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Abstract

Inositol phosphates are naturally occurring compounds that regulate diverse cellular processes including apoptosis. Apoptosis is a mechanism by which cells undergo natural death to maintain cellular homeostasis. It causes cell death in areas during a state that is harmful to the body. It also regulates cellular development. Previous work has shown that exogenously administered, as well as endogenously manipulated inositol phosphates bring about apoptotic changes. It has been demonstrated that cellular levels of inositol phosphates, particularly higher inositol phosphates such as inositol hexakis-phosphate (IP6) and diphosphoinositol pentakis-phosphate (IP7) levels increase during apoptotic conditions. In this study, we have attempted to separate and identify higher inositol phosphates such as IP6 by polyacrylamide gel electrophoresis (PAGE) and shown that changes in inositol phosphate levels can be detected by this method. Cells were treated with etoposide to induce apoptosis, and apoptotic cells were observed under UV light following ethidium bromide/acridine orange staining. This staining showed that IP3 - IP6 induced apoptosis in SW480 cells with IP6 being the most effective inducing agent. The extracts from apoptotic and control cells were then loaded onto the polyacrylamide gel and run along with standard IP6. Results showed that IP6 could be detected using the PAGE method and that cellular levels of IP6 were increased in SW480 cells, in which apoptosis had been induced by etoposide. Our results demonstrated that this technique could be utilized instead of the laborious radioactive labeling and HPLC separation method to study the changes in cellular levels of inositol phosphates particularly IP6.

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