Determining the driving factors and molecular flow-through that define the switch from favorable to aggressive high-risk disease is critical to the betterment of neuroblastoma cure. In this study, we examined the cytogenetic and tumorigenic physiognomies of distinct population of metastatic site- derived aggressive cells (MSDACs) from high-risk tumors, and showed the influence of acquired genetic rearrangements on poor patient outcomes. Karyotyping in SH-SY5Y and MSDACs revealed trisomy of 1q, with additional non-random chromosomal rearrangements on 1q32, 8p23, 9q34, 15q24, 22q13 (additions), and 7q32 (deletion). Array CGH analysis of individual clones of MSDACs revealed genetic alterations in chromosomes 1, 7, 8, and 22, corresponding to a gain in the copy numbers of LOC100288142, CD1C, CFHR3, FOXP2, MDFIC, RALYL, CSMD3, SAMD12-AS1, and MAL2, and a loss in ADAM5, LOC400927, APOBEC3B, RPL3, MGAT3, SLC25A17, EP300, L3MBTL2, SERHL, POLDIP3, A4GALT, and TTLL1. QPCR analysis and immunoblotting showed a definite association between DNA-copy number changes and matching transcriptional/translational expression in clones of MSDACs. Further, MSDACs exert a stem-like phenotype. Under serum-free conditions, MSDACs demonstrated profound tumorosphere formation ex vivo. Moreover, MSDACs exhibited high tumorigenic capacity in vivo and prompted aggressive metastatic disease. Tissue microarray analysis coupled with automated IHC revealed significant association of RALYL to the tumor grade in a cohort of 25 neuroblastoma patients. Clinical outcome association analysis showed a strong correlation between the expression of CFHR3, CSMD3, MDFIC, FOXP2, RALYL, POLDIP3, SLC25A17, SERHL, MGAT3, TTLL1, or LOC400927 and overall and relapse-free survival in patients with neuroblastoma. Together, these data highlight the ongoing acquired genetic rearrangements in undifferentiated tumor-forming neural crest cells, and suggest that these alterations could switch favorable neuroblastoma to high-risk aggressive disease, promoting poor clinical outcomes.

Heterogeneously distributed hypoxic areas are a characteristic property of locally advanced breast cancers (BCa) and generally associated with therapeutic resistance, metastases, and poor patient survival. About 50% of locally advanced BCa, where radiotherapy is less effective are suggested to be due to hypoxic regions. In this study, we investigated the potential of bioactive phytochemicals in radio-sensitizing hypoxic BCa cells. Hypoxic (O2-2.5%; N2-92.5%; CO2-5%) MCF-7 cells were exposed to 4 Gy radiation (IR) alone or after pretreatment with Curcumin (CUR), curcumin analog EF24, neem leaf extract (NLE), Genistein (GEN), Resveratrol (RES) or raspberry extract (RSE). The cells were examined for inhibition of NFκB activity, transcriptional modulation of 88 NFκB signaling pathway genes, activation and cellular localization of radio-responsive NFκB related mediators, eNos, Erk1/2, SOD2, Akt1/2/3, p50, p65, pIκBα, TNFα, Birc-1, -2, -5 and associated induction of cell death. EMSA revealed that cells exposed to phytochemicals showed complete suppression of IR-induced NFκB. Relatively, cells exposed EF24 revealed a robust inhibition of IR-induced NFκB. QPCR profiling showed induced expression of 53 NFκB signaling pathway genes after IR. Conversely, 53, 50, 53, 53, 53 and 53 of IR-induced genes were inhibited with EF24, NLE, CUR, GEN, RES and RSE respectively. In addition, 25, 29, 24, 16, 11 and 21 of 35 IR-suppressed genes were further inhibited with EF24, NLE, CUR, GEN, RES and RSE respectively. Immunoblotting revealed a significant attenuating effect of IR-modulated radio-responsive eNos, Erk1/2, SOD2, Akt1/2/3, p50, p65, pIκBα, TNFα, Birc-1, -2 and −5 with EF24, NLE, CUR, GEN, RES or RSE. Annexin V-FITC staining showed a consistent and significant induction of IR-induced cell death with these phytochemicals. Notably, EF24 robustly conferred IR-induced cell death. Together, these data identifies the potential hypoxic cell radio-sensitizers and further implies that the induced radio-sensitization may be exerted by selectively targeting IR-induced NFκB signaling.

Studying the potential role of tumour necrosis factor (TNF)alpha in the initiation of genomic instability is necessary to understand whether TNFalpha can serve as a signalling mediator of radiation-induced genomic instability in non-irradiated bystander cells. In this study, we examined whether TNFalpha could initiate processes through oxidative stress signalling that lead to DNA damage and genomic instability in primary vascular endothelium. In these cells, low linear energy transfer (LET) radiation (0.1-2 Gy) induced the secretion of TNFalpha into the culture medium. When added ectopically, TNFalpha at concentrations ranging from 0.1 ng ml(-1) to 10 ng ml(-1) increased (twofold to threefold) intracellular oxidative stress. Next, to examine whether TNFalpha induces genetic damage, cells were treated with TNFalpha for 5 h and analysed immediately using the single cell gel electrophoresis assay or after 3 days, 12 days and 20 days using solid stain chromosomal analysis. Cells exposed to 0.1 Gy, 1 Gy or 2 Gy or treated with 100 microM H2O2 were used as positive controls. The results showed that TNFalpha as low as 0.1 ng ml(-1) could initiate increased DNA damage compared with untreated controls. When examined in the progeny cells after several generations, the chromosomal instability appeared to be carried over even after day 12 and day 20. The increased genetic damage is inhibited in cells that are pre-incubated with the antioxidant enzyme catalase, the antioxidant N-acetyl-L-cysteine or the metal chelator pyrrolidine dithiocarbamate. These results clearly indicate that TNFalpha at concentrations at which no cytotoxicity is observed could induce genetic damage through free radical generation, which could, in turn, lead to the delayed events associated with genomic instability.

Our objective was to understand the mechanism through which cells that initially survive irradiation could acquire survival advantage. In this study, we show evidence that low-linear energy transfer gamma-radiation can induce telomerase enzyme activity in primary aortic endothelial cells, and that an upstream regulator, nuclear factor kappa B (NF-kappaB), controls this activation. Telomeric repeat amplification protocol (TRAP) assay showed that cells exposed to a dose of 2 Gy induce telomerase activity. Subsequent analysis revealed that radiation-induced telomeric activity is regulated at the transcriptional level by triggering activation of the promoter of the telomerase catalytic subunit, telomerase reverse transcriptase (TERT). A mechanistic study revealed that NF-kappaB becomes functionally activated upon radiation exposure and mediates the upregulation of telomerase activity by binding to the kappaB-binding region in the promoter region of the TERT gene. More significantly, elimination of the NF-small ka, CyrillicB recognition site on the telomerase promoter or inhibition of NF-small ka, CyrillicB by ectopically expressing the inhibitor protein IkappaBalpha mutant (Ismall ka, CyrillicBalpha(S32A/S36A))) compromises radiation-induced telomerase promoter activation. Consistent with the notion that NF-kappaB mediates gamma-ray-induced telomerase responses, TRAP assay revealed that ectopically expressed IkappaBalpha(S32A/S36A)) also attenuated telomerase enzyme activity. These findings indicate that NF-kappaB activation following ionizing radiation exposure may elicit a survival advantage by upregulating and maintaining telomerase activity.