) The objective is to test whether ionizing radiation (IR) induced Ca2+ transients are critical for activation of cell cycle checkpoints; and if genetic manipulation of Ca2+ homeostasis will blunt IR-induced Ca2+ transients, short circuit IR induced cell cycle checkpoints and alter radio-sensitivity. Ca2+ homeostasis of two breast carcinoma cell lines, MCF-7 and MDA-MB-231, has been genetically manipulated by stable transfection with the B-lymphocyte Ca2+ channel, CD20, under control of an inducible promoter. An initial characterization reveals that CD20 expression increases cytosolic free [Ca2+], shortens G1 of the cell cycle, and blocks the IR-induced G1 arrest.
Three aims are proposed: (1) Continue characterization of the CD20-expressing clones to include measurements of cytosolic free [Ca2+], growth and cell cycle analysis. Representative clones and a null point assay developed to determine at what extracellular [Ca2+], the radio- biological and cell-cycle control properties of the parental and non- induced CD20 transfected cell lines are recovered in the CD20 expressing sensitive responses to IR; (2) Test whether expression of CD20 alters the IR-induced Ca2+ oscillations and the size of internal Ca2+ stores and establish the linkage between the IR-induced Ca2+ transients and cell cycle checkpoints; (3) Define Ca2+-sensitive IR-induced cell cycle checkpoints. Initially, the G1/S checkpoint of MCF7 cells will be examined since this is abrogated in CD20 Rb phosphorylation checkpoint to the Ca2+- sensitive event. Studies will focus on the synthesis, stabilities, and activities of key regulatory proteins, e.g. cyclin dependent kinases and their endogenous inhibitors, the goal being to define the most immediate, Ca2+-dependent event necessary for checkpoint activation. Based on results from Aim #1, Ca2+-sensitive S-or G2-checkpoints will be examined with the overall approach guided by the analysis of the G1/S checkpoint. Results from this experimentation may indicate possible targets for therapeutic manipulation of tumor cell radio-sensitivity.
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