a1_Proteinase-activated receptor-2 (PAR-2) is a ubiquitous surface molecule participating in many biological processes. It belongs to the family of G protein-coupled receptors activated by the site-specific proteolysis of trypsin and similar proteases. Altered function of PAR-2 has been described in different malignant tumors. In the present study, we investigated the expression of PAR-2 in breast cancer surgical specimens and the role of trypsin in breast cancer cell line MDA MB-231 proliferation and metabolism. A total of 40 surgical samples of infiltrative ductal breast cancer and breast cancer cell line were included in this study. We analyzed PAR-2 expression by immunohistochemistry, RT-PCR and western blot. Activation of PAR-2 on cell line MDA MB-231 was measured using calcium mobilization assay determined by flow cytometry. MTT cell metabolism assay and cell count analysis were used to assess the trypsin influence on breast cancer cell line MDA MB-231 proliferation. Immunohistochemical examination showed the expression of PAR-2 in all samples of breast cancer surgical specimens and high levels of cell lines which was confirmed by RT-PCR and western blot., a2_Calcium mobilization assay corroborated the activation of PAR-2 on cell line MDA MB-231 either by trypsin or by an agonistic peptide. Cell metabolism assay and cell count analysis showed significant differences of proliferative activity of breast cancer cells dependent on the presence or absence of trypsin and serum in the culture medium. PAR-2 is expressed by high levels in infiltrative ductal breast cancer tissue specimens. PAR-2 is also strongly expressed in studied breast cancer cell lines. PAR-2 is activated by trypsin and also by agonistic peptide in the model of breast cancer cell line MDA MB-231. Activation of PAR-2 in vitro influences proliferative and metabolic activity of breast cancer cell line MDA MB-231. The action of trypsin is modified by the presence of serum which is a potential source of protease inhibitors., R. Matěj, P. Manďáková, I. Netíková, P. Poučková, T. Olejár., and Obsahuje biblografii a bibliografické odkazy
The aim of the present study was to introduce methods for exome sequencing of two ATP-binding cassette (ABC) transporters ABCC8 and ABCD2 recently suggested to play a putative role in breast cancer progression and prognosis of patients. We performed next generation sequencing targeted at analysis of all exons in ABCC8 and ABCD2 genes and surrounding noncoding sequences in blood DNA samples from 24 patients with breast cancer. The revealed alterations were characterized by in silico tools. We then compared the most frequent functionally relevant polymorphism rs757110 in ABCC8 with clinical data of patients. In total, the study identified 113 genetic alterations (>70 % novel ones) in both genes. Of these alterations, 83 were noncoding, 13 synonymous, 10 frameshifts and 7 were missense alterations. Four in silico programs predicted pathogenicity of two polymorphisms and four newly identified alterations. Rs757110 polymorphism in ABCC8 did not significantly associate with clinical data of the patients. In conclusion, exome sequencing identified several functionally relevant alterations in ABCC8 and ABCD2 genes that may further be used for a larger follow-up study aiming to assess their clinical significance., P. Soucek, V. Hlavac, K. Elsnerova, R. Vaclavikova, R. Kozevnikovova, K. Raus., and Obsahuje bibliografii