Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-07-25
2004-06-08
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S029000, C435S032000, C435S254110, C435S483000, C435S484000
Reexamination Certificate
active
06746843
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the fields of cell biology, molecular biology and oncology. More particularly, it concerns the use of
Dictyostelium discoideum
cells in the screening of chemical agents for use in cancer therapy and cancer prevention.
2. Description of Related Art
Cells are continually exposed to a variety of extracellular insults that can damage their DNA, such as UV light, ionizing radiation and chemicals. DNA damage can lead to mutations and ultimately cancer. It is ironic that the radio- and chemotherapies that are used to treat many malignances will eventually result in the selection of resistant tumor cells, or will cause secondary tumors by virtue of their DNA damaging capacity. Clearly, a full understanding of the mechanisms of DNA damage, DNA repair and drug resistance is essential for the effective prevention and treatment of cancer.
All species have mechanisms for repairing DNA damage. These mechanisms are highly specific, and allow the cell to recognize a specific type of damage and mount an appropriate response. Moreover, these systems are highly conserved throughout species, and information that is obtained from studying model organisms is relevant to humans.
SUMMARY OF THE INVENTION
Thus, in accordance with the present invention, there is provided a method of screening agents for use in the prevention or treatment of cancer comprising (a) contacting a vegetative cell of
Dictyostelium discoideum
with a test agent; (b) assessing the cytotoxicity of said test agent; (c) assessing the effect of said test agent on the expression of one or more of repB, repD and APE gene products; and (d) comparing said cytotoxicity and said expression in the presence of said test agent with a vegetative cell of
Dictyostelium discoideum
not exposed to said test agent; wherein (i) a test agent that is cytotoxic but does not induce expression of one or more of repB, repD and APE gene products will be useful as a chemotherapeutic; (ii) a test agent that is not cytotoxic but does induce expression of one or more of repB, repD and APE gene products will be useful as a chemopreventative; and (iii) a test agent that inhibits the expression of one or more of repB, repD and APE gene products will be useful as a chemotherapeutic when applied in combination with a DNA damaging agent.
Assessing expression may comprise assessing repB expression, assessing repD expression, assessing APE expression, assessing repB and repD expression, assessing repB and APE expression, assessing repD and APE, or assessing repB, repD and APE expression. In an additional step, one may measure, in a vegetative cell of
Dictyostelium discoideum
not treated with said test agent, the expression of the same gene or genes as set forth above.
Cytoxocity may be assessed by measuring viability by clonal plating, trypan blue exclusion, phyloxine B dye exclusion, and degradation/laddering of DNA. Expression may be assessed by hybridization of a probe to a target nucleic acid, including RT-PCR™ and “real time” PCR™. One or more probes may comprise a label, such as a radiolabel, a fluorophore label, a chemilluminescent label, an enzyme label or a ligand (such as biotin, where the ligand is detected by contacting with enzyme-conjugated avidin or streptavidin and a detectable enzyme substrate). The method may also comprise binding target nucleic acid to a substrate, such as a nylon or nitrocellulose membrane.
Expression also may be assessed by means of an expression cassette stably transformed into said a vegetative cell of
Dictyostelium discoideum
, said expression cassette comprising a nucleic acid segment encoding a detectable reporter enzyme under the transcriptional control of a repB, repD or APE promoter region. The detectable reporter enzyme may encode &bgr;-galactosidase, &bgr;-glucuronidase, luciferase or green fluorescent protein.
The assay may further comprise a positive control for inhibition of expression of one or more of repB, repD and APE gene products, a positive control for induction of expression of one or more of repB, repD and APE gene products, and/or a positive control for cytotoxicity.
The assay may further comprise a negative control for inhibition of expression of one or more of repB, repD and APE gene products, a negative control for induction of expression of one or more of repB, repD and APE gene products, and/or a negative control for cytotoxicity.
The test agent may be a naturally-occurring molecule, a synthetic molecule, or a synthetic derivative of a naturally-occurring molecule. The method also may further comprise assessing DNA damage in said cell, for example, by mass spectroscopy.
In another embodiment, there is provided a vegetative cell of
Dictyostelium discoideum
stably transformed with an expression cassette comprising a nucleic acid segment encoding a detectable reporter enzyme under the transcriptional control of a repB, repD or APE promoter region.
In still yet another embodiment, there is provided a method of making a compound for use in the prevention or treatment of cancer comprising (a) contacting a vegetative cell of
Dictyostelium discoideum
with said compound; (b) assessing the cytotoxicity of said compound; (c) assessing the effect of said compound on the expression of one or more of repB, repD and APE; (d) comparing said cytotoxicity and said expression in the presence of said compound with a vegetative cell of
Dictyostelium discoideum
not exposed to said compound; and (e) making said compound.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
Cancer is a leading cause of mortality in both industrialized and non-industrialized countries, with over one million new cases identified each year in the U.S. alone. Progress has been made in the identification of new drugs that, alone or in combination, work to inhibit cancer cells. However, tumors frequently become resistant to chemotherapeutic drugs. Thus, there is a constant need to identify new and more powerful drugs for use in cancer therapy.
1. The Present Invention
The cellular slime mold
Dictyostelium discoideum
offers an amenable system for the analysis of the cellular response to chemotherapeutic drugs. Although a relatively simple haploid organism, this organism exhibits many of the aspects of cellular physiology and development seen in more complex organisms, thus allowing the study of complicated genetic responses that would be impossible using higher organisms. The present inventors propose to utilize their knowledge of Dictyostelium molecular biology to created screening assays for agents that can be used against cancers as well as benign hyperproliferative diseases.
The assays will focus on the expression of two different genes in the Nucleotide Excision Repair (NER) pathway—repB, repD—and the Base Excision Repair (BER) gene APE. The expression of these genes will be correlated to the cytotoxcity of the agent. Three different outcomes for the assays are envisioned:
compounds that are cytotoxic but do not induce expression of repB, repD or APE: compounds will be useful as anticancer drugs because they do not induce expression of repair genes;
compounds that are not cytotoxic but induce expression of repB, repD or APE: compounds of this class will be anti-cancer drug candidates used to prevent cancer by inducing DNA repair; and
compounds that suppress the expression of repB, repD or APE: compounds of this class would preferentially effect rapidly growing cells, and would render them more susceptible to DNA damaging agents
The exploitation of this assay is described in detail in the following pages.
2.
Dictyostelium discoideum
Dictyostelium is a relatively simple eukaryotic organism, but exhibits cellular and developmental properties of higher multicellular organisms. There is a high degree of evolutionary conservation of individual proteins, enzymes and biochemical pathways, including DNA replication and cell cycle control between Dictyostelium and humans. It has a relatively small genome (35 mb), and like human cells,
Alexander Hannah
Alexander Stephen
Fulbright & Jaworski LLP
Ketter James
The Curators of The University of Missouri
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