Differential expression of stress and immune response pathway transcripts and miRNAs in normal human endothelial cells subjected to fractionated or single-dose radiation.

UNLABELLED: Although modern radiotherapy technologies can precisely deliver higher doses of radiation to tumors, thus, reducing overall radiation exposure to normal tissues, moderate dose, and normal tissue toxicity still remains a significant limitation. The present study profiled the global effects on transcript and miR expression in human coronary artery endothelial cells using single-dose irradiation (SD, 10 Gy) or multifractionated irradiation (MF, 2 Gy × 5) regimens. Longitudinal time points were collected after an SD or final dose of MF irradiation for analysis using Agilent Human Gene Expression and miRNA microarray platforms. Compared with SD, the exposure to MF resulted in robust transcript and miR expression changes in terms of the number and magnitude. For data analysis, statistically significant mRNAs (2-fold) and miRs (1.5-fold) were processed by Ingenuity Pathway Analysis to uncover miRs associated with target transcripts from several cellular pathways after irradiation. Interestingly, MF radiation induced a cohort of mRNAs and miRs that coordinate the induction of immune response pathway under tight regulation. In addition, mRNAs and miRs associated with DNA replication, recombination and repair, apoptosis, cardiovascular events, and angiogenesis were revealed. IMPLICATIONS: Radiation-induced alterations in stress and immune response genes in endothelial cells contribute to changes in normal tissue and tumor microenvironment, and affect the outcome of radiotherapy.

mRNA Expression Profiles for Prostate Cancer following Fractionated Irradiation Are Influenced by p53 Status.

We assessed changes in cell lines of varying p53 status after various fractionation regimens to determine if p53 influences gene expression and if multifractionated (MF) irradiation can induce molecular pathway changes. LNCaP (p53 wild-type), PC3 (p53 null), and DU145 (p53 mutant) prostate carcinoma cells received 5 and 10 Gy as single-dose (SD) or MF (0.5 Gy x 10, 1 Gy x 10, and 2 Gy x 5) irradiation to simulate hypofractionated and conventionally fractionated prostate radiotherapies, respectively. mRNA analysis revealed 978 LNCaP genes differentially expressed (greater than two-fold change, P < .05) after irradiation. Most were altered with SD (69%) and downregulated (75%). Fewer PC3 (343) and DU145 (116) genes were induced, with most upregulated (87%, 89%) and altered with MF irradiation. Gene ontology revealed immune response and interferon genes most prominently expressed after irradiation in PC3 and DU145. Cell cycle regulatory (P = 9.23 x 10(-73), 14.2% of altered genes, nearly universally downregulated) and DNA replication/repair (P = 6.86 x 10(-30)) genes were most prominent in LNCaP. Stress response and proliferation genes were altered in all cell lines. p53-activated genes were only induced in LNCaP. Differences in gene expression exist between cell lines and after varying irradiation regimens that are p53 dependent. As the duration of changes is ≥24 hours, it may be possible to use radiation-inducible targeted therapy to enhance the efficacy of molecular targeted agents.

Radiation survivors: understanding and exploiting the phenotype following fractionated radiation therapy.

Radiation oncology modalities such as intensity-modulated and image-guided radiation therapy can reduce the high dose to normal tissue and deliver a heterogeneous dose to tumors, focusing on areas deemed at highest risk for tumor persistence. Clinical radiation oncology produces daily doses ranging from 1 to 20 Gy, with tissues being exposed to 30 or more daily fractions. Hypothesizing the cells that survive fractionated radiation therapy have a substantially different phenotype than the untreated cells, which might be exploitable for targeting with molecular therapeutics or immunotherapy, three prostate cancer cell lines (PC3, DU145, and LNCaP) and normal endothelial cells were studied to understand the biology of differential effects of multifraction (MF) radiation of 0.5, 1, and/or 2 Gy fraction to 10 Gy total dose, and a single dose of 5 and 10 Gy. The resulting changes in mRNA, miRNA, and phosphoproteome were analyzed. Significant differences were observed in the MF radiation exposures including those from the 0.5 Gy MF that produces little cell killing. As expected, p53 function played a major role in response. Pathways modified by MF include immune response, DNA damage, cell-cycle arrest, TGF-ß, survival, and apoptotic signal transduction. The radiation-induced stress response will set forth a unique platform for exploiting the effects of radiation therapy as "focused biology" for cancer treatment in conjunction with molecular targeted or immunologically directed therapy. Given that more normal tissue is treated, albeit to lower doses with these newer techniques, the response of the normal tissue may also influence long-term treatment outcome.

Fractionated radiation alters oncomir and tumor suppressor miRNAs in human prostate cancer cells.

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 microarray. 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.

Fractionated radiation therapy can induce a molecular profile for therapeutic targeting.

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.

NS-398, ibuprofen, and cyclooxygenase-2 RNA interference produce significantly different gene expression profiles in prostate cancer cells.

Cyclooxygenase-2 (COX-2) plays a significant role in tumor development and progression. Nonsteroidal anti-inflammatory drugs (NSAID) exhibit potent anticancer effects in vitro and in vivo by COX-2-dependent and COX-2-independent mechanisms. In this study, we used microarray analysis to identify the change of expression profile regulated by a COX-2-specific NSAID NS-398 (0.01 and 0.1 mmol/L), a nonspecific NSAID ibuprofen (0.1 and 1.5 mmol/L) and RNA interference (RNAi)-mediated COX-2 inhibition in PC3 prostate cancer cells. A total of 3,362 differentially expressed genes with 2-fold change and P<0.05 were identified. Low concentrations of NSAIDs and COX-2 RNAi altered very few genes (1-3%) compared with the higher concentration of NS-398 (17%) and ibuprofen (80%). Ingenuity Pathway Analysis was used for distributing the differentially expressed genes into biological networks and for evaluation of functional significance. The top 3 networks for both NSAIDs included functional categories of DNA replication, recombination and repair, and gastrointestinal disease. Immunoresponse function was specific to NS-398, and cell cycle and cellular movement were among the top functions for ibuprofen. Ingenuity Pathway Analysis also identified renal and urologic disease as a function specific for ibuprofen. This comprehensive study identified several COX-2-independent targets of NSAIDs, which may help explain the antitumor and radiosensitizing effects of NSAIDs. However, none of these categories were reflected in the identified networks in PC3 cells treated with clinically relevant low concentrations of NS-398 and ibuprofen or with COX-2 RNAi, suggesting the benefit to fingerprinting preclinical drug concentrations to improve their relevance to the clinical setting.

PX-478, an inhibitor of hypoxia-inducible factor-1alpha, enhances radiosensitivity of prostate carcinoma cells.

Overexpression of hypoxia-inducible factor-1alpha (HIF-1alpha) in human tumors is associated with poor prognosis and poor outcome to radiation therapy. Inhibition of HIF-1alpha is considered as a promising approach in cancer therapy. The purpose of this study was to test the efficacy of a novel HIF-1alpha inhibitor PX-478 as a radiosensitizer under normoxic and hypoxic conditions in vitro. PC3 and DU 145 prostate carcinoma cells were treated with PX-478 for 20 hr, and HIF-1alpha protein level and clonogenic cell survival were determined under normoxia and hypoxia. Effects of PX-478 on cell cycle distribution and phosphorylation of H2AX histone were evaluated. PX-478 decreased HIF-1alpha protein in PC3 and DU 145 cells. PX-478 produced cytotoxicity in both cell lines with enhanced toxicity under hypoxia for DU-145. PX-478 (20 mumol/L) enhanced the radiosensitivity of PC3 cells irradiated under normoxic and hypoxic condition with enhancement factor (EF) 1.4 and 1.56, respectively. The drug was less effective in inhibiting HIF-1alpha and enhancing radiosensitivity of DU 145 cells compared to PC3 cells with EF 1.13 (normoxia) and 1.25 (hypoxia) at 50 mumol/L concentration. PX-478 induced S/G2M arrest in PC3 but not in DU 145 cells. Treatment of PC3 and DU 145 cells with the drug resulted in phosphorylation of H2AX histone and prolongation of gammaH2AX expression in the irradiated cells. PX-478 is now undergoing Phase I clinical trials as an oral agent. Although the precise mechanism of enhancement of radiosensitivity remains to be identified, this study suggests a potential role for PX-478 as a clinical radiation enhancer.

Microarray and protein analysis of human pterygium.

PURPOSE: Pterygium is a sunlight-related, ocular-surface lesion that can obscure vision. In order to identify specific genes that may play a role in pterygium pathogenesis, we analyzed the global gene expression profile of pterygium in relation to autologous conjunctiva. METHODS: Oligonucleotide microarray hybridization was used to compare the gene expression profile between human whole pterygium and autologous conjunctiva. Selected genes were further characterized by RT-PCR, western blot, and immunohistochemistry, and comparisons were made with limbal and corneal tissues. RESULTS: Thirty-four genes exhibited a 2 fold or greater difference in expression between human whole pterygium and autologous conjunctiva. Twenty-nine transcripts were increased and five transcripts were decreased in pterygium. Fibronectin, macrophage-inflammatory protein-4 (MIP-4), and lipocalin 2 (oncogene 24p3; NGAL) were increased 9, 5, and 2.4 fold, respectively, while Per1 and Ephrin-A1 were decreased 2 fold in pterygium. Western blots showed that fibronectin and MIP-4 were increased in pterygium compared to limbus, cornea, and conjunctiva. Immunohistochemical analysis showed fibronectin in the stroma; lipocalin 2 in the basal epithelial cells, basement membrane, and extracellular stroma; and MIP-4 in all areas of the pterygium. CONCLUSIONS: These data show both novel and previously identified extracellular-matrix-related, proinflammatory, angiogenic, fibrogenic, and oncogenic genes expressed in human pterygium. Comparisons of selected genes with limbal and corneal tissues gave results similar to comparisons between pterygium and normal conjunctiva. The increased expression of lipocalin 2, which activates matrix metalloproteinases (MMP), is consistent with our previous findings that MMP-9 and other MMPs are highly expressed in pterygium basal epithelium.

Accumulation of p53 protein in pterygia is not accompanied by TP53 gene mutation.

Previously, we reported that pterygial epithelial cells show positive p53 staining by immunohistochemistry, and that they do not demonstrate apoptosis. We wished to determine whether the accumulation of p53 protein was caused by missense mutations in exons 5-8 of the TP53 gene, as is frequently the case in malignant tumours that contain high levels of abnormal p53. From 11 pterygia, epithelial cells were isolated by laser capture microdissection, or manually, in order to reduce the contribution of TP53 from normal cells. DNA from pterygial epithelial cells was amplified across exons 5-8 in 10 pterygia and across exons 5,7 and 8 in another pterygium. In 2 pterygia, all translated exons (2-11) were sequenced. No mutations were found, although normal polymorphisms in codon 72 were readily detected in 2 pterygia. RT-PCR was used to compare amounts of TP53 mRNA isolated from normal conjunctiva and pterygia from eight additional patients. We detected an approximate two-fold increase of TP53 RNA in pterygia compared to that in normal conjunctiva. Western blotting was used to compare amounts of p53 protein in pterygia and normal conjunctiva. Consistent with our previous immunohistochemical studies, amounts of p53 protein in pterygia, detected by the western blotting, were elevated compared to those detected in normal conjunctiva and corneal limbal epithelium. However, the TP53 gene in pterygia is not mutated, and therefore, the elevated levels of p53 protein must result from a different mechanism than that seen in malignant tumours containing TP53 missense mutations. The increased amount of p53 protein in pterygial cells does not cause apoptosis or block cell proliferation, suggesting that these normal p53 functions are inactivated in pterygia.