Molecular biologyDifferential quantitative zinc-induced expression of human metallothionein isogenes in haematopoietic precursor cell lines
Introduction
Zinc (Zn) is an essential trace element for all forms of life. It plays an important role in growth and development, immune response, neurological function, and reproduction. On the cellular level, Zn has a catalytic role in Zn-dependent enzymes, a structural role in Zn fingers, other proteins and cell membranes, and a regulatory role in transcription factors, cell signalling and apoptosis [1]. The supply of Zn in important cellular processes is maintained in a well-regulated Zn homeostasis [2].
Metallothioneins (MTs) are highly conserved, low molecular weight (6–7 kDa), cysteine-rich proteins. Containing 20 cysteine residues per molecule, each thionein which is the metal-free form of MT, can bind up to seven Zn or cadmium ions, or up to 12 copper ions [3], [4]. Biological functions of MT include maintenance of the metabolically active cellular Zn pool, protection against various forms of oxidative damage and tolerance to metal toxicity [2], [5].
In human, MTs are encoded by a family of 10 functional MT isogenes and the encoded proteins are conventionally subdivided into four groups: MT-1, MT-2, MT-3, and MT-4 [6]. In mammals, MT-1 and MT-2 are widely expressed in all organs; MT-3 is expressed mainly in the brain and the reproductive system and MT-4 is most abundant in certain stratified tissues [7], [8], [9]. A set of MT-1 genes (MT-1A, MT-1B, MT-1E, MT-1F, MT-1G, MT-1H, and MT-1X) encodes the MT-1 group of proteins while a single MT-2A gene encodes the MT-2 protein [6].
Metals, glucocorticoids, cytokines and a variety of chemical and physical stress conditions induce MT-1 and MT-2 expression. The promotor regions of MT-1 and MT-2 genes contain several metal and glucocorticoid responsive elements, MRE and GRE, respectively, as well as elements involved in basal level transcription [10]. Elevated zinc concentrations are required for efficient DNA binding of metal-responsive transcription factor-1 (MTF-1) to a promotor proximal MRE [11]. Differential expression of both MT-1 and MT-2 isoforms in nasopharyngeal and breast cancer cell lines, as well as anaplastic thyroid carcinoma cells has previously been investigated [12], [13], [14].
In the present study four human erythroleukemia cell lines, namely K562, DAMI, MEG-01, and ELF-153 were investigated for their ability to transcribe and translate functional MT. Based on the cell surface antigen expression, K562 is considered more mature, whereas DAMI and MEG-01 are considered as moderately mature and ELF-153 as an immature haematopoietic precursor cell line [15], [16], [17], [18], [19]. Depending on the stimulus, the K562 cell line can undergo further differentiation in either megakaryocytic or erythroid lineage: phorbol-12 myristate-13 acetate (PMA) and hemin stimulate differentiation towards megakaryocytic and erythroid lineages, respectively [20]. The effect of PMA and hemin on the expression of the MT gene family has been examined. Analysis of various isogenes transcription at various concentration of Zn was performed by quantitative real-time-PCR to provide a more detailed view on the promising role of MTs.
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Cell culture
K562, DAMI, MEG-01, and ELF-153 megakaryocytic cell lines were grown in RPMI 1640 medium (Gibco BRL, Invitrogen, Paisley, UK) supplemented with 10% (v/v) heat inactivated foetal calf serum (FCS) (Gibco-BRL), 2 mmol/L l-glutamine (Gibco), 50 mg/L gentamycin (Gibco), and 0.025% (w/v) glucose, and 10 ng/mL of interleukin-3 (IL-3) (PeProTech EC, London, UK) only in ELF-153 cell cultures to enhance the growth of this cell line. IL-3 has no effect on induction of MT gene transcription in the erythroid
Zn induction of MT isogenes transcription
The cell lines were incubated either at different concentrations of Zn (50 or 75 μmol/L) or without Zn (control) for 24 h. In K562, DAMI, and MEG-01 cell lines, transcripts of MT-1A, MT-1E, MT-1F, MT-1G, MT-1H, MT-1X, and MT-2A isogenes were detected in control cultures, while the addition of Zn resulted in additional expression of MT-1B mRNA and an increased transcription of the above-mentioned isogenes (Fig. 1A). A representative photograph of an agarose gel with amplified MT transcripts of
Discussion
The present study was undertaken to reveal the expression profile and quantitative measurement of 10 functional human MT isogenes transcription in four different haematopoietic precursor cell lines. The expression profile of MT isogenes in haematopoietic precursors is key to revealing potential roles MT might play in proliferation and differentiation. We have used four haematopoietic precursor cell lines namely, K562, DAMI, MEG-01, and ELF-153. The expression pattern of MT isoforms in these
Acknowledgments
We are grateful to K.U. Leuven for a research fellowship (P.M.B.). We thank Pieter Chys for his help in the statistical analyses and Iris Govaerts for the technical assistance. We thank Dr. Nurul Islam Siddiqui and Caroline Greenman for their help in preparing the manuscript.
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