Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1996-10-11
1998-11-10
Knode, Marian C.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 721, 435 30, 436546, C12Q 168, G01N 33567
Patent
active
058341960
DESCRIPTION:
BRIEF SUMMARY
This application is a U.S. national stage application of international application PCT/NL95/00134, filed on Apr. 11, 1995, and also claims priority benefit of European Patent Application 94200968.9, filed Apr. 11, 1994.
FIELD OF THE INVENTION
The subject invention lies in the field of medicine and pharmacology. In particular, the present invention relates to a procedure to measure apoptosis in physiology and pathology in order to support clinical diagnosis and to evaluate and monitor efficacies of drugs and therapies.
BACKGROUND OF THE INVENTION
The total number of cells of an organism is the result of processes, that lead to the formation of new cells (proliferation) on the one hand and processes, that lead to the breakdown of existing cells (cell death) on the other hand. Apoptosis is the major process responsible for the breakdown of existing cells (1).
Apoptosis is also known as programmed cell death or cell suicide, a process that is characterized by a sequence of distinct events ultimately leading to cell death. Cells, that enter into apoptosis, break up junctions with neighbouring cells if present. The cytoplasm condenses, the nucleus coalesces and breaks up into fragments. At the onset of apoptosis chromatin compacts and segregates against the nuclear envelope. Cytoplasm condenses and the nuclear and cellular membranes begin to convolute. In late stage apoptosis nuclear fragmentation occurs and the cell surface developes protuberances. Apoptotic bodies form that are phagocytosed by adjacent cells. During apoptosis the plasma membrane characteristics alter. For example a modification in carbohydrate composition and a change in membrane hydrophobicity and charge take place. The endoplasmic reticulum transforms into vesicles that fuse with the plasma membrane and loss of intracellular fluids and ions occurs. In that final stage the cell breaks up into a number of small apoptotic bodies.
Cell death through apoptosis affects single cells within a population in an unsynchronised manner and occurs rather inconspicuously without any inflammatory response (2).
Normal tissue homeostasis requires that for every cell that is added one must die. Apoptosis is an essential process of physiology and proceeds in a well regulated manner in homeostatic balance with its counterpart proliferation (3,4). If the processes of proliferation and apoptosis are out of balance this may result in pathogenesis. It is now becoming evident that an increasing number of pathological situations can be related to aberrant apoptosis. There is currently a lot of research directed at anti-cancer therapies as it is postulated that cancer occurs when cells refuse to die, i.e. when a defect in the apoptotic pathway occurs. An anti-cancer treatment would therefore consist of inducing naturally occurring suicide pathways to yield biotech cancer cures. Apoptosis is furthermore considered to be potentially relevant for a large number of diseases such as ischemia, stroke, heart disease and autoimmunity. It is considered to be such a fundamental biological phenomenon that the challenge is in fact to find a process it is not involved with (13). For example the macrophages dying in the lungs of patients suffering from cystic fibrosis are undergoing apoptosis. The lung-clogging viscosity of the DNA is characteristic of apoptotic death (12). Apoptosis has also been used as diagnostic in autoimmune diseases such as systemic lupus erythematosus (SLE) (13). B-cell malignancies such as B-cell leukemia lymphoma display growth of tumor cells not because of an increased proliferation rate but due to defective apoptosis. Tumor cell metastasis is succesful if apoptosis of the migrating tumor cells is suppressed. The ratio between the rate of proliferation and the rate of apoptosis of tumor cells determines the rate of tumor growth and hence its life threatening character.
From these considerations it is clear that modulation of apoptosis in vivo by drugs is a promising strategy for future anti-cancer therapies. In order to diagnose the proliferation: ap
REFERENCES:
patent: 5627036 (1997-05-01), Reutelingsperger
Tait et al; "Phospholipid Binding Properties of Human Piacental Anticoagulant Protein-I, a Member of the Lipocortin Family", Journal of Biological Chemistry, 264 (14): 7044-49, May 15, 1989.
Andree et al., "Binding of Vascular Anticoagulant .alpha. (VAC.alpha.) to Planar Phospholipid Bilayers", Journal of Biological Chemistry, 256(9): 4923-4928, Mar. 25, 1990.
Fadok et al., Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition by macrophages, J. Immunol. 148(7):2207-2216, 1992.
Umeda et al., Effective production of monoclonal antibodies against phosphatidylserine: stereo-specific recognition of phosphatidylserine by monoclonal antibody, J. Immunol. 143(7):2273-2279, 1989.
Rote et al., Immunologic detection of phosphatidylserine externalization during thrombin-induced platelet activation. Clin. Immunol. Immunopathol. 66(3):193-200, 1993.
Thiagarajan et al., Binding of Annexin V/Placental Anticoagulant Protein I to Platelets. Evidence for phosphatidylserine exposure in the procoagulant response of activated platelets. J. Biol. Chem. 265(29):17420-17423, 1990.
Knode Marian C.
Nexins Research B.V.
Wortman Donna C.
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