Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-02-29
2003-01-21
Caputa, Anthony C. (Department: 1642)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S004000
Reexamination Certificate
active
06509162
ABSTRACT:
TECHNICAL FIELD
The present invention, based on the discovery of a new biological phenomena, provides methods and compositions for use in identifying agents that modulate the phosphorylation of survivin, the interation between survivin and p34
cdc2
cyclin B1 kinase complex, and the interaction between survivin and caspase-9. Related methods and compositions can be used to modulate survivin regulated apoptosis.
BACKGROUND OF THE INVENTION
A. The Role of Survivin in Programmed Cell Death
Programmed cell death (sometimes referred to as apoptosis) is distinguishable, both morphologically and functionally, from necrosis. Programmed cell death is a natural form of death that organisms use to dispose of cells. Cells dying by programmed cell death usually shrink, rarely lyse, and are efficiently removed from the organism (rapidly recognized and engulfed by macrophages) without the appearance of inflammation (Michael Hengartner, “
Cell Death and Aging, Molecular Mechanisms of
,” In Molecular Biology and Biotechnology 158-62 (ed. R. A. Meyers, 1995)).
Apoptosis was initially used to describe a subset of programmed cell deaths sharing a particular set of morphological features which include membrane blebbing, shrinkage of cytoplasm, chromatic condensation and formation of a “DNA ladder.” During apoptosis, cells lose their cell junctions and microvilli, the cytoplasm condenses and nuclear chromatin marginates into a number of discrete masses. While the nucleus fragments, the cytoplasm contracts and mitochondria and ribosomes become densely compacted. After dilation of the endoplasmic reticulum and its fusion with the plasma membrane, the cell breaks up into several membrane bound vesicles, referred to as apoptotic bodies, which are usually phagocytosed by adjacent cells. Activation of particular genes such as tumor suppressor genes in vertebrates is thought to be necessary for apoptosis to occur. Apoptosis induced by numerous cytotoxic agents can be suppressed by expression of the gene bcl-2, which produces a cytoplasmic protein Bcl-2 (The Encyclopedia of Molecular Biology 67 (ed. John Kendrew et al., Blackwell Science; Oxford, England, 1994).
Survivin has recently been identified as a novel inhibitor of apoptosis (IAP). The gene encoding survivin is located on chromosome 17q25. Survivin is a 16.5 kD cytoplasmic protein containing a single partially conserved BIR domain, and a highly charged carboxyl-terminus coiled-coil region instead of a RING finger, which inhibits apoptosis induced by growth factor (IL-3) withdrawal when transferred in B cell precursors (Ambrosini, G. et al., 1997). Unlike other members of the IAP family, survivin has only one BIR domain and does not have a carboxy-terminal RING finger. Instead, survivin has a carboxy-terminus coiled-coil region. Based on overall sequence conservation, the absence of a carboxy terminus RING finger and the presence of a single, partially conserved, BIR domain, survivin is the most distantly related member of the IAP family, sharing the highest degree of similarity with NAIP (Roy, N. et al., 1995). Additionally, unlike other IAP proteins, survivin is undetectable in adult tissues, but becomes prominently expressed in all the most common human cancers of lung, colon, breast, pancreas, and prostate, and in ~50% of high-grade non-Hodgkin's lymphomas, in vivo.
Expression of survivin in embryonic and fetal development may contribute to tissue homeostasis and differentiation that is independent of bcl-2 (Adida et al., 1998) . Aberrations of this survivin-associated developmental pathway may result in prominent re-expression of survivin in neoplasia and abnormally prolonged cell viability (Adida et al., 1998).
Deregulation of programmed cell death has emerged as a primary mechanism contributing to the pathogenesis of various human diseases including cancer. While the impact of anti-apoptosis genes in neoplasia has focused, for example, on the role of bcl-2 in follicular lymphoma, a potential distribution of IAP proteins, such as survivin, has only begun to been investigated. Survivin is rarely present in adult tissues but has been detected in adenocarcinoma of the pancreas, breast adenocarcinoma, colon cancer, head and neck squamous cell carcinoma, neuroblastoma, malignant thymoma, prostate cancer, and benign prostate hyperplasia (see U.S. Ser. No. 08/975,080). This expression pattern suggests that overexpression of survivin or alterations in survivin gene regulation may commonly occur during tumorigenesis.
B. Cell Cycle
Living organisms are composed of cells, whose growth and division require a regular sequence of events and processes that comprise the cell cycle. Some cell cycle events are continuous (e.g., synthesis of proteins and lipids), whereas others are discontinuous (e.g., DNA synthesis). Two discontinuous processes for cell survival are the replication of DNA and the segregation of chromosomes to the daughters of cell division during mitosis. If either of these steps are performed inaccurately, the daughter cells will be different from each other and will almost certainly be flawed. Segregation of chromosomes occurs during mitosis, normally a relatively brief period in the cell cycle, which culminates in the highly visible act of cell division (e.g., cytokinesis). The remainder of cell cycle comprises interphase, during which growth occurs. Chromosome replication occurs in eukaryotic cells only during interphase; and replication and segregation are mutually exclusive processes.
Interphase is subdivided into the S phase when DNA synthesis occurs and the gaps separating S phase from mitosis. G1 is the gap after mitosis, before DNA synthesis starts; G2 is the gap after DNA synthesis is complete, before mitosis and cell division. Cellular constituents direct the cell cycle by acting as regulatory elements.
C. Checkpoint Mechanisms for Apoptosis and Cell Cycle
One of the central functions of apoptosis (programmed cell death) in maintaining homeostasis is the elimination of damaged and potentially harmful cells (Vaux and Korsmeyer, 1999). For this process to be effective, the apoptotic machinery must constantly couple to surveillance mechanisms, i.e. “checkpoints”, sensing DNA damage, adverse environmental conditions, and oncogene or viral transformation (Hunter, 1997; Paulowich et al., 1997). Checkpoint activation under these conditions initiates apoptosis (Evan and Littlewood, 1998) via the assembly of an evolutionary conserved “apoptosome”, which in mammalian cells comprises an upstream cell death protease, caspase-9, the adapter/cofactor protein Apaf-1, mitochondria-derived cytochrome c and dATP/ATP (Green, 1998). Although it is debated how apoptosome assembly promotes caspase-9 catalytic activity (Rodriguez and Lazebnik, 1999; Zou et al., 1999), this process culminates with downstream activation of effector caspases and cleavage of critical cellular substrates (Salvesen and Dixit, 1997; Thomberry and Lazebnik, 1998). A similar paradigm linking apoptosis control to checkpoint activation (Levine, 1997), has been extended to surveillance mechanisms presiding over cell cycle transitions (Pines, 1999), assembly of a bipolar mitotic apparatus (Merdes and Cleveland, 1997), preservation of ploidy (Nicklas, 1997), and timing of cytokinesis (Field et al., 1999). In this context, dysregulated expression of apoptosis inhibitors bcl-2 and bcl-
XL
has been shown to restrain S phase entry (Linette et al., 1996), promote cell cycle exit (Huang et al., 1997), and cause aneuploidy (Minn et al., 1996), further demonstrating a role of the apoptotic machinery in cell-cycle progression.
Survivin is expressed in G2/M in a cell cycle-dependent manner, and localized to mitotic spindle microtubules and intercellular acto-myosin bridges, i.e. midbodies, during cell division (Li et al., 1998). Interference with this topography, or blocking survivin expression, caused increased caspase-3 activity in G2/M (Li et al., 1998), and a profound dysregulation of mitotic progression (Li et al., 1999), suggesting that survivin may regulate a novel apoptotic checkpo
Caputa Anthony C.
Davis Natalie
Morgan & Lewis & Bockius, LLP
Yale University
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