Biochemical and Biophysical Research Communications
Activation of GPR54 promotes cell cycle arrest and apoptosis of human tumor cells through a specific transcriptional program not shared by other Gq-coupled receptors
Section snippets
Materials and methods
Cell lines. The human breast cancer cell line MDA-MB-435S was obtained from the American Type Culture Collection (ATCC) and grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum. Kisspeptin-10 and bradykinin were purchased from Calbiochem and Neosystem, respectively. The cDNA coding regions of human GPR54 and bradykinin B2 receptors [3], [6] cloned in the pEFIN3 vector, as well as the empty vector pEFIN3, were transfected into MDA-MB-435S cells using Fugene 6 as
Expression of recombinant GPR54 in a human breast carcinoma cell line
We established a recombinant expression system in the human MDA-MB-435S cell line, originating from a metastatic ductal breast carcinoma, allowing to compare in a rigorous way cells expressing or not GPR54, as well as other receptors reported to be coupled to the same intracellular pathways. This cell line was tested negative for the presence of GPR54 transcripts, and it did not respond functionally to kisspeptin-10. The full-length GPR54 coding sequence was inserted into the bicistronic
Discussion
The involvement of GPR54 and its ligands in the control of the metastatic potential of tumoral cells has been described previously [4], [17], [18]. However, the mechanisms by which KiSS-1 expression or GPR54 activation restrains the metastatic properties of cells are largely unknown. In order to investigate this process, we have analyzed the transcriptional program regulated by GPR54, in a human breast carcinoma cell line. In agreement with previous reports [2], [3], [4], stimulation of human
Acknowledgments
This work was supported by a grant from the Actions de Recherche Concertées of the Communauté Française de Belgique, the Centre de Recherche Interuniversitaire en Vaccinologie, the Belgian program on Interuniversity Poles of attraction initiated by the Belgian State, Prime Minister’s Office, Science Policy Programming, the Cell Factory (Grant QLK3-CT2000-00237) and LifeSciHealth (Grant LSHB-CT-2003-503337) programs of the European Community, the Fonds de la Recherche Scientifique Médicale of
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2014, Molecular and Cellular EndocrinologyCitation Excerpt :This coupling was further confirmed by the observation that KISS1R stimulation induces the formation of IP3 through a PLC-dependent mechanism (Kotani et al., 2001; Stafford et al., 2002). Additional information on the intracellular signaling was derived from studies performed either in cell lines (HEK-293, CHO, B16-Bl6, COS-7, NIH-3T3, MDA-MB-435S, NPA) transfected with the receptor (Kotani et al., 2001; Muir et al., 2001; Ohtaki et al., 2001; Stafford et al., 2002; Becker et al., 2005; Stathatos et al., 2005) or in immortalized cell lines natively expressing the receptor (GT1-7, Human thyroid carcinoma ARO, AsPC-1, PANC-1) (Masui et al., 2004; Stathatos et al., 2005; Babwah et al., 2012). These studies demonstrated that, aside from Ca2+ mobilization, other intracellular events are associated with KISS1R activation by Kp.