Metallothioneins (MTs) belong to a family of cysteine-rich, metal-binding intracellular proteins, which have been linked with cell proliferation. In this study, expression levels of the 8 known MT-1 and MT-2 functional isoforms in human invasive ductal breast cancer specimens were determined by RT-PCR. The expression profiles of the MT protein and MT-2A mRNA were further evaluated in 79 cases of human invasive ductal breast carcinoma by immunohistochemistry and in situ hybridization, and correlated with cancer cell proliferation (determined by Ki-67 nuclear antigen immunolabeling). MT-1A, MT-1E, MT-1F, MT-1G, MT-1H, MT-1X and MT-2A but not MT-1B, were detected in breast cancer tissue samples. The MT-2A mRNA transcript was the highest among all the isoforms detected. A positive correlation was observed between MT-2A mRNA and MT protein expression with Ki-67 labeling (P = 0.0003 and P < 0.0001, respectively) but not with apoptosis (P = 0.1244 and P = 0.8189, respectively). Co-localization of the MT protein and Ki-67 nuclear antigen in breast cancer cells was demonstrated by double immunofluorescence staining. There was also significantly higher MT protein and MT-2A mRNA expression in histological grade 3 tumors than in histological grade 1 and 2 tumors. The finding that MT 2A appears to be the main isoform associated with cell proliferation in invasive ductal breast cancer tissues, may have therapeutic implications.

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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.

Influenza A virus has caused a number of pandemics in past decades, including the recent H1N1-2009 pandemic. Viperin is an interferon (IFN)-inducible protein of innate immunity, and acts as a broad-spectrum antiviral protein. We explored the antiviral activities and mechanisms of viperin during influenza virus (IFV) infection in vitro and in vivo. Wild-type (WT) HeLa and viperinexpressing HeLa cells were infected with influenza A/WSN/33/H1N1 (WSN33) virus, and subjected to virological, light and electron microscopic analyses. Viperin expression reduced virus replication and titres, and restricted viral budding. Young and old viperin-knockout (KO) mice and WT control animals were challenged with influenza WSN33 at lethal doses of 10 3 and 10 4 p.f.u.via the intratracheal route. Lungs were subjected to histopathological, virological and molecular studies. Upon lethal IFV challenge, both WT and KO mice revealed similar trends of infection and recovery with similar mortality rates. Viral quantification assay and histopathological evaluation of lungs from different time points showed no significant difference in viral loads and lung damage scores between the two groups of mice. Although the in vitro studies demonstrated the ability of viperin to restrict influenza H1N1 virus replication, the viperin-deficient mouse model indicated that absence of viperin enhanced neither the viral load nor pulmonary damage in the lungs of infected mice. This may be due to the compensation of IFN-stimulated genes in the lungs and/or the influenza non-structural protein 1-mediated IFN antagonism dampening the IFN response, thereby rendering the loss of viperin insignificant. Nevertheless, further investigations that exploit the antiviral mechanisms of viperin as prophylaxis are still warranted.

Immunohistochemical expression of metallothioneins (MTs), a group of intracellular metal-binding proteins, is well documented in breast cancer. However, there is a paucity of information on the expression of the different MT isoforms in breast cancer tissues. The dichotomous association of MT overexpression with tumour types and progression led us to examine the role of the MT-1F mRNA isoform in breast cancer. We evaluated MT expression in 48 primary invasive ductal breast cancer tissues by immunohistochemistry, and the corresponding MT-1F mRNA expression via a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay. The specificity of the RT-PCR products was confirmed by direct cycle sequencing and restriction enzyme digestion. Immunohistochemical analysis of MT revealed a significantly higher MT expression in histological grade 3 tumours as compared to grade 1 and 2 tumours (p = 0.021). Similarly, MT-1F mRNA expression was found to be significantly higher in grade 3 tumours (p < 0.001). The results suggest that the MT-1F isoform influences histological differentiation in invasive ductal breast cancer. The converse is also true in that the histological grade may determine the level of MT-1F expression in breast cancer.

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Metallothioneins (MTs), a group of cysteine-rich proteins with a small molecular mass, are known to have metalloregulatory functions. MT gene expression has been demonstrated to be cell type-specific and differentially regulated (possibly related to their germ layer origin and different functional states). In vitro studies suggest that MT-2A, MT-IE, and MT-1F isoforms may be related to breast cancer. In this study, data on MT-2A, MT-1E, MT-1F mRNA analysis via reverse transcription-polymerase chain reaction in invasive ductal breast cancer tissues and their adjacent benign breast tissues from 27 mastectomies are presented. Expression of mRNA in all the three MT isoforms was detected in both cancerous and adjacent benign breast tissues (with MT-2A mRNA expression being the highest). MT-1F expression was significantly higher in benign breast tissues compared with the breast cancers (P=0.017). In situ hybridization confirmed the expression of MT-2A mRNA in the myoepithelial cells of the breast tissues. Immunohistochemical localization of the MT protein revealed that myoepithelial cells consistently expressed the MT protein, while the cancer cells expressed MT with great variation. Based on our immunohistochemical and mRNA analysis, it is likely that the three MT isoforms are specifically expressed in myoepithelial cells of benign breast tissues and cancer cells of the invasive ductal breast cancer tissues. As MT expression occurs in myoepithelial cells and ductal breast cancer cells, our finding supports the proposition that loss of myoepithelial cells in invasive mammary cancers may be compensated in part by changes in the tumor cells, which may subsequently be the basis for studying the role of MT in breast physiology and carcinogenesis. Differential MT-1F expression in breast myoepithelial cells warrants further study.