Regulation of metallothionein gene expression in human blood precursor cells and cell lines.

The main biological role of metallothionein (MT) is still not fully understood more than fifty years after its discovery. MTs are responsible, first of all, for maintenance of metal homeostasis and detoxification processes. In addition MTs control several intracellular processes, including proliferation, apoptosis and cell differentiation. Only limited study has been done in association with the relationship between MT expression and cell differentiation. During my PhD program, the expression pattern of functional members of the MT gene family was studied in haematopoietic precursor cell lines namely, K562, DAMI, MEG-01, and ELF-153 in order to strengthen the proposed function of MT in differentiation. We found that relative quantitative transcription of MT isogenes and MT proteinbiosynthesis were higher in the mature cell lines, such as K562, DAMI, and MEG-01, than in the immature ELF-153 cell line. Moreover, we detected the effect of hematopoietic differentiation stimuli on expression of MT isogenes in K562 cells and we reported the role of MT in differentiation of human haematopoietic precursor cell lines. To further addressthis possibility, we focused on megakaryocyte differentiation (Maghdooni Bagheri et al., 2009).Megakaryocyte research is hampered by the fact that human megakaryocytes constitute only 0.03 0.06% of nucleated bone marrow cells, and are difficult to isolate and maintain in culture. So, we used a megakaryocytic cell line (DAMI), induced differentiation, and verified the quantitative MT isogenes transcription in this condition. MT isogene transcription was significantly increased in phorbol-12-myristate-13-acetate (PMA) incubated leukemic DAMI cells, which supports its role in cellular differentiation. To further address this possibility, we constructed one stable MT-2A overexpressing DAMI cell line. Increase of cell size, intracellular granulation and megakaryocytic specificantigen expression such as CD41and CD42, and arresting cell proliferation have validated the role of MT in differentiation in this cell line (Maghdooni Bagheri et al., 2010).In the next part of the project we studied the expression of MT in immature human umbilical CD34+ haematopoietic cells, in order to obtain further information about the possible role of MT in the differentiation of blood cells. Human umbilicalCD34+ immature hematopoietic cells were rapidly and efficiently obtained from light density mononuclear cells (MNC) by magnetic cell sorting (MACS). An ex vivo expanded population of CD34+ was cultured in serumfree medium supplemented with cytokines flt3 ligand, stem cell factor (SCF), and thrombopoietin (TPO) in order to obtain a sufficient number ofCD34+ cells. CD34+ cells expanded from cord blood for 7 days were demonstrated to increase in the absolute number of CD34+ cells by 5.1 ± 2.5 fold (mean ± SD, n = 3). Flow cytometric analysis demonstrated that the percentage of CD34 antigen expression after expansion of the culture was 97.8 ± 1.0 %, whereas it was 69.4 ± 10.4 % innon-expanded CD34+ cells (mean ± SD, n = 3), thus defining a system that allowed extensive amplification accompanied by no maturation. We examined the expression pattern of functional members of the MT gene family in immature CD34+ cells and compared it with more mature CD34- cells in order to strengthen the proposed function of MT in differentiation. Cells were cultured in RPMI 1640 medium, with or without different zinc supplements for 24 h. Relative quantitative expression of MT isogenes in the mature CD34- cells was higher than in the immature CD34+ cells. Immunohistochemical staining revealed an increased MT protein biosynthesis in CD34- cells, greater than in CD34+ cells. Therefore, to our knowledge, the role of MT in differentiation of human haematopoietic progenitor cells from human cord blood was reported for the first time (Maghdooni Bagheri and De Ley, 2011).There is little information on the subcellular distribution of MTs and their role during cell differentiation. Localization of proteins in subcellularstructures is important for the study of their functions. In the last part of the study, we investigated the changes in MT distribution, especially its nuclear/cytoplasmic localization during differentiation. We constructed a stable recombinant vector that expresses a fusion protein consisting of a green fluorescent protein (GFP) and MT-1X isoformand we verified the intracellular localization of MT by confocal microscopy. We verified the effect of different stimuli including PMA, IL-3, serum deprivation, serum supplementation and H2O2 onGFP-MT fusion protein localization in transfected haematopoietic precursor cells. Our studies demonstrated translocation of GFP-MT fusion proteins from nucleus to the cytoplasm during differentiation and late exposed H2O2 (for 30 min) and very rarely in the small cells treatedwith high Zn concentration. We proposed that accumulation of GFP-MT fusion proteins in the cytoplasm is related to anti-apoptosis effect of MT during differentiation, and cytoplasmic expression of MT decreases the level of intracellular reactive oxygen species in the presence of oxidizing agent such as H2O2 and protects the lipid membrane against peroxidation. It may be necessary to have more protection against oxidants and heavy metals by MT in cytoplasm of the small cells as compared to the bigger cells.status: publishe