DNA damaging agents in combination with tyrosine kinase...

Chemistry: molecular biology and microbiology – Treatment of micro-organisms or enzymes with electrical or...

Reexamination Certificate

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C435S173400, C435S212000, C424S094500, C514S025000, C514S027000, C514S456000, C514S457000, C600S001000

Reexamination Certificate

active

06524832

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the field of biochemical pathways. More particularly, it concerns the pathways connecting DNA damage and phosphorylation by tyrosine kinases.
2. Description of the Related Art
Current treatment methods for cancer, including radiation therapy alone, surgery and chemotherapy, are known to have limited effectiveness. Cancer mortality rates will therefore remain high well into the 21st century. The rational development of new cancer treatment methods will depend on an understanding of the biology of the cancer cell at the molecular level.
Certain cancer treatment methods, including radiation therapy, involve damaging the DNA of the cancer cell. The cellular response to DNA damage includes activation of DNA repair, cell cycle arrest and lethality (Hall, 1988). The signaling events responsible for the regulation of these events, however, remain unclear.
Several checkpoints in cell cycle progression control growth in response to diverse positive and negative regulatory signals (Lau & Pardee, 1982). Ionizing radiation, for example, slows growth by inducing delays in G
1
/S and G
2
phases of the cell cycle. The available evidence suggests that G
2
arrest in necessary for repair of DNA damage before entry into mitosis (Steinman et al., 1991; Weinert & Hartwell, 1988). Genetic studies in Saccharomyces cerevisiae have demonstrated that the RAD9 protein controls G
2
arrest induced by DNA damage (Schiestl et al., 1989; Murray, 1989). Mutants of the rad9 locus are unable to delay entry into mitosis following exposure to genotoxic agents and thereby replicate damaged DNA. Although the mammalian homolog of rad9 remains unidentified, other studies in various eukaryotic cells have demonstrated that entry into mitosis is regulated by a 34 kD serine/threonine protein kinase, designated p34
cdc2
(Nurse, 1990; Pines & Hunter, 1989; Russell & Nurse, 1987).
Recent studies have shown that exposure of eukaryotic cells to ionizing radiation is associated with induction of certain early response genes that code for transcription factors. Members of the jun/fos and early growth response (EGR) gene families are activated by ionizing radiation (Sherman et al., 1990; Datta et al., 1992a). Expression and DNA binding of the nuclear factor kB (NF-kB) are also induced in irradiated cells (Brach et al., 1991; Uckun et al., 1992a). Other studies have shown that levels of the tumor suppressor p53 protein increase during X-ray-induced arrest of cells in Gi phase (Kastan et al., 1991; 1992). The activation of these transcription factors presumably represents transduction of early nuclear signals to longer term changes in gene expression that constitute the response to irradiation. Ionizing radiation also induces protein kinase C (PKC) and protein tyrosine kinase activities (Hallahan et al., 1990; Uckun et al., 1993). However, the specific kinases responsible for these activities and their substrates require further study.
Mitomycin C (MMC) is an antitumor antibiotic isolated from Streptomyces caespitosus that covalently binds to DNA (Tomasz et al., 1988). This agent induces both monofunctional and bifunctional DNA lesions (Carrano et al., 1979). Other studies have demonstrated that MMC stimulates the formation of hydroxyl radicals (Dusre et al., 1989). Although the precise mechanism of action of this agent is unclear, MMC-induced cytotoxicity has been attributed to DNA alkylation and the formation of interstrand cross-links (Carrano et al., 1979; Dusre et al., 1989; Tomasz et al., 1988). Treatment of mammalian cells with MMC is associated with inhibition of DNA synthesis and induction of sister-chromatid exchange (Carrano et al., 1979). Previous work has demonstrated that MMC also enhances transcription of HIV-1 and collagenase promoter constructs transfected into HeLa cells (Stein et al., 1989). These studies indicated that AP-1 is involved in MMC-induced activation of the collagenase enhancer. However, little is known about the effects of this agent on other signaling events.
Protein tyrosine phosphorylation contributes to the regulation of cell growth and differentiation. Protein tyrosine kinases can be divided into receptor-type and nonreceptor-type (Src-like) kinases (Cantley et al., 1991; Hanks et al., 1988; Bonni et al., 1993; Larner et al., 1993; Ruff-Jamison et al., 1993). Several protein tyrosine kinases have been purified from the cytosolic fractions of various tissues (Nakamura et al., 1988; Wong & Goldberg, 1984; Zioncheck et al., 1986).
The Src-like kinases, which can associate with receptors at the plasma membrane, induce rapid tyrosine phosphorylation and/or activation of effectors such as phospholipase C-&ggr;1 (PLC&ggr;1) (Carter et al., 1991), PLC&ggr;2 (Hempel et al., 1992), mitogen-activated protein (MAP) kinase (Casillas et al., 1991), GTPase activating protein (GAP) (Gold et al., 1992a) and phosphati-dylinositol 3-kinase (PI3-K) (Gold et al., 1992b). Recent studies have demonstrated an increase in tyrosine phosphorylation following irradiation of B-lymphocyte precursors (Uckun et al., 1993). Studies of p59
fyn
, p56/p53
lyn
, p55
blk
and p56
lck
activity demonstrated that these Src-family tyrosine kinases were not responsible for radiation-induced tyrosine phosphorylation (Uckun et al., 1992a). These findings suggested that other protein tyrosine kinases, perhaps of the receptor-type, are involved in the response of cells to ionizing radiation.
Varying the environmental conditions following exposure to ionizing radiation or DNA damaging agents can influence the proportion of cells that survive a given dose due to the expression or repair of potentially lethal damage (PLD). The damage is potentially lethal because while under normal circumstances it causes cell death, manipulation of the post-irradiation environment can modify the cell response. Studies show that cell survival can be increased if the cells are arrested in the cell cycle for a protracted period of time following radiation exposure, allowing repair of DNA damage. (Hall, 1988). Thus, PLD is repaired and the fraction of cells surviving a given dose of x-rays is increased if conditions are suboptimal for growth, such that cells do not have to undergo mitosis while their chromosomes are damaged.
For some diseases, e.g., cancer, ionizing radiation is useful as a therapy. Methods to enhance the effects of radiation, thereby reducing the necessary dose, would greatly benefit cancer patients. Therefore, methods and compositions were sought to enhance radiation effects by increasing the sensitivity of cells to damage from ionizing radiation and DNA damaging agents such as alkylating compounds. Cells that are irradiated or treated with DNA damaging agents halt in the cell cycle at G
2
, so that an inventory of chromosome damage can be taken and repair initiated and completed before mitosis is initiated. By blocking the stress or survival response in these cells, they undergo mitosis with damaged DNA, express the mutations, and are at a greater risk of dying.
SUMMARY OF THE INVENTION
In one aspect, the present invention provides a process of selectively activating the lyn family of Src-like tyrosine kinases in a cell that expresses the lyn gene. In accordance with that process, the cell is exposed to an effective activating dose of ionizing radiation. In a preferred embodiment, p56/p53
lyn
is activated.
In another aspect, the present invention provides a process of stimulating tyrosine phosphorylation of Src-like tyrosine kinase substrates. In such a process, a cell that expresses the lyn gene is exposed to an effective activating dose of ionizing radiation. Exemplary Src-like tyrosine kinase substrates include phospholipase C-&ggr;1, phospholipase C-&ggr;2, mitogen-activated protein kinase, GTPase activating protein, phosphatidylinositol 3-kinase and enolase.
In another aspect, the present invention provides a process of inhibiting ionizing radiation induced tyrosine phosphorylation in a cell that expresses the lyn gene comprising inhibit

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