ELF3 plays an important role in defining TRAIL sensitivity in breast cancer by modulating the expression of decoy receptor 2 (DCR2).

BACKGROUND: TRAIL protein on binding to its cognate death receptors (DR) can induce apoptosis specifically in breast tumor cells sparing normal cells. However, TRAIL also binds to decoy receptors (DCR) thereby inhibiting the apoptotic pathways thus causing TRAIL resistance. Also, one of the barriers due to which TRAIL-based therapy could not become FDA-approved might be because of resistance to therapy. Therefore, in the current study we wanted to explore the role of transcription factors in TRAIL resistance with respect to breast cancer. METHODS: Microarray data from TRAIL-sensitive (TS) and TRAIL-resistant (TR) MDA-MB-231 cells were reanalyzed followed by validation of the candidate genes using quantitative PCR (qPCR), immunoblotting and immunofluorescence technique. Overexpression of the candidate gene was performed in MDA-MB-231 and MCF7 cells followed by cell viability assay and immunoblotting for cleaved caspase-3. Additionally, immunoblotting for DCR2 was carried out. TCGA breast cancer patient survival was used for Kaplan-Meier (KM) plot. RESULTS: Validation of the candidate gene i.e. ELF3 using qPCR and immunoblotting revealed it to be downregulated in TR cells compared to TS cells. ELF3 overexpression in MDA-MB-231 and MCF7 cells caused reversal of TRAIL resistance as observed using cell viability assay and cleaved caspase-3 immunoblotting. ELF3 overexpression also resulted in DCR2 downregulation in the MDA-MB-231 and MCF7 cells. Furthermore, KM analysis found high ELF3 and low DCR2 expression to show better patient survival in the presence of TRAIL. CONCLUSION: Our study shows ELF3 to be an important factor that can influence TRAIL-mediated apoptosis in breast cancer. Also, ELF3 and DCR2 expression status should be taken into consideration while designing strategies for successful TRAIL-based therapy.

Co-administration of L-Ascorbic Acid and α-Tocopherol Alleviates Arsenic-Induced Immunotoxicities in the Thymus and Spleen by Dwindling Oxidative Stress-Induced Inflammation.

Herein, we investigated whether L-ascorbic acid (L-AA) and α-tocopherol (α-T) co-administration has the potential to alleviate arsenic-induced immunotoxicities in the thymus, spleen, and circulating leukocytes. Forty-eight adult male Wistar rats were randomly divided into four groups before the treatment: group I (control); group II (sodium arsenite, 3 mg/kg/day/rat); group III (sodium arsenite + L-AA (200 mg/kg/day/rat) and α-T (400 mg/kg/day/rat)); group IV (L-AA and α-T). The result showed that sodium arsenite exposure (consecutive 30 days) caused weight reduction, structural alterations in the thymus and spleen, accompanied by a decrease in thymocyte and splenocyte count. Decreased superoxide dismutase and catalase activity, increased malondialdehyde and protein-carbonyl content, reduced Nrf2 and Bcl2 expression, and increased p-ERK, NF-kß, Bax, and cleaved-caspase-3 expression were also observed in the thymus and spleen of arsenic-exposed rats. Enhanced plasma ACTH and corticosterone, ROS-induced apoptosis of lymphocytes were also observed. L-AA and α-T co-administration has the potential to abrogate the deleterious impact of arsenic on the thymus, spleen, and circulating lymphocytes. Whole transcriptome analysis of leukocytes revealed that arsenic treatment augmented the expression of Itga4, Itgam, and MMP9 genes, which might help in transient migration of the leukocytes through the endothelial cell layer. Co-administration with L-AA and α-T maintained Itga4, Itgam, and MMP9 gene expression within leukocytes at a lower level.

Ascorbic acid modulates immune responses through Jumonji-C domain containing histone demethylases and Ten eleven translocation (TET) methylcytosine dioxygenase.

Ascorbic acid is a redox regulator in many physiological processes. Besides its antioxidant activity, many intriguing functions of ascorbic acid in the expression of immunoregulatory genes have been suggested. Ascorbic acid acts as a co-factor for the Fe+2 -containing α-ketoglutarate-dependent Jumonji-C domain-containing histone demethylases (JHDM) and Ten eleven translocation (TET) methylcytosine dioxygenasemediated epigenetic modulation. By influencing JHDM and TET, ascorbic acid facilitates the differentiation of double negative (CD4- CD8- ) T cells to double positive (CD4+ CD8+ ) T cells and of T-helper cells to different effector subsets. Ascorbic acid modulates plasma cell differentiation and promotes early differentiation of hematopoietic stem cells (HSCs) to NK cells. These findings indicate that ascorbic acid plays a significant role in regulating both innate and adaptive immune cells, opening up new research areas in Immunonutrition. Being a water-soluble vitamin and a safe micro-nutrient, ascorbic acid can be used as an adjunct therapy for many disorders of the immune system.

Anion-directed structural tuning of two azomethine-derived Zn2+ complexes with optoelectronic recognition of Cu2+ in aqueous medium with anti-cancer activities: from micromolar to femtomolar sensitivity with DFT revelation.

Herein, two novel mononuclear transition metal Zn2+ complexes i.e. [Zn(HL)(N3)(OAc)] (NS-1) & [Zn(HL)2(ClO4)2] (NS-2) have been synthesised using a tridentate clickable Schiff base ligand, HL (2-methyl-2-((pyridin-2-ylmethyl)amino)propan-1-ol), and the polyatomic monoanions N3- and ClO4- for NS-1 and NS-2 respectively. Interestingly, NS-1 and NS-2 have been explored for the detection of Cu2+ with an LOD of 48.6 fM (response time ∼6 s) and 2.4 µM respectively through two mutually independent pathways that were studied using sophisticated methods like UV-Vis, cyclic voltammetry, ESI-MS etc. with theoretical DFT support. Herein, both chemosensors are equally responsive towards the detection of Cu2+ in aqueous as well as other targeted real field samples with appreciable recovery percentage (74.8-102%), demonstrating their practical applicability. Moreover, the detection of unbound Cu2+ in a human urine specimen was also analysed which may be helpful for the diagnosis of Cu2+-related disorders like Wilson's disease. Taking one step ahead, TLC strips have been employed for on-field detection of the targeted analytes by contact mode analysis. Additionally, the anti-cancer activity of these complexes has also been studied on breast cancer cells with the help of the MTT assay. It has been found that at a 0.5 mM dose, both NS-1 and NS-2 could kill 81.4% and 73.2% of cancer cells respectively. However, it has been found that NS-1 destroys normal cells together with cancer cells. Hence, NS-2 could be administered as a better anticancer drug for MDA-MB-231 cancer cells in comparison with NS-1. In a nutshell, the present work describes how anion-directed synthesis of two architecturally different metal complexes leads toward the detection of the same analyte via an independent chemodosimetric pathway along with their anti-cancer activities on breast cancer cells.

CDH1 overexpression sensitizes TRAIL resistant breast cancer cells towards rhTRAIL induced apoptosis.

PURPOSE: Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is well known for its unique ability to induce apoptosis in cancer cells but not normal cells. However, a subpopulation of cancer cells exist that does not respond to toxic doses of TRAIL. In this study, we aimed to identify key factors regulating TRAIL resistance in breast cancer. METHODS: rhTRAIL (recombinant human TRAIL) resistant cells (TR) isolated from TRAIL sensitive MDA-MB-231 parental cells (TS) were confirmed using trypan blue assay, cell viability assay and AO/EtBr (acridine orange/ethidium bromide) staining. Microarray was performed followed by analysis using DAVID and Cytoscape bioinformatics software to identify the candidate hub gene. Gene expression of the candidate gene was confirmed using real-time PCR and western blot. Candidate gene was overexpressed via transient transfection to identify its significance in the context of rhTRAIL. Breast cancer patient data was obtained from The Cancer Genome Atlas (TCGA) database. RESULTS: Whole transcriptome analysis identified 4907 differentially expressed genes (DEGs) between TS and TR cells. CDH1 was identified as the candidate hub gene, with 18-degree centrality. We further observed CDH1 protein to be downregulated, overexpression of which increased apoptosis in TR cells after rhTRAIL treatment. TCGA patient data analysis also showed CDH1 mRNA to be low in TRAIL resistant patient group compared to TRAIL sensitive group. CONCLUSION: CDH1 overexpression sensitizes TR cells towards rhTRAIL induced apoptosis. Therefore, we can hypothesize that CDH1 expression should be taken into account while performing TRAIL therapy in breast cancer.

Association Study of Transforming Growth Factor Beta 1 + 29 T/C exon 1 Polymorphism in Breast Cancer Patients from North Indian Population.

BACKGROUND: TGFB1 cytokine is involved in normal mammary epithelial development as well as in breast tumorigenesis. It has role in both breast tumor suppression and progression. TGFB1 gene has several single nucleotide polymorphisms (SNPs) many of which modulate the activity of TGFB1. Our aim in this study was to analyze TGFB1 + 29 polymorphism in breast cancer individuals from North Indian population. METHODS: TGFB1 + 29 T/C polymorphism was analyzed using Sanger sequencing in 285 breast cancer patients and age matched 363 healthy controls from North Indian population. Next, transcript expression of 13 apoptotic genes, TRAIL, DR4, DR5, DcR1, DcR2, Bcl2, cytochrome c, Casp8L, Casp8, FlipS, FlipL, Casp3s and Casp3 were carried out in 77 breast tumor tissues obtained from 77 individuals. RESULTS: TGFB1 + 29 CC genotype provided protection against the development of breast cancer (P = 0.012). This was mainly attributable to higher age group (> 45 years) women (P = 0.016). Individuals having CC protector genotype showed significantly higher expression of TGFB1 transcript compared to the TT and TC risk genotypes (P = 0.044). Furthermore, we observed that TGFB1 + 29 CC genotype showed increased TRAIL mediated apoptosis via the extrinsic pathway in breast tumor patients with age greater than 45 years (P = 0.027). CONCLUSION: TGFB1 + 29 homozygous mutant CC genotype is related to protection against breast cancer in North Indian women population greater than 45 years of age.

Chloroplast Engineering: Fundamental Insights and Its Application in Amelioration of Environmental Stress.

Chloroplasts are specialized organelle that are responsible for converting light energy to chemical energy, thereby driving the carbon dioxide fixation. Apart from photosynthesis, chloroplast is the site for essential cellular processes that determine the plant adaptation to changing environment. Owing to the presence of their own expression system, it provides an optimum platform for engineering valued traits as well as site for synthesis of bio-compounds. Advancements in technology have further enhanced the scope of using chloroplast as a multifaceted tool for the biotechnologist to develop stress-tolerant plants and ameliorate environmental stress. Focusing on chloroplast biotechnology, this review discusses the advances in chloroplast engineering and its application in enhancing plant adaptation and resistance to environmental stress and the development of new bioproducts and processes. This is accomplished through analysis of its biogenesis and physiological processes, highlighting the chloroplast engineering and recent developments in chloroplast biotechnology. In the first part of the review, the evolution and principles of structural organization and physiology of chloroplast are discussed. In the second part, the chief methods and mechanisms involved in chloroplast transformation are analyzed. The last part represents an updated analysis of the application of chloroplast engineering in crop improvement and bioproduction of industrial and health compounds.

Changes in ecological conditions may influence intraguild competition: inferring interaction patterns of snow leopard with co-predators.

Background: Large-scale changes in habitat conditions due to human modifications and climate change require management practices to consider how species communities can alter amidst these changes. Understanding species interactions across the gradient of space, anthropogenic pressure, and season provide the opportunity to anticipate possible dynamics in the changing scenarios. We studied the interspecific interactions of carnivore species in a high-altitude ecosystem over seasonal (summer and winter) and resource gradients (livestock grazing) to assess the impact of changing abiotic and biotic settings on coexistence. Methods: The study was conducted in the Upper Bhagirathi basin, Western Himalaya, India. We analyzed around 4 years of camera trap monitoring data to understand seasonal spatial and temporal interactions of the snow leopard with common leopard and woolly wolf were assessed in the greater and trans-Himalayan habitats, respectively. We used two species occupancy models to assess spatial interactions, and circadian activity patterns were used to assess seasonal temporal overlap amongst carnivores. In addition, we examined scats to understand the commonalities in prey selection. Results: The result showed that although snow leopard and wolves depend on the same limited prey species and show high temporal overlap, habitat heterogeneity and differential habitat use facilitate co-occurrence between these two predators. Snow leopard and common leopard were spatially independent in the summer. Conversely, the common leopard negatively influences the space use of snow leopard in the winter. Limited prey resources (lack of livestock), restricted space (due to snow cover), and similar activity patterns in winter might result in strong competition, causing these species to avoid each other on a spatial scale. The study showed that in addition to species traits and size, ecological settings also play a significant role in deciding the intensity of competition between large carnivores. Climate change and habitat shifts are predicted to increase the spatial overlap between snow leopard and co-predators in the future. In such scenarios, wolves and snow leopards may coexist in a topographically diverse environment, provided sufficient prey are available. However, shifts in tree line might lead to severe competition between common leopards and snow leopards, which could be detrimental to the latter. Further monitoring of resource use across abiotic and biotic environments may improve our understanding of how changing ecological conditions can affect resource partitioning between snow leopards and predators.

Mitochondrial cytochrome b indicates the presence of two paraphyletic diverged lineages of the blue sheep Pseudois nayaur across the Indian Himalaya: conservation implications.

BACKGROUND: Populations exhibit signatures of local adaptive traits due to spatial and environmental heterogeneity resulting in microevolution. The blue sheep is widely distributed across the high Asian mountains and are the snow leopard's principal prey species. These mountains differ in their evolutionary history due to differential glaciation and deglaciation periods, orography, and rainfall patterns, and such factors causes diversification in species. METHODS AND RESULTS: Therefore, we assess the phylogeographic status of blue sheep using the mitochondrial cytochrome b gene (220 bp) across the Indian Himalayan region (IHR) and its relationship with other populations. Of the observed five haplotypes, two and three were from the western Himalayas (WH) and eastern Himalayas (EH) respectively. One of the haplotypes from WH was shared with the population of Pamir plateau, suggesting historical maternal connectivity between these areas. The phylogenetic analyses split the blue sheep into two paraphyletic clades, and western and eastern populations of IHR were within the Pamir and Tibetan plateau clades, respectively. We observed a relatively higher mean sequence divergence in the EH population than in the WH. CONCLUSION: We propose five 'Evolutionary Significant Units' across the blue sheep distribution range based on observed variation in the species' ecological requirements, orography, climatic conditions, and maternal lineages, viz.; Western Himalaya-Pamir plateau (WHPP); Eastern Himalaya-Tibetan plateau (EHTP); Qilian mountains; Helan mountains and Hengduan mountains population. Despite the small sample size, population divergence was observed across the IHR, therefore, we suggest a transboundary, collaborative study on comparative morphology, anatomy, ecology, behaviour, and population genetics using harmonized different genetic markers for identifying the overall taxonomic status of the blue sheep across its range for planning effective conservation strategies.

Reactive oxygen species-dependent upregulation of death receptor, tumor necrosis factor receptor 1, is responsible for theophylline-mediated cytotoxicity in MDA-MB-231 breast cancer cells.

Theophylline, a methylxanthine drug, has been used as a therapy for respiratory diseases. Recently, it has also been shown to have a potential in treating different cancers. Also, it has shown promising results in clinical trials for AML in combination therapy. Subsequently, studies have shown theophylline to kill breast cancer cells but not normal breast cells. Therefore, in this study, we have explored the molecular mechanism underlying the cytotoxic effect of theophylline on breast cancer cells. Theophylline-treated cancer cells were analyzed for the transcript and protein expression of candidate apoptotic genes such as TNFR1, caspase-8, -9, -3 using qPCR and immunoblotting, respectively. Cell viability and apoptosis was measured in the presence or absence of TNFR1 inhibitor, R7050, using AO/EtBr staining and MTT assay, respectively. Similarly, oxidative stress was studied by analyzing ROS in the presence or absence of ROS inhibitor, NAC, using DCFDA assay. Theophylline caused reduced cell viability in cancer but not normal cells. Theophylline-treated breast cancer cells showed increased expression of death receptor, TNFR1, along with elevated levels of active caspase-8, -9 and -3. Inhibition of TNFR1 reduced caspase-dependent apoptosis even in the presence of theophylline. Theophylline further caused increased ROS generation, inhibition of which resulted in reduced TNFR1-mediated apoptosis. Theophylline also increased cathepsin activity, which was reduced on exposure of cells to TNFR1 inhibitor, R7050. We conclude that ROS-mediated activation of TNFR1 is responsible for caspase-3 and cathepsin-dependent cell death in breast cancer cells on exposure to theophylline.

Enhanced expression of death receptor 5 is responsible for increased cytotoxicity of theophylline in combination with recombinant human TRAIL in MDA-MB-231 breast cancer cells.

Background: Theophylline has been reported to induce cytotoxicity and cell cycle arrest in cancer cells. On the other hand, TRAIL, a secretory ligand, is known for its unique ability to induce cell death only in tumor cells. In the present study, we elucidated the mechanism behind the cytotoxic effect of theophylline in combination with recombinant human TRAIL (rhTRAIL) on cancer cell line MDA-MB-231. Materials and Methods: Cytotoxicity of theophylline in combination with TRAIL was measured via trypan blue assay and MTT assay. Protein levels were assessed using Western hybridization. Reactive oxygen species (ROS) levels were measured using 2',7'-dichlorofluorescin diacetate and mitochondrial membrane potential (MMP) assay was conducted using tetramethylrhodamine, ethyl ester. Results: We observed theophylline in combination with rhTRAIL to be significantly cytotoxic to the cancer cells in comparison to theophylline and rhTRAIL alone. Next, western hybridization showed combination treatment to upregulate cleaved form of caspase-8, 9 and 3, in comparison to the cells treated with rhTRAIL and theophylline alone. Theophylline in combination also increased the levels of ROS and reduced MMP. Interestingly, combination treatment increased the protein level of death receptor 5 (DR5), sensitizing the cells towards TRAIL-induced apoptosis. Conclusion: Theophylline in combination with TRAIL significantly increases cytotoxicity in the MDA-MB-231 breast cancer cell line when compared to theophylline and rhTRAIL alone via upregulation of DR5 levels.

Exploring the Molecular Mechanism of Cinnamic Acid-Mediated Cytotoxicity in Triple Negative MDA-MB-231 Breast Cancer Cells.

BACKGROUND: Cinnamic Acid (CA), also known as 3-phenyl-2-propenoic acid, is a naturally occurring aromatic fatty acid found commonly in cinnamon, grapes, tea, cocoa, spinach and celery. Various studies have identified CA to have anti-proliferative action on glioblastoma, melanoma, prostate and lung carcinoma cells. OBJECTIVE: Our objective was to investigate the molecular mechanism underlying the cytotoxic effect of CA in killing MDA-MB-231 triple negative breast cancer cells. METHODS: We performed MTT assay and trypan blue assay to determine cell viability and cell death, respectively. Comet analysis was carried out to investigate DNA damage of individual cells. Furthermore, AO/EtBr assay and sub-G1 analysis using flow cytometry were used to study apoptosis. Protein isolation followed by immunoblotting was used to observe protein abundance in treated and untreated cancer cells. RESULTS: Using MTT assay, we have determined CA to reduce cell viability in MDA-MB-231 breast cancer cells and tumorigenic HEK 293 cells but not in normal NIH3T3 fibroblast cells. Subsequently, trypan blue assay and comet assay showed CA to cause cell death and DNA damage, respectively, in the MDA-MB-231 cells. Using AO/EtBr staining and sub-G1 analysis, we further established CA to increase apoptosis. Additionally, immunoblotting showed the abundance of TNFA, TNF Receptor 1 (TNFR1) and cleaved caspase-8/-3 proapoptotic proteins to increase with CA treatment. Subsequently, blocking of TNFA-TNFR1 signalling by small molecule inhibitor, R-7050, reduced the expression of cleaved caspase-8 and caspase-3 at the protein level. CONCLUSION: Thus, from the above observations, we can conclude that CA is an effective anticancer agent that can induce apoptosis in breast cancer cells via TNFA-TNFR1 mediated extrinsic apoptotic pathway.

Revisiting the Woolly wolf (Canis lupus chanco) phylogeny in Himalaya: Addressing taxonomy, spatial extent and distribution of an ancient lineage in Asia.

Of the sub-species of Holarctic wolf, the Woolly wolf (Canis lupus chanco) is uniquely adapted to atmospheric hypoxia and widely distributed across the Himalaya, Qinghai Tibetan Plateau (QTP) and Mongolia. Taxonomic ambiguity still exists for this sub-species because of complex evolutionary history anduse of limited wild samples across its range in Himalaya. We document for the first time population genetic structure and taxonomic affinity of the wolves across western and eastern Himalayan regions from samples collected from the wild (n = 19) using mitochondrial control region (225bp). We found two haplotypes in our data, one widely distributed in the Himalaya that was shared with QTP and the other confined to Himachal Pradesh and Uttarakhand in the western Himalaya, India. After combining our data withpublished sequences (n = 83), we observed 15 haplotypes. Some of these were shared among different locations from India to QTP and a few were private to geographic locations. A phylogenetic tree indicated that Woolly wolves from India, Nepal, QTP and Mongolia are basal to other wolves with shallow divergence (K2P; 0.000-0.044) and high bootstrap values. Demographic analyses based on mismatch distribution and Bayesian skyline plots (BSP) suggested a stable population over a long time (~million years) with signs of recent declines. Regional dominance of private haplotypes across its distribution range may indicate allopatric divergence. This may be due to differences in habitat characteristics, availability of different wild prey species and differential deglaciation within the range of the Woolly wolf during historic time. Presence of basal and shallow divergence within-clade along with unique ecological requirements and adaptation to hypoxia, the Woolly wolf of Himalaya, QTP, and Mongolian regions may be considered as a distinct an Evolutionary Significant Unit (ESU). Identifying management units (MUs) is needed within its distribution range using harmonized multiple genetic data for effective conservation planning.

Commensal in conflict: Livestock depredation patterns by free-ranging domestic dogs in the Upper Spiti Landscape, Himachal Pradesh, India.

In human-populated landscapes worldwide, domestic dogs (Canis lupus familiaris) are the most abundant terrestrial carnivore. Although dogs have been used for the protection of livestock from wild carnivores, they have also been implicated as predators of livestock. We used a combination of methods (field surveys, interview surveys, and data from secondary sources) to examine the patterns and factors driving livestock depredation by free-ranging dogs, as well as economic losses to local communities in a Trans-Himalayan agro-pastoralist landscape in India. Our results show that livestock abundance was a better predictor of depredation in the villages than local dog abundance. Dogs mainly killed small-bodied livestock and sheep were the most selected prey. Dogs were responsible for the majority of livestock losses, with losses being comparable to that by snow leopards. This high level of conflict may disrupt community benefits from conservation programs and potentially undermine the conservation efforts in the region through a range of cascading effects.

CUX2 protein functions as an accessory factor in the repair of oxidative DNA damage.

CUX1 and CUX2 proteins are characterized by the presence of three highly similar regions called Cut repeats 1, 2, and 3. Although CUX1 is ubiquitously expressed, CUX2 plays an important role in the specification of neuronal cells and continues to be expressed in postmitotic neurons. Cut repeats from the CUX1 protein were recently shown to stimulate 8-oxoguanine DNA glycosylase 1 (OGG1), an enzyme that removes oxidized purines from DNA and introduces a single strand break through its apurinic/apyrimidinic lyase activity to initiate base excision repair. Here, we investigated whether CUX2 plays a similar role in the repair of oxidative DNA damage. Cux2 knockdown in embryonic cortical neurons increased levels of oxidative DNA damage. In vitro, Cut repeats from CUX2 increased the binding of OGG1 to 7,8-dihydro-8-oxoguanine-containing DNA and stimulated both the glycosylase and apurinic/apyrimidinic lyase activities of OGG1. Genetic inactivation in mouse embryo fibroblasts or CUX2 knockdown in HCC38 cells delayed DNA repair and increased DNA damage. Conversely, ectopic expression of Cut repeats from CUX2 accelerated DNA repair and reduced levels of oxidative DNA damage. These results demonstrate that CUX2 functions as an accessory factor that stimulates the repair of oxidative DNA damage. Neurons produce a high level of reactive oxygen species because of their dependence on aerobic oxidation of glucose as their source of energy. Our results suggest that the persistent expression of CUX2 in postmitotic neurons contributes to the maintenance of genome integrity through its stimulation of oxidative DNA damage repair.

The function of CUX1 in oxidative DNA damage repair is needed to prevent premature senescence of mouse embryo fibroblasts.

Despite having long telomeres, mouse embryo fibroblasts (MEFs) senesce more rapidly than human diploid fibroblasts because of the accumulation of oxidative DNA damage. The CUX1 homeodomain protein was recently found to prevent senescence in RAS-driven cancer cells that produce elevated levels of reactive-oxygen species. Here we show that Cux1-/- MEFs are unable to proliferate in atmospheric (20%) oxygen although they can proliferate normally in physiological (3%) oxygen levels. CUX1 contains three domains called Cut repeats. Structure/function analysis established that a single Cut repeat domain can stimulate the DNA binding, Schiff-base formation, glycosylase and AP-lyase activities of 8-oxoguanine DNA glycosylase 1, OGG1. Strikingly and in contrast to previous reports, OGG1 exhibits efficient AP-lyase activity in the presence of a Cut repeat. Repair of oxidative DNA damage and proliferation in 20% oxygen were both rescued in Cux1-/- MEFs by ectopic expression of CUX1 or of a recombinant Cut repeat protein that stimulates OGG1 but is devoid of transcription activation potential. These findings reinforce the causal link between oxidative DNA damage and cellular senescence and suggest that the role of CUX1 as an accessory factor in DNA repair will be critical in physiological situations that generate higher levels of reactive oxygen species.

Microsatellite instability: an indirect assay to detect defects in the cellular mismatch repair machinery.

The DNA mismatch repair (MMR) pathway plays a prominent role in the correction of errors made during DNA replication and genetic recombination and in the repair of small deletions and loops in DNA. Mismatched nucleotides can occur by replication errors, damage to nucleotide precursors, damage to DNA, or during heteroduplex formation between two homologous DNA molecules in the process of genetic recombination. Defects in MMR can precipitate instability in simple sequence repeats (SSRs), also referred to as microsatellite instability (MSI), which appears to be important in certain types of cancers, both spontaneous and hereditary. Variations in the highly polymorphic alleles of specific microsatellite repeats can be identified using PCR with primers derived from the unique flanking sequences. These PCR products are analyzed on denaturing polyacrylamide gels to resolve differences in allele sizes of >2 bp. Although (CA)n repeats are the most abundant class among dinucleotide SSRs, trinucleotide and tetranucleotide repeats are also frequent. These polymorphic repeats have the advantage of producing band patterns that are easy to analyze and can be used as an indication of a possible MMR defect in a cell. The presumed association between such allelic variation and an MMR defect should be confirmed by molecular analysis of the structure and/or expression of MMR genes.

RAS transformation requires CUX1-dependent repair of oxidative DNA damage.

The Cut homeobox 1 (CUX1) gene is a target of loss-of-heterozygosity in many cancers, yet elevated CUX1 expression is frequently observed and is associated with shorter disease-free survival. The dual role of CUX1 in cancer is illustrated by the fact that most cell lines with CUX1 LOH display amplification of the remaining allele, suggesting that decreased CUX1 expression facilitates tumor development while increased CUX1 expression is needed in tumorigenic cells. Indeed, CUX1 was found in a genome-wide RNAi screen to identify synthetic lethal interactions with oncogenic RAS. Here we show that CUX1 functions in base excision repair as an ancillary factor for the 8-oxoG-DNA glycosylase, OGG1. Single cell gel electrophoresis (comet assay) reveals that Cux1⁺/⁻ MEFs are haploinsufficient for the repair of oxidative DNA damage, whereas elevated CUX1 levels accelerate DNA repair. In vitro base excision repair assays with purified components demonstrate that CUX1 directly stimulates OGG1's enzymatic activity. Elevated reactive oxygen species (ROS) levels in cells with sustained RAS pathway activation can cause cellular senescence. We show that elevated expression of either CUX1 or OGG1 prevents RAS-induced senescence in primary cells, and that CUX1 knockdown is synthetic lethal with oncogenic RAS in human cancer cells. Elevated CUX1 expression in a transgenic mouse model enables the emergence of mammary tumors with spontaneous activating Kras mutations. We confirmed cooperation between Kras(G12V) and CUX1 in a lung tumor model. Cancer cells can overcome the antiproliferative effects of excessive DNA damage by inactivating a DNA damage response pathway such as ATM or p53 signaling. Our findings reveal an alternate mechanism to allow sustained proliferation in RAS-transformed cells through increased DNA base excision repair capability. The heightened dependency of RAS-transformed cells on base excision repair may provide a therapeutic window that could be exploited with drugs that specifically target this pathway.

miR-24-2 controls H2AFX expression regardless of gene copy number alteration and induces apoptosis by targeting antiapoptotic gene BCL-2: a potential for therapeutic intervention.

INTRODUCTION: New levels of gene regulation with microRNA (miR) and gene copy number alterations (CNAs) have been identified as playing a role in various cancers. We have previously reported that sporadic breast cancer tissues exhibit significant alteration in H2AX gene copy number. However, how CNA affects gene expression and what is the role of miR, miR-24-2, known to regulate H2AX expression, in the background of the change in copy number, are not known. Further, many miRs, including miR-24-2, are implicated as playing a role in cell proliferation and apoptosis, but their specific target genes and the pathways contributing to them remain unexplored. METHODS: Changes in gene copy number and mRNA/miR expression were estimated using real-time polymerase chain reaction assays in two mammalian cell lines, MCF-7 and HeLa, and in a set of sporadic breast cancer tissues. In silico analysis was performed to find the putative target for miR-24-2. MCF-7 cells were transfected with precursor miR-24-2 oligonucleotides, and the gene expression levels of BRCA1, BRCA2, ATM, MDM2, TP53, CHEK2, CYT-C, BCL-2, H2AFX and P21 were examined using TaqMan gene expression assays. Apoptosis was measured by flow cytometric detection using annexin V dye. A luciferase assay was performed to confirm BCL-2 as a valid cellular target of miR-24-2. RESULTS: It was observed that H2AX gene expression was negatively correlated with miR-24-2 expression and not in accordance with the gene copy number status, both in cell lines and in sporadic breast tumor tissues. Further, the cells overexpressing miR-24-2 were observed to be hypersensitive to DNA damaging drugs, undergoing apoptotic cell death, suggesting the potentiating effect of mir-24-2-mediated apoptotic induction in human cancer cell lines treated with anticancer drugs. BCL-2 was identified as a novel cellular target of miR-24-2. CONCLUSIONS: mir-24-2 is capable of inducing apoptosis by modulating different apoptotic pathways and targeting BCL-2, an antiapoptotic gene. The study suggests that miR-24-2 is more effective in controlling H2AX gene expression, regardless of the change in gene copy number. Further, the study indicates that combination therapy with miR-24-2 along with an anticancer drug such as cisplatin could provide a new avenue in cancer therapy for patients with tumors otherwise resistant to drugs.

Functional implication of TRAIL -716 C/T promoter polymorphism on its in vitro and in vivo expression and the susceptibility to sporadic breast tumor.

Recently, TRAIL function has been elucidated beyond its known classical role of mediating cellular homeostasis and immune surveillance against transformed cells. Here, we show how CC genotype of -716 TRAIL promoter SNP rendered risk for sporadic breast cancer as compared to the CT and TT genotypes (P (recessive model) = 0.018, OR = 1.4, 95% CI = 1.1-1.9; P (allele model) = 0.010, OR = 1.3, 95% CI = 1.1-1.7). The in silico prediction of the introduction of core Sp1/Sp3-binding motif suggested the functional significance of the SNP variation. This functional implication was validated by luciferase assay in HeLa (P = 0.026), MCF-7 (P = 0.022), HepG2 (P = 0.024), and HT1080 (P = 0.030) cells and also by real-time expression studies on tumor tissues (P = 0.01), revealing the transcriptionally repressed status of -716 T when compared to -716 C allele. The SNP-SNP interactions reflected an enhanced protective effect of CT and TT genotypes with the protective genetic backgrounds of TP53-BRCA2 (P = 0.002, OR = 0.2, 95% CI = 0.1-0.6), IFNG (P = 0.0000002, OR = 0.3, 95% CI = 0.2-0.4), and common variant Casp8 (P = 0.0003, OR = 0.5, 95% CI = 0.3-0.7). Interestingly, a comparison with clinical parameters showed overrepresented CT and TT genotypes in progressing (P = 0.041) and ER/PR negative tumors (P = 0.024/0.006). This was explained by increased apoptotic index, calculated as a ratio of selected pro-apoptotic and anti-apoptotic gene expression profiles, in CC genotyped tumors, favoring either intrinsic (P = 0.008,0.018) or extrinsic (P = 0.025,0.217) pathway depending upon the ER/PR status. Our study reveals for the first time that a promoter SNP of TRAIL functionally modulates the gene and consequently its role in breast cancer pathogenesis, cautioning to consider the -716 TRAIL SNP status in patients undergoing TRAIL therapy.