Upregulation Of DNA Repair Genes Research Articles

Retinoic Acid and Ascorbate Remodel the Epigenome of Leukemia Cells to Improve Therapeutic Efficacy By Enhancing TET Activity

Ten-eleven translocation (TET) enzymes are commonly mutated in leukemia, leading to aberrant DNA methylation and impaired hydroxymethylation. All-trans retinoic acid (ATRA) is a vitamin A derivative and cofactor of retinoic acid receptors (RARs) which, in combination with arsenic trioxide, can curatively treat acute promyelocytic leukemia. ATRA has also been shown to upregulate expression of Tet2 and Tet3 in murine embryonic stem cells, and retinol and ascorbic acid (vitamin C) have been shown to enhance pluripotent stem cell reprogramming through increasing hydroxymethylcytosine and promoting DNA demethylation. Our previous work has demonstrated ascorbate can slow disease progression in vivo and, combined with the PARP inhibitor Olaparib, promote cell cycle dysregulation and induce cellular differentiation in acute myeloid leukemia (AML) models both in vitro and in vivo. Here, we show ATRA can induce TET2 and/or TET3 expression in AML cells, and, combined with ascorbate, increase genome-wide oxidized methylcytosine in a TET-dependent manner. Combination treatment with ATRA and ascorbate also induces transcriptional reprogramming, leading to upregulation of DNA repair genes and pathways leading to increased expression of myeloid differentiation markers. Additionally, we observe chromatin remodeling associated with an enrichment for ETS motifs including genes and regions regulated by the transcription factor PU.1, an essential modulator of hematopoietic cell fate. We also show enrichment of retinoic acid receptor A (RARA) at the TET2 locus and at several RAR elements in response to ATRA in AML cells. Using very low dose ascorbate and ATRA, we show that the combination treatment can enhance the efficacy of Olaparib and Venetoclax to alter the cell cycle, promote differentiation, and induce apoptosis of AML cells. These data demonstrate the potential for ATRA and ascorbate to increase TET activity and drive myeloid differentiation and death of AML cells that can be exploited as an adjuvant therapy for the treatment leukemia.

Transcriptomic and genomic effects of gamma-radiation exposure on strains of the black yeast Exophiala dermatitidis evolved to display increased ionizing radiation resistance.

Melanized fungi thrive in extreme environments, including those with high levels of ionizing radiation. To understand the role that melanin may play in ionizing radiation resistance, we previously performed an adaptive laboratory evolution experiment in which we used melanized and non-melanized strains of the yeast Exophiala dermatitidis to develop evolved lines that exhibit increased ionizing radiation resistance. In this study, we further characterized these evolved lines by analyzing their response to ionizing radiation at the transcriptomic and genomic levels. RNA sequencing showed that the response to gamma irradiation in both unevolved and evolved strains involved the induction of DNA repair genes. However, in the melanized lines evolved to exhibit increased ionizing radiation resistance, DNA-associated genes were constitutively expressed, compared to their expression levels in wild type. Non-melanized lines that were evolved to be resistant to ionizing radiation, on the other hand, exhibited upregulation of genes involved in redox homeostasis, even under non-irradiated conditions. Additionally, we characterized genome-wide mutations induced by a single high dose of gamma radiation in these evolved lines and observed that while melanin did not directly affect survival after gamma radiation exposure, melanized lines that evolved to exhibit higher ionizing radiation resistance experienced fewer mutations, whereas similarly evolved, non-melanized lines accumulated more mutations, similar to the parent, non-melanized strain. These results underscore the complex yet measurable role of melanin in the response to ionizing radiation in E. dermatitidis. Furthermore, this study enhances our understanding of the mechanisms underlying the recovery after ionizing radiation exposure in melanized fungi and offers insights into the potential therapeutic applications of melanin and other redox molecules for protecting against ionizing radiation-induced damage. IMPORTANCE Ionizing radiation poses a significant threat to living organisms and human health, given its destructive nature and widespread use in fields such as medicine and the potential for nuclear disasters. Melanized fungi exhibit remarkable survival capabilities, enduring doses up to 1,000-fold higher than mammals. Through adaptive laboratory evolution, we validated the protective role of constitutive upregulation of DNA repair genes in the black yeast Exophiala dermatitidis, enhancing survival after radiation exposure. Surprisingly, we found that evolved strains lacking melanin still achieved high levels of radioresistance. Our study unveiled the significance of robust activation and enhancement of redox homeostasis, as evidenced by the profound transcriptional changes and increased accumulation of mutations, in substantially improving ionizing radiation resistance in the absence of melanin. These findings underscore the delicate balance between DNA repair and redox homeostasis for an organism's ability to endure and recover from radiation exposure.

Upregulation of DNA repair genes and cell extrusion underpin the remarkable radiation resistance of Trichoplax adhaerens.

Trichoplax adhaerens is the simplest multicellular animal with tissue differentiation and somatic cell turnover. Like all other multicellular organisms, it should be vulnerable to cancer, yet there have been no reports of cancer in T. adhaerens or any other placozoan. We investigated the cancer resistance of T. adhaerens, discovering that they are able to tolerate high levels of radiation damage (218.6 Gy). To investigate how T. adhaerens survive levels of radiation that are lethal to other animals, we examined gene expression after the X-ray exposure, finding overexpression of genes involved in DNA repair and apoptosis including the MDM2 gene. We also discovered that T. adhaerens extrudes clusters of inviable cells after X-ray exposure. T. adhaerens is a valuable model organism for studying the molecular, genetic, and tissue-level mechanisms underlying cancer suppression.

Ddx41 loss R-loops in cGAS to fuel inflammatory HSPC production

In this issue of Developmental Cell, Weinreb etal. reveal that loss of the DEAD-box helicase Ddx41 unexpectedly triggers an R-loop-mediated sterile inflammatory cascade which drives HSPC production during embryonic development. Human studies suggest mechanistic conservation for inflammation in DDX41-associated hematologic disease, uncovering a potential route for future therapeutic intervention.

Monosomy 7 and Co-Occurrent Genomic Aberrations Determine Chemotherapy Response in Acute Myeloid Leukemia (AML) Patients Using the Cellworks Omics Biology Model (CBM): Mycare-021-04

Background: Monosomy 7/Del 7 (-7) or its long arm (del(7q)) is one of the most common cytogenetic abnormalities in pediatric and adult myeloid malignancies, particularly in adverse-risk acute myeloid leukemias (AMLs). In general, (-7) is associated with poor response to induction chemotherapy (PMID 12393746). At the same time, not all patients fare poorly so the ability to identify responders and non-responders remains a high priority. Aim: To predict the response for induction chemotherapy in AML patients with (-7) and identify novel genomic signatures of response and resistance. Methods: Genomic data from 13 consecutive patients with (-7) were analyzed using the Cellworks Omics Biology Model (CBM) to generate patient-specific protein network models. All data was anonymized, de-identified and exempt from IRB review. For each model, disease simulations were performed and patients were segregated into HOXA-upregulated and HOXA-downregulated cohorts based on the simulation levels of HOXA5 and HOXA9. Digital drug simulations for induction chemotherapy were accomplished by measuring the impact of drug effect on a cell growth score, a composite of cell proliferation, viability and apoptosis indices. Each patient-specific model was analyzed to identify mechanisms underlying treatment outcomes. Results: 7/13 (54%) of (-7) patients failed to achieve remission after induction chemotherapy (Table 1) which highlighted that (-7) alone does not confer resistance to chemotherapy. CBM identified other genomic alterations that determine chemotherapy response, including DNA repair deficiency genes, mismatch repair (MMR), and homologous recombination repair (HRR) genes. DNA methylation and histone methylation (H3K27me) impacting HOXA gene expression, mainly HOXA5 and HOXA9, were identified as upstream regulators of DNA repair genes. Loss or reduced levels of EZH2 is associated with lower H3K27 methylation and thereby higher expression of HOXA5 and HOXA9 gene targets. High active levels of HOXA correlated with low rates of successful remission induction (22%, n=7) and lower activity levels of HOXA correlated with successful induction chemotherapy (100%, n=4) (Table 1). CBM identified that (-7) results in a decreased expression of EZH2, CARD11, EIF3, PMS2, HUS1, KMT2C (MLL3), CDK5 and IKZF1 genes. Since EZH2 abnormalities alone are not implicated in poor prognosis for AML patients, we sought other aberrations that prevent HOXA upregulation. Using CBM, we identified multiple accompanying aberrations which regulate HOXA genes. Deletions of KAT6A, ASXL1, DNMT3B, DNMT3L genes and high KMT2A-partial tandem duplication correspond to HOXA-upregulation whereas EED amplification, gain of function mutations in DNMT3A, mutations in IDH1/2, MYC and KAT6A amplification and KDM4A deletion result in HOXA-downregulation. Conclusion: Alterations of chromatin regulation have consequences for transcription factors that regulate expression of DNA repair genes. Under conditions where DNA repair is enhanced, induction chemotherapy was 78% less likely to effect remission in (-7) AML patients undergoing induction chemotherapy. Loss of H3K27 methylation associated with loss of PRC2 function by any means resulted in HOXA-upregulation and upregulation of DNA repair genes induced resistance to induction therapy. On the other hand, CBM analysis identified genetic signatures associated with a 100% remission rate from AML induction therapy despite the presence of (-7). Generation of H3K27me caused by PRC2 activation resulting from numerous mechanisms led to HOXA-downregulation and 100% response to induction therapy. Stratification of patients harboring (-7) by HOXA biomarker analysis could inform treatment planning, avoid drug-related adverse events and reduce treatment costs after validation with a larger prospective dataset. Disclosures Castro: Cellworks Group Inc: Consultancy. Watson:Cellworks Group Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cellmax Life Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Mercy Bioanalytics, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; SEER Biosciences, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; BioAI Health Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees. Kumar:Cellworks Research India Private Limited: Current Employment. Nair:Cellworks Research India Private Limited: Current Employment. Grover:Cellworks Research India Private Limited: Current Employment. Sahu:Cellworks Research India Private Limited: Current Employment. Mohapatra:Cellworks Research India Private Limited: Current Employment. G:Cellworks Research India Private Limited: Current Employment. Agarwal:Cellworks Research India Private Limited: Current Employment. Suseela:Cellworks Research India Private Limited: Current Employment. Ganesh:Cellworks Research India Private Limited: Current Employment. Sauban:Cellworks Research India Private Limited: Current Employment. Kumar:Cellworks Research India Private Limited: Current Employment. Raman:Cellworks Research India Private Limited: Current Employment. Singh:Cellworks Research India Private Limited: Current Employment. Basu:Cellworks Research India Private Limited: Current Employment. Lunkad:Cellworks Research India Private Limited: Current Employment. Mundkur:Cellworks Group Inc.: Current Employment. Macpherson:Cellworks Group Inc.: Current Employment. Kapoor:Cellworks Research India Private Limited: Current Employment. Howard:Servier: Consultancy, Other: Speaker; Boston Scientific: Consultancy; Sanofi: Consultancy, Other: Speaker; EUSA Pharma: Consultancy; Cellworks: Consultancy.

Abstract 2596: Toll like receptor 4 as a potential DNA repair modulator in U87MG-GBM cells

Abstract Toll like receptor 4 (TLR4) has a dual role in cellular fate, depending on the stimuli and microenvironment context. Its activation may promotes cell survival and proliferative phenotype via MyD88 with NFkB activation, or leads to cell death via TRIF. Glioblastoma (GBM), grade IV astrocytoma, is the most common and aggressive primary malignancy in the brain. Surgical tumor cytoreduction followed by radiotherapy and adjunct chemotherapy with Temozolomide (TMZ) have been considered the standard of care for GBM. And, in spite of several combinatory rescue therapeutic trials, the median overall survival of GBM patients continue to be approximately 15 months. In this context, new therapeutic strategies are urgent, and immune modulation has demonstrated optimistic results in several tumor types. Therefore, the aim of this study was to analyze the role of TLR4 in human astrocytomas. First, we evaluated TLR4 expression in 140 human astrocytoma of different grades of malignancy, by qRT-PCR. TLR4 molecular signaling pathways were investigated through LPS stimuli in U87MG-GBM cell line. In astrocytoma samples, TLR4 was up-regulated when compared to brain non-neoplastic samples (p<0.05, Kruskall Wallis and Dunn tests), with increased expression in Mesenchymal compare to Classical and Proneural subtypes. U87MG cells stimulation with LPS (10ug/ml) lead to NF-kB (p65) nuclear translocation after 12hr through western blot and immunofluorescence, which was unexpected as such translocation through the MyD88/TRAF6 canonical pathway occurs within 30min to 2hr after LPS stimulation. TRIF, IL1B, RIPK1/3 expressions were increased after LPS stimulation, suggesting an activation of inflammasome and ripoptosome pathways. Moreover, the combined LPS treatment with TMZ showed a higher apoptotic rate after 48hr when compared to TMZ alone. However, the transcriptomic analysis by RNASeq of U87MG cells after LPS stimulation showed an upregulation of DNA repair genes, particularly MBD4 and PLAUR. Both genes are upregulated in mesenchymal GBM subtype in the TCGA RNASeq dataset, and they present correlated expression with TLR4 (r=0.406, p=0.004 and r=0.338, p=0.019 by Spearman's test). Our results suggest that the anti-tumoral effect by TLR4 pathway activation by enhancement of tumor apoptosis might be more efficient with combined inhibition of DNA repair cascade. In fact, OSI-296 and BKM-120 have shown to decrease MBD4 expression, and increased overall survival was observed in a phase II clinical trial for solid tumor. Citation Format: Isabele F. Moretti, Antonio Lerario, Sueli M. Oba-Shinjo, Suely K. Marie, Marina Trombetta Lima. Toll like receptor 4 as a potential DNA repair modulator in U87MG-GBM cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2596.

P53 Activation by Cr(VI): A Transcriptionally Limited Response Induced by ATR Kinase in S-Phase.

Cellular reduction of carcinogenic chromium(VI) causes several forms of Cr-DNA damage with different genotoxic properties. Chromate-treated cultured cells have shown a strong proapoptotic activity of the DNA damage-sensitive transcription factor p53. However, induction of p53 transcriptional targets by Cr(VI) in rodent lungs was weak or undetectable. We examined Cr(VI) effects on the p53 pathway in human cells with restored levels of ascorbate that acts as a principal reducer of Cr(VI) in vivo but is nearly absent in standard cell cultures. Ascorbate-restored H460 and primary human cells treated with Cr(VI) contained higher levels of p53 and its Ser15 phosphorylation, which were induced by ATR kinase. Cr(VI)-stimulated p53 phosphorylation occurred in S-phase by a diffusible pool of ATR that was separate from the chromatin-bound pool targeting DNA repair substrates at the sites of toxic mismatch repair (MMR) of Cr-DNA adducts. Even when more abundantly present than after exposure to the radiomimetic bleomycin, Cr(VI)-stabilized p53 showed a much more limited activation of its target genes in two types of primary human cells. No increases in mRNA were found for nucleotide excision repair factors and a majority of proapoptotic genes. A weak transcription activity of Cr(VI)-upregulated p53 was associated with its low lysine acetylation in the regulatory C-terminal domain, resulting from the inability of Cr(VI) to activate ATM in ascorbate-restored cells. Thus, p53 activation by ascorbate-metabolized Cr(VI) represents a limited genome-protective response that is defective in upregulation of DNA repair genes and proapoptotic transcripts for elimination of damaged cells.

TGF-β/Smads Signaling Affects Radiation Response and Prolongs Survival by Regulating DNA Repair Genes in Malignant Glioma.

To understand the molecular mechanism underlying the causal relationship between aberrant upregulation of transforming growth factor beta (TGF-β) and radio-resistance in glioma. The mouse glioma cell GL261 was irradiated, and relative expression of TGF-β/Smad signaling genes was determined by real-time PCR and western blotting. The DNA repair response on exogenous TGF-β or LY2109761 was evaluated by quantification of diverse genes by real-time PCR and western blotting. Xenograft mice were employed for in vivo investigation to assess the response to irradiation and LY2109761 either alone or in combination. The expression of DNA repair genes was further determined in the xenograft tumor. The TGF-β/Smad signaling pathway was activated by radiation in the GL261 cell line. The exogenous complement of TGF-β significantly stimulated DNA repair response. Administration of LY2109761 suppressed DNA repair genes. Simultaneous treatment with LY2109761 abrogated the upregulation of DNA repair genes in GL261. In the xenograft tumor model, LY2109761 synergistically improved the therapeutic effect of radiation via improvement of sensitivity. Our data suggested that LY2109761 treatment re-sensitized glioma to radiation via antagonizing TGF-β/Smad-induced DNA repair.

Epigenetic alterations mediate iPSC-induced normalization of DNA repair gene expression and TNR stability in Huntington's disease cells.

Huntington's disease (HD) is a rare autosomal dominant neurodegenerative disorder caused by a cytosine-adenine-guanine (CAG) trinucleotide repeat (TNR) expansion within the HTT gene. The mechanisms underlying HD-associated cellular dysfunction in pluripotency and neurodevelopment are poorly understood. We had previously identified downregulation of selected DNA repair genes in HD fibroblasts relative to wild-type fibroblasts, as a result of promoter hypermethylation. Here, we tested the hypothesis that hypomethylation during cellular reprogramming to the induced pluripotent stem cell (iPSC) state leads to upregulation of DNA repair genes and stabilization of TNRs in HD cells. We sought to determine how the HD TNR region is affected by global epigenetic changes through cellular reprogramming and early neurodifferentiation. We find that early stage HD-affected neural stem cells (HD-NSCs) contain increased levels of global 5-hydroxymethylation (5-hmC) and normalized DNA repair gene expression. We confirm TNR stability is induced in iPSCs, and maintained in HD-NSCs. We also identify that upregulation of 5-hmC increases ten-eleven translocation 1 and 2 (TET1/2) protein levels, and show their knockdown leads to a corresponding decrease in the expression of select DNA repair genes. We further confirm decreased expression of TET1/2-regulating miR-29 family members in HD-NSCs. Our findings demonstrate that mechanisms associated with pluripotency induction lead to a recovery in the expression of select DNA repair gene and stabilize pathogenic TNRs in HD.

Adaptive upregulation of DNA repair genes following benzo(a)pyrene diol epoxide protects against cell death at the expense of mutations.

A coordinated and faithful DNA damage response is of central importance for maintaining genomic integrity and survival. Here, we show that exposure of human cells to benzo(a)pyrene 9,10-diol-7,8-epoxide (BPDE), the active metabolite of benzo(a)pyrene (B(a)P), which represents a most important carcinogen formed during food preparation at high temperature, smoking and by incomplete combustion processes, causes a prompt and sustained upregulation of the DNA repair genes DDB2, XPC, XPF, XPG and POLH. Induction of these repair factors on RNA and protein level enhanced the removal of BPDE adducts from DNA and protected cells against subsequent BPDE exposure. However, through the induction of POLH the mutation frequency in the surviving cells was enhanced. Activation of these adaptive DNA repair genes was also observed upon B(a)P treatment of MCF7 cells and in buccal cells of human volunteers after cigarette smoking. Our data provide a rational basis for an adaptive response to polycyclic aromatic hydrocarbons, which occurs however at the expense of mutations that may drive cancer formation.

Distinctive adaptive response to repeated exposure to hydrogen peroxide associated with upregulation of DNA repair genes and cell cycle arrest

Many environmental and physiological stresses are chronic. Thus, cells are constantly exposed to diverse types of genotoxic insults that challenge genome stability, including those that induce oxidative DNA damage. However, most in vitro studies that model cellular response to oxidative stressors employ short exposures and/or acute stress models. In this study, we tested the hypothesis that chronic and repeated exposure to a micromolar concentration of hydrogen peroxide (H2O2) could activate DNA damage responses, resulting in cellular adaptations. For this purpose, we developed an in vitro model in which we incubated mouse myoblast cells with a steady concentration of ~50μM H2O2 for one hour daily for seven days, followed by a final challenge of a 10 or 20X higher dose of H2O2 (0.5 or 1mM). We report that intermittent long-term exposure to this oxidative stimulus nearly eliminated cell toxicity and significantly decreased genotoxicity (in particular, a >5-fold decreased in double-strand breaks) resulting from subsequent acute exposure to oxidative stress. This protection was associated with cell cycle arrest in G2/M and induction of expression of nine DNA repair genes. Together, this evidence supports an adaptive response to chronic, low-level oxidative stress that results in genomic protection and up-regulated maintenance of cellular homeostasis.

Structural chromosome abnormalities, increased DNA strand breaks and DNA strand break repair deficiency in dermal fibroblasts from old female human donors

Dermal fibroblasts provide a paradigmatic model of cellular adaptation to long-term exogenous stress and ageing processes driven thereby. Here we addressed whether fibroblast ageing analysedex vivo entails genome instability. Dermal fibroblasts from human female donors aged 20-67 years were studied in primary culture at low population doubling. Under these conditions, the incidence of replicative senescence and rates of age-correlated telomere shortening were insignificant. Genome-wide gene expression analysis revealed age-related impairment of mitosis, telomere and chromosome maintenance and induction of genes associated with DNA repair and non-homologous end-joining, most notably XRCC4 and ligase 4. We observed an age-correlated drop in proliferative capacity and age-correlated increases in heterochromatin marks, structural chromosome abnormalities (deletions, translocations and chromatid breaks), DNA strand breaks and histone H2AX-phosphorylation. In a third of the cells from old and middle-aged donors repair of X-ray induced DNA strand breaks was impaired despite up-regulation of DNA repair genes. The distinct phenotype of genome instability, increased heterochromatinisation and (in 30% of the cases futile) up-regulation of DNA repair genes was stably maintained over several cell passages indicating that it represents a feature of geroconversion that is distinct from cellular senescence, as it does not encompass a block of proliferation.

Transcriptional regulation of human DNA repair genes following genotoxic stress: trigger mechanisms, inducible responses and genotoxic adaptation

DNA repair is the first barrier in the defense against genotoxic stress. In recent years, mechanisms that recognize DNA damage and activate DNA repair functions through transcriptional upregulation and post-translational modification were the focus of intensive research. Most DNA repair pathways are complex, involving many proteins working in discrete consecutive steps. Therefore, their balanced expression is important for avoiding erroneous repair that might result from excessive base removal and DNA cleavage. Amelioration of DNA repair requires both a fine-tuned system of lesion recognition and transcription factors that regulate repair genes in a balanced way. Transcriptional upregulation of DNA repair genes by genotoxic stress is counteracted by DNA damage that blocks transcription. Therefore, induction of DNA repair resulting in an adaptive response is only visible through a narrow window of dose. Here, we review transcriptional regulation of DNA repair genes in normal and cancer cells and describe mechanisms of promoter activation following genotoxic exposures through environmental carcinogens and anticancer drugs. The data available to date indicate that 25 DNA repair genes are subject to regulation following genotoxic stress in rodent and human cells, but for only a few of them, the data are solid as to the mechanism, homeostatic regulation and involvement in an adaptive response to genotoxic stress.

TLR agonist treatment elicits an increase in DNA repair machinery (117.19)

Abstract The toll-like receptor (TLR) pathway is stimulated by pathogen associated molecular patterns (PAMPs) and damage associated molecular patterns (DAMPs). Activation of this pathway leads to an inflammatory and immunomodulating response appropriate for the stimulus by which it was activated. Recently a body of literature has emerged indicating that TLR stimulation may not only result in an inflammatory response, but may also lead to an up-regulation of DNA repair genes and an increase in functional DNA repair after injury. These conclusions are drawn from studies conducted in the mouse animal model, however the human DNA repair response to TLR activation has yet to be investigated. Using an in vitro cell culture system, we find that human DNA repair genes are similarly up-regulated. Previously, only TLR 7 and TLR 9 agonists have been shown in mice to stimulate increased DNA repair; in this study we expand the panel of TLR agonists investigated and show that the induction of this phenomenon may not be unique to the previously studied TLR agonists. This functional increase in repair after injury may lead to a wide range of outcomes for the cells involved and warrants further study into the ever broadening role of TLR signaling in controlling cell fate.

Abstract LB-281: Inhibition of Hedgehog results in altered regulation of c-jun and of ERCC1 in cisplatin-resistant A2780-CP70 human ovarian cancer cells

Abstract Cancer stem cells are presumed to be highly resistant to chemotherapy. Activation of the Hedgehog pathway has been reported as necessary, for the cancer stem cell state. A key gene in the Hedgehog pathway is Gli1. Resistance to platinum compounds is due in part, to up-regulated nucleotide excision repair, NER. ERCC1 is a key marker for NER activity. Transcriptional control of ERCC1 occurs in part, through c-Jun. Upon treatment with cisplatin, cisplatin-resistant A2780-CP70 cells normally up-regulate c-jun and c-fos, which then up-regulate ERCC1 and other NER genes. More specifically, the c-jun Ser63/73 cascade is up-regulated, with up-regulation of activator protein 1 (Reed, JBC 1998). We studied the relationship between Gli1 expression, ERCC1 expression, and cisplatin resistance in the human ovarian cancer cell lines A2780 and A2780-CP70. A2780-CP70 cells are 10-15 fold more resistant to cisplatin than A2780 cells. We used two different probes to modulate the Hedgehog pathway: a) cyclopamine as a pharmacologic inhibitor of the Smoothened; and, b) an anti-Gli1 shRNA to specifically inhibit Gli1. At baseline, cisplatin-resistant cells, expressed > 10-fold more Gli1 protein than cisplatin-sensitive cells. In cisplatin-resistant cells, Gli1 protein was down-regulated in response to cyclopamine, and to anti-Gli1 shRNA. When A2780-CP70 cisplatin-resistant cells are pretreated with cyclopamine, or anti-Gli1 shRNA, the following events occur. The c-jun Thr91/93 cascade is upregulated, which is associated with pro-apoptotic processes. The c-jun Ser63/73 cascade is suppressed. This cascade is associated with pro-growth processes. If cisplatin is given after cyclopamine or anti-Gli1 pretreatment; Gli1 protein is down-regulated; the c-jun Thr91/93 cascade is upregulated; the c-jun Ser63/73 cascade is suppressed; and cells do not up-regulate ERCC1, XPD, or XRCC1. Thus, the normal cisplatin-induced up-regulation of these three genes involved in NER (ERCC1, XPD) and in base excision repair (XRCC1), is suppressed by suppression of the Hedgehog pathway. We conclude that Gli1 and/or other genes involved in the Hedgehog pathway (which is critical for cancer stem cells), may play key roles in the up-regulation of DNA repair genes, specifically in response to DNA damaging agents such as cisplatin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-281.

Abstract CN4-3: Factors that influence the regulation of ERCC1 in drug-resistant cells

Abstract ERCC1 is an excellent marker for nucleotide excision (NER) repair activity. ERCC1 expression in tumor tissues has been closely correlated with clinical resistance to platinum coordination complexes in lung cancer, ovarian cancer, gastric cancer, and other malignancies. We have previously demonstrated that up-regulation of ERCC1 is coordinated with up-regulation of other genes in the NER excision repair protein complex, such as XPA, XPD, XPB, etc (Reed and colleagues; Mut Res 1993, J Clin Invest 1994, Cancer Res 1995, Biochem Pharm 2000). The positive transcriptional regulator for ERCC1 is activator protein 1, or AP1, a heterodimer of c-jun and c-fos (Reed, JBC 1998). The negative transcriptional regulator for ERCC1 is the myeloid zinc finger gene MZF1 (Reed, Biochem Pharm 2006). Cancer stem cells are presumed to be highly resistant to chemotherapy. Activation of the Hedgehog pathway has been reported as necessary, for the cancer stem cell state. A key gene in the Hedgehog pathway is Gli1. Resistance to platinum compounds is due in part, to up-regulated nucleotide excision repair, NER. Upon treatment with cisplatin, cisplatin-resistant A2780-CP70 cells normally up-regulate c-jun and c-fos, which then up-regulate ERCC1 and other NER genes. More specifically, the c-jun Ser63/73 cascade is up-regulated, with up-regulation of activator protein 1 (Reed, JBC 1998). We therefore studied the relationships between Gli1 expression, c-jun expression, ERCC1 expression, and cisplatin resistance in the human ovarian cancer cell lines A2780 and A2780-CP70. A2780-CP70 cells are 10–15 fold more resistant to cisplatin than A2780 cells. We used two different probes to modulate the Hedgehog pathway: a) cyclopamine as a pharmacologic inhibitor of the Smoothened; and, b) an anti-Gli1 shRNA to specifically inhibit Gli1. At baseline, cisplatin-resistant cells, expressed >10-fold more Gli1 protein than cisplatin-sensitive cells. In cisplatin-resistant cells, Gli1 protein was down-regulated in response to cyclopamine, and to anti-Gli1 shRNA. When A2780-CP70 cisplatin-resistant cells are pretreated with cyclopamine, or anti-Gli1 shRNA, the following events occur. The c-jun Thr91/93 cascade is upregulated, which is associated with pro-apoptotic processes. The c-jun Ser63/73 cascade is suppressed. This cascade is associated with pro-growth processes. If cisplatin is given after cyclopamine or anti-Gli1 pretreatment; Gli1 protein is down-regulated; the c-jun Thr91/93 cascade is upregulated; the c-jun Ser63/73 cascade is suppressed; and cells do not up-regulate ERCC1, XPD, or XRCC1. Thus, the normal cisplatin-induced up-regulation of these three genes involved in NER (ERCC1, XPD) and in base excision repair (XRCC1), is suppressed by suppression of the Hedgehog pathway. We conclude that Gli1 and/or other genes involved in the Hedgehog pathway (which is critical for cancer stem cells), may play key roles in the up-regulation of DNA repair genes, specifically in response to DNA damaging agents such as cisplatin. Citation Information: Clin Cancer Res 2010;16(7 Suppl):CN4-3

Upregulation of DNA repair genes in active cirrhosis associated with hepatocellular carcinoma

Phenotypic changes in injured livers involve complex network of genes whose interplays may lead to fibrosis and cirrhosis, a major risk of hepatocellular carcinoma. Gene expression profiles in fibrotic livers were analyzed by using cDNA microarray, hierarchical clustering and gene ontology. Analyses of a major cluster of upregulated genes in cirrhosis identified a new set of genes involved in DNA repair and damage. The upregulation of DNA repair genes was confirmed by real-time quantitative polymerase chain reaction and associated with necroinflammatory activity ( P < 0.001). Increased DNA repair activity in cirrhosis with inflammatory activity may reflect increased DNA damages as a consequence of chronic liver injury.

Microarray Analysis of Temporal Gene Responses to Ionizing Radiation in Two Glioblastoma Cell Lines: Up-regulation of DNA Repair Genes

To determine the patterns of gene expression responsible for the radiosensitivity of glioblastoma cells, we analyzed transcriptional changes after ionizing radiation in different cell lines. After completing clonogenic survival assays, we selected two glioblastoma cell lines with different radiosensitivities. Subsequently, they were investigated by using the technique of DNA microarray, and we then categorized the upregulated genes into 10 groups. Between the two cell lines, the difference in the percentage of DNA repair/replication category was the largest, and this category was present at a greater percentage with radioresistant cell line U87MG. Moreover, among the commonly upregulated genes, the DNA repair/replication category was present in the largest percentage. These genes included G22P1 (Ku70) and XRCC5 (Ku80) genes known as important members of the nonhomologous end-joining (NHEJ) pathway of DNA double strand break (DSB) repair. Furthermore, cell line that specifically upregulated genes included the members of major pathways of DNA DSB or single strand damage repair. These pathways were not only NHEJ, but also homologous recombination (HR) and postreplication repair (PRR). In conclusion, the distribution of genes involved in the DNA repair/replication category was most different between two human glioblastoma cell lines of different radiosensitivities. Among commonly upregulated genes, the DNA repair/replication category was present in the largest percentage.

522 Cytochrome P450 2E1, but not NADPH oxidase is required for ethanol-induced DNA damage and upregulation of DNA repair genes in mouse liver

522 Cytochrome P450 2E1, but not NADPH oxidase is required for ethanol-induced DNA damage and upregulation of DNA repair genes in mouse liver