Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-10-13
2003-07-15
Fredman, Jeffrey (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091500, C435S091200, C436S501000, C536S024300, C536S024330, C536S026600
Reexamination Certificate
active
06593084
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The present invention is drawn to the field of biotechnology and more specifically to biomedical applications. Further, this invention relates to novel assays for testing whether a compound is a carcinogen. Differential patterns of gene expression for test chemical treated and untreated mammalian cells are compared to those patterns established for known carcinogens.
BACKGROUND OF THE INVENTION
The current “gold standard” for testing whether a compound is carcinogenic is performed with both sexes of two animal species that are treated chronically for two years with a dose of compound at or near the maximum tolerated dose. The end point of the assay is tumor formation. These assays are both expensive and laborious. The aim of this invention is to greatly shorten the time required to determine whether a chemical compound is a carcinogen.
Carcinogens act upon a cell in several ways, with the common result by definition being induction of neoplasia or tumor formation. Carcinogens may be chemical or biological agents. Chemical carcinogens include, for example, compounds of: DMBA; benzo(a)pyrene; dibenzanthracene; acetoxyaminofluoren, methyl N. nitrosogreoxidine; nitroquinoline; dibromomethane; dibromoethane; furan; and 12-O-tetradecanoyil-13-phorbol-acetate (TPA). Biological carcinogens include oncogenic viruses, for example, such as members of the family of papilloma virus. Chemical compounds and biological agents have the shared property that they are each able to cause mutations of the genetic material of an animal cell or otherwise alter the level of expression of non-mutated genes. Despite the different mechanisms of action of carcinogens, these compounds each produce changes in gene expression that occur in the transition from normal to tumor cell. Changes in the transcription level may be detected by differential displays of mRNA isolated from a cell treated with a carcinogen. The changes in the level of translation of the mRNA may also be detected in the differential display of proteins or peptides synthesized in the treated cell.
Differential display of mRNA has been articulated as a means of evaluating the gene expression profile of a cell for the identification of cancer related genes and elucidation of their role in tumorigenesis. For example, more than 100 candidate tumor suppressor genes have been described. (Sager, R.,
Proc. Natl. Acad. Sci. USA
(1997) 94:952-955). Some of these have been identified using differential display of cDNA prepared from isolated mRNA. While more than 100 oncogenes have been identified in animal cells, only a small subset have been found consistently as mutated genes in human cancer (Copper G. M. (1995)
Oncogenes;
Jones & Bartlett, Seedbury); Weinberg, R. A.
Sci. Am.
(1996) 275:62-70; and Bishop, J. M.
Cell
(1991) 64:235-248). The progression of neoplasia development and transformation into malignant tumors is not well understood. However, since cancer genes are defined by their phenotypic expression, research into their differential phenotypic expression has been undertaken as a means to further elucidate the process of carcinogenic induction of tumorigenesis.
For example, carcinogen-mediated changes in cell metabolism may differentially alter the expression of oncogenes, tumor suppressor genes, cell cycle regulators, DNA replication genes (e.g., encoding repair enzymes, receptors), growth factors, stress proteins or known tumor markers. Such differential gene expression may be represented by an increase or decrease of levels of expression or may result in modifications to the gene product expressed, that is, altered forms of proteins, which function differently than proteins in normal cells (e.g., greater or lesser binding affinity; requirement or lack of requirement of co-factors).
Many studies have addressed the changes in genes (mutations) and the changes in gene expression in tumors. These alterations in sequence and synthesis have also been examined as markers of tumorigenesis. However, no one has examined the early changes in gene expression in the first days or weeks after the beginning of treatment with a test compound to determine whether a subset of the changes that occur early in carcinogen treatment are useful to predict whether a tested compound is a carcinogen. The pattern that is developed need not be similar to that in the tumor but need only be predictive of tumorigenic potential.
Seven cDNA fragments of gene transcripts that were overexpressed in mammary carcinomas in the rat model induced by 1-methyl-1-nitrosourea have been identified by differential display of mRNA and molecular cloning (Lu, J., et al.,
Molecular Carcinogenesis
(1997) 20:204-215). However, this research involved the comparison of the latter stage of tumorigenesis (2 months following chemical carcinogenic induction) with control mammary tissue. Lu et al. did not disclose any methods for the differential display of gene transcript fragments associated with the earlier stages of tumorigenesis, that is, prior to actual tumor formation.
Similarly, there are numerous reports in the literature of the identification and role of various oncogenes and tumor suppressor genes. For example, the use of differential display to study gene expression during development of squamous cell carcinomas and the important role played by c-fos (or v-fos) in the transformation to the malignant phenotype in keratinocytes has been reported (Rutberg, S., et al.,
Molecular Carcinogenesis
(1997) 20:88-98). Rutberg et al., as did Lu et al., used the latter stage endpoint of tumor formation in the comparison of differential gene expression in establishing the role of oncogene transformation of epithelial tissue.
There is a clear need for assays which predict at an early stage whether an unknown compound is a carcinogen. Current assays are designed to determine whether an agent is carcinogenic by using one or more surrogate markers. For example, the widely used Ames test represents an analysis of the effects of a test compound upon the frequency of reverse mutation of bacterial test strains, such as the histidine auxotrophic strains of
Salmonella typhimurium
(
Manual of Methods for General Bacteriology,
Gerhardt et al, American Society for Microbiology Press, Washington, D.C. (1981) at page 237). This test is not absolute, since a compound under this assay may be mutagenic and not be carcinogenic or alternatively a compound may not evidence mutagenicity in this assay but be carcinogenic as determined using other tests.
Current short-term assays are based mostly on correlation between mutagenic and carcinogenic potential of compound that was established in earlier studies. This correlation is exploited directly in Salmonella mutation assay (Ames test), in the mouse tk
+/−
assay, and CHO hprf assay. There are also assays based on abilities of carcinogens to interact with DNA or chromatin directly, like DNA adducts assay and single cell gel assay for DNA strand breaks. The mechanisms underlying other assays are less clearly defined, like for micronucleus assay, and cytogenetic assays (FISH, aneuploidy or sister chromatid exchange assays) but are thought to reflect at least in part, the disturbance of the DNA or chromosome organization. These assays show good sensitivity for mutagenic carcinogens but as a rule cannot detect effectively activity of the nongenotoxic carcinogens.
Recently a new test, the Syrian Hamster Embryo (SHE) Cell Transformation assay was developed and refined, which is based on the ability of carcinogens to induce immortalization and transformation (morphological and malignant) of the SHE cells in culture (Isfort and LeBoeuf,
Mutation Research
(1996) 365:161-173). This assay shows good sensitivity and accuracy for mutagenic carcinogens as well as nongenotoxic carcinogens.
Application of the battery of mentioned short-term tests increases the validity of prediction of whether compounds may be carcinogens. However, tumorigenic transformation of a cell in an organism is a result of the complex interaction of th
Bird Robert Earl
Borellini Flavia
Glebov Oleg K.
Jacobson-Kram David
Ostrove Jeffrey M.
Bird Robert E.
Burns Doane Swecker & Mathis L.L.P.
Chakrabarti Arun Kr.
Fredman Jeffrey
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