Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
2000-01-27
2001-11-13
Myers, Carla J. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S006120, C435S007100, C435S007400, C435S007700, C530S350000, C536S023500, C536S024310
Reexamination Certificate
active
06316213
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
Generally, the present invention relates to the fields of molecular biology and medicine. More specifically, the present invention is in the field of ovarian and other cancer diagnosis.
2. Background of the Invention
To date, ovarian cancer remains the number one killer of women with gynecologic malignant hyperplasia. Approximately 75% of women diagnosed with such cancers are already at an advanced stage (III and IV) of the disease at their initial diagnosis. During the past 20 years, neither diagnosis nor five year survival rates have greatly improved for these patients. This is substantially due to the high percentage of high-stage initial detections of the disease. Therefore, the challenge remains to develop new markers that improve early diagnosis and thereby reduce the percentage of high-stage initial diagnoses.
Extracellular proteases have already been implicated in the growth, spread and metastatic progression of many cancers, due to the ability of malignant cells not only to grow in situ, but to dissociate from the primary tumor and to invade new surfaces. The ability to disengage from one tissue and re-engage the surface of another tissue is what provides for the morbidity and mortality associated with this disease. Therefore, extracellular proteases may be good candidates for markers of neoplastic development.
In order for malignant cells to grow, spread or metastasize, they must have the capacity to invade local host tissue, dissociate or shed from the primary tumor, and for metastasis to occur, enter and survive in the bloodstream, implant by invasion into the surface of the target organ and establish an environment conducive for new colony growth (including the induction of angiogenic and growth factors). During this progression, natural tissue barriers have to be degraded, including basement membranes and connective tissue. These barriers include collagen, laminin, proteoglycans and extracellular matrix glycoproteins, including fibronectin. Degradation of these natural barriers, both those surrounding the primary tumor and at the sites of metastatic invasion, is believed to be brought about by the action of a matrix of extracellular proteases.
Proteases have been classified into four families: serine proteases, metallo-proteases, aspartic proteases and cysteine proteases. Many proteases have been shown to be involved in the human disease process and these enzymes are targets for the development of inhibitors as new therapeutic agents. Additionally, certain individual proteases have been shown to be induced and overexpressed in a diverse group of cancers, and as such, are potential candidates for markers of early diagnosis and possible therapeutic intervention. A group of examples are shown in Table 1.
TABLE 1
Known proteases expressed in various cancers
Gastric
Brain
Breast
Ovarian
Serine
uPA
uPA
NES-1
NES-1
Proteases:
PAI-1
PAI-1
uPA
uPA
tPA
PAI-2
Cysteine
Cathepsin B
Cathepsin L
Cathepsin B
Cathepsin B
Proteases:
Cathepsin L
Cathepsin L
Cathepsin L
Metallo-
Matrilysin*
Matrilysin
Stromelysin-3
MMP-2
proteases:
Collagenase*
Stromelysin
MMP-8
Stromelysin-1*
Gelatinase B
MMP-9
Gelatinase A
uPA, Urokinase-type plasminogen activator; tPA, Tissue-type plasminogen activator; PAI-I, Plasminogen activator 0 inhibitors; PAI-2, Plasminogen activator inhibitors; NES-1, Normal epithelial cell-specific-1; MMP, Matrix P metallo-protease. *Overexpressed in gastrointestinal ulcers.
Significantly, there is a good body of evidence supporting the downregulation or inhibition of individual proteases and the reduction in invasive capacity or malignancy. In work by Clark et al., inhibition of in vitro growth of human small cell lung cancer was demonstrated using a general serine protease inhibitor. More recently, Torres-Rosedo et al., [
Proc. Natl. Acad. Sci. USA,
90, 7181-7185 (1993)] demonstrated an inhibition of hepatoma tumor cell growth using specific antisense inhibitors for the serine protease hepsin gene. Metastatic potential of melanoma cells has also been shown to be reduced in a mouse model using a synthetic inhibitor (batimastat) of metallo-proteases. Powell et al. [
Cancer Research,
53, 417-422 (1993)] presented evidence to confirm that the expression of extracellular proteases in relatively non-invasive tumor cells enhances their malignant progression using a tumorgenic, but non-metastatic, prostate cell line. Specifically, enhanced metastasis was demonstrated after introducing and expressing the PUMP-1 metallo-protease gene. There is also a body of data to support the notion that expression of cell surface proteases on relatively non-metastatic cell types increases the invasive potential of such cells.
Thus, the prior art is deficient in a good tumor marker useful as an indicator of early disease, particularly for ovarian cancers. The present invention fulfills this long-standing need and desire in the art.
SUMMARY OF THE INVENTION
This invention allows for the detection of cancer, especially ovarian cancer, by screening for PUMP-1 mRNA in tissue, which is indicative of the PUMP-1 protease, which is shown herein to be specifically associated with the surface of 80 percent of ovarian and other tumors. Proteases are considered to be an integral part of tumor growth and metastasis, and therefore, markers indicative of their presence or absence are useful for the diagnosis of cancer. Furthermore, the present invention is useful for treatment (i.e., by inhibiting PUMP-1 or expression of PUMP-1), for targeted therapy, for vaccination, etc.
In one embodiment of the present invention, there is provided a method of diagnosing cancer in an individual, comprising the steps of (a) obtaining a biological sample from an individual; and (b) detecting PUMP-1 in the sample. Usually, the presence of PUMP-1 in the sample is indicative of the presence of cancer in the individual, and the absence of PUMP-1 in the sample is indicative of the absence of cancer in the individual.
In one embodiment of the present invention, there is provided a method for detecting malignant hyperplasia in a biological sample, comprising the steps of (a) isolating mRNA from the sample; and (b) detecting PUMP-1 mRNA in the sample. Typically, the presence of the PUMP-1 mRNA in the sample is indicative of the presence of malignant hyperplasia, and the absence of the PUMP-1 mRNA in the sample is indicative of the absence of malignant hyperplasia.
In one embodiment of the present invention, there is provided a method for detecting malignant hyperplasia in a biological sample, comprising the steps of (a) isolating protein from the sample; and (b) detecting PUMP-1 protein in the sample. Generally, the presence of the PUMP-1 protein in the sample is indicative of the presence of malignant hyperplasia, and the absence of the PUMP-1 protein in the sample is indicative of the absence of malignant hyperplasia.
In one embodiment of the present invention, there is provided a method of inhibiting expression of endogenous PUMP-1 in a cell, comprising the step of (a) introducing a vector into a cell, wherein the vector comprises a PUMP-1 gene in opposite orientation operably linked to elements necessary for expression. Upon expression of the vector, the cell produces PUMP-1 antisense mRNA, which hybridizes to endogenous PUMP-1 mRNA, thereby inhibiting expression of endogenous PUMP-1 in the cell.
In one embodiment of the present invention, there is provided a method of inhibiting PUMP-1 protein in a cell, comprising the step of (a) introducing an antibody into a cell which is specific for a PUMP-1 protein or a fragment thereof. Generally, binding of the antibody to the PUMP-1 protein inhibits the PUMP-1 protein.
In one embodiment of the present invention, there is provided a method of targeted therapy to an individual, comprising the step of (a) administering a compound to an individual, wherein the compound has a targeting moiety and a therapeutic moiety. Specifically, the targeting moiety is specific for PUMP-1.
In one embodiment of the present inv
Adler Benjamin Aaron
Myers Carla J.
The Board of Trustees of the University of Arkansas
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