Apoptotic pathways controlled by the Rel/NF-kB family of transcription factors may regulate the response of cells to DNA damage. Here, we have examined the NFkB status of several prostate tumor cell lines. In the androgen-independent prostate tumor cells PC-3 and DU-145, the DNA-binding activity of NF-kB was constitutively activated and IkB-a levels were decreased. In contrast, the androgen-sensitive prostate tumor cell line LNCaP had low levels of NF-kB which were upregulated following exposure to cytokines or DNA damage. The activity of the IkB-a kinase, IKKa, which mediates NF-kB activation, was also measured. In PC-3 cells, IKKa activity was constitutively active, whereas LNCaP cells had minimal IKKa activity that was activated by cytokines. The anti-in¯ammatory agent ibuprofen inhibited the constitutive activation of NF-kB and IKKa in PC-3 and DU-145 cells, and blocked stimulated activation of NF-kB in LNCaP cells. However, ibuprofen did not directly inhibit IkB-a kinase. The results demonstrate that NF-kB is constitutively activated in the hormone-insensitive prostate tumor cell lines PC-3 and DU-145, but not in the hormone responsive LNCaP cell line. The constitutive activation of NF-kB in prostate tumor cells may increase expression of anti-apoptotic proteins, thereby decreasing the e ectiveness of anti-tumor therapy and contributing to the development of the malignant phenotype.

We have previously demonstrated that prostate carcinoma cells exposed to fractionated radiation differentially expressed more genes compared to single-dose radiation. To understand the role of miRNA in regulation of radiation-induced gene expression, we analyzed miRNA expression in LNCaP, PC3 and DU145 prostate cancer cells treated with single-dose radiation and fractionated radiation by micro-array. Selected miRNAs were studied in RWPE-1 normal prostate epithelial cells by RT-PCR. Fractionated radiation significantly altered more miRNAs as compared to single-dose radiation. Downregulation of oncomiR-17-92 cluster was observed only in the p53 positive LNCaP and RWPE-1 cells treated with single-dose radiation and fractionated radiation. Comparison of miRNA and mRNA data by IPA target filter analysis revealed an inverse correlation between miR-17-92 cluster and several targets including TP53INP1 in p53 signaling pathway. The base level expressions of these miRNAs were significantly different among the cell lines and did not predict the radiation outcome. Tumor suppressor miR-34a and let-7 miRNAs were upregulated by fractionated radiation in radiosensitive LNCaP (p53 positive) and PC3 (p53-null) cells indicating that radiation-induced miRNA expression may not be regulated by p53 alone. Our data support the potential for using fractionated radiation to induce molecular targets and radiation-induced miRNAs may have a significant role in predicting radiosensitivity

To examine the possibility of using fractionated radiation in a unique way with molecular targeted therapy, gene expression profiles of prostate carcinoma cells treated with 10 Gy radiation administered either as a single dose or as fractions of 2 Gy × 5 and 1 Gy × 10 were examined by microarray analysis. Compared to the single dose, the fractionated irradiation resulted in significant increases in differentially expressed genes in both cell lines, with more robust changes in PC3 cells than in DU145 cells. The differentially expressed genes (>twofold change; P < 0.05) were clustered and their ontological annotations evaluated. In PC3 cells genes regulating immune and stress response, cell cycle and apoptosis were significantly up-regulated by multifractionated radiation compared to single-dose radiation. Ingenuity Pathway Analysis (IPA) of the differentially expressed genes revealed that immune response and cardiovascular genes were in the top functional category in PC3 cells and cell-to-cell signaling in DU145 cells. RT-PCR analysis showed that a flexure point for gene expression occurred at the 6th-8th fraction and AKT inhibitor perifosine produced enhanced cell killing after 1 Gy × 8 fractionated radiation in PC3 and DU145 cells compared to single dose. This study suggests that fractionated radiation may be a uniquely exploitable, non-oncogene-addiction stress pathway for molecular therapeutic targeting.