Spontaneously immortalised bovine mammary epithelial cells exhibit a distinct gene expression pattern from the breast cancer cells

<p>Abstract</p> <p>Background</p> <p>Spontaneous immortalisation of cultured mammary epithelial cells (MECs) is an extremely rare event, and the molecular mechanism behind spontaneous immortalisation of MECs is unclear. Here, we report the establishment of a spontaneously immortalised bovine mammary epithelial cell line (BME65Cs) and the changes in gene expression associated with BME65Cs cells.</p> <p>Results</p> <p>BME65Cs cells maintain the general characteristics of normal mammary epithelial cells in morphology, karyotype and immunohistochemistry, and are accompanied by the activation of endogenous <it>bTERT </it>(bovine Telomerase Reverse Transcriptase) and stabilisation of the telomere. Currently, BME65Cs cells have been passed for more than 220 generations, and these cells exhibit non-malignant transformation. The expression of multiple genes was investigated in BME65Cs cells, senescent BMECs (bovine MECs) cells, early passage BMECs cells and MCF-7 cells (a human breast cancer cell line). In comparison with early passage BMECs cells, the expression of senescence-relevant apoptosis-related gene were significantly changed in BME65Cs cells. P16^INK4awas downregulated, p53 was low expressed and Bax/Bcl-2 ratio was reversed. Moreover, a slight upregulation of the oncogene <it>c-Myc</it>, along with an undetectable level of breast tumor-related gene <it>Bag-1 </it>and <it>TRPS-1</it>, was observed in BME65Cs cells while these genes are all highly expressed in MCF-7. In addition, <it>DNMT1 </it>is upregulated in BME65Cs. These results suggest that the inhibition of both senescence and mitochondrial apoptosis signalling pathways contribute to the immortality of BME65Cs cells. The expression of <it>p53 </it>and <it>p16</it>^{<it>INK4a </it>}in BME65Cs was altered in the pattern of down-regulation but not "loss", suggesting that this spontaneous immortalization is possibly initiated by other mechanism rather than gene mutation of <it>p53 </it>or <it>p16</it>^{<it>INK4a</it>}.</p> <p>Conclusions</p> <p>Spontaneously immortalised BME65Cs cells maintain many characteristics of normal BMEC cells and exhibit non-malignant transformation. Although this cell line displays altered patterns of gene expression, it is clearly distinct from malignant breast cancer cell line. It showed that co-inhibition of cellular senescence and mitochondrial apoptosis pathways coordinates BME65Cs cells immortalisation. Additionally, mechanisms other than gene mutation are likely to be involved in regulation of cellular functions. This study provides an insight into the relationship between cell senescence and immortalisation. BME65Cs cells will be useful in future studies of cellular senescence and tumorigenesis.</p

Selection of DNA aptamers using atomic force microscopy

Atomic force microscopy (AFM) can detect the adhesion or affinity force between a sample surface and cantilever, dynamically. This feature is useful as a method for the selection of aptamers that bind to their targets with very high affinity. Therefore, we propose the Systematic Evolution of Ligands by an EXponential enrichment (SELEX) method using AFM to obtain aptamers that have a strong affinity for target molecules. In this study, thrombin was chosen as the target molecule, and an ‘AFM-SELEX’ cycle was performed. As a result, selected cycles were completed with only three rounds, and many of the obtained aptamers had a higher affinity to thrombin than the conventional thrombin aptamer. Moreover, one type of obtained aptamer had a high affinity to thrombin as well as the anti-thrombin antibody. AFM-SELEX is, therefore, considered to be an available method for the selection of DNA aptamers that have a high affinity for their target molecules