Browsing by Author "Kato, Takamitsu, committee member"

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Open Access
Evaluation of dose enhancement due to CuATSM uptake in hypoxic environments with external radiation
(Colorado State University. Libraries, 2019) Martinez, Stephen K., author; Leary, Del, advisor; Boss, Keara, committee member; Brandl, Alexander, committee member; De Long, Susan, committee member; Kato, Takamitsu, committee member
Most solid tumors contain areas of chronic hypoxia caused by limited diffusion of oxygen from tumor microvasculature. Hypoxic regions have been found to be radioresistant and their presence results in a worse prognosis for tumor control. Metal radiosensitizers have been employed to alleviate the radioresistance in hypoxic tumors by increasing dose through additional photoelectrons and Auger cascades. In recent years, gold nanoparticles (GNP) have been explored for their potential as an enhancer of external beam radiation and become the standard-bearer for the treatment modality; however, GNP have lower cellular uptake in anoxic and hypoxic conditions than under normoxic conditions. Additionally, the large size of nanoparticles decreases their diffusivity, reducing their ability to penetrate into tumor tissue distant from vasculature. The chelator diacetylbis (N(4)-methylthiosemicarbazonato) copper II (CuATSM) provides the potential to overcome the hypoxic barrier by preferentially depositing copper into tumor regions previously inaccessible to treatment. The characteristics of CuATSM have led to its utilization in positron emission tomography (PET) imaging of hypoxic regions. These PET images have also been investigated as a method for dose painting, amplification of intensity modulated radiation therapy (IMRT) dose to hypoxic regions. Additionally, radioactive ⁶⁴CuATSM has been investigated for implementation in brachytherapy for hypoxic tumors. The Auger electrons ejected upon decay of the radioisotope have been shown to be highly damaging to DNA. The intention of this study is to evaluate the potential of CuATSM as an external beam radiosensitizer. This project investigates radiosensitization of CuATSM by Monte Carlo (MC) modeling of different energy external beam spectra available clinically with Electron Gamma Shower (EGSnrc) and Geometry and Tracking (Geant4) and evaluating dose enhancement with CuATSM. These MC models are informed by and evaluated against cell models. Research indicates that CuATSM at high concentrations with low energy photons has efficacy for enhancing dose in hypoxic tumor regions.
Open Access
Functional redundancy between the RAD51 accessory proteins RAD51AP1 and RAD54 in homologous recombination DNA repair
(Colorado State University. Libraries, 2021) Selemenakis, Platon, author; Wiese, Claudia, advisor; Argueso, Lucas, committee member; Kato, Takamitsu, committee member; Kim, Seonil, committee member
Cancer is responsible for the death of millions of people annually. Factors that increase the risk of tumorigenesis are endogenous challenges and exogenous compounds. These insults are responsible for the generation of DNA lesions, the most toxic one of which is a DNA double-strand break (DSB). DSBs can be repaired by several different DNA repair pathways, among which homologous recombination (HR) is the least error prone. In HR, DNA strand exchange is mediated by the RAD51 recombinase which forms a nucleoprotein filament on single-stranded DNA for strand invasion. RAD51-mediated strand invasion is supported by the DNA motor protein RAD54 and by the RAD51-Associated Protein 1 (RAD51AP1). While the pre-synaptic steps of HR in human cells have been studied extensively, there are still extensive knowledge gaps with respect to the molecular mechanisms of synapsis and post-synapsis and the roles of RAD51AP1 and RAD54 in these later steps of HR. Here, I hypothesized that RAD51AP1 and RAD54 may exhibit functional redundancy in human cells. Also, I speculated that Rad51ap1 disruption in mice would be associated with an increased susceptibility of these mice to radiation carcinogenesis. Finally, I hypothesized that post-translational modification and, more specifically, phosphorylation may regulate the activity of human RAD51AP1. To test for functional redundancy between RAD51AP1 and RAD54, we investigated the impact of simultaneous RAD51AP1 and RAD54 disruption in human cancer cell lines and in response to DNA-damaging agents in cell survival and DNA replication assays. We found that cells lacking both RAD51AP1 and RAD54 (i.e., double KO cells) are more sensitive to the cytotoxic effects of mitomycin C (MMC) or olaparib exposure than cells lacking either RAD51AP1 or RAD54. Accordingly, double KO cells exhibit a more pronounced G2/M arrest, higher levels of chromosomal aberrations and increased sensitivity to DNA replication stress as determined by DNA combing experiments. These results show that RAD51AP1 and RAD54 can compensate for each other in human cancer cell lines. To investigate the consequences of RAD51AP1 loss in mice we utilized a novel mouse model that lacks Rad51ap1 and determined the susceptibility of these mice to radiation carcinogenesis. We found that compared to wild type mice, loss of Rad51ap1 does not affect the survival of mice after whole body IR. We speculate that functional redundancy between RAD51AP1 and RAD54 may also exist in mice, and that Rad51ap1-/- Rad54-/- double KO mice may exhibit pronounced susceptibility to radiation carcinogenesis. Finally, we sought to characterize the regulation of RAD51AP1 activity by post-translational modification. To achieve this objective, we identified two critical residues in RAD51AP1 that appear to be regulated by phosphorylation, S277 and S282. We found that mutation of these residues to the non-phosphorylatable S277A and S282A compromises RAD51AP1 function as measured by DNA replication and cell survival assays. These results suggest that phosphorylation of S277 and/or S282 is crucial for RAD51AP1 function. Collectively, our studies clarify one aspect of functional redundancy within the HR pathway, and the role of post-translational modification of RAD51AP1. Our results provide new insights on the mild phenotypes associated with RAD51AP1 or RAD54 deficiency in human cells and mice. Our findings highlight the importance of development of personalized approaches for cancer treatment.
Open Access
Long-term hematopoietic response in leukocyte counts and differentials for rhesus macaques (Macaca mulatta) from acute whole-body radiation exposure
(Colorado State University. Libraries, 2023) Chino, Yuiko, author; Johnson, Thomas, advisor; Bailey, Susan, committee member; Kato, Takamitsu, committee member; Walrond, John, committee member
Accumulating evidence from A-bomb survivors and radiation therapy patients suggest that survivors are at risk of developing delayed effects of acute radiation exposure (DEARE). In contrast to acute radiation syndrome (ARS), the underlying mechanisms of DEARE are largely unknown. Better understanding of DEARE is vital for improving estimates of risk and predictions of long-term health outcomes following a variety of radiation exposure scenarios, whether accidental or intentional, and including nuclear accidents, cancer treatment, and space travel. The hematopoietic system is highly sensitive to ionizing radiation (IR) exposure; leukocyte counts reach a nadir in days to several weeks post-acute exposure, followed by a recovery period from 4-8 weeks to a year. Accumulating evidence from the A-bomb survivor cohort and animal studies suggests residual damage in the hematopoietic system persists for a long time. Long-term effects in hematopoietic system are very likely the underlying cause of DEARE disease, although there is limited understanding of the process. In this study, archival leukocyte counts and differentials from the Non-Human Primate Radiation Late Effects Cohort (RLEC), were analyzed to evaluate long-term effects. The RLEC cohort consists of over 200 Rhesus Macaques (Macaca mulatta) previously exposed to acute whole body irradiation from 1.14 to 8.5 Gy and approximately 50 control animals. The dataset was created from blood sampling started approximately 1 year post-exposure and continued every 2-6 months. Linear mixed models were developed for total leukocyte count and the differentials including neutrophil, lymphocyte, and monocyte counts and their percentages. Preliminary analysis was conducted for animals with the same dose level, sex, and age at the time of exposure and age and sex matched control animals. The linear mixed models had statistically significant elevations in leukocyte and neutrophil counts and neutrophil% in irradiated animals compared to the controls. Lymphocyte% was significantly lower in irradiated animals. Longitudinal trends for both control and irradiated animals were consistent with expected trends of aging in hematopoiesis, which is skewed towards production of myeloid lineage cells such as neutrophils and monocytes rather than lymphoid cells. There was no statistical difference among the longitudinal trends of control and irradiated animals. Next analysis was extended from the preliminary analysis with a larger dataset including animals with different dose, sex, age at the time of exposure, as well as mitigator assignment. Longitudinal trends were estimated for different dose levels (control, LD50; 6.8 to 8.5 Gy), and adjusted for sex, age at the time of exposure, and status of mitigator use. All models suggested that dose levels were a statistically significant factor for the longitudinal trends of leukocytes and the differentials. Controls showed a slight decrease of total leukocyte count and monocyte skewed differentiation, consistent with changes estimated from aging in hematopoietic system. The LD50 animals than controls and
Open Access
Molecular regulation of glial inflammation in Parkinson's disease
(Colorado State University. Libraries, 2014) De Miranda, Briana R., author; Tjalkens, Ronald, advisor; Kato, Takamitsu, committee member; Legare, Marie, committee member; Mykles, Donald, committee member
Parkinson's disease (PD) is the most prevalent movement disorder that affects adults, the primary pathology of which includes the loss of dopamine producing neurons from the substantia nigra (SN) and inflammatory activation of immune mediators in the brain. Reactive astrocytes and microglia have been implicated in driving the progressive phase of dopamine neuron loss in PD, the result of which leads to worsening neurodegeneration over time that no current therapy can inhibit. A significant need exists for small molecule therapeutics that reach the CNS, decrease glial activation, and confer neuroprotection over the progressive phase of PD. A novel class of anti-inflammatory compounds, originally shown to have anticancer properties, are examined here as potential therapeutic agents to decrease glial inflammation in a progressive mouse model of PD. Derived from the condensation product of indol-3-carbinol produced in cruciferous vegetables, para-phenyl substituted diindolylmethanes or C-DIMs have been shown to induce orphan nuclear receptor family NR4A in several different cancer lines, and have reduced inducible nitric oxide synthase (NOS2) expression in primary murine astrocytes. In order to examine the neuroprotective benefit selected C-DIM compounds have on the survival of dopamine neurons during the inflammatory progressive phase of PD, several experimental studies were conducted, and reported here. The initial development of a method using immunofluorescent sereological counts of TH-positive neurons in the SN, resulted in the ability to measure early neuronal degeneration with sensitivity to small changes in neuron loss through design-based 3D modeling of the SN. Additionally, a pharmacokinetic analysis of four C-DIM compounds determined that structural variations changed the metabolism of these compounds in mice, however all C-DIMs tested were orally bioavailable, distributed to the brain, and displayed pharmacokinetic profiles equivalent to current PD therapies. Initial neuroprotective studies demonstrated that three of the selected C-DIMs are able to limit the progression of dopamine neuron loss from the SN after the onset of a lesion, in a progressive PD mouse model using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, 80 mg/kg) and the drug clearance inhibitor probenecid (250 mg/kg, expressed as MPTPp). A post lesion model was utilized in transgenic NF-κB-EGFP adult mice, where MPTPp treatment was given for 7 days (MPTPp7d) and animals were given either corn oil (vehicle) or one C-DIM (DIM-C-pPhOCH3, DIM-C-pPhOH, DIM-C-pPhCl) via daily oral gavage for the next 7 days (MPTPp14d). Further investigation into the mechanism by which CDIMs reduced dopamine neuron loss revealed that activation of microglia and astrocytes was reduced in the SN from MTPPp7d to MPTP14d in animals given daily oral gavage of the compounds, compared to corn oil control. NF-κB-EGFP intrinsic reporter detection of NF-κB expression was reduced in C-DIM treated animals compared to animals that received no treatment from MPTPp7d to MPTPp14d. Reduction in NF-κB related cytokines (TNF, IL-1α, IFNγ) were also seen after C-DIM treatment. As a final investigation into C-DIM mechanism of action, DIM-C-pPhCl was examined in the BV-2 microglia cell line treated with LPS to stimulate cytokine production. DIM-C-pPhCl treatment reduced the expression of inflammatory proteins such NOS2, TNFα, and IFNγ after LPS treatment, and its effectiveness was dependent on the nuclear receptor transcription factor NR4A2 (Nurr1). In addition, DIM-C-pPhCl treatment increased the amount of Nurr1 present at the NOS2 promoter, suggesting that it enhances the Nurr1-dependent transrepression of NF-κB, recently described as a novel mechanism in glial cells to reduce inflammatory protein and protect against the progression of PD.
Open Access
Neuroinflammation and the two-hit hypothesis of Parkinson's disease
(Colorado State University. Libraries, 2019) Bantle, Collin M., author; Tjalkens, Ronald B., advisor; Zabel, Mark, committee member; Moreno, Julie, committee member; Kato, Takamitsu, committee member; Randall, Elissa K., committee member
The ever-increasing prevalence of neurodegenerative diseases, Alzheimer's Disease (AD) and Parkinson's disease (PD), impose one the most significant medical and public health threats throughout the world. Characteristic PD symptoms include loss of voluntary motor control due to α-synuclein protein-aggregation, neuroinflammatory glial activation, mitochondrial dysfunction, oxidative stress, and progressive neuronal loss. There are currently no disease-modifying therapies for the disease nor has the etiology of PD been elucidated. Epidemiologic and experimental evidence suggests that genetic susceptibility, environmental pesticide exposure, and viral infections are possible risk-factors for PD, but a clear understanding of the environmental links to PD and how these factors can act in concert remains extremely limited. Research is beginning to shed light on neuroinflammation as a converging and coalescing pathway in the pathogenesis and pathophysiology of genetic, sporadic, and postencephalitic PD. While it has been appreciated since the late 1980s that brain inflammation is a hallmark of PD and other age-related neurodegenerative diseases, the immunological role of glia and the key trophic and inflammatory factors and pathways responsible for neurotoxicity and neuronal death in PD have not been clearly elucidated. Understanding how these pathways are regulated in glia during genetic, sporadic, and postencephalitic PD, and how they can directly or secondarily affect the onset and progression of PD is of keen interest. Therefore, the subject of this work will be to explore mechanisms by which glial cells modulate neuronal injury in genetic, sporadic, and postencephalitic cases of PD, with an emphasis on the role of neuroinflammatory activation of glia in single and two-hit models of PD.
Open Access
Telomere length, telomerase activity, and structural variants as biomarkers of extreme environments
(Colorado State University. Libraries, 2017) McKenna, Miles J., author; Bailey, Susan M., advisor; Dow, Steven, committee member; Kato, Takamitsu, committee member; Simon, Steven, committee member; Thamm, Douglas, committee member
Mammals, and in particular humans, are masterful at overcoming and adapting to extreme environments. Whether astronauts in low earth orbit aboard the International Space Station (ISS) or U.S. military veterans exposed to nuclear fallout from atomic weapons testing, humans can persist through a wide range of physical, psychological, and environmental stressors. The overall goal of the studies presented here was to evaluate the biological influences of extreme environments not commonly experienced by the general population. Whether spaceflight or exposure to nuclear fallout, results improve our understanding of short- and long-term effects of low gravity environments, exposure to ionizing radiation (IR) of mixed qualities, as well as low dose and low dose effects of IR. We explored these scenarios by evaluating biomarkers of stress, specifically telomere length dynamics, and biomarkers of DNA damage, specifically transmissible structural rearrangements. Telomeres are not only regarded as valuable biomarkers of aging and age-related degenerative pathologies like cardiovascular disease and cancer, and so are reflective of overall health status, they also serve as "hallmarks" of radiosensitivity. Stable chromosomal structural rearrangements (translocations and inversions) persist with time and so provide informative signatures of IR exposure as well. During the 1950's United States military personnel and weathermen, collectively known as the atomic veterans, were unintentionally exposed to nuclear fallout during atomic bomb testing following WWII. Here, directional Genomic Hybridization (dGH) for high-resolution detection of IR-induced chromosomal inversions and translocations was assessed as a more sensitive, quantitative retrospective biodosimetry approach. The influence of IR exposure on telomere length dynamics was also evaluated to determine the long-term influence of such exposures. Our results illustrate that even for nuclear events that occurred six decades in the past, evidence of exposure is still present. We find that although translocations and inversions are reliable biodosimeters independently, a combined approach provides a more sensitive measurement of past radiation exposure. We also report, for the first time, the influence of age and smoking on background inversion frequencies. Furthermore, telomere length was inversely related to IR dose, suggesting that a single acute exposure to nuclear fallout may lead to persistent long-term effects on overall health. Telomere length dynamics and structural rearrangements were also monitored longitudinally in monozygotic twin and unrelated astronauts. NASA astronauts are a unique group of individuals who experience an extreme environment that the human body is not adapted for. Little is known about the biological health effects of a low gravity environment with increased IR exposure including galactic cosmic rays (GCRs), solar particle events, and secondary neutrons. Not only do astronauts have shorter telomeres than age-/gender-matched controls at baseline (pre-flight), but a transient increase in telomere length during space flight was also observed. Results suggest this unexpected finding may be due to an upregulation of telomerase, the enzyme responsible for maintaining telomere length. Moreover, telomerase activity also increased post-flight in both twins, Scott and Mark Kelly. Although not spaceflight specific, this is the first report of telomerase upregulation in humans due to a psychologically traumatic event. A dose dependent increase in inversions, and to a lesser extent, translocations, as a consequence of IR exposure on the ISS was also seen in Scott Kelly. Collectively, the studies presented here demonstrate a profound influence of extreme environments, particularly those involving low dose IR, on human biological responses. Telomere length dynamics and chromosome aberration frequencies (e.g. translocations and inversions) provide insight into the long-term health effects and implications of spaceflight and exposure to nuclear events.
Open Access
Telomeric double strand breaks undergo resection - but not repair - in G1 human cells
(Colorado State University. Libraries, 2017) Nelson, Christopher Boulanger, author; Bailey, Susan, advisor; Argueso, Lucas, committee member; Kato, Takamitsu, committee member; Wiese, Claudia, committee member; Miller, Benjamin, committee member; Chicco, Adam, committee member
Telomeres are specialized G-rich repetitive regions at the ends of eukaryotic chromosomes (TTAGGGn in mammalian cells). Telomeres function to prevent double strand break (DSB) repair activities at chromosome ends, in order to avoid fusion events which result in lethal dicentric chromosomes. Telomeric repeats make up an appreciable amount of genomic DNA (1-15kb per chromosome end). Therefore, an interesting question becomes, how is the inevitable DSB occurring within a telomere dealt with by the cell? It has been suggested that DSBs within telomeric DNA may not be repaired at all, as DSB DNA damage response (DDR) foci at telomeres do not resolve following large amounts of global DNA damage (e.g. ionizing radiation). Such studies also suggest that telomere repair may be inhibited specifically in G1, as the majority of surviving cells with unresolved telomere damage responses were senescent (a G1 phenotype). On the other hand, studies on the fragmentation of telomeric DNA following cutting with a telomere-targeted endonucleases indicate that repair of telomere-specific DSBs involves Homologous Recombination (HR) and Break-Induced Replication (BIR). However, a marker of telomeric DSB DDRs was only observed in cells with BrdU incorporation, in support of the view that repair of telomeric DSBs is an S/G2-related process, which does not occur in G1. To follow up on these studies, we investigated telomeric DDRs and DSB repair in individual G1 cells using ionizing radiation (IR) and a targeted telomere-cutting endonuclease. IR exposure could potentially induce loss of telomere function, such that persistent DDRs may not represent actual DSBs. To rule out this possibility, we evaluated whether persistent telomeric DDRs following IR occurred at telomeres that were critically short or lacking TRF2. We found that persistent telomeric DDRs occurred at telomeres of normal length and TRF2 status, in support of the conclusion that G1 telomeric DSBs are irreparable. Additionally, using the telomere-targeted endonuclease we observed that telomeric DSBs in G1 cells elicited a relatively conventional DSB DDR – with one important exception – G1 telomeric DDRs failed to recruit 53BP1, an event implicated in the completion of DSB repair by most pathways, but especially, canonical non-homologous end joining (cNHEJ). Further, shRNA knockdown and kinase inhibition of the cNHEJ factor DNA-PKcs, provided evidence that cNHEJ is not responsible for repair of telomeric DSBs, and that DNA-PKcs does not influence recruitment of 53BP1 to telomeric DSBs in G1. Partial deprotection of telomeres, achieved by siRNA depletion of TRF2, also failed to alleviate inhibition of 53BP1 recruitment to G1 telomeric DSBs, suggesting that 53BP1 recruitment to telomeric DSBs may require full deprotection of telomeres. However, as 53BP1 recruitment occurs at de-protected telomeres, this idea would be difficult to test. Most likely related to the lack of 53BP1 recruitment, an abundance of bidirectionally occurring single-stranded DNA was observed at G1 telomeric DSBs, a characteristic of long-range repair-associated resection. In support of long-range resection, RPA70 and phospho-RPA32 were observed at G1 telomeric DSBs. Additionally, conventional DSB repair-associated resection machinery, including MRE11 and EXO1, but not the telomere processing exonuclease Apollo, promoted resection at telomeric DSBs. We then investigated whether long-range resection-dependent repair was occurring at G1 telomeric DSBs via RAD51 or RAD52 foci, and DNA synthesis (S/G2 related processes). Despite activity resembling long-range repair-associated resection at G1 telomeric DSBs, no evidence for repair by these pathways was found. Taken together, the results presented here provide strong evidence in support of the view that telomeric DSBs in G1 are unrepairable. Therefore, the extensive resection observed at telomeric DSBs must be reflective of an alternative, non-repair related function, perhaps related to structural end-protection. We speculate that resection at G1 telomeric DSBs may serve to prevent 53BP1 recruitment, thereby circumventing a full DDR and activation of cNHEJ, a scenario that would create a serious threat to genome stability.