The ability of organisms to prevent cells which have damaged or amplified DNA from replicating, to repair DNA damage, or to terminate cells which have irreparable DNA damage are important defenses against cancer. Cells which have unrepaired mutations and are permitted to divide may form tumors.
Certain human diseases arise from an inherited inability to repair DNA, to prevent DNA damage or to prevent propagation of cells with damaged DNA. As a result, victims have a high rate of cancer. When these cells are contacted by carcinogens, they are unable to repair the DNA damage and the cells are tumorigenically transformed. For example, xeroderma pigmentosum, Cockayne's syndrome and trichothiodystrophy arise from an inability of a cell to perform nucleotide excision repair. These cells are unable to repair damage from alkylating agents and other agents which induce bulky additions to DNA. Fanconi's anemia induces a sensitivity to crosslinking agents such as nitrogen mustard, mitomycin C, and cisplatin. Cells from patients with ataxia-telangiectasia are sensitive to ionizing radiation and to oxidative stress because they are able to continue to divide despite unrepaired DNA damage. Cells from patients with amyotrophic lateral sclerosis are unable to repair oxidative damage caused by active oxygen free radicals because they lack superoxide dismutase. Hereditary nonpolyposis colon cancer occurs as a result of failure of excision repair and mismatch repair.
DNA damage occurs as a result of exposure to mutagens or as a longer term result of exposure to genotoxic or nongenotoxic carcinogens. Mutagenic carcinogens are usually electrophiles or are capable of metabolic conversion to electrophiles which attack DNA causing base alteration and mutation. Nonmutagenic carcinogens induce cell proliferation and DNA synthesis by a variety of biochemical mechanisms eventually resulting in genome alteration; but they are not initially mutagenic. Some metal cations such as vanadate act as mitogens or alter protein phosphorylation.
New chemicals are constantly produced either for consumer use or as by-products into the environment. These potential human carcinogens are tested in cultures of prokaryotes or lower eukaryotes, in living rodents and in mammalian cells in tissue culture. Although these tests are reproducible, reliable, quick, relatively inexpensive and do not sacrifice higher animals, they are inadequate for testing human carcinogens.
Cell transformation assays can detect both mutagenic and nonmutagenic carcinogens. Therefore, presumably, a chemical that induces or promotes transformation is a carcinogen. To investigate chemical carcinogenesis and mechanisms or transformation, several assays have been developed which rely on cell transformation. (See, e.g., DiPaolo, J. A. et al. (1969) "Quantitative Studies of in vitro Transformation by Chemical Carcinogens," J. Natl. Cancer Inst., 42:867; Reznikoff, C. A. et al. (1973) "Establishment and Characterization of a Cloned Line of C3H Mouse Embryo Cells Sensitive to Postconfluence Inhibition of Cell Division," Cancer Res. 33:3231; Kakunaga, T. (1973) "A Quantitative System for Assay of Malignant Transformation by Chemical Carcinogens Using a Clone Derived from BALB/c3T3," Intl. J. Cancer, 12:463). Transformed foci are the endpoint in these assays.
These tests, however, suffer from lack of reproducibility from laboratory to laboratory, technical difficulties, and difficulties in scoring foci as there are several different types of foci. Due to the low transformation frequency, large numbers of plates must be used to obtain statistically significant results for weak carcinogens. Moreover, these tests do not specifically test for human carcinogens, nor are these prior tests based on the inability of test cells to repair DNA, to prevent DNA damage or to prevent propagation of cells with damaged DNA.
A few assays have been described which can be used to detect potential mutagens in mammalian or specifically, human, cells (See, for example, Calos, 1988, U.S. Pat. No. 4,753,874, Schiestl, 1993, U.S. Pat. No. 5,273,880, Reddel et al., 1989, U.S. Pat. No. 4,885,238, Skopek et al., 1981, U.S. Pat. No. 4,302,535; Harris et al., 1996, U.S. Pat. No. 5,506,131; Crespi et al., 1995, U.S. Pat. No. 5,429,948; and States et al., 1993, U.S. Pat. No. 5,180,666). The assays, however, do not use transformation as an endpoint, but rely on more complex endpoints for detection of carcinogens which are only useful for detecting genotoxic or mutagenic carcinogens. Therefore, the assays described prior to the present invention are not designed to detect non-genotoxic carcinogens and/or tissue-specific carcinogens. These assays also suffer from technical complexity and limited commercial availability. Moreover, these assays do not identify the biochemical mechanism of carcinogenicity of carcinogenic compounds. Therefore, there exists a need for improved transformation assays for rapid and reliable screening for mutagenic, genotoxic, nongenotoxic and tissue-specific carcinogens.
USPTO Backgrounds
USPTO Backgrounds