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Methods and products related to metabolic interactions in disease

Methods and products related to metabolic interactions in disease description/claims

This application is a continuation of application of Ser. No. 09/277,575, filed Mar. 27, 1999, entitled METHODS AND PRODUCTS RELATED TO METABOLIC INTERACTIONS IN DISEASE, and now pending, which claims priority to U.S. Provisional application Ser. No. 60/082,250, filed Apr. 17, 1998; 60/101,580, filed Sep. 24, 1998; and 60/094,519, filed Jul. 29, 1998, now abandoned, each of which the entire contents are incorporated by reference.

This invention was made with Government support under Grant/Contract No. AI 33470 awarded by the National Institute of Health. The Government may retain certain rights in this invention.

This invention relates to methods of regulating cell growth and division to control disease processes by manipulating mitochondrial metabolism and the expression of cell surface immune proteins. The invention also relates to compositions and screening assays.

Normal tissue develops and is maintained by normal processes of cell division and cell death. In many diseases, such as cancer, diabetes mellitus Type I, and autoimmune disease, the normal balance between cell division and cell death is disrupted causing either a rapid growth of unwanted and potentially dangerous cells or a loss of cells which are essential to maintaining the functions of tissue.

Cell division occurs by a process known as mitosis. During mitosis dividing cells use glucose cytolytically at an increased rate as the primary source for energy (ATP) production in a process referred to as glycolysis (Brand, K. A., and U. Hermfisse. 1997. Aerobic glycolysis by proliferating cells: a protective strategy against reactive oxygen species. Faseb J 11, no. 5:388-95). Glycolysis occurs in the cytosol and is required for mitochondrial energy production. An increased rate of glycolysis occurs when cells divide, providing more of the ATP from cytosolic glycolysis. Mitochondrial synthesis of ATP proceeds through coupling of electron transport-dependent oxido-reductive reactions to ATP synthetase (oxidative phosphorylation) (Harper, M. E. 1997. Obesity research continues to spring leaks. Clinical Investigations in Medicine 20, no. 4:239-244). During this process, a proton gradient is generated by the pumping of protons out of the mitochondria (Himms-Hagen, J. 1992. Brown Adipose Tissue. Obesity, eds. P. Bjorntorp and B. N. Brodoff. 1 vols. J. B. Lippincott, Philadelphia. 1 pp), increasing mitochondrial membrane potential. Uncoupling proteins (UCPs) reversibly uncouple oxidative phosphorylation from electron transport and thereby can decrease mitochondrial membrane potential (Harper, M. E. 1997. Obesity research continues to spring leaks. Clinical Investigations in Medicine 20, no. 4:239-244). Elevating glucose concentrations can increase mitochondrial membrane potential (Harper, M. E. 1997. Obesity research continues to spring leaks. Clinical Investigations in Medicine 20, no. 4:239-244).

Cell death is a physiologic process that ensures homeostasis is maintained between cell production and cell turnover in self-renewing tissues and is essential to the proper functioning of the immune system. Physiological cell death occurs through the processes of apoptosis and necrosis. The boundaries between these processes, once thought to be distinct, have blurred with the explosion of information on the role of cell death in development, tissue modeling, regenerative processes, and in the immune system. However, it is widely accepted that necrotic cell death (sometimes called oncosis) typically results in the osmotic rupture of a cell, followed by an inflammatory response, while apoptotic death involves cell shrinkage, fragmentation of the cell, and phagocytosis of the fragments often without inflammation. Most cells die in a form of suicide characteristically apoptotic and tightly regulated by complex signals (Zakeri, Z., W. Bursch, M. Tenniswood, and R. A. Lockshin. 1995. Cell Death: Programmed, apoptosis, necrosis, or other. Cell Death and Differentiation 2:87-96). Apoptotic cell death is particularly important in the reticulo-endothelial system where the balance between mitosis and cell death may determine the effectiveness and the nature of an immune response (Zakeri, Z., W. Bursch, M. Tenniswood, and R. A. Lockshin. 1995. Cell Death: Programmed, apoptosis, necrosis, or other. Cell Death and Differentiation 2:87-96). Failure results in autoimmune disease or in a lack of immune surveillance.

Inappropriate cell division or cell death results in serious life-threatening diseases. Diseases associated with increased cell division include cancer and atherosclerosis. Disease resulting from increased cell death include AIDS, neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa), aplastic anemia, atherosclerosis (e.g., myocardial infarction, stroke, reperfusion injury), and toxin induced liver disease. Many methods for treating these disorders have been proposed Although these diseases share the common physiological trait of either excess cell division or premature cell death, strategies for identifying potential therapeutic treatments have been individualized rather than searching for a common mechanism. It would be desirable to identify a common mechanism by which cell division could be interrupted or cell death could be promoted to treat all of these diseases.

PC12 cells, a cell line derived from rat pheochromocytoma (Greene and Tischler, 1976) have been extensively used as a model for the study of nerve growth factor (NGF)-induced neuronal differentiation and dependency (Mills et al., 1997), and of neuronal cell apoptosis resulting from serum and/or trophic factor withdrawal (Mesner et al., 1995, Fulle et al., 1997), oxidative stress (Vinard et al., 1996) and, the addition of calcium ionophores (Fulle et al, 1997). NGF promotes differentiation, neurite outgrowth and the acquisition of a mature sympathetic neuronal morphology on PC12 cells. Withdrawal of NGF, however, results in apoptosis of the PC12 cells which is characterized by prototypic changes, i.e., chromatin degradation, nuclear fragmentation, acidification, alterations of surface lipids, cell fragmentation, blebbing and nucleosome formation (Gottlieb et al, 1997).

PC12 transfected variants such as TrkA have been developed to elucidate the role of NGF and signal transduction in neuronal function. Nerve growth factor (NGF) binds to two synergistic receptors, tyrosine kinase A (TrkA) and p75NGRF (Canossa et al., 1996). The PC12 TrkA cell line overexpresses TrkA, a 140 kDa protein with intrinsic tyrosine kinase activity (Kaplan et al., 1991) and responds more vigorously than native PC12 cells to NGF stimulation. It is believed that the NGF-TrkA complex acts as a messenger that delivers the growth signal from axon terminals to sympathetic neuronal cell bodies (Riccio et al., 1997).

Epidermal growth factor (EGF) has different effects on PC12 cells than NGF. When tenative PC12 cells are treated with EGF they are induced to undergo proliferation rather than differentiation. In contrast, EGF stimulation of the v-Crk and TrkA cell lines induce neuronal differentiation (Teng et al., 1995).

Fas, a member of the tumor necrosis receptor family that includes the nerve growth factor receptor, mediates apoptotic cell death in several instances, including TCR (T cell receptor)/CD3-induced T cell activation (Nagata et. Al., Science). When the Fas molecule interacts with Fas ligand or an appropriate anti-Fas antibody, cellular death can ensue (Gottlieb et al., 1997). Fas was originally described on the membrane surface of hematopoietic lineage cells (Itoh et al., 1991), but its presence has been documented on endothelial cells (Richardson et al., 1994), hepatocytes (Tanaka et al., 1998) and oligodendrocytes in multiple sclerosis lesions (Bonetti and Raine, 1997).

The B7 molecules, B7.1 (CD80) and B7.2 (CD86) are known for their ability to co-stimulate T cell proliferation (Linsley et al., 1991), the production of interleukin-2 (Freeman et al., 1992) and the expression of interleukin-2 receptors (Razi-Wolf et al., 1996). Expression of these co-stimulatory molecules on immune cells also may play an important role in the pathogenesis and response to several bacterial, parasitic and viral infections as well as autoimmune disease (Reiser and Stadecker, 1996) such as systemic lupus erythematosus (Folzenlogen et al., 1997), experimental allergic encephalomyelitis (Perrin et al., 1996) and in the rejection phase of alloimmune responses (Akalin et al., 1997).

B7.1 and B7.2 are members of the immunoglobulin gene superfamily and include a V-like and a C2-like extracellular domain. Although originally described on B cells, B7.1 and B7.2 have also been described on monocytes, dendritic cells and activated T cells (June et al., 1994). B7.1 (CD80) and particularly B7.2 (CD86) are upregulated on the B lymphocyte surface of patients with systemic lupus erythematosus (SLE) (Folzenlogen at al., 1997).

The invention involves the finding that mitochondrial metabolism plays an essential role in regulating cellular division and cell death occurring in various diseases. It was found according to the invention that the status of the cellular proton motor force which can be assessed by the coupling relationship between electron transport and oxidative phosphorylation plays an important role in the signal which determines whether a cell will undergo cellular division, cellular differentiation or cellular death. This finding has important implications for treating diseases associated with excessive cellular division, aberrant differentiation, and premature cellular death, e.g., for the treatment of cancers, autoimmune disease, neurodegenerative diseases, etc.

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