Our previous work and that of other investigators strongly suggest a relationship between the upregulation of metalloproteinase-9 (MMP-9) and urokinase-type plasminogen activator receptor (uPAR) in tumor angiogenesis and metastasis. In this study, we evaluated the role of MMP-9 and uPAR in medulloblastoma cancer cell resistance to ionizing irradiation (IR) and tested the antitumor efficacy of siRNA (short interfering RNA) against MMP-9 [plasmid siRNA vector for MMP-9 (pM)] and uPAR [plasmid vector for uPAR (pU)] either alone or in combination [plasmid siRNA vector for both uPAR and MMP-9 (pUM)]. Cell proliferation (BrdU assay), apoptosis (in situ TUNEL for DNA fragmentation), and cell-cycle (FACS) analyses were carried out to determine the effect of siRNA either alone or in combination with IR on G 2 /M cell-cycle arrest in medulloblastoma cells. IR upregulated MMP-9 and uPAR expression in medulloblastoma cells; pM, pU, and pUM in combination with IR effectively reduced both MMP-9 and uPAR expression, thereby leading to increased radiosensitivity of medulloblastoma cells. siRNA treatments (pM, pU, and pUM) also promoted IR-induced apoptosis and enhanced IR-induced G 2 /M arrest during cell-cycle progression. While IR induces G 2 /M cell-cycle arrest through inhibition of the pCdc2-and cyclin B-regulated signaling pathways involving p53, p21/WAF1, and Chk2 gene expression, siRNA (pM, pU, and pUM) alone or in combination with IR induced G 2 /M arrest mediated through inhibition of the pCdc2-and cyclin B1-regulated signaling pathways involving Chk1 and Cdc25A gene expression. Taken together, our data suggest that downregulation of MMP-9 and uPAR induces Chk1-mediated G 2 /M cell-cycle arrest, whereas the disruption caused by IR alone is dependent on p53-and Chk2-mediated G 2 /M cell-cycle arrest. Mol Cancer Res; 9(1); 51-66. Ó2010 AACR.