Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-01-20
2001-05-15
Eyler, Yvonne (Department: 1646)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C530S388100, C530S389100, C530S324000, C435S810000, C435S006120, C424S138100
Reexamination Certificate
active
06232086
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a novel gene sequence coding for a protein which is related to cell proliferation and programmed cellular death (apoptosis), and specifically relates to cDNA and amino acid sequences for this cellular apoptosis susceptibility (CAS) gene and protein.
BACKGROUND OF THE INVENTION
Apoptosis or programmed cell death is a regulated network of biochemical events which lead to cell death. It is a physiological process involved in cell differentiation, organ development and maintenance of cellular populations in multicellular organisms (Cohen, J. J.,
Immunol. Today
14: 126-15 130, 1993). Furthermore, apoptosis is a reaction to various external stimuli and cell damage (e.g. induced by drugs).
Apoptotic cells generally shrink and are phagocytosed by other cells. In contrast, necrotic cells are characterized by swelling, especially of the mitochondria which become dysfunctional, which usually results in cell lysis.
Molecular events characteristic of apoptotic cells include nuclear collapse with condensation of chromatin and loss of nucleoli. Later, the chromatin becomes fragmented into units of single or multiple nucleosomes which present a “ladder” appearance when separated by size on a gel matrix (Compton, M. M.,
Cancer Metast. Rev
. 11: 105-119, 1992). Activation of an endogenous endonuclease causes the chromatin fragmentation. Intracellular RNA, especially mRNA, is also degraded early during apoptosis.
Apoptosis can be triggered in various ways, including virus infection, growth factor withdrawal, DNA damage resulting from irradiation, exposure to glucocorticoids and certain chemotherapy drugs, or by signals such as TNF binding to its receptor or crosslinking the Fas receptor with anti-Fas antibodies (Cohen, J. J.,
Immunol. Today
14:126-130, 1993; Williams, G. T., & Smith, C. A., Cell 74:777-779, 1993; Suda et al.,
Cell
75:1169-1178, 1993; Smith, et al.,
Cell
76:959-962, 1994; Lowe et al.,
Nature
362:847-849, 1993; Sentman et al., Cell 67:879-888, 1991). The mechanism of apoptosis is not well understood, but the observed molecular changes that occur in apoptotic cells suggest that endogenous genes are responsible for apoptosis. The proteins produced from these induced genes lead to destruction of RNA and DNA ultimately leading to cell death.
Just as cell death by apoptosis is involved in normal regulation of cellular populations, cell proliferation is also required to maintain homeostasis of tissues and organs. However, in some cells, proliferation is aberrant leading to cancer. Cancer cells can be invasive, metastatic and highly anaplastic.
Although the mechanisms of tumor formation are still not completely and well defined, genetic elements, including oncogenes, have been shown to increase cell proliferation and relieve cells of normal check-points in the cell division cycle. Genes that control cell cycle check points have been identified in multicellular organisms and in yeast where they play an essential role in regulating the cell cycle. It is even possible that cell cycle check points may serve as switch points for choosing between cell proliferation and apoptosis. Thus, when a gene that controls a checkpoint is deleted, mutated, amplified in the genome, or otherwise aberrantly expressed in the cell, it may divert the cell into aberrant proliferation. Similarly, when a gene that normally controls a cell cycle switch point leading to apoptosis is aberrantly expressed, it may result in abnormal cell proliferation.
Some mammalian genes and proteins that have been simultaneously implicated in the regulation of cell proliferation and apoptosis including the genes for p53, BCL-2 or Myc. Although the pathways leading to apoptosis have not been fully elucidated, several genes that play a role in apoptosis have also been shown to play an important role in cancer. The p53 gene, coding for a tumor suppressor, is required for radiation-induced apoptosis (Lowe et al., Nature 362:847-849, 1993). BCL-2 inhibits apoptosis in many cells (Sentman et al.,
Cell
67:879-888, 1991; Vanhaesebroeck et al.,
Oncogene
8: 1075-1081, 1993) and furthermore, increased BCL-2 gene expression has been detected in primary breast cancer tissue without bcl-2 gene amplification (Nathan et al.,
Ann. Oncol
. 5:409-414, 1994).
Apoptosis can also be induced by exposing cells to Diphtheria toxin (DT) or Pseudomonas toxin (PE) (Kochi, S. K., and Collier, R. J.,
Exp. Cell Res
. 208: 296-302, 1993; Chang, M. P., et al.,
J. Biol. Chem
. 264: 15261-15267, 1989; Morimoto, H., and Bonavida,
B., J. Immunol
. 149: 2089-2094, 1992). Both of these bacterial toxins inhibit eukaryotic protein synthesis by inactivating elongation factor 2 by ADP-ribosylation (Carroll, S. F. and Collier, R. J.,
J. Biol. Chem.
262: 8707-8711, 1987). Although the toxin-specific mechanism by which these toxins induce apoptosis is unknown, it is not simply due to inhibition of protein synthesis because other protein synthesis inhibitors do not induce apoptosis (Chang, M. P., et al.,
J. Biol. Chem
. 264: 15261-15267, 1989; Morimoto, H., and Bonavida, B.,
J. Immunol
. 149: 2089-2094, 1992).
SUMMARY OF THE INVENTION
According to one aspect of the invention, there is provided a purified and isolated cDNA coding for a human CAS protein having the sequence of SEQ ID NO:1.
According to another aspect of the invention, there is provided a purified and isolated cDNA sequence coding for a portion of a human CAS protein consisting of nucleotides 2100 to 2536 of SEQ ID NO:1.
According to another aspect of the invention, there is provided a purified and isolated human CAS protein having of the amino acid sequence of SEQ ID NO:2.
According to yet another aspect of the invention, there is provided a purified and isolated human CAS protein consisting of the amino acid sequence corresponding to amino acids at positions 700-845 of SEQ ID NO:2.
According to yet another aspect of the invention, there is provided a method of detecting cancerous cells, comprising measuring expression of a human CAS gene at a level higher than expression of a CAS gene in normal noncancerous human cells. In one embodiment of the method, the CAS gene expression is measured by detecting the level of CAS mRNA in cells. The CAS mRNA can be detected by hybridization with a complementary DNA or by a polymerase chain reaction. In another embodiment, CAS gene expression is measured by detecting the level of CAS protein in cells. In a preferred embodiment, the CAS protein is detected by binding of antibody that recognizes CAS protein.
According to another aspect of the invention, there is provided a method of detecting cancer cells, including measuring the number of copies of a CAS gene present in cells. In this method, the number of copies of a CAS gene is higher in cancer cells than the number of copies of a CAS gene in normal noncancer cells. In one embodiment of the method, the number of copies of a CAS gene is measured by detecting the amount of hybridization of a DNA complementary to SEQ ID NO: 1 or any portion of 25 or more nucleotides therein. In another embodiment, the method further includes measuring the number of copies of a single copy gene present in both cancerous and normal noncancerous cells and comparing the number of the single copy gene to the number of copies of a CAS gene in both cancerous and normal noncancerous cells. In a preferred embodiment, the single copy gene is an actin gene. In another embodiment of the method, the number of copies of a CAS gene is determined by a polymerase chain reaction that amplifies either a 5′ portion of the CAS gene, a 3′ portion of the CAS gene, or both a 5′ and a 3′ portion of the CAS gene. In preferred embodiments of the method, the cancer cells are breast tissue cells or colon cells.
According to another aspect of the invention, there is provided a method of treating cancer in a mammal in need of treatment, including administering to the mammal an agent that decreases CAS protein activity in cells. In one embodiment of the method, the agent is a
Brinkmann Ulrich
Pastan Ira
Andres Janet L
Eyler Yvonne
Knobbe Martens Olson & Bear LLP
The United States of America as represented by the Department of
LandOfFree
Method for detecting cancer cells does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method for detecting cancer cells, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method for detecting cancer cells will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2556652