology International, New Brunswick, NJ) while cells were superfused at a constant perfusion rate of 2 ml/min with HCO3--buffered solution equilibrated with 95% O2-5% CO2 to maintain a pH of 7.4. All experiments were performed at 37°C. The excitation wavelength alternated between 340 and 380 nm, and emission fluorescence was recorded at 510 nm. The values of [Zn2+]i were calculated by using a modification of the equation described by Grynkiewicz et al. (12): [Zn2+]i = Kd · Sf2/Sb2[(R - Rmin)/(Rmax - R)], in which Kd of magfura-2 for Zn2+ was assumed to be 20 nM (36), R is the observed 340/380 fluorescence ratio, Rmin is the 340/380 fluorescence ratio value determined in mag-fura-2-loaded HT-29 cells exposed to 100 µM TPEN, Rmax is the 340/380 fluorescence ratio value [obtained by adding 20 µM 1-hydroxypyridine-2-thione (pyrithione; Sigma, St. Louis, MO), a Zn2+ ionophore, in the presence of 1 mM Zn2+], Sf2 is the fluorescence intensity at 380 nm at Rmin, and Sb2 is the fluorescence intensity at 380 nm at Rmax.
Statistical analysis. The statistical significances of differences between the positive- and negative-regulated groups were determined by using a one-way ANOVA and Bonferroni's test. A P value <0.05 was considered statistically significant. The results are expressed as means ± SD.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
The level of DNA synthesis was measured by determining the extent of [3H]thymidine incorporation and was found to increase in cells by more than twofold on adding 10 µM ZnCl2 to MSSM (Fig. 1C). The amount of thymidine incorporated by 10 µM ZnCl2 was only slightly lower than that level acquired by growing cells in medium containing 10% FBS without zinc treatment (Fig. 1C). However, the thymidine incorporation level was reduced by adding 100 µM ZnCl2 to cells grown in MSSM.
Positive and negative regulation of zinc-treated HT-29 cell growth by PD-98059 in MSSM. The ERK pathway is involved in cell cycle arrest (7, 35, 40) and is well known to be a function of cell cycle progression (4, 7). The ERKs were previously found to be strongly activated in a prolonged manner by 100 µM ZnCl2 treatments in MSSM, and this activation was related to the antiproliferation of colorectal cancer cells, including HT-29 cells (28). However, ERK activities were also found to be transiently and weakly activated by extracellular zinc treatment (14, 33). This growth regulation was not further investigated with respect to differential ERK regulation. In this study, we investigated the growth and proliferation induced by the different zinc concentrations and their relationship with the ERK activation profiles.
Cell numbers, which had been previously increased by 10 µM ZnCl2 in MSSM, were dose-dependently decreased by treatment with the MEK inhibitor PD-98059 (Fig. 2A), suggesting that ERK activation affects the proliferation of HT-29 cells by 10 µM ZnCl2 in MSSM. Surprisingly, cell numbers reduced by 100 µM ZnCl2 treatment in MSSM were increased by subsequently treating the cells with 1 µM PD-98059 (Fig. 2B). These results suggest that the ERK activation by 100 µM ZnCl2 in MSSM, which caused growth inhibition (28), was blocked by 1 µM PD-98059. A further increase in PD-98059 to 5 and 10 µM in the cells treated with 100 µM ZnCl2 resulted in a further reduction in the number of cells (Fig. 2B). These results suggest that the ERK pathway may be involved in both the positive and negative regulation of cell growth and further suggest that the zinc-induced growth regulation of HT-29 cells may be highly sensitive to the level of ERK activation. The upregulation of cell numbers by PD-98059 in 100 µM ZnCl2-treated cells in MSSM confirms that 100 µM ZnCl2 is not a toxic concentration to HT-29 cells. The thymidine incorporation level, which was increased by adding 10 µM ZnCl2, was also dose-dependently inhibited by pretreating the cells with PD-98059 in MSSM (Fig. 2C). However, the thymidine incorporation level was also increased by the 1 µM PD-98059 cotreatment in the cells treated with 100 µM ZnCl2 and was reduced by cotreatment with 10 µM PD-98059 (Fig. 2C) compared with 1 µM PD-98059 cotreatment. Therefore, cell numbers in this system were found to be highly correlated with proliferation status. The differential modulations of cell growth and proliferation by PD-98059 suggest that the ERK pathway participates in both the proliferation and the antiproliferation of cellular growth.
|
The cyclin D1 and p21Cip/WAF1 are differentially induced by 10 and 100 µM ZnCl2 in MSSM and are dependent on ERK activations. The ERK pathway is involved in the progression and inhibition of the G1-to-S phase progression by the differential activations of Raf-1 MEK
ERK cascade kinases, which is followed by the differential induction of cell cycle regulators such as cyclin D1 and p21Cip/WAF1 (35, 40).
In this study, we monitored the inductions of cyclin D1 and p21Cip/WAF1 at 10 min and 9 h after treating HT-29 cells with 10 and 100 µM ZnCl2 in MSSM, which induced transient and prolonged ERK activations, respectively (28). Cyclin D1 was induced 9 h after treatment with 10 or 100 µM ZnCl2, and this was higher in cells treated with the higher concentration of ZnCl2 (Fig. 3). On the other hand, the p21Cip/WAF1 was significantly induced only in cells treated with 100 µM ZnCl2 (Fig. 3). The cyclin D1 and p21Cip/WAF1 inductions by 100 µM ZnCl2 were reduced by subsequently treating cells with PD-98059 (Fig. 4), which suggests that both cyclin D1 and p21Cip/WAF1 inductions are dependent on ERK activations.
|
|
The effects of zinc on ERK activation, as well as cyclin D1 and p21Cip/WAF1 induction, resulted in different sensitivities to the zinc concentrations in an extracellular serum concentration-dependent manner. In contrast to the cells grown in MSSM, we did not observe inhibition of cell growth by 100 µM ZnCl2 in 10% FBS medium (Fig. 1B). To further characterize the role of ERK activation in zinc-induced growth regulation, we measured the effects of 100 µM ZnCl2 on ERK activation and the subsequent cyclin D1 and p21Cip/WAF1 inductions in media containing 10% FBS. We did not observe prolonged ERK activation by 100 µM ZnCl2 in this medium; only transient ERK activation was observed 10 min after the zinc treatment (Fig. 5). Furthermore, we observed the weak induction of cyclin D1 9 h after treatment with 100 µM ZnCl2 in medium containing 10% FBS, without significant p21Cip/WAF1 induction. These observations of the effects of zinc, including prolonged ERK activation and p21Cip/WAF1 induction in MSSM, may have been caused by an inhibitory factor(s) present in the serum or because the effect of zinc may have been attenuated by signaling homeostasis of the cells grown in 10% FBS medium. Both the transient and prolonged ERK activations, detected within 10 min and 9 h of zinc treatment, respectively, were redetected in cells grown in 10% FBS medium containing 500 µM ZnCl2 (Fig. 5). In addition, cyclin D1 and p21Cip/WAF1 inductions were also observed in cells grown in 10% FBS medium containing 500 µM ZnCl2. The patterns of ERK activations and subsequent cyclin D1 and p21Cip/WAF1 inductions by 100 and 500 µM ZnCl2 in 10% FBS medium were similar to the patterns observed in cells grown in MSSM containing 10 and 100 µM ZnCl2, respectively (Fig. 3A).
|
Differential growth regulations by zinc in 10% FBS medium were associated with prolonged ERK activation and subsequent p21Cip/WAF1 induction. To measure the differential effects of zinc in more detail, HT-29 cells were treated with a range of ZnCl2 concentrations (0, 100, 200, 300, and 500 µM) in 10% FBS medium, and the effects of different zinc concentrations on cell growth were measured. As observed in MSSM, the positive and negative regulations of cell growth were also observed by the higher zinc concentrations in the 10% FBS medium. The number of HT-29 cells was increased by 100 µM ZnCl2 (Fig. 6A). However, the cell numbers were unaffected by 200 and 300 µM ZnCl2 and were reduced as a result of the 300 and 500 µM ZnCl2 treatments (Fig. 6A). p21Cip/WAF1 protein levels were found to be increased in 10% FBS medium by only 200-500 ZnCl2, and this occurred concomitantly with prolonged ERK activation (Fig. 6B). On the other hand, cyclin D1 protein induction was weak at 100 µM ZnCl2 and at higher zinc concentrations (200-500 µM ZnCl2) in 10% FBS medium. Both cyclin D1 and p21Cip/WAF1 inductions by zinc in 10% FBS medium were inhibited by pretreatment with 10 µM PD-98059 (Fig. 6B).
|
Extracellular zinc was not significantly taken up into HT-29 cells. To identify uptake level of extracellular zinc in HT-29 colorectal cancer cells, we measured the intracellular zinc levels after adding 10 or 100 µM ZnCl2 to cells grown in MSSM and after adding 500 µM ZnCl2 to cells grown in 10% FBS medium. Intracellular zinc levels were not found to be significantly increased by treating cells grown in MSSM with 10 and 100 µM ZnCl2 or in cells grown in 10% FBS containing 500 µM ZnCl2 (Fig. 7). However, the intracellular zinc levels started to increase soon after adding the Zn2+ ionophore pyrithione to cells previously treated with 100 or 500 µM ZnCl2. Therefore, the high levels of zinc applied to the culture medium were not significantly taken up by HT-29 colorectal cancer cells regardless of the serum levels, and ERK activation may have been effected via an extracellular zinc sensor such as ZnR (15) rather than via the activation of intracellular zinc-sensing protein(s).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We recently identified growth arrest in colorectal cancer cells grown in MSSM containing 100 µM ZnCl2 and found that this may have been mediated by the activation of p21Cip/WAF1 via ERK pathway activation (28). In the current study, we also observed the growth stimulation of HT-29 colorectal cancer cells in MSSM containing 10 µM ZnCl2 and in MSSM with 10% FBS containing 100 µM ZnCl2. Therefore, extracellular zinc is involved in both the positive and negative regulation of colorectal cancer cell growth, and the direction of this regulation is dependent on the levels of extracellular zinc. The pH was slightly changed during the culture period and was not significantly affected by zinc or FBS concentrations. Therefore, the differential cell growth induced by zinc may not have been caused by nonspecific effects such as pH change.
To identify an intracellular signaling mechanism for the differential regulation of cell growth by extracellular zinc, we used HT-29 colorectal cancer cells, which retain putative ZnR (15). These observations of the positive and negative regulations of HT-29 cell growth and proliferation by 10 and 100 µM ZnCl2 in MSSM are surprising and suggest that the extracellular zinc-mediated signal transduction in colorectal cancer cells is highly dependent on the extracellular zinc level. Moreover, in MSSM, this cell growth switching point lies in the range of 10-20 µM ZnCl2. The positive and negative regulations of HT-29 cell growth by 10 and 100 µM ZnCl2 were further modulated by subsequent treatment with PD-98059, suggesting the involvement of the ERK pathway. The growth inhibition of HT-29 cells by 100 µM ZnCl2 in MSSM was abrogated by 1 µM PD-98059, which suggests that at 100 µM ZnCl2 is not toxic to HT-29 cells. Moreover, the ERK pathway is known to be involved in both cellular proliferation and growth arrest (35, 40). In those studies, only cyclin D1 was weakly induced by the moderate activation of the ERK pathway, and this induced G1-to-S phase cell cycle progression. However, both cyclin D1 and p21Cip/WAF1 were substantially induced by strong ERK pathway activation, and this blocked G1-to-S phase progression (40).
The patterns of cyclin D1 and p21Cip/WAF1 inductions by 10 and 100 µM ZnCl2 in MSSM were similar to those reported in a study of the transient and sustained activation of the ERK pathway (40). The inhibition of cyclin D1 and p21Cip/WAF1 induction by PD-98059 suggest that these inductions occur via ERK activation. Furthermore, the regulation of cell growth and proliferation by different zinc treatments was highly correlated with the G1-to-S phase progression observed for different levels of ERK pathway activation (16, 40). Therefore, we suggest that the ERK pathway is involved in the positive and negative regulation of cell growth and proliferation by extracellular zinc and that this regulation is achieved via the differential inductions of cell cycle regulators, such as cyclin D1 and p21Cip/WAF1. It is not known why cyclin D1 was induced with p21Cip/WAF1 in growth- and proliferation-inhibited cells. The effects of zinc on ERK activation and cyclin D1 and p21Cip/WAF1 induction were found to be highly sensitive to serum levels and were not observed in cells grown in 10% FBS medium containing 10 and 100 µM ZnCl2. However, the positive and negative regulation of cell growth were reobserved in 10% FBS medium containing 100 and 500 µM ZnCl2. These results suggest that the lack of the differential zinc effects in cells treated with 10 and 100 µM ZnCl2 in 10% FBS medium may be due to an intracellular homeostasis, which reduces zinc sensitivity.
Correlation between p21Cip/WAF1 induction and prolonged ERK activation and the inhibition of p21Cip/WAF1 induction by PD-98059 confirmed that p21Cip/WAF1 induction occurs after prolonged ERK activation. The serum-modulated effects of zinc on cyclin D1 and p21Cip/WAF1 induction were found to correlate well with cellular growth. For example, cellular growth was not inhibited but slightly enhanced by 100 µM ZnCl2 in 10% FBS medium, and under this condition cyclin D1 was induced and p21Cip/WAF1 was not. On the other hand, the level of cell growth was reduced by treatment with 300-500 µM ZnCl2, where the p21Cip/WAF1 levels were most increased.
The zinc concentrations (100-500 µM of ZnCl2) used in the present study to activate ERKs and differential cell growth in media containing 10% FBS were higher than the zinc levels in plasma and in the luminal surface of enterocytes, where relatively high zinc concentration was observed (11). However, we found that intracellular zinc levels were not increased by treatment with 100-500 µM ZnCl2. These results suggest that the zinc-mediated differential growth regulation of HT-29 colorectal cancer cells may be achieved via extracellular receptor-mediated ERK pathway activation and the subsequent induction of cell cycle regulators such as p21Cip/WAF1.
Our identification of positive and negative growth regulation of colorectal cancer cells by extracellular zinc and other epidemiological studies related to zinc (9, 10, 26, 37) suggest that an adequate intake and the right dosage of zinc is important to maintain good health and prevent disease.
![]() |
DISCLOSURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
FOOTNOTES |
---|
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.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |