Browsing by Subject "DNA damage"
Now showing 1 - 8 of 8
Results Per Page
Sort Options
Item ATM promotes apoptosis and suppresses tumorigenesis in response to Myc(2008-12) Pusapati, Raju V. L. N., 1969-; Johnson, David, 1963-; Richburg, John H.Precancerous lesions from a variety of human tissues display markers of DNA damage suggesting that genetic instability occurs early during the process of carcinogenesis. Consistent with this, several oncogenes can activate ATM and other components of the DNA damage response pathway when expressed in cultured cells. Here we demonstrate that preneoplastic epithelial tissues from four different transgenic mouse models expressing the oncogenes c-myc, SV40 T antigen, human papilloma virus (HPV) E7, or E2F3a display [gamma]-H2AX foci and other markers of DNA damage. Moreover, transgenic expression of these oncogenes leads to increased levels of damaged DNA as measured by the comet assay. In at least the Myc transgenic model, the formation of [gamma]-H2AX foci is dependent on functional ATM. Inactivation of Atm also impairs p53 activation and reduces the level of apoptosis observed in transgenic tissue overexpressing Myc. This correlates with accelerated tumor development in Myc transgenic mice lacking ATM. To understand the mechanism by which oncogenes induce DNA damage, we employed an adenoviral overexpression system. Under conditions in which Myc or E2F3a induced replication is inhibited, we see a reduction in the DNA damage induced by these oncogenes both by comet assay and levels of [gamma]-H2AX. Moreover, Myc and E2F3a induced increased levels of the Cdt1 protein, a replication origin- licensing factor implicated in aberrant DNA replication. Taken together, these findings suggest that deregulated oncogenes induce unscheduled DNA replication leading to DNA damage and activation of the ATM DNA damage response pathway, which is important for the activation of p53, induction of apoptosis and the suppression of tumorigenesis.Item Cave and cliff swallows as indicators of exposure and effects of environmental contaminants on birds from the Rio Grande, Texas(Texas A&M University, 2004-11-15) Musquiz, DanielCave (Petrochelidon fulva) and cliff swallows (Petrochelidon pyrrhonota) were collected along the Rio Grande and evaluated as potential indicators of environmental contamination. The Rio Grande receives toxic substances from agricultural, industrial, municipal, and non-point sources; consequently, high levels of contaminants have been detected in birds, mammals, fishes and sediments. Swallows were obtained from 8 sites between Brownsville and El Paso, as well as from a reference site in Burleson County, 320 miles north of the nearest site of the Rio Grande. Blood samples were analyzed by flow cytometry, a technique that allows the detection of DNA damage in blood and other tissues. Plasma samples were analyzed for thyroid hormones using a radioimmunoassay technique. Organochlorines and trace metal analysis was limited to a few samples. DDE and PCB levels were below levels known to cause reduced hatching, embryo mortality, and deformities, Hg, Pb, and As were below detection, and Se, Ni and Cr concentrations were lower than levels known to cause harm in birds. Neither species showed sex-related differences in chromosome damage. Cave swallows from the Del Rio area had the highest levels of DNA variation, which may be indicative of DNA damage, possibly from PAHs exposure. Previous studies indicate that sediment samples from tributaries near Del Rio have high levels of chromium compared to other sites along the Rio Grande. A significant increase in DNA variation between sampling years was detected in cave swallows from Llano Grande Lake. Wildlife samples collected from Llano Grande Lake have recorded high levels of DDE and PCBs; in addition, this urban/agricultural contaminant sink appears to be affected by PAH exposure. T3 levels were below the detection limit of the radioimmunoassay. There were no gender related differences in T4 levels in cave swallows. Cave swallows sampled from Laredo had significantly higher T4 levels than those from birds at other sites during 1999. It was not possible to determine thyroid hormone disruption in plasma samples. Thyroid hormone and flow cytometry data were useful in establishing baseline data. Areas of concern based on genotoxic data include Llano Grande Lake, Del Rio, and El Paso.Item Cytotoxicological Response to Engineered Nanomaterials: A Pathway-Driven Process(2012-07-16) Romoser, Amelia AntoniaNanoparticles, while included in a growing number of consumer products, may pose risks to human health due to heavy metal leaching and/or the production of reactive oxygen species following exposures. Subcellular mechanisms of action triggered as a result of exposure to various nanoparticles are still largely unexplored. In this work, an effort to elucidate such toxicological parameters was accomplished by evaluating oxidative stress generation, changes in gene and protein expression, and cell cycle status after low-dose exposures to a variety of metal and carbon-based nanomaterials in primary human dermal cells. Additionally, mitigation of nanoparticle toxicity via microencapsulation was investigated to assess the feasibility of utilizing nanomaterials in dermally implantable biosensor applications. Cellular immune and inflammatory processes were measured via qPCR and immunoblotting, which revealed gene and protein expression modulation along the NF-kappaB pathway after a variety of nanoparticle exposures. The role of immunoregulatory transcription factor NF-kappaB was examined in an oxidative stress context in cells exposed to a panel of nanoparticles, whereby glutathione conversion and modulation of oxidative stress proteins in normal and NF-kappaB knockdown human dermal fibroblasts were monitored. Results revealed decreased antioxidant response and corresponding increased levels of oxidative stress and cell death in exposed normal cells, compared to NF-kappaB incompetent cells. However, reactive oxygen species production was not an absolute precursor to DNA damage, which was measured by the comet assay, gamma-H2AX expression, and flow cytometry. Protein analysis revealed that map kinase p38, rather than p53, was involved in the halting of the cell cycle in S-phase after ZnO exposures, which caused DNA double strand breaks. Microencapsulation of fluorescent quantum dot nanoparticles, specifically, was utilized as a method to improve system functionality and surrounding cellular viability for the purpose of a dermal analyte detection assay. In vitro results indicated a functional localization of nanoparticles, as well as cessation of cellular uptake. Subsequently, cellular metabolism was unaffected over the range of time and concentrations tested in comparison to unencapsulated quantum dot treatments, indicating the usefulness of this technique in developing nanoparticle-driven biomedical applications.Item DNA Damage Causes p27^(Kip1) Accumulation Through COP1 Signaling(2014-05-06) Choi, Hyun Hop27 is a critical CDK inhibitor involved in cell cycle regulation, but its response to DNA damage remains unclear. Constitutive photomorphogenesis 1 (COP1), a p53- targeting E3 ubiquitin ligase, is downregulated by DNA damage, but the biological consequences of this phenomenon are poorly understood. Here, we report that p27 levels were elevated after DNA damage, with concurrent reduction of COP1 levels. Mechanistic studies showed that COP1 directly interacted with p27 through a VP motif on p27 and functions as an E3 ligase of p27 to accelerate the ubiquitin-mediated degradation of p27. Also, COP1 overexpression lead to cytoplasmic distribution of p27, thereby accelerating p27 degradation. COP1 overexpression resulted in elevation of Aurora A kinase. COP1 and Aurora A levels were positively correlated in patient samples and associated with poor overall survival. We found that COP1 expression promoted cell proliferation, cell transformation, and tumor progression, manifesting its role in cancer promotion whereas p27 negatively regulated COP1 function and prevented tumor growth in a mouse xenograft model of human cancer. Together, these findings define a mechanism for posttranslational regulation of p27 after DNA damage that can explain the correlation between COP1 overexpression and p27 downregulation during tumorigenesis.Item E2F3a functions as an oncogene and induces DNA damage response pathway mediated apoptosis(2007) Paulson, Qiwei Xia, 1974-; Johnson, David, 1963-; Bratton, Shawn B.Mutation or inactivation of RB occurs in most human tumors and results in the deregulation of several E2F family transcription factors. Among the E2F family, E2F3 has been implicated as a key regulator of cell proliferation and E2f3 gene amplification and overexpression is detected in some human tumors. To study the role of E2F3a in tumor development, we established a transgenic mouse model expressing E2F3a in a number of epithelial tissues via a keratin 5 (K5) promoter. Transgenic expression of E2F3a leads to hyperproliferation, hyperplasia and increased levels of p53-independent apoptosis in transgenic epidermis. Consistent with data from human cancers, the E2f3a transgene is found to have a weak oncogenic activity on its own and to enhance the response to a skin carcinogenesis protocol. While E2F3a induces apoptosis in the absence of p53, the inactivation of both p53 and p73, but not p73 alone, significantly impairs apoptosis induced by E2F3a. This suggests that both p53 and p73 contribute to E2F3a induced apoptosis but that their function is compensatory. Even though data suggest that E2F3a carries out its unique apoptotic activity in part through another E2F family member E2F1, unlike E2F1, the ARF tumor suppressor is required for E2F3a-induced apoptosis. While both E2F3a and E2F1 require ATM for apoptosis, E2F3a activates ATM through a distinct mechanism from E2F1. The overexpression of E2F3a results in the accumulation of DNA damage in K5 transgenic keratinocytes and normal human fibroblasts (NHFs). In response to this, the DNA damage checkpoint kinase ATM is activated, and phosphorylation of the downstream targets p53 and the histone variant H2AX are significantly increased. Additional studies show that increased Cdk activity and aberrant DNA replication contributes to DNA damage, ATM activation and apoptosis in response to deregulated E2F3a, which suggest that aberrant replication imposed by deregulated E2F3a plays an important role in the activation of the ATM DNA damage response pathway. Activation of ATM by E2F3a is not affected by loss of ARF or E2F1. Meanwhile, E2F3a-induced ARF upregulation is not affected by E2F1 loss. The above results indicate that E2F3a engages several parallel pathways involving E2F1, ARF and the ATM kinase, and these pathways cooperate to promote apoptosis.Item The induction of apoptosis by the E2F1 transcription factor and the emergence of a role for E2F1 in the DNA double strand break response(2006) Powers, John Thomas; Tucker, Philip W.Item Replication-associated base excision repair Of oxidized bases in the mammalian genome(2009-10-31) Corey Allen Theriot; John Papaconstantinou, Ph.D.; Tapas Hazra, Ph.D.; Sankar Mitra, Ph.D.; Isvan Boldogh, Ph.D.; Cornelis Elferink, Ph.D.; Alan Tomkinson, Ph.D.Reactive oxygen species (ROS), the most pervasive endogenous and radiation-induced genotoxic agents induce strand breaks and a plethora of base lesions in DNA that (except double-strand breaks) are repaired via the DNA base excision repair (BER) pathway. Four mammalian DNA glycosylases, namely, OGG1 and NTH1 in the Nth family, and NEIL1 and NEIL2 in the Nei family, with overlapping substrate range initiate BER by excising oxidized base lesions and cleaving the DNA strand. NEIL1 prefers oxidized pyrimidines or ring-opened purines as substrates and is upregulated at the mRNA and protein level during S-phase. NEIL1 also demonstrates the unique able to excise base lesions from forked or single-stranded DNA substrates that mimic intermediates generated during DNA replication. This suggests a direct linkage of NEIL1’s repair activity to genome replication. In addition, inactivating mutations in the NEIL1 gene have been epidemiologically linked with gastric cancer, NEIL1-downregulation induces a mutator phenotype and NEIL1 KO mice display symptoms of the human metabolic syndrome such as obesity, dyslipidemia, and fatty liver disease. These observations lead us to develop the working hypothesis that NEIL1 is involved in a preferential repair pathway for oxidized base damage in the replicating genome where repair of both template strands is equally important because an unrepaired base lesion in either strand could induce mutations. Thus, specific involvement of NEIL1 with the DNA replication machinery may be required to effectively and efficiently accomplish this. In support of our hypothesis, we have identified several new NEIL1 interacting proteins that are components of the DNA replication machinery, including Replication Protein A (RPA), Proliferating Cell Nuclear Antigen (PCNA), Flap Endonuclease 1 (FEN1), DNA Polymerase ä, Replication Factor C (RFC), and DNA Ligase I as well as the stress responsive Rad9-Rad1-Hus1 (9-1-1) DNA sliding clamp. We mapped the overlapping binding sites for all of these interacting protein partners to a small disordered region near the unconserved C-terminus of NEIL1 that is dispensable for its enzymatic activity. In support of the biological significance of these interactions, we showed that the DNA polymerase processivity factor and sliding clamp, PCNA, stimulates NEIL1’s activity on various DNA substrates including forked and single-stranded DNA. We also investigated NEIL1’s association with the DNA damage activated alternative sliding clamp 9-1-1 and showed direct interaction as well as stimulation of NEIL1 activity in a similar fashion as PCNA. In contrast, the RPA complex inhibits NEIL1’s activity when the damage is in the single-stranded region of a DNA primer-template structure, inhibition that is relieved in the presence of PCNA. These results suggest that PCNA and RPA, along with other proteins, collaborate to regulate a replication-associated repair pathway in mammalian cells that not only maintains efficient and proper replication but also repair of oxidative DNA damage to prevent mutagenesis and maintain genomic integrity.Item Telomere Regulation in Arabidopsis thaliana by the CST Capping Complex and DNA Damage Response Proteins(2013-09-11) Boltz, Kara A.The ends of chormosomes are capped by telomeres, which distinguish the termini from damaged DNA. Paradoxically, DNA repair proteins are also required for telomere maintenance. How DNA repair pathways are regulated to maintain telomeres while remaining competent to repair DNA damage throughout the genome is unknown. In this dissertation, I used a genetic approach to investigate how critical components of telomerase and the telomere protein complex interact with the DNA damage response (DDR). In the flowering plant, Arabidopsis thaliana telomeres are bound by the CST (CTC1/STN1/TEN1) heterotrimer. Loss of any CST component results in telomere shortening, telomere fusions, increased G-overhang length and telomere recombination. To understand the phenotypes caused by CST deficiency, I examined telomeres from plants lacking CTC1 or STN1 and TERT or KU. My analysis showed that CST acts in a separate genetic pathway for telomere length regulation from both KU and TERT. Further, I found that KU and CST act in separate genetic pathways for regulation of G-overhang formation. These demonstrate that multiple pathways are used to maintain telomere length and architecture in plants. My study of the interaction of telomere components with the DDR revealed ATR promotes genome stability and telomere length maintenance in the absence of CTC1, probably by activating programmed cell death of stem cells with high amounts of DNA damage. I also found that poly(ADP-ribosylation) is not required for maintenance of Arabidopsis telomeres, in contrast to human telomeres. Finally, I found an unexpected connection between the DDR and telomerase. My research showed that ATR maintains telomerase activity levels. Further, induction of double- stranded DNA breaks in seedlings led to a rapid decrease in telomerase activity, which correlated with increased abundance of TER2, an alternate Arabidopsis telomerase RNA. I hypothesize that TER2 inhibits telomerase to prevent its inappropriate action at internal sites in chromosomes. These data reveal two ways that DDR pathways work in concert with telomerase to promote genome integrity.