Differential gene expression following ionizing radiation in multicellular spheroid depending on p53 status: Identification of potential targets and prediction of responsive signaling pathways

Ionizing radiation (IR) therapy is a potential treatment option of several solid tumors; however, the molecular responses of carcinoma cells to IR are not yet clarified. A multicellular spheroid (MCS)-in vivo mimic model was established in which allow us to in situ study of cellular response to IR in human carcinoma cells with different intrinsic p53 status. Here, relative cell survival rate was determined in p53-wild and-null type cells of MCS in comparison to that of monolayer culture in response to 10 Gy IR by trypan blue exclusion assay. It obviously showed that under MCS culture system radioresistance phenotype was dependent on p53 status since p53-null type MCS exhibited significantly higher cell survival rate, in contrast to p53-wild type MCS. In order to screen molecular targets in p53-proficient cancer cells upon IR exposure, we conducted microarray under 10 Gy IR to observe gene expression pattern change in MCS compared to monolayer cells, with and without p53. In addition, potential molecular network was analyzed using Pathway Studio software to define responsive signaling and interactions. Total 478 genes were notably altered at transcript level toward the IR treatment. Discovered genes were participated in several cellular major processes, including apoptosis (both caspase-dependent and-independent pathways), cell migration and proliferation. Among them ANXA11, C2, KCNE2, KIF3C, MSH5, and OSCAR were differentially expressed in p53-proficient MCS but constant in p53-deficient MCS. These genes might be considered as target molecules for evaluation of IR efficiency in p53-proficient MCS as a typical 3D-in vivo mimic model. Our findings demonstrate complex responses of p53-carcinoma cells following IR exposure including putative signaling pathways, leading to emphasis on the importance of in vivo mimic MCS model rather than conventional monolayer culture system.