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Cell Biology International (2005) 29, 843–848 (Printed in Great Britain)
Effect of metallothionein on cell viability and its interactions with cadmium and zinc in HEK293 cells
Jie Li, Ying Liu and Binggen Ru*
National Key Laboratory of Protein Engineering, College of Life Sciences, Peking University, Beijing 100871, China


Abstract

Metallothioneins (MTs) are thought to participate in a wide variety of physiological roles, but the mechanisms involved are still unclear. The study was designed to examine the possible factors related to these mechanisms. Methods, including transfection, MTT assay and flow cytometry, were used to investigate the effect of MTs on cell viability and their interactions with cadmium and zinc in HEK293 cells. The results showed that transient overexpression of human MT1A, MT2 and MT3 genes dynamically affected cell viability, and the effect was influenced by zinc and cadmium ions. Overexpressed MTs with added zinc showed a greater inhibitory effect on cell viability. Overexpressed MTs protected cells against low concentrations of cadmium ions (10μM), but increased cell death in response to high concentrations (20–50μM). Out of the three MTs, MT1A was more efficient than MT2 and MT3 in its resistance to cadmium (10μM), and MT3 together with zinc showed more cell growth inhibition than MT1 and MT2. These results indicate that both of the divalent metal ions that could bind MTs, as well as the individual MT isoforms, affect the role of MTs on cell viability, which may explain in part why the comprehensive effect of MTs on the cells was elusive.


Keywords: Cell viability, HEK293, Divalent metal ion, Metallothionein isoforms, Overexpression.

*Corresponding author. Tel./fax: +8610 62751842.


1 Introduction

Metallothioneins (MTs) are a family of cystein-rich and low-molecular weight proteins. There are currently four isoforms of human MT: MT1, MT2, MT3, and MT4. Human MT1 has 10 sub-isoforms while human MT2 has only one active form, and both of them are fairly ubiquitous in most normal and cancer tissues or cells. MT3, the main isoform in neurons (Masters et al., 1994), also exists in many other tissues, including prostate, peripheral blood lymphocytes, kidney, and cancers such as bladder, breast and gastric.

In most of the reports about MT overexpression (Taylor et al., 2004) or downregulation (Tekur and Ho, 2002; Abdel-Mageed and Agrawal, 1997) through transfection, antisense inhibition or inducing agents, the function of MTs is to help protect cells against apoptosis or damage caused by stress, such as cadmium (Wlostowski, 1992; McAleer and Tuan, 2001), oxidative injury (Pitt et al., 1997; You et al., 2002), free radicals (Zhang et al., 1992; Kumari et al., 1998) etc. However, MT overexpression or added MT is also thought to make cells more sensitive to death or growth inhibition, when alone or combined with other factors (Borghesi and Lynes, 1996; Cui et al., 2003; Kang et al., 2001).

Reports about the functions of MTs in tumors are also inconsistent. Studies show the involvement of MT in resistance to platinum compounds (Hosokawa et al., 2000) and other chemotherapy (Taylor et al., 2004), but its precise influence on the susceptibility of tumor cells to chemotherapy also remains elusive, because of some opposing reports (Papouli et al., 2002). Clinical studies investigating possible associations between MT expression in tumor tissue in response to chemotherapy and survival time have obtained conflicting results. For example, in some tumors, overexpressed MTs were found to indicate a bad prognosis or correlate with histopathological parameters (Sens et al., 2000; Saga et al., 2002). Downregulation of MTs induces growth arrest and apoptosis in carcinoma cells (Dutta et al., 2002; Abdel-Mageed and Agrawal, 1997). But in many tumors, MTs overexpression does not have a definite correlation with survival time (Janssen et al., 2002; Cherian, 1994; Sutoh et al., 2000).

The reported opposing effects of MTs indicate the complexity of their functional mechanisms. To elucidate these mechanisms, further study on MTs is needed. In order to investigate the possible factors related to the mechanism, three MT isoforms (MT1–3) were transiently transfected into human kidney cell HEK293 and the effect of divalent metal ions (zinc and cadmium) on the function of MTs was investigated. Both of the ions can induce and bind MTs (Wlostowski, 1992; Garrett et al., 1998a, 1998b). The cell line was selected mainly because all three MT isoforms (MT1–3) are reportedly expressed in human embryonic kidney or renal cells (Nguyen et al., 2000; Garrett et al., 1999) and none of them have a known distinct function. The results showed that the effects of overexpressed MTs on cell viability were different with different MT isoforms and different metals, which may give some clues to elucidate the mechanism underlying the complex function of MTs.

2 Materials and methods

2.1 Culture of HEK293 cells

Human embryonic kidney cells (HEK293) were obtained from Dr. Chen Danying. The cells were grown in monolayer culture in Dulbeco's modified Eagle's medium (Gibco-BRL) supplemented with 10% bovine fetal serum, 50μg/ml penicillin and 10μg/ml streptomycin (Gibco-BRL) in an atmosphere of 5% CO2 at 37°C.

2.2 MTT cell viability assay

One hundred microlitres of HEK293 cells were plated at 11×105cells/ml in 96-well plates (Costar) the night before treatment. Following treatment with transfection and/or metal ions for certain times, 20μl MTT (5mg/ml) was added to each well, followed by incubation for an additional 2h. The media were removed carefully and the resulting formazan was dissolved in 100μl dimethylsulfoxide. Absorbance was measured at 570nm using a Rainbow Spectra ELISA microplate reader (TECAN). Cell viability was defined relative to corresponding control cells (i.e. relative cell viability=absorbance of treated sample/absorbance of control sample).

2.3 Cell transfection

Plasmid vectors pcDNA3.1 embedded with MT1A, MT2, MT3 genes (about 200bp) were constructed separately. Plasmids used for transfection were extracted with high purity plasmid extraction kit (TIANWEI). HEK293 cells were plated at &007E;1×105cells/ml in group-well plates the night before treatment. Cells were transfected with the same amount of transfection complex of plasmid vectors containing one of the three test genes or the control plasmid pCDNA3.1. The transfection kit was Lipofectamine™2000 (Invitrogen) and all of the performance and the amount of DNA used followed the standard procedure provided by the manufacturer for 96-well and 24-well plates. The medium was changed 6h after transfection. Cells were treated with MTT or collected for flow cytometry and Western blotting over different time periods.

2.4 Measurement of cell viability by flow cytometry (FACS)

After treating with transfection and cadmium, cells were collected, trypsin digested, PBS washed three times, and then resuspended in binding buffer. Annexin V-FITC and propidium iodide staining agents (BSC) were added according to the manufacturer's instructions. After incubation for 20min in the dark, cells were counted by flow cytometry (FACS). For each analysis, 10,000 events were counted.

2.5 Western blotting

Cells grown in 24-well culture plates were removed, centrifuged and rinsed with PBS. The pellets were resuspended in 50μl SDS-PAGE loading buffer. After freeze and thawing, the mixture was boiled for 10min, and 15μl of the suspension was loaded onto 16.9% SDS-polyacrylamide gels. After electrophoresis, the proteins were transferred to nitrocellulose membranes at 100V for 1h. The membranes were blocked with TTBS for 1h, probed overnight at 4°C with rabbit anti-human MT1/2 and MT3 polyclonal antibodies (self-made) separately. Because human MT1/2 and control rabbit MT1 can interact with the same polyclonal antiserum, only one set of controls for MT1 and MT2 was used. The second antibody was alkaline phosphatase-conjugated goat anti-rabbit IgG. BCIP/NBT was the chromogenic substrate.

2.6 Statistical analysis

Data are expressed as means±SD. Statistical analysis was performed by one-way analysis of Student's t-test. P-values of <0.05 and <0.001 were considered significant.

3 Results

3.1 Transfection efficiency assayed by Western blot

MTs transfection efficiency was determined by Western blot (Fig. 1). Extracts of cells transfected with MT genes showed stronger immunoreaction than untransfected controls, which proved that MT genes were transfected into cells and overexpressed. In the preparation process for electrophoresis, MTs from cells that were transfected with MTs genes formed diverse disulfide linkages due to the notorious instability of MTs because of their high cystein content and the repeated freeze–thawing of cells for protein extraction. As shown in Fig. 1, MTs in greater concentrations formed more types of multimers. MT1, MT2 and MT3 showed varying stability during the process, which is discussed elsewhere.


Fig. 1

Transfection efficiency assayed by Western blot. (A) The first antibody was rabbit anti-human MT1, which also cross-reacts with rabbit MT1 and human MT2. ck+: Positive control of rabbit MT1; ck−: extract of cells transfected with the control plasmid; MT1 and MT2: extracts of cells transfected with the plasmids embedded with human MT1A, MT2 genes. (B) The first antibody was rabbit anti-human MT3 polyclonal antibody. ck+: Positive control of human recombinant MT3 control; ck−: extract of cells transfected with the control plasmid; MT3: extract of cells transfected with the plasmid embedded with human MT3 gene. Note: because of the instability of MTs, they showed diverse disulfide linked multimers in the electrophoresis.


3.2 Effects of MTs overexpression on cell viability with/without metal ions over different time periods

3.2.1 Effects of MTs overexpression on cell viability without metal ions

The MTT assay was used to measure cell viability because it is a sensitive and quantitative colorimetric assay based on the capacity of mitochondrial succinyl dehydrogenase in living cells to convert yellow substrate into a dark blue formazan product. MTT assay results showed that HEK293 cells with MTs overexpression and without adding metal ions displayed significantly increased cell viability at 30h (Table 1A). However, the effect was decreased and even showed reduced viability at 54h, close to significance levels (Table 1B). The different results at different times indicate the change in MT effects due to different active conditions over time.


Table 1.

Effect of MTs overexpression with/without metal ions on relative cell viability assayed by MTT

Metal ions added (concentration, μM)MT1AMT2MT3ControlPt-test (MTs/control)Pt-test (MT3/control)
A. Relative cell viability 30 h after transfection
No ion1.106 ± 0.1121.130 ± 0.0471.086 ± 0.0621 ± 0.0600.002**
Zn (10)0.986 ± 0.1090.979 ± 0.1130.909 ± 0.1071 ± 0.0620.1130.0394*
Zn (20)0.958 ± 0.0750.938 ± 0.0770.862 ± 0.0531 ± 0.0250.001**0.0004**
Cd (10)1.068 ± 0.1081.065 ± 0.0931.036 ± 0.0931 ± 0.0880.046*
Cd (20)0.989 ± 0.0690.972 ± 0.0931.019 ± 0.0861 ± 0.080
Metal ions added (concentration, μM)MT1AMT2MT3ControlPt-test (MTs/control)Pt-test (MT1/control)
B. Relative cell viability 54 h after transfection
No ion0.970 ± 0.1280.959 ± 0.1100.945 ± 0.1151 ± 0.0870.068
Zn (10)0.981 ± 0.0820.977 ± 0.0821.024 ± 0.0881 ± 0.0840.425
Zn (20)0.909 ± 0.0700.975 ± 0.0900.909 ± 0.0361 ± 0.0940.035*
Cd (10)1.178 ± 0.0831.032 ± 0.0530.977 ± 0.0601 ± 0.1250.1120.003**
Cd (20)0.888 ± 0.1580.815 ± 0.0540.907 ± 0.0801 ± 0.0790.001**


3.2.2 Effects of MTs overexpression on cell viability with addition of zinc ions

Ten and twenty micromolars of zinc added to the medium increased cell viability to some degree at 12–54h (data not shown), which indicated that the physiologically essential zinc ions at these two concentrations were not harmful to cell viability.

After addition of 20μM zinc following transfection of MT genes, the viability of cells detected at 30h and 54h was inhibited significantly and specifically, whilst the effect was less with 10μM zinc (Table 1A, B). Among the three MTs, the inhibition effect of MT3 combining with the addition of zinc ions was stronger than MT1A and MT2 at 30h.

3.2.3 Effects of MTs overexpression on cell viability with addition of cadmium ions

Cadmium (10–50μM) added to the medium decreased HEK293 cell's viability. The viability loss was positively associated with the concentration of cadmium (data not shown). Compared with the effect of MTs overexpression with zinc ion addition, the effect of MTs overexpression with cadmium ions displayed a more complex character. The results showed that MTs overexpression protected cells from damage caused by 10μM cadmium, which was supported by both MTT assay (Table 1A, B) and FACS assay (Table 2). Among the three MT isoforms, MT1A protected cells better than MT2 and MT3 at 54h (Table 1B and Table 2). However, MTs overexpression did not display a protective effect from damage caused by cadmium at 20μM or more, compared with control, but exacerbated the cell death caused by high concentrations of cadmium (Table 1A,B and Table 2). The results indicate that overexpressed MTs decrease cell resistance to high concentrations of cadmium, but provide cells more resistance to cadmium at lower concentrations.


Table 2.

Effect of overexpression of MT1A, MT2 and MT3 genes on the number of viable HEK293 cells detected by FACS

Treatments
Relative viable cell events
MT1AMT2MT3Control
(A) 30 h after transfection and with 10 μM Cd for 24 h1.2211.2031.2511
(B) 54 h after transfection and with 10 μM Cd for 48 h1.2341.0861.0731
(C) 54 h after transfection and with 20 μM Cd for 24 h0.7730.7790.8201
(D) 54 h after transfection and with 50 μM Cd for 6 h0.9110.8770.9081


4 Discussion

Although MTs have been studied for decades, the effect of the binding metal ions on their function has scarcely been investigated. The present study showed that overexpressed MTs without the addition of metal ions increased cell viability. However, the cell viability upregulation effect of MTs overexpression was decreased with the addition of 10μM zinc and was changed to an inhibitory effect with the addition of 20μM zinc. These results support the idea that zinc–MT inhibits cell proliferation, whereas apo-MTs (MTs binding no metals) increase cell proliferation. The results also suggest that the inhibitory effect following MTs overexpression was not caused by the possible relative scarcity of metal ions, but was due to the function of zinc–MTs themselves.

The relative cell viability with 10μM cadmium and MTs overexpression were between the values with no ion addition and those with zinc addition, which is very likely to be the comprehensive results of the complex properties of MTs functions: to protect cells from damage caused by cadmium and to inhibit cell proliferation. The results support the conclusion indirectly about the cell growth inhibitory effect of metal-containing MTs.

MT1, MT2 and MT3 have shown complicated expression patterns in various cells and tissues. Their functions in tumors and brain tissue are particularly of note. MTs expression levels were upregulated or downregulated in different tumors and their functions, or the association between MTs expression and tumor progression, were inconsistent, not only in different tumors (Cherian, 1994), but also in cases of the same type of tumor (Janssen et al., 2002), despite a few reports about quite definite correlations between MTs overexpression and tumor histopathology (Jin et al., 2001; Saga et al., 2002). The present study provides a possible explanation for the phenomenon. Both divalent metal ion levels and MTs expression levels influence the proportion of MTs to apo-MTs. It is possible that the elusive effect of MTs in tumors on cell proliferation is influenced by the relative state of MTs (MT/apo-MT).

The negative effect of MTs on cell growth has been shown in some studies. MTs overexpression or added MT was also reported to make cells more sensitive to death or growth inhibition, either alone or combined with other factors (Borghesi and Lynes, 1996; Cui et al., 2003; Kang et al., 2001; Hecht et al., 2002; Papouli et al., 2002). Downregulation of metallothionein-2A expression occurs at immortalization (Duncan and Reddel, 1999). Transgenic mice that express MT3 ecotopically in pancreata often die at 2–3 months of age (Quaife et al., 1998). These reports show the negative effect of excess MTs on cell proliferation. In this study MTs overexpression alone at 54h caused minor cell viability loss but significantly increased cell death when high concentrations of cadmium were added. The mechanism underlying it may be similar to the effect of excess MTs added or overexpressed in cells. In addition, the effect is consistent with the report that neither metallothionein overexpression nor supplemental dietary zinc provides further protection to metallothionein transgenic mice against carbon tetrachloride treatment (Davis et al., 2001). The results further complement the previous explanation about MTs function in tumors.

Metallothionein has several isoforms that share many common properties, but a few relatively distinct roles of individual MT isoforms have been reported. MT isoforms provide neuroprotection against oxidative stress (Wanpen et al., 2004) and MT3 is a neuron growth inhibition factor, together with another protein (Kang et al., 2001). In this study, the three MTs showed quite similar effects on cell viability, while MT1 was a bit more resistant to 10μM cadmium than MT2 and MT3 (Table 1B and Table 2), and MT3 with zinc showed more cell proliferation inhibition than MT1 and MT2 (Table 1A). This result indicates that the effect of MTs on cell viability is their common function, but they differ in the extent of their effect. The difference could originate from their individual specific structures. The functional difference between individual MT isoforms affects the comprehensive effect of MTs in tumors, and may be another reason that MT3 was found to be the main isoform (Masters et al., 1994) and the proliferation inhibition factor in neurons, rather than MT1 and MT2.

Largely as stressor proteins against a wide range of inducing factors, MTs usually display mild but diverse effects on cell viability in most tissues, except in some extreme conditions, which were reconfirmed in this study. This may be one of the main reasons for the difficulty in defining their functions compared with many other proteins.

In conclusion, many aspects of the MTs effect on cell viability were observed in this experiment. It is the first time that the effect of different MTs overexpression over different time periods with and without different concentrations of divalent ions on cell growth has been reported. The results link the diverse functions of MTs with the metals binding them and their individual characters, which would be helpful for further elucidating the mechanism of MT function in the future.

Acknowledgements

We thank Dr. Chen Danying for providing the cell line and his kindly suggestions on cell culture. We thank Ms. Zhang Ting for doing the flow cytometry detection and Han Tiegang for revising the paper.

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Received 13 January 2005/19 March 2005; accepted 20 May 2005

doi:10.1016/j.cellbi.2005.05.008


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
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