FUNCTIONAL IMAGING OF INTRACELLULAR METABOLIC COFACTORS IN HUMAN NORMAL AND CANCER BREAST CELLS
Open Access
- Author:
- Yu, Qianru
- Graduate Program:
- Bioengineering
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- June 15, 2009
- Committee Members:
- Ahmed A. Heikal, Committee Chair/Co-Chair
Cheng Dong, Committee Member
William O. Hancock, Committee Member
Emine Koc, Committee Member
William O Hancock, Committee Member - Keywords:
- NADH
- Abstract:
- Oxidative phosphorylation pathway in the inner membrane of mitochondria provides the majority of energy needs of eukaryotic cells. Reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) are key metabolic cofactors in energy metabolism and redox reactions in all living cells. The enzymatic activities of these biochemical reactions are dependent on the concentration and distribution of these NADH and FAD coenzymes. Mutations of mitochondrial DNA in diseased cells lead to structural mutations of the electron transport chain complexes (I-IV) and, therefore, numerous health problems such as cancer, neurodegenerative diseases, cardiomyopathy, and ageing. Mitochondria also participate in programmed cell death (apoptosis), generation of free radicals and oxidative stress. As a result, intracellular coenzymes have the potential to serve as natural biomarkers for the respiratory state activities and mitochondria-related health problems. Conventional biochemical methods have provided the bulk information on the concentration of these intrinsic cofactors using cell lysates and, therefore, a snapshot of the enzymatic activities in both normal and diseased cells. These biochemical techniques, however, are incapable of providing the morphological context and molecular conformation dynamics inherent in living cells or tissues. Other steady-state fluorescence-based techniques have been developed since the pioneering work of Britton Chance in the 1960’s. However, these techniques are limited by the ambiguous distinction between intrinsic NADH concentration and alteration of cellular autofluorescence, which is sensitive to the molecular conformation (i.e., free or enzyme-bound) as well as the complex cell environment. To overcome these limitations, we have developed integrated multimodal fluorescence microscopy and spectroscopy techniques, on a single platform, to differentiate between normal and cancer breast cells using intracellular NADH and FAD as natural biomarkers. Human breast cancer cell line (Hs578T) and its non-transformed fibroblast counterpart (Hs578Bst) from the same patient were used as model systems. To examine the sensitivity of our assay to different cell lines, a non-tumorigenic mammalian epithelial cell line (MCF10A) and its transformed counterpart (MCF7) from different patients were also used. Complementary studies on primary and ras-transduced keratinocyte cells isolated from mice enabled us to assess the sensitivity of our experimental approach and these coenzymes to targeted alteration of key regulatory proteins. The environmental heterogeneity of intracellular cofactors (i.e., how cellular environment may influence the autofluorescence quantum yield) was investigated using two-photon fluorescence lifetime imaging. These fluorescence lifetime and intensity images, recorded using a calibrated microscope, were analyzed to construct concentration images of the endogenous coenzymes at the single cell level. Time-resolved fluorescence anisotropy images, using polarization-analyzed autofluorescence, were then used to obtain direct measurements of the fluorescence fraction of free and enzyme-bound NADH at the single-cell level. Complementary studies were also conducted using potassium cyanide to inhibit ETC enzymes in these model cells. Control experiments include NADH and FAD interactions with representative enzymes (e.g. lactate dehydrogenase, mitochondrial malate dehydrogenase, and lipoamide dehydrogenase) in solution. Using our integrated fluorescence assay, we also investigated the local environment and complexity of the inner membrane of mitochondria, where oxidative phosphorylation takes place, using rhodamine-123 as mitochondrial label. Our results are discussed in terms of the cell biology, cancer pathology, and related literature studies. The potential of our fluorescence-dynamics assay and future outlook are also outlined.