Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving transferase
Reexamination Certificate
2001-05-17
2004-05-25
Weber, Jon P. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving transferase
C435S029000
Reexamination Certificate
active
06740497
ABSTRACT:
BACKGROUND OF INVENTION
1. Field of the Invention
The invention relates to the field of micro-analytical chemistry in the areas of cellular biochemical and biomedical analysis, and in particular to a class of chemical compounds and method and apparatus for assaying the activity of oncoproteins in a single cell or selected cells.
2. Description of the Prior Art
In biology, the individual cell can be thought of as the fundamental unit of life. phenomena that we observe in living organisms. Most intracellular chemical processes are mediated by proteins. Typically these proteins are enzymes that catalytically enhance the rates of specific chemical reactions. Very often, intracellular enzymes participate in cascades of chemical reactions known as signal transduction pathways. Each of these signal transduction pathways is composed of sequentially acting enzymes, frequently of a class known as kinases. These enzymes not only interact with and modify the behavior of other proteins within the same pathway, but also influence the operation of other signal transduction pathways. These interacting cascades of signaling molecules and chemical reaction products form complex networks that ultimately regulate processes of cell growth, proliferation, quiescence, and programmed cell death.
Inappropriate signaling within a cell can give rise to defects in any of these processes and is implicated as the basis for many forms of cancer. To further complicate matters, individual cells display a high degree of heterogeneity in their internal biochemical signaling. Malignant tumors are typically composed of such a heterogeneous group of cells, and cells within the same tumor often utilize disparate and aberrant growth signaling pathways. This heterogeneity makes it necessary to analyze individual cells to elucidate their unique errors in signaling. Where only a small proportion of cells show aberrant signaling, analysis of an entire population will produce an average signal more reflective of the majority rather than of any individual aberrant cell. Thus, the measurement “averages out” the very errors one seeks to detect.
Often there is a genetic basis, in the form of mutations in genes for signaling proteins, for inappropriate signaling. Mutations in the genes for signaling proteins result in the presence of structurally altered, aberrantly acting, signaling proteins. Currently, there exist methods to analyze the DNA and/or RNA of a single cell to detect the presence of signaling protein mutations and thereby infer the presence of mutant signaling proteins. However, it is not currently possible to predict or understand errors in signaling purely from knowledge of the genes or mutations in the genes that occur within a tumor. For example, the aberrant activity of mutant signaling proteins in one pathway may be counteracted or modified by the activities of other proteins, mutant or normal, involved in other pathways. This is very likely since the interior of a tumor cell is a complex mixture of signaling proteins generally with hundreds to thousands of different kinds present simultaneously and participating in pathways that frequently influence one another. To assess the function of any single kind of a signaling protein, its net activity must be measured from within a single living cell.
Further, to completely describe the function of an entire pathway, measurements of the activities of all the signaling proteins involved within a pathway are needed. Such knowledge would be exceptionally useful in the individualized diagnosis and treatment of diseases that involve faulty intracellular signaling since signaling proteins are often prime targets for therapeutic drug intervention.
Further, such a capability would revolutionize the ability to conduct research on basic cellular physiology. As noted, kinases represent an exemplary class of protein enzymes that commonly play a key role in intracellular signaling. In fact, aberrant activation of growth promoting kinases is a general feature of tumor cells. Thus protein kinases are promising targets for cancer therapies and the development of kinase antagonist drugs is an area of intense research. These enzymes catalytically enhance the rate at which a covalent chemical bond is formed between a phosphate group and a molecular substrate molecule, frequently, a different protein. This process is termed “phosphorylation”. When the substrate itself is an enzyme, such phosphorylation generally either enhances or suppresses the substrate enzyme's chemical activity.
Kinases generally demonstrate substrate specificity such that the preferred substrates of one kind of kinase are not efficiently phosphorylated by other kinds of kinases. Frequently, the preferred substrates for kinases are different kinases; thus, the inappropriate activity of one kinase can result in changes in the activity of multiple downstream kinases within a signaling pathway.
A general method to measure kinase activity within a cell could, in many instances, yield an abundance of information about an entire signaling pathway. However, in order to make such a measurement strict criteria must be met. To begin with, the number of copies per cell of many enzymatic proteins, such as kinases and kinase substrates, can be as low as 100 to 1,000,000 molecules, or about 150 pM to 1.5 &mgr;M in concentration in a typical mammalian cell with a volume of 1 pl. In terms of moles, this is equivalent to a necessary limit range of approximately 1.7×10
−18
to 1.7×10
−22
.
Further, since the concentrations of phosphorylated substrates in cells change on time scales of the order of seconds, the time resolution of the measurement, from the instant the contents of a cell are obtained to the time that the biochemical reactions are terminated, must be sub-second. Most conventional biochemical assays meet neither the temporal resolution nor the sensitivity limits required for these single cell measurements. The temporal resolution requirement can be met through the use of apparatus described in application Ser. No. 09/036,706 filed Mar. 6, 1998, now U.S. Pat. No. 6,156,576, and entitled, “Fast Controllable Laser Lysis of Cells for Analysis”, to which this continuation-in-part application is related and which parent application is herein expressly incorporated by reference. The necessary degree of sensitivity can be achieved with traditional capillary electrophoresis (CE) methods. Lacking, until now, has been the molecular means to accurately determine the intracellular activity of one or more kinase species or other enzymes.
Current techniques for kinase measurements can generally be divided into three methodologies. The first method uses the phosphorylation of kinase substrates by cellular extracts to estimate the kinase activity that occurs within intact cells There are a number of major drawbacks to such a method. Because it is not sensitive, the internal contents of large numbers of cells must be pooled. Since the cells are not synchronous with respect to their activation status, a time averaged level of kinase activity is actually measured. Furthermore, during the time required to generate a cellular extract, a time that may span many seconds to minutes, many chemical reactions continue to proceed. This results in a highly distorted representation of the relative amounts of reactants and products as they occurred within actual cells.
Even beyond these difficulties, it is virtually impossible to reproduce the unique chemical microenvironments that occur within cells; thus, the chemical activity observed in extracts can differ greatly from that which actually occurs within intact cells.
A second methodology relies on labeling the kinase within a cell with specific antibodies, kinase inhibitors, or fluorescent tags and then observing the cells with a fluorescence microscope. Both inactive and active kinase molecules are labeled by this method, and an attempt is then made to infer the activity level of the kinase from its actual location within a cell. However, it is extremely risky to att
Allbritton Nancy
Sims Christopher
Sonneschein Nath & Rosenthal LLP
Weber Jon P.
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