Method for extracting quantitative information relating to...

Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid

Reexamination Certificate

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C435S366000, C435S320100, C435S325000, C435S354000, C435S357000, C435S358000, C435S365000, C435S367000, C800S013000, C536S023100, C536S023500

Reexamination Certificate

active

06518021

ABSTRACT:

FIELD OF INVENTION
The present invention relates to a method and tools for extracting quantitative information relating to an influence, on a cellular response, in particular an influence caused by contacting or incubating the cell with a substance influencing a cellular response, where the cellular response is manifested in redistribution of at least one component in the cell. In particular, the invention relates to a method for extracting quantitative information relating to an influence on an intracellular pathway involving redistribution of at least one component associated with the pathway. The method of the invention may be used as a very efficient procedure for testing or discovering the influence of a substance on a physiological process, for example in connection with screening for new drugs, testing of substances for toxicity, identifying drug targets for known or novel drugs. Other valuable uses of the method and technology of the invention will be apparent to the skilled person on the basis of the following disclosure. In a particular embodiment of the invention, the present invention relates to a method of detecting intracellular translocation or redistribution of biologically active polypeptides, preferably an enzyme, affecting intracellular processes, and a DNA construct and a cell for use in the method.
BACKGROUND OF THE INVENTION
Intracellular pathways are tightly regulated by a cascade of components that undergo modulation in a temporally and spatially characteristic manner. Several disease states can be attributed to altered activity of individual signalling components (i.e. protein kinases, protein phosphatases, transcription factors). These components therefore render themselves as attractive targets for therapeutic intervention.
Protein kinases and phosphatases are well described components of several intracellular signalling pathways. The catalytic activity of protein kinases and phosphatases are assumed to play a role in virtually all regulatable cellular processes. Although the involvement of protein kinases in cellular signalling and regulation have been subjected to extensive studies, detailed knowledge on e.g. the exact timing and spatial characteristics of signalling events is often difficult to obtain due to lack of a convenient technology.
Novel ways of monitoring specific modulation of intracellular pathways in intact, living cells is assumed to provide new opportunities in drug discovery, functional genomics, toxicology, patient monitoring etc.
The spatial orchestration of protein kinase activity is likely to be essential for the high degree of specificity of individual protein kinases. The phosphorylation mediated by protein kinases is balanced by phosphatase activity. Also within the family of phosphatases translocation has been observed, e.g. translocation of PTP2C to membrane ruffles [(Cossette et al. 1996)], and likewise is likely to be indicative of phosphatase activity.
Protein kinases often show a specific intracellular distribution before, during and after activation. Monitoring the translocation processes and/or redistribution of individual protein kinases or subunits thereof is thus likely to be indicative of their functional activity. A connection between translocation and catalytic activation has been shown for protein kinases like the diacyl glycerol (DAG)-dependent protein kinase C (PKC), the cAMP-dependent protein kinase (PKA) [(DeBernardi et al. 1996)] and the mitogen-activated-protein kinase Erk-1 [(Sano et al. 1995)].
Commonly used methods of detection of intracellular localisation/activity of protein kinases and phosphatases are immunoprecipitation, Western blotting and immunocytochemical detection.
Taking the family of diacyl glycerol (DAG)-dependent protein kinase Cs (PKCs) as an example, it has been shown that individual PKC isoforms that are distributed among different tissues and cells have different activator requirements and undergo differential translocation in response to activation. Catalytically inactive DAG-dependent PKCs are generally distributed throughout the cytoplasm, whereas they upon activation translocate to become associated with different cellular components, e.g. plasma membrane [(Farese, 1992),(Fulop Jr. et al. 1995)] nucleus [(Khalil et al. 1992)], cytoskeleton [(Blobe et al. 1996)]. The translocation phenomenon being indicative of PKC activation has been monitored using different approaches: a) immunocytochemistry where the localisation of individual isoforms can be detected after permeabilisation and fixation of the cells [(Khalil et al. 1992)]; and b) tagging all DAG-dependent PKC isoforms with a fluorescently labelled phorbol myristate acetate (PMA) [(Godson et al. 1996)]; and c) chemical tagging PKC b1 with the fluorophore Cy3 [(Bastiaens & Jovin 1996)] and d) genetic tagging of PKC&agr; ([Schmidt et al. 1997]) and of PKC&ggr; and PKC &egr; ([Sakai et at 1996]). The first method does not provide dynamic information whereas the latter methods will. Tagging PKC with fluorescently labelled phorbol myristate acetate cannot distinguish between different DAG-dependent isoforms of PKC but will label and show movement of all isoforms. Chemical and genetic labelling of specific DAG-dependent PKCs confirmed that they in an isoform specific manner upon activation move to cell periphery or nucleus.
In an alternative method, protein kinase A activity has been measured in living cells by chemical labelling one of the kinase's subunit (Adams et al. 1991). The basis of the methodology is that the regulatory and catalytic subunit of purified protein kinase A is labelled with fluorescein and rhodamine, respectively. At low cAMP levels protein kinase A is assembled in a heterotetrameric form which enables fluorescence resonance energy transfer between the two fluorescent dyes. Activation of protein kinase A leads to dissociation of the complex, thereby eliminating the energy transfer. A disadvantage of this technology is that the labelled protein kinase A has to be microinjected into the cells of interest. This highly invasive technique is cumbersome and not applicable to large scale screening of biologically active substances. A further disadvantage of this technique as compared to the presented invention is that the labelled protein kinase A cannot be inserted into organisms/animals as a transgene. Recently it was discovered that Green Fluorescent Protein (GFP) expressed in many different cell types, including mammalian cells, became highly fluorescent [(Chalfie et al. 1994)]. WO95/07463 describes a cell capable of expressing GFP and a method for detecting a protein of interest in a cell based on introducing into a cell a DNA molecule having DNA sequence encoding the protein of interest linked to DNA sequence encoding a GFP such that the protein produced by the DNA molecule will have the protein of interest fused to the GFP, then culturing the cells in conditions permitting expression of the fused protein and detecting the location of the fluorescence in the cell, thereby localizing the protein of interest in the cell. However, examples of such fused proteins are not provided, and the use of fusion proteins with GFP for detection or quantitation of translocation or redistribution of biologically active polypeptides affecting intracellular processes upon activation, such as proteins involved in signalling pathways, e.g. protein kinases or phosphatases, has not been suggested. WO 95/07463 further describes cells useful for the detection of molecules, such as hormones or heavy metals, in a biological sample, by operatively linking a regulatory element of the gene which is affected by the molecule of interest to a GFP, the presence of the molecules will affect the regulatory element which in turn will affect the expression of the GFP. In this way the gene encoding GFP is used as a reporter gene in a cell which is constructed for monitoring the presence of a specific molecular identity.
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