Browsing by Subject "NADH"

Now showing 1 - 3 of 3
  • Thumbnail Image
    Item
    Macromolecular Crowding Effects on Cellular NADH-enzyme Binding Kinetics
    (2017-08) Wilson, Shane
    Reduced nicotinamide adenine dinucleotide (NADH) is a major cofactor for a large number of biological enzymes that are essential in a myriad of metabolic pathways such as glycolytic and oxidative phosphorylation pathways. In addition, NADH is intrinsically fluorescent and therefore has the potential of serving as a biomarker to monitor mitochondrial dysfunctions associated with aging, cancer, and apoptosis. In this thesis, we investigate how macromolecular crowding may affect the biochemical reaction kinetics of NADH interaction with lactate dehydrogenase (LDH) as a model system in biomimetic crowding (e.g., Ficoll-enriched buffer at 0 ̶ 400 g/L). Using noninvasive, quantitative two-photon fluorescence lifetime and associated anisotropy, we exploit the sensitivities of NADH fluorescence lifetime and rotational diffusion to protein binding. To differentiate between viscosity and crowding effects on the reaction kinetics, we also conducted complementary measurements in glycerol-enriched buffer. Additionally, we are investigating the sensitivity of cellular NADH interaction with dehydrogenases to metabolic manipulations. Our quantitative and non-invasive methodology complements the traditional biochemical and thermodynamics techniques without the destruction of live cells. Intracellular NADH also exists as a mixture of free and enzyme-bound populations at dynamic equilibrium throughout living cells, which can be imaged using fluorescence lifetime imaging for both quantitative and noninvasive assessment of cellular metabolism. 2P-fluorescence lifetime imaging microscopy (FLIM) and 2P- fluorescence anisotropy of intrinsic NADH was measured in cultured mouse embryonic cells under both resting conditions and metabolic-manipulation.
  • Thumbnail Image
    Item
    Metabolic adaptations in proliferating cells and cancer
    (2017-06) Hanse, Eric Allan
    The studies described in this dissertation focus on metabolic adaptations that occur during cell proliferation. In the first part of the thesis we focus on the Warburg effect which is a major hallmark of proliferating cells. We provide evidence that increased glucose consumption in proliferating cells requires malate dehydrogenase 1 (MDH1) to help regenerate the NAD required to sustain high levels of glycolysis. This NAD regeneration has previously only been attributed to lactate dehydrogenase (LDH). We found LDH was dispensable for proliferation whereas MDH1 was not. We also report the MDH1 gene is amplified in a significant number of human tumors and correlates with poor prognosis. We go on to show glutamine, not glucose is a carbon source for MDH1 during proliferation, which allows more of the glucose carbon consumed to go toward biomass production. The second part of the thesis focuses on glutamine metabolism. The increased consumption and metabolism of glutamine leads to increased cellular concentrations of alpha-ketoglutarate (αKG) a tumor suppressor metabolite that affects gene expression via non-metabolic co-factor functions. One of these functions is the activation of the cytosine demethylase, Tet. Increased αKG concentrations therefore change the tumor cell’s epigenetic landscape which can cause differentiation and growth arrest. We reasoned cancer cells must have mechanisms in place to prevent the tumor suppressor activity of αKG. We demonstrate that over-expression of the one carbon metabolism pathway which generates methyl groups and the cytosine methyl-transferase, DNMT1 provide an αKG resistance mechanism. We find combining αKG treatment with low concentrations of the DNMT1 inhibitor decitabine causes significant cell death and reveals a potential therapeutic vulnerability in glutamine addicted cancers. Taken together, our studies provide significant insight into the general biology of proliferative metabolism. Through these insights, our work opens new avenues for targeting the energy and biomass producing pathways cancer cells depend on to proliferate.
  • Thumbnail Image
    Item
    Metabolic-response assessment of metastatic murine breast cancer in 2D and 3D cultures using intrinsic NADH as a natural biomarker
    (2019-08) Cong, Anh
    The majority of in vitro studies of living cells are routinely conducted in a two-dimensional (2D) monolayer culture towards pathophysiological investigation, drug screenings, and cancer diagnostics. There is strong evidence, however, that suggests cellular behavior and metabolism in 2D cell culture is too simplistic of a model as compared with those in vivo tumor cells. In this project, we hypothesize that cancer cell metabolism and metabolic responses to external stimuli (e.g. drug treatments) are distinctly different in threedimensional (3D), tumor-like model as compared with that of the conventional 2D monolayer culture. To test this hypothesis, we employed two-photon (2P) fluorescence lifetime imaging microscopy (2P-FLIM) and time-resolved 2P-fluorescence anisotropy of the reduced nicotinamide adenine dinucleotide (NADH) in metastatic murine breast cancer cells 4T1. In addition, we investigated the cellular metabolic response of 4T1 cells in 2D monolayer and 3D collagen matrix cultures to drug treatment using two novel metabolic drugs, namely MD1 and TPPBr. Both 2P-FLIM and complementary time-resolved anisotropy approaches reveal significant differences between metabolic activities of 4T1 cells in 2D and 3D cultures. Our results suggest that these 4T1 cells in 3D culture adapt an oxidative shift but glycolysis dominances the metabolic state of 2D cells. In addition, 4T1 cells in 3D culture appear to adapt more quickly and exhibit enhanced metabolic activities in response to drug treatment. In contrast, 4T1 cells in 2D monolayer culture exhibit a mute response and are less sensitive to drug treatments. While the tumor-like 3D collagen matrix model may not be an exact replica of in vivo tumors, these studies represent a critical step towards the development of a fundamental understanding of cellular behaviors and metabolism in the more complex in vivo models. These studies would also help advance our understanding of how the cancer cell heterogeneity and microenvironmental conditions impact metabolism and metabolic plasticity in tumor growth and metastatic progression.