IR-induced Gene Research Articles

Effects of cyclophosphamide and mitomycin C on radiation-induced transcriptional biomarkers in human lymphoblastoid cells

Purpose Ionizing radiation (IR)-induced transcriptional changes are considered a potential biodosimetry for dose evaluation and health risk monitoring of acute or chronic radiation exposure. It is crucial to understand the impact of confounding factors on the radiation-responsive gene expressions for accurate and reproducible dose assessment. This study aims to explore the potential influence of exposures to chemotherapeutic agents such as cyclophosphamide (CP) and mitomycin C (MMC) on IR-induced transcriptional biomarkers. Methods The human B lymphoblastoid cells (AHH-1) were exposed to 0, 20, 50, 100, 200 and 500 μg/ml CP or 0, 0.025, 0.05, 0.1 and 1 μg/ml MMC, respectively. The appropriate concentrations of CP and MMC were added for 1 h before irradiation with 0, 2, 4 and 6 Gy of 60Co γ-rays at a dose rate of 1 Gy/min. Cell viability was evaluated by CCK-8 assay. The gene expression responses of 18 radiation-induced transcriptional biomarkers were examined at 24 h after exposures to CP and MMC, respectively. The expression levels of five crucial DNA interstrand crosslinks (ICLs) repair genes were also evaluated. The biodosimetry models were established based on the specific radiation-responsive gene combinations. Results The baseline transcriptional levels of the 18 selected genes were slightly affected by CP treatment in the absence of IR, while the transcript responses to IR could be inhibited as the concentration of CP up to 50 μg/ml. MMC treatment up-regulated the background levels in most radiation-responsive gene expressions. Of 18 genes, only the relative mRNA expression levels of CDKN1A and BBC3 were repressed after treatment with IR and MMC in combination. The relative mRNA level of RAD51 was significantly up-regulated after exposure to CP, while the expression of FANCD2, RAD51 and BLM showed an overall increase in response to MMC treatment. After irradiation, the relative mRNA expression levels of FANCD2, BRCA2 and RAD51 exhibited dose-dependent increases in IR alone and MMC treatment groups. In addition, the biodosimetry models were established using 2-4 radiation-responsive genes based on different radiation exposure scenarios. Conclusion Our findings suggested that IR-induced gene expression changes were slightly affected after exposure to a relatively low concentration of CP and MMC. Gene expression combinations might improve the broad applicability of transcriptional biodosimetry across diverse radiation exposures.

HGG-12. Rapid PTEFb-dependent transcriptional reorganization underpins the glioma adaptive response to radiotherapy

BACKGROUND: Dynamic regulation of gene expression is fundamental for cellular adaptation to exogenous stressors. PTEFb-mediated promoter proximal pause-release of Pol II is a conserved regulatory mechanism for synchronous transcriptional induction best described in response to heat shock, but this pro-survival role has not been examined in the applied context of cancer therapy. DESIGN/METHOD: In order to examine the dynamics of chromatin reorganization following radiotherapy, we performed a combination of ChIP-, ATAC-, and RNA-seq in model systems of diffuse intrinsic pontine glioma (DIPG) and other pediatric high-grade gliomas (pHGG) following IR exposure. We interrogated IR-induced gene expression in the presence or absence of PTEFb blockade, including both mechanistic and functional consequences of concurrent inhibition or genetic depletion. We utilized culture models with live cell imaging to assess the therapeutic synergy of PTEFb inhibition with IR, as well as the therapeutic index of this intervention relative to normal controls. Finally, we employed orthotopic models of pHGG treated with conformal radiotherapy and CNS-penetrant PTEFb inhibitors in order to assess tolerability and anti-tumor effect in vivo. RESULTS: Rapid genome-wide redistribution of active chromatin features and PTEFb facilitates Pol II pause-release to drive nascent transcriptional induction within hours of exposure to therapeutic ionizing radiation. Concurrent inhibition of PTEFb imparts a transcription elongation defect, abrogating canonical adaptive programs such as DNA damage repair and cell cycle regulation. This combination demonstrates a potent, synergistic therapeutic potential agnostic of glioma subtype, leading to a marked induction of tumor cell apoptosis and prolongation of xenograft survival. CONCLUSION: These studies reveal a central role for PTEFb underpinning the early adaptive response to radiotherapy, opening new avenues for combinatorial treatment in these lethal malignancies.

Transcriptional Dynamics of DNA Damage Responsive Genes in Circulating Leukocytes during Radiotherapy.

Simple SummaryIn this study, the transcriptional response of a panel of radiation responsive genes was monitored over time in blood samples after radiation exposure in vivo. For this aim, cancer patients treated by radiotherapy were recruited after consent forms were obtained. Following the first fraction of radiotherapy, 2 mL blood samples were collected at different time points during the first 24h hours (before the second fraction was delivered) and at mid and end of treatment. Amongst the 9 genes studied, the gene FDXR stood out as the most sensitive and responsive to the low dose of radiation received from the localised radiation treatment by the circulating white blood cells. The activation of FDXR was found to depend on the volume of the body exposed with a peak of expression around 8–9 hours after irradiation was delivered. Finally results obtained ex vivo confirmed the results obtained in vivo.External beam radiation therapy leads to cellular activation of the DNA damage response (DDR). DNA double-strand breaks (DSBs) activate the ATM/CHEK2/p53 pathway, inducing the transcription of stress genes. The dynamic nature of this transcriptional response has not been directly observed in vivo in humans. In this study we monitored the messenger RNA transcript abundances of nine DNA damage-responsive genes (CDKN1A, GADD45, CCNG1, FDXR, DDB2, MDM2, PHPT1, SESN1, and PUMA), eight of them regulated by p53 in circulating blood leukocytes at different time points (2, 6–8, 16–18, and 24 h) in cancer patients (lung, neck, brain, and pelvis) undergoing radiotherapy. We discovered that, although the calculated mean physical dose to the blood was very low (0.038–0.169 Gy), an upregulation of Ferredoxin reductase (FDXR) gene transcription was detectable 2 h after exposure and was dose dependent from the lowest irradiated percentage of the body (3.5% whole brain) to the highest, (up to 19.4%, pelvic zone) reaching a peak at 6–8 h. The radiation response of the other genes was not strong enough after such low doses to provide meaningful information. Following multiple fractions, the expression level increased further and was still significantly up-regulated by the end of the treatment. Moreover, we compared FDXR transcriptional responses to ionizing radiation (IR) in vivo with healthy donors’ blood cells exposed ex vivo and found a good correlation in the kinetics of expression from the 8-hours time-point onward, suggesting that a molecular transcriptional regulation mechanism yet to be identified is involved. To conclude, we provided the first in vivo human report of IR-induced gene transcription temporal response of a panel of p53-dependant genes. FDXR was demonstrated to be the most responsive gene, able to reliably inform on the low doses following partial body irradiation of the patients, and providing an expression pattern corresponding to the % of body exposed. An extended study would provide individual biological dosimetry information and may reveal inter-individual variability to predict radiotherapy-associated adverse health outcomes.

Radiation-Induced Gene Expression Changes in High and Low Grade Breast Cancer Cell Types.

Background: There is extensive scientific evidence that radiation therapy (RT) is a crucial treatment, either alone or in combination with other treatment modalities, for many types of cancer, including breast cancer (BC). BC is a heterogeneous disease at both clinical and molecular levels, presenting distinct subtypes linked to the hormone receptor (HR) status and associated with different clinical outcomes. The aim of this study was to assess the molecular changes induced by high doses of ionizing radiation (IR) on immortalized and primary BC cell lines grouped according to Human epidermal growth factor receptor (HER2), estrogen, and progesterone receptors, to study how HR status influences the radiation response. Our genomic approach using in vitro and ex-vivo models (e.g., primary cells) is a necessary first step for a translational study to describe the common driven radio-resistance features associated with HR status. This information will eventually allow clinicians to prescribe more personalized total doses or associated targeted therapies for specific tumor subtypes, thus enhancing cancer radio-sensitivity. Methods: Nontumorigenic (MCF10A) and BC (MCF7 and MDA-MB-231) immortalized cell lines, as well as healthy (HMEC) and BC (BCpc7 and BCpcEMT) primary cultures, were divided into low grade, high grade, and healthy groups according to their HR status. At 24 h post-treatment, the gene expression profiles induced by two doses of IR treatment with 9 and 23 Gy were analyzed by cDNA microarray technology to select and compare the differential gene and pathway expressions among the experimental groups. Results: We present a descriptive report of the substantial alterations in gene expression levels and pathways after IR treatment in both immortalized and primary cell cultures. Overall, the IR-induced gene expression profiles and pathways appear to be cell-line dependent. The data suggest that some specific gene and pathway signatures seem to be linked to HR status. Conclusions: Genomic biomarkers and gene-signatures of specific tumor subtypes, selected according to their HR status and molecular features, could facilitate personalized biological-driven RT treatment planning alone and in combination with targeted therapies.

Transcriptional and post-transcriptional regulation of the ionizing radiation response by ATM and p53

In response to ionizing radiation (IR), cells activate a DNA damage response (DDR) pathway to re-program gene expression. Previous studies using total cellular RNA analyses have shown that the stress kinase ATM and the transcription factor p53 are integral components required for induction of IR-induced gene expression. These studies did not distinguish between changes in RNA synthesis and RNA turnover and did not address the role of enhancer elements in DDR-mediated transcriptional regulation. To determine the contribution of synthesis and degradation of RNA and monitor the activity of enhancer elements following exposure to IR, we used the recently developed Bru-seq, BruChase-seq and BruUV-seq techniques. Our results show that ATM and p53 regulate both RNA synthesis and stability as well as enhancer element activity following exposure to IR. Importantly, many genes in the p53-signaling pathway were coordinately up-regulated by both increased synthesis and RNA stability while down-regulated genes were suppressed either by reduced synthesis or stability. Our study is the first of its kind that independently assessed the effects of ionizing radiation on transcription and post-transcriptional regulation in normal human cells.

Abstract 4912: Hyperthermia induced NO repressed NFκB regulates IR-induced EMT, CSC self-renewal and pluripotency

Abstract Based on clinical evidence, hyperthermia treatment (HT) in combination with radiation (IR) achieves superior local control for recurrent chest wall breast cancer (BCa). The effect of HT combined with IR is due partly to its independent cytotoxic effects, and partly to its radiosensitizing effect. Recently, we have shown that apoptosis upon hyperthermia of BCa cells is regulated by NO-mediated suppression of NFκB. Also, we have demonstrated that tumor cells that survive a course of IR are instrumental in tumor relapse and progression. Accordingly, we investigated whether HT exerts NO-NFκB axis dependent regulation of IR-induced alterations in factors that drive the EMT, CSCs self-renewal capacity and pluripotency maintenance in surviving BCa Cells. Human breast adenocarcinoma (MCF-7) cells either mock-irradiated or exposed IR (2Gy) with or without HT (43°C for 1h) were analyzed for transcriptional regulation of 93 stem cell related molecules using QPCR profiling. NFκB (p50/p65) overexpression, muting by ΔIκBα (with IR) and NO induction by SNP (with HT) approach were used to delineate the role of HT-induced NO and the selective targeting of IR-induced NFκB. Nanog, Sox-2, ABCG-2, N-Cadherin and Myc expressions were examined with immunoblotting. IR profoundly increased the transactivation of 80 stem-cell related molecules. Interestingly, HT reverted the activation of 79 IR-induced genes. Attenuation of all 80 IR-induced genes observed after blocking NFκB with ΔIκBα and by increasing intracellular NO with SNP demonstrated the significant role of NO dependent NFκB suppression in HT-induced inhibition of IR modulated stem-cell related transcriptome. Expression levels of Nanog, Sox-2, ABCG-2, N-Cadherin and Myc validates the potential of HT in mitigating IR-induced stemness. Together this data demonstrates that HT attenuates IR-induced stemness in BCa cells and further imply that HT induced increase in NO may alleviate stemness by selectively targeting IR-induced NFκB. Citation Format: Corinne Dinges, Mohan Natarajan, Sheeja Aravindan, Terence S. Herman, Natarajan Aravindan. Hyperthermia induced NO repressed NFκB regulates IR-induced EMT, CSC self-renewal and pluripotency. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4912. doi:10.1158/1538-7445.AM2014-4912

Molecular basis of ‘hypoxic’ breast cancer cell radio-sensitization: phytochemicals converge on radiation induced Rel signaling

BackgroundHeterogeneously 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.MethodsHypoxic (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.ResultsEMSA 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.ConclusionsTogether, 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.

Abstract P2-07-02: Low-LET γ-Radiation Induced Protein S-Nitrosylation Regulates Tumor Invasion and Metastatic Transcriptional Response

Abstract Local-regional radiotherapy is one of the major therapeutic means in the management of breast cancer. However, besides the therapeutic effect, studies have shown that irradiation promotes malignant behavior of cancer cells both in vitro and in vivo by activating several tumor invasion and metastatic molecules. In this regard, ascertaining the molecular alterations contributing to this radiation-induced response is imperative. Accordingly, we investigated the role of radiation-induced NO-dependent nitrosylation in the transcription of invasion and metastatic genes in human breast adenocarcinoma cells. Human MCF-7 cells either mock irradiated, exposed to 2Gy (IR), treated with L-NAME (NOS inhibition) or BAY-11-7082 (phosphorylation inhibition) and then exposed to 2Gy, were examined for modulations in the transcriptional response of 93 tumor invasion and metastasis signaling genes. Cells treated with β-estradiol and mock-irradiated MDA-MB-231 cells were used as positive controls. Compared to mock irradiated cells, real time QPCR profiling revealed that 20 of 29 IR-induced genes including Birc3, Casp8, Ctgf, Ctsk, Htatip2, Kras, Map2k7, Mgat5, Mta2, Nme1, Nme2, Nme4, Pax5, Pten, Runx1, Serpinb5, Syk, Timp1, Timp2 and Timp4 were completely suppressed with NOS inhibition. Conversely, as opposed to NOS inhibition, IR-induced phosphorylation inhibition either failed to suppress or further induced the expression of these IR-induced genes. Furthermore, β-estradiol treatment revealed upregulation of 16 of these 20 IR-induced nitrosylation-dependent molecules and serves as a positive control for the study. More importantly, the highly metastatic, MDA-MB-231 cell line showed a robust induction in the expression of 15 of these 20 IR-induced nitrosylation-dependent genes. Taken together, these data imply that IR induces the expression of tumor invasion and metastatic genes and more importantly, may regulate the radiation-activated invasion and metastatic phenotype in breast adenocarcinoma cells. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-07-02.

Genome-Wide Expression Changes in Saccharomyces cerevisiae in Response to High-LET Ionizing Radiation

To understand the yeast response to high-linear energy transfer (LET) ionizing radiation (IR), we investigated global gene expression in yeast irradiated by three types of high-LET IR (fast neutrons, heavy ions, and thermal neutrons) and gamma rays using DNA microarray analysis. Stationary cells were irradiated by each IR and recultured in yeast-peptone-dextrose medium to allow repair for 40 min. RNA was then isolated from three independent samples of irradiated yeast. Genes involved in the Mec1p kinase pathway, which functions in DNA damage response, were induced by all forms of high-LET IR and by gamma rays. Some genes related to oxidative stress and the cell wall were induced by all forms of high-LET IRs. Gene expression patterns as a function of each type of high-LET IR were examined statistically by one-way analysis of variance. This analysis demonstrated the existence of irradiation-specific responses. For example, genes involved in ribosomal DNA synthesis were specifically induced by fast neutron irradiation, while the ubiquitin-proteasome system and heat shock response were specifically induced by thermal neutron irradiation. The study characterizes high-LET IR-induced gene expression and provides a molecular understanding of subsequent adaptation in yeast.

Transcriptional profiling in C. elegans suggests DNA damage dependent apoptosis as an ancient function of the p53 family

BackgroundIn contrast to the three mammalian p53 family members, p53, which is generally involved in DNA damage responses, and p63 and p73 which are primarily needed for developmental regulation, cep-1 encodes for the single C. elegans p53-like gene. cep-1 acts as a transcription activator in a primordial p53 pathway that involves CEP-1 activation and the CEP-1 dependent transcriptional induction of the worm BH3 only domain encoding genes egl-1 and ced-13 to induce germ cell apoptosis. EGL-1 and CED-13 proteins inactivate Bcl-2 like CED-9 to trigger CED-4 and CED-3 caspase dependent germ cell apoptosis. To address the function of p53 in global transcriptional regulation we investigate genome-wide transcriptional responses upon DNA damage and cep-1 deficiency.ResultsExamining C. elegans expression profiles using whole genome Affymetrix GeneChip arrays, we found that 83 genes were induced more than two fold upon ionizing radiation (IR). None of these genes, with exception of an ATP ribosylase homolog, encode for known DNA repair genes. Using two independent cep-1 loss of function alleles we did not find genes regulated by cep-1 in the absence of IR. Among the IR-induced genes only three are dependent on cep-1, namely egl-1, ced-13 and a novel C. elegans specific gene. The majority of IR-induced genes appear to be involved in general stress responses, and qRT-PCR experiments indicate that they are mainly expressed in somatic tissues. Interestingly, we reveal an extensive overlap of gene expression changes occurring in response to DNA damage and in response to bacterial infection. Furthermore, many genes induced by IR are also transcriptionally regulated in longevity mutants suggesting that DNA damage and aging induce an overlapping stress response.ConclusionWe performed genome-wide gene expression analyses which indicate that only a surprisingly small number of genes are regulated by CEP-1 and that DNA damage induced apoptosis via the transcriptional induction of BH3 domain proteins is likely to be an ancient DNA damage response function of the p53 family. Interestingly, although the apoptotic response to DNA damage is regulated through the transcriptional activity of CEP-1, other DNA damage responses do not appear to be regulated on the transcriptional level and do not require the p53 like gene cep-1.

Stratification by DNA Damage-Induced Apoptotic and Transcriptional Responses Provides Insight into the Molecular Mechanisms Underlying Blast Clearance Rates in Paediatric B-Precursor ALL.

Although there have been dramatic improvements in the treatment of paediatric ALL in recent years, a significant proportion of these patients still experience relapse. Early treatment responses, indicated by the rate and degree of blast clearance, are good markers for the likelihood of ALL relapse. However, despite their clinical predictive value, the molecular basis underlying these observations remains unknown.We aimed to elucidate the molecular mechanisms behind treatment resistance in paediatric ALL by analysing cellular responses to IR-induced damage. Using western blotting, we classified 82 paediatric diagnostic ALL samples into two categories on the basis of their activation of apoptosis following DNA damage. We found that 31 ALL tumours had an apoptotic resistant phenotype, failing to cleave PARP1 and caspases 3, 7 and 9 by 24h after ionising radiation (IR), in contrast to 51 tumours classified as apoptotic responsive. Strikingly, we observed that our DNA damage apoptotic response stratification of ALL tumours closely correlated with blast clearance following treatment in ALL patients. Among apoptotic resistant patients, 69% still had more than 25% blasts by morphological assessment on day 8 or 15, and 64% had a blast population that could be detected by molecular minimal residual disease (MRD) at day 28, indicating high risk of relapse. In contrast, in the apoptotic responsive group only 8% had more than 25% blasts at day 8 or 15, and just 6% were positive by MRD at day 28.Differential baseline expression of specific genes has been previously linked with drug resistance in paediatric ALL. To provide further insight into the biology of resistance to DNA damaging agents we analysed changes in transcriptional profiles following exposure of blast cells to IR in 22 representative tumours, 11 apoptotic responsive and 11 apoptotic resistant. We identified over 300 IR-induced genes that could discriminate between the two categories, including genes involved in cellular proliferation and the regulation of apoptosis. When we considered only the subset with TEL/AML1 fusion, we found that the expression of phospholipase C epsilon (PLCe), which is implicated in the Ras cellular survival pathway, provided powerful discrimination between the apoptotic resistant and responsive ALL tumours. We have subsequently confirmed that PLCe is also differentially expressed at the protein level at 24h after IR between apoptotic resistant and responsive ALLs, regardless of TEL/AML1 status. Finally, we have identified model pre-B ALL cell lines for each of the two response categories, and in these we have shown that Ras is activated in response to DNA damage and associates with PLCe only in the apoptotic resistant cells.In conclusion, we have shown that differences in cellular apoptotic response to DNA damage closely correlate with the rate of blast clearance in paediatric ALL. Furthermore, we believe that alteration in the Ras survival pathway is one mechanism to account for differential apoptotic responses to DNA damage in both primary ALL tumour cells and ALL cell lines. These findings provide new insight into the factors influencing clinical outcome in ALL patients and are likely to have important implications for the development of future targeted treatments in paediatric ALL.

Co-activation of ERK, NF-κB, and GADD45β in Response to Ionizing Radiation

NF-kappaB has been well documented to play a critical role in signaling cell stress reactions. The extracellular signal-regulated kinase (ERK) regulates cell proliferation and survival. GADD45beta is a primary cell cycle element responsive to NF-kappaB activation in anti-apoptotic responses. The present study provides evidence demonstrating that NK-kappaB, ERK and GADD45beta are co-activated by ionizing radiation (IR) in a pattern of mutually dependence to increase cell survival. Stress conditions generated in human breast cancer MCF-7 cells by the administration of a single exposure of 5 Gy IR resulted in the activation of ERK but not p38 or JNK, along with an enhancement of the NF-kappaB transactivation and GADD45beta expression. Overexpression of dominant negative Erk (DN-Erk) or pre-exposure to ERK inhibitor PD98059 inhibited NF-kappaB. Transfection of dominant negative mutant IkappaB that blocks NF-kappaB nuclear translocation, inhibited ERK activity and GADD45beta expression and increased cell radiosensitivity. Interaction of p65 and ERK was visualized in living MCF-7 cells by bimolecular fluorescence complementation analysis. Antisense inhibition of GADD45beta strikingly blocked IR-induced NF-kappaB and ERK but not p38 and JNK. Overall, these results demonstrate a possibility that NF-kappaB, ERK, and GADD45beta are able to coordinate in a loop-like signaling network to defend cells against the cytotoxicity induced by ionizing radiation.

Expression Profiling of IR-induced Genes in A Human Normal Fibroblast

Expression Profiling of IR-induced Genes in A Human Normal Fibroblast