Joseph P. Kao - US grants

The long-term goal of the proposed research is to understand the mechanisms by which Ca2+ regulates mitotic progression in cells. To achieve this goal, novel fluorescent probes of Ca2+ and Ca2+-dependent signalling processes will be synthesized and applied in mitotic cells. Two types of probes are proposed for synthesis: 1) a non-metabolizable fluorescent aqueous-soluble polymer-supported Ca2+ indicator which based on fura-2. A polymer-bound indicator, by evading cellular transport mechanisms, can be used for monitoring Ca2+ concentration in mitotic cells over the entire course of mitosis without artifacts arising from extrusion or sequestration of the indicator into subcellular organelles. 2) Fluorescent peptide probes that specifically detect the activation of protein kinase C, multifunctional Ca2+/calmodulin-dependent protein kinase, and calmodulin. Each peptide is a specific recognition sequence, either for phosphorylation by a specific kinase or for binding to calmodulin, flanked by independently synthesized fluorescent amino acid residues that form a donor-acceptor pair for resonant energy transfer (RET). RET efficiency will be sensitive to the peptide conformation which , in turn, changes in response to phosphorylation or calmodulin binding. By measuring RET in a specific peptide probe spectroscopically, one can monitor activation of calmodulin or a specific kinase. Application of these probes in mitotic cells will yield information on the pathways by which Ca2+ exerts influence on various stages of mitosis.

[unreadable] DESCRIPTION (provided by applicant): A "caged" molecule is a photosensitive, but temporarily inert, precursor of a biologically active molecule. Light absorption transforms the precursor into a molecule with full bioactivity. Because a light beam can be easily focused and steered, and photochemical reactions are extremely fast, caged molecules are versatile tools for using light to manipulate biology with exceptionally high spatial and temporal resolution. The long-term objective is to develop a broad spectrum of novel photochemical tools that will enable cellular physiologists to use light to probe and control dynamic signaling processes in living cells and tissues. The proposed research has four foci: 1) Develop new "cages" that a) are strongly activated by light, b) show fast kinetics of product release on photolysis, c) give high yield of product when photolyzed, and d) are chemically and metabolically stable in the absence of light. 2) Develop new probes for cellular signaling, specifically cage neurotransmitters, lipid messengers, as well as probes that permit "photochemical knock-out" of neurotransmitter receptors and transporters, and ion channels. 3) Develop and optimize two probes of intracellular calcium signaling: a caged calcium and a caged "anti- calcium" for using light to rapidly generate and ablate intracellular calcium signals, respectively. 4) Apply the developed probes to cellular physiology research in the areas of signal processing by nerve terminals and dendrites, synaptic plasticity, and calcium regulation of cell excitability. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant):The goal of this project is to develop the tools and the method for using light to control the expression of genes in living cells or tissues in a spatially and temporally specific manner.

In keeping with the mission of the NIH to support research ?with respect to the cause, diagnosis, prevention, and treatment of cancer? (NCI) and ?of new biomedical imaging and bioengineering techniques and devices to fundamentally improve the detection, treatment, and prevention of disease? (NIBIB), the overarching goal of this proposal is to leverage metabolic alterations in cancer cells for the noninvasive diagnosis and characterization of glioblastoma (GBM), the most deadly and common primary brain cancer in adults. GBM takes more than 13,000 lives in the USA each year and is defined by multiple, complex genetic subtypes and tumor invasion into adjacent brain tissue. Emerging dilemmas in the management of patients with GBM include characterizing the tumor-specific alterations that occur over time and in response to therapies. One strategy is to define and analyze these alterations using real-time information on the molecular level acquired with advanced imaging techniques. This strategy has the potential to differentiate genetic and loco-regional tumor variations as well as evaluate and monitor treatment responses. Specifically, the in vivo visualization of tumor- specific metabolic pathways is likely to add greatly to the diagnostic information used to effectively manage patients with GBM. One such pathway is the conversion of 5-aminolevulinic acid (5-ALA, a naturally occurring substrate) to protoporphyrin IX (PpIX, the fluorescent product) during heme biosynthesis. This pathway is highly and selectively upregulated in 90% of GBMs and increasing levels of PpIX have been identified within regions of increasing tumor grade. The recent development of hyperpolarized 13C magnetic resonance spectroscopy (MRS) enables for the first time the real-time non-invasive measurement of critical dynamic metabolic processes in vivo. Here, we propose to develop a hyperpolarized 13C MRS-based approach for noninvasive assessment of GBM by exploiting the altered porphyrin metabolism of cancer cells. Firstly, we will synthesize 5-ALA 13C-substituted in specific positions and optimize the substrate formulation to achieve maximum polarization, which directly translates into higher signal amplification when used as an imaging agent (Aim 1). Secondly, we will evaluate the feasibility of using hyperpolarized 13C-5-ALA as molecular imaging agent to measure the altered porphyrin metabolism in GBM, first in a series of in vitro cell experiments and then in a rat model of GBM (Aim 2). Given that hyperpolarized 13C MRS is being actively investigated in patients with numerous diseases, the successful completion of this high-risk/high-reward project has the potential to provide a noninvasive approach for the diagnosis, monitoring, and therapeutic evaluation of GBM patients.