br Conclusions In this paper we describe the recombinant exp
Conclusions In this paper we describe the recombinant expression and characterization of ParI, a C5-DNA-MTase from P. arcticus 273–4. To our knowledge, this is the first characterization of an orphan C5-DNA-MTase from a psychrophilic bacterium. The C5-DNA-MTase could not be expressed in regular E. coli expression strains, likely due to the presence of McrBC SJB3-019A that restricted methylated DNA. Successful expression was obtained using an McrBC-negative E. coli expression strain. To improve solubility, an MBP tag was added as a fusion partner between the His-tag and ParI. This contributed to an overall increased yield and solubility. Despite originating from P. arcticus 273–4, ParI did not exhibit expected cold-adapted features such as a low melting temperature. We hypothesize that the reason for this is ParI's origin from a phage that is not cold-adapted. Using two different in vivo assays where ParI-methylated genomic DNA was treated with McrBC, we showed that both native and recombinant ParI is able to methylate genomic DNA. Using a radiolabeled methyltransferase assay, we showed that ParI possesses methyltransferase activity also in vitro but only as a fusion construct with His-MBP and/or His-tag, while the untagged protein showed no activity.
Acknowledgements This work has been funded by an internal PhD grant at UiT The Arctic University of Norway.
Introduction Breast cancer is the most prevalent and the second particular cause of cancer-related deaths after ovarian cancer among women world wild (Rofaiel et al., 2010; Zafra-Ceres et al., 2013). Estrogen receptor (ER) and progesterone receptor (PR) are effective diagnostic features which reflect disease etiology and predict response to endocrine therapy. About 80% of breast tumors are ER positive (ER+). Estrogen plays a leading role in the pathogenesis of breast cancer through ER (Yager and Davidson, 2006). The most important therapeutic aims in Estrogen receptor positive (ER+) patients are reduction of estrogen levels and blocking signaling pathway through ER to disrupt tumor growth (Yager and Davidson, 2006; Herynk and Fuqua, 2007). Tamoxifen was approved by the US Food and Drug Administration in 1977 (Del Re et al., 2012) and it has been used as an agent of choice in the treatment of hormone responsive breast tumors for over four decades. Furthermore, this drug is prescribed as a chemo-preventive agent for high-risk women who have a familial history of breast cancer (Mandlekar and Kong, 2001). Tamoxifen pharmacologically belongs to Selective Estrogen Receptor Modulators (SERM) and is the most frequently used estrogen antagonist in ER+ patients in neoadjuvant or adjuvant treatment protocol (Osborne, 1998). Although, tamoxifen improves patient's survival but unfortunately about one-third of patients do not respond to treatment initially or over times (Johansson et al., 2013). In clinic, predictions the probability of resistance incidence in the beginning of treatment based on immunohistochemical characteristics can leads to modification of disease treatment protocol, prevent disease recurrence and provides better survival for patients. The potential mechanisms include in progression of tamoxifen resistance are really complicated and are not completely clear to date, however mainly involved of genetic and epigenetic modifications. Epigenetic modifications include DNA methylation, histone acetylation, chromatin remodeling processes and microRNAs. Recently, the importance of epigenetic modifications especially DNA methylation have been established in disease recurrence in tamoxifen-treated patients. DNA methylation is the most leading cause of epigenetic modification and plays essential role in setting gene expression programs during development. Three major types of DNA methyltransferases have been found to be involved in regulation of maintenance (DNMT1) and de novo (DNMT3A and DNMT3B) DNA methylation pattern. There is strong evidence suggesting that the hypermethylation of estrogen and progesterone receptor promoters (Bardou et al., 2003; Cui et al., 2005; Herynk and Fuqua, 2007), and tumor suppressor gene promoters (e.g. Phosphatase and tensin homolog (PTEN) (Phuong et al., 2011) contribute to tamoxifen refractory. Although, DNMTs are overexpressed in multiple malignancies, however little is known about their roles in drug resistance during cancer treatment. In the present study, we try to exhibit if immunohistochemical analyses confirm the contribution of DNMTs' protein expression in the progression of tamoxifen resistance in recruited breast carcinoma patients or not.