Master of Science - Research
School of Biological Sciences
Gadeock, Safina, Expression and function of the P2X7 receptor on human malignant cell lines, Master of Science - Research thesis, School of Biological Sciences, University of Wollongong, 2010. http://ro.uow.edu.au/theses/3248
The P2X7 receptor is a ligand-gated ion channel present on normal and malignant cells of hematopoietic and epithelial origins. Activation of the P2X7 receptor by extracellular adenosine 5’-triphosphate (ATP) causes the rapid flux of Na+, K+ and Ca2+. Continued activation of P2X7 allows the formation of large, non-specific pores, allowing the uptake of organic molecules including cation dyes. In addition, activation of P2X7 leads to a number of downstream signalling events which includes the maturation and release of interlukin (IL)-1β, as well as cell proliferation and death. As a result of these latter roles of P2X7 in cell survival, this receptor is attracting considerable interest for its growth-promoting and growth-inhibitory roles in cancer. Therefore, the aim of this project was to study the expression and function of the P2X7 receptor in myeloid leukemic and epithelial malignant cancer cell lines.
Previous work in our laboratory demonstrated that the immunomodulatory cytokine, transforming growth factor (TGF)-β1 prevents the up-regulation of P2X7 in the human myeloid leukemic cell line, THP-1 by interferon (IFN)-γ and lipopolysaccharide (LPS). The mechanism of action of TGF-β1 in this process however was never elucidated. Therefore, the first part of my study aimed to determine the mechanism of action of TGF-β1 on the up-regulation of P2X7 expression and function in THP-1 cells differentiated with IFN-γ and LPS. Cell-surface molecules including P2X7 were examined by immunofluorescence staining. Total P2X7 protein and mRNA was assessed by immunoblotting and RT-PCR respectively. P2X7 function was evaluated by ATP-induced cation dye uptake measurements. IL-1β release was measured by ELISA. Cell-surface P2X7 was present on THP-1 cells differentiated for 3 days with IFN-γ and LPS but not on undifferentiated THP-1 cells. Similarly, ATP induced ethidium+ uptake into differentiated but not undifferentiated THP-1 cells. Coincubation of cells with TGF-β1 plus IFN-γ and LPS prevented the up-regulation of P2X7 expression and ATP-induced ethidium+ uptake Moreover, ATP-induced YOPRO- 12+ uptake and IL-1β release were abrogated in IFN-γ and LPS-treated cells coincubated with TGF-β1. Of note, TGF-β1 abrogated the amount of total P2X7 protein and mRNA induced by IFN-γ and LPS. Finally, TGF-β1 prevented the up-regulation of cell-surface CD86, but not other differentiation markers (CD14 and MHC class II), by IFN-γ and LPS. Collectively, these results indicate that TGF-β1 prevents the upregulation of P2X7 by IFN-γ and LPS in THP-1 monocytes by preventing P2X7 transcription and subsequent translation. Moreover, this effect of TGF-β1 is not due to general impairment of THP-1 cell differentiation by IFN-γ and LPS. This suggests that TGF-β1 may limit P2X7-mediated processes during inflammation and immunity, as well as in cancer cells.
Previous work by others has indicated that ATP and 2(3’)-O-(4-benzoylbenzoyl) adenosine 5’-triphosphate (BzATP) induces the death of the human epithelial colon carcinoma cell lines, HCT-8 and Caco-2 in a manner characteristic of P2X7 activation. However a direct role for this receptor in this process was not established. Therefore, the second part of my study aimed to determine if HCT-8 and Caco-2 cells expressed functional P2X7. Cell-surface and total P2X7 was examined by immunofluorescence staining and immunoblotting respectively. P2X7 function was evaluated by ATP- and BzATP-induced ethidium+ uptake measurements. Reduction in cell numbers, as an indirect measure of cell death, was evaluated using a tetrazolium-based colorimetric assay. The human multiple myeloma cell line, RPMI 8226, was used as a positive control. HCT-8 and Caco-2 cells expressed low levels of cell-surface P2X7. Whole lysates of these cells expressed low levels of the full length (75 kDa) P2X7, but higher levels of a 42 kDa P2X7 variant. In contrast, RPMI 8226 cells expressed relatively high levels of cell-surface P2X7, as well as full-length P2X7. ATP and BzATP consistently failed to induce ethidium+ uptake in HCT-8 and Caco-2 despite extended incubation times of up to 30 min, and the use of several media known to potentiate P2X7 activation. ATP and BzATP also failed to cause significant death in both the cell lines. In contrast, ATP and BzATP induced ethidium+ uptake and death in RPMI 8226 cells. IFN-γ failed to upregulate P2X7 expression and function in HCT-8 cells. Collectively, these results indicate the absence of functional P2X7 in HCT-8 and Caco-2 cells despite the presence of low levels of cell-surface P2X7. The high proportion of a 42 kDa P2X7 variant provides a possible explanation for the lack of functional P2X7 in these cells.
The presence of functional P2X7 in primary myeloid leukemias has been reported by others; however cell line models of myeloid leukemia that constitutively expresses functional P2X7 are limited. Therefore, the final part of my study aimed to determine if the human myeloid leukemic cell line KG-1 expresses functional P2X7. Cell-surface P2X7 was examined by immunofluorescence staining. Total P2X7 protein and mRNA was assessed by immunoblotting and RT-PCR respectively. P2X7 function was evaluated by ATP-induced ethidium+ uptake measurements. Reduction in cell numbers was evaluated using a tetrazolium-based colorimetric assay and cell death was confirmed using a trypan blue exclusion assay and morphological analysis. KG-1 cells expressed low levels of cell-surface P2X7, as well as low levels of P2X7 protein and mRNA. Both, ATP and BzATP consistently induced ethidium+ uptake in KCl and sucrose but not NaCl medium. ATP-induced ethidium+ uptake in a concentration dependant manner with a maximal response at 100 μM and with an EC50 of 8.5 μM which is ~ 10 fold lower than that reported for recombinant P2X7-induced cation fluxes. Moreover, the non-P2X7 agonist, α-β-methyl adenosine 5’-triphosphate (αβmeATP), as well as ATP, BzATP and adenosine 5'-O-[3-thiotriphosphate] (ATP-γ-S) induced ethidium+ uptake in KG-1 cells. The P2X7 antagonists, KN-62, AZ10606120 and A- 438079, each impaired ATP-induced ethidium+ uptake. ATP induced KG-1 cell death and morphological analysis indicated that this process was characteristic of apoptosis. ATP-induced apoptosis was impaired by KN-62 and AZ10606120. Collectively, these results indicate that KG-1 cells express functional P2X7 with an atypical pharmacological profile.