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Research Paper
RNA Interference Targeting of A1 Receptor-Overexpressing Breast Carcinoma Cells Leads To Diminished Rates of Cell Proliferation and Induction of Apoptosis
Asra Mirza, Andrea Basso, Stuart Black, Michael Malkowski, Lia Kwee, Jonathan A. Pachter, Jean E. Lachowicz, Yan Wang and Suxing Liu
volume 4 | issue 12
december 2005Pages: 1355-1360
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To determine if A1 adenosine receptors mediate breast tumorigenesis, we evaluated A1 receptor expression in human tumor cell lines and human primary breast tumor tissues using both quantitative RT-PCR and Western blot analysis. A1 receptor mRNA expression is up-regulated in all breast tumor cell lines examined (n = 7) compared to normal mammary epithelial cells/cell lines (n = 3) as determined by quantitative RT-PCR analysis. Western blot analysis indicates that protein expression of A1 adenosine receptor is higher in 15 (62.5%) of 24 human primary breast tumor tissues than in matched normal breast tissue. To explore its cellular function, the A1 adenosine receptor was depleted by small interfering RNA (siRNA) in MDA-MB-468 human breast tumor cells. Depletion of A1 receptors in MDA-MB-468 breast tumor cells attenuated both cell growth and cell proliferation as measured by cell number counts and [14C]-thymidine incorporation, respectively. Cell cycle analysis indicated that depletion of A1 receptors by siRNA impairs G1 checkpoint, leading to marked accumulation of cells in G2/M phase, in agreement with the inhibitory effect on cell proliferation. Further supporting this finding, synchronization studies of Hela cells in various cell cycle phases suggest that A1 receptor expression is suppressed in G2/M cells and depletion of A1 receptor expression by siRNA produced differential expression of several key cell cycle regulators, i.e. accumulation of the cyclin-dependent kinase inhibitor p27 with concomitant reduction of CDK4 and cyclin E proteins. In addition to the impact on cell cycle progression, depletion of A1 receptors by siRNA results in substantial cell death and apoptosis as determined by FACS analysis and annexin V staining method. Together these findings suggest that the A1 adenosine receptor may contribute to tumor cell growth and survival in breast tumor cells.
We now provide open access to journal articles published online for one year or more. This article may be downloaded at the following link:
Download PDFIf the document does not open, please right-click on the link (control-click on a Macintosh) and select the option to save the file to disk.