Metallothionein (MT), a low-molecular weight protein with pleiotropic functions, is believed to play an important role in tumorigenesis. The aim of this study was to compare the expression of functional MT-1 and MT-2 mRNA isoforms in five breast cancer cell lines ranging from noninvasive MCF7 breast cancer cells to highly aggressive MDA-MB-231 breast cancer cells together with breast myoepithelial cells in vitro by conventional semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR. The MT-2A isoform was observed to be differentially upregulated in the invasive phenotype. The MT-1E isoform was found to be present in estrogen receptor-negative breast cancer cell lines (MDA-MB-231 and Hs578T) but not detectable in the estrogen receptor-positive cell lines (T47D, MCF7, and ZR75-1 cells). Only the myoepithelial cells exhibited the presence of the MT-1G transcript. Direct sequencing of the RT-PCR products revealed the occurrence of a variant MT-1H isoform with changes in amino acid residues in the protein sequence and notable differences in the predicted secondary protein structure. The observations in this study are relevant to the development of novel approaches to metastatic breast cancer disease, and may herald the search for novel MT mutants and the elucidation of their biological roles.
Metallothionein (MT) is a cysteine-rich protein with pleiotropic functions and a high binding affinity for heavy metals. The present study was designed to examine the relationship between MT expression and tissue zinc levels in conjunction with cell proliferation in nasopharyngeal cancer (NPC). Proliferative activity in NPC was quantified by Ki67 immunolabelling and MT expression was determined by immunohistochemistry. Total zinc and subcellular zinc fractions were analysed by flame atomic absorption spectrometry. MT immunostaining was observed in the nuclei of NPC cells, with the percentage MT immunopositivity ranging from 3.0 to 59.7%. Thirteen tumours displayed weak MT staining and the remaining 11 showed moderate to strong immunostaining. There was a significant positive correlation between MT and Ki67 positivity (P ⍧ 0.0127). Tissue zinc levels were higher in NPC as compared with benign nasopharyngeal tissues (4.800 ⍨ 0.4610 versus 2.889 ⍨ 0.4045 µg/g dry wt tissue, respectively; P ⍧ 0.0122). Nuclear zinc levels in NPC were significantly higher than levels in membrane and cytosolic fractions (mean zinc levels 1.4840 ⍨ 0.1489, 0.6286 ⍨ 0.0789 and 0.3014 ⍨ 0.0250 µg/mg protein, respectively). A linear relationship was also observed between nuclear zinc levels and MT immunostaining (P ⍧ 0.0024) as well as with Ki67 immunopositivity (P ⍧ 0.0123). Our results show that MT and zinc are correlated with proliferative activity in NPC, providing further insights into the biology of this enigmatic and aggressive tumour.Metallothioneins (MTs) are a family of intracellular cysteinerich proteins involved in a variety of cellular processes (1-3). The pleiotropic physiological roles of MT include metalloregulatory functions in cell growth, differentiation, repair and protection against oxidative stress-induced injury and apoptosis (1,3,4). Cell proliferation is believed to be a putative function of MT as there have been reports indicating MT to be a cell cycle-dependent protein. MT-positive nuclei were observed to be present specifically in the S to G 2 M phases of the cell cycle of regenerating hepatocytes (5). A 2-to 3-fold increase in MT expression (quantitated by enzyme-linked immunosorbent Abbreviations: EGF, epidermal growth factor; MT, metallothionein; NPC, nasopharyngeal carcinoma.© Oxford University Press 1809 assay) was observed in proliferating human colonic HT-29 cells (6).The proliferative potential of MT has also been linked to zinc, a trace element which participates in the activity of a number of DNA and RNA polymerases (2,7,8). MT has a high binding affinity for zinc and Zn-MT has been hypothesized to play important roles in transmission and expression of genetic information, in response to signals for cell activation (2,8). Zinc ions are sequestered in the metal thiolate clusters of the metalloprotein (7). In this study, our aim was to investigate the relationship of MT and zinc with proliferative activity in undifferentiated nasopharyngeal carcinoma (NPC), a cancer which is common among the Chinese popul...
The expression of metallothionein (MT), an intracellular ubiquitous low molecular weight protein thiol with antioxidant properties, was studied in nasopharyngeal cancer (NPC) and correlated with the apoptotic index. Immunohistochemical staining of randomly selected, formalin-fixed and paraffin-embedded normal and malignant nasopharyngeal tissues were analysed for the expression of MT using the commercially available E9 antibody directed against MT I and MT II isoforms. The corresponding apoptosis labelling indices were evaluated by the TUNEL method. Localization of MT at the ultrastructural level was studied by immunogold labelling. All the tumour sections (17 specimens) showed MT-immunopositivity. A direct correlation between the percentage of MT-positive cells and the staining intensity was noted (P< 0.001; Pearson's r = 0.95). There was absence of cytoplasmic staining and only nuclear staining (with localization in the nucleoplasm) was demonstrated in the tumour cells. In normal epithelium of the nasopharynx, the basal layer was stained. An inverse relationship was observed between the level of MT expression and the apoptotic index in the NPC tissues (P = 0.0059; Pearson's r = –0.6380). The results suggest that overexpression of MT in NPC may protect the tumour cells from entering into the apoptotic process and thereby contribute to tumour expansion. Preferential localization of MT in the nuclei of NPC cells may possibly enhance radioresistance since radiotherapy is known to eradicate tumour cells by free radical-induced apoptosis. © 2000 Cancer Research Campaign
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