Abstract 4971: Detection of receptor and non receptor tyrosine kinases in the culture filtrates of astrocytes and cancer cells

Abstract

Abstract As demonstrated in our previous work, meningioma cells shed their membrane receptors and these receptors function in a cell-free environment. The purpose of this study was to determine if receptor and non-receptor kinases were similarly being released in different cancers. Using antibody arrays, cell culture filtrates from glioma and prostate cancer cell lines were assessed to determine if solubilized receptors and kinases were present. Supernatants from cells treated with Brefeldin-A were analyzed similarly. Further, culture filtrates concentrated by ultrafiltration were subjected to luminescence based kinase assays and western blotting to assess the active forms. Cell viability was measured with Trypan blue staining in all the experiments. Our results demonstrate that an array of receptors are solubilized into culture filtrates of brain and prostate cancer cell lines within the first 24 hours. Simultaneously, soluble receptors in the culture filtrates of astrocytes within 48 hours of culture were also detected. Compared to astrocytes, cancer cells showed elevated levels of receptors and kinases released at 48 hours. As many as 15 receptor or non-receptor tyrosine kinases including Src family of kinases were found in the culture filtrates of the brain and prostate cancer cell lines. Treatment with Brefeldin-A inhibited the release of receptors and kinases partially, but markedly, within first 24 hours suggesting a secretory pathway for their release. Additionally, phosphorylation of tyrosine kinases was detected in the western blots within 24 hours. Kinase assays further confirmed active nature of the extracellular kinases, particularly, when checked for the active form of Lyn kinase in the culture filtrates, phosphorylation on Tyrosine-507 was detected along with the total forms. Cell viability assays on the cultured cells revealed more than 98% viability, thus eliminating the possibility of the release of detected kinases from dead cells. In light of this study, we propose a new niche for solubilized receptor and tyrosine kinase functioning that might have a role in intercellular signaling in cell-free conditions both in disease and physiological statuses. Note: This abstract was not presented at the meeting. Citation Format: Venkateswara Rao Gogineni. Detection of receptor and non receptor tyrosine kinases in the culture filtrates of astrocytes and cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4971. doi:10.1158/1538-7445.AM2015-4971