Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-01-07
2002-02-05
Crouch, Deborah (Department: 1632)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S029000, C435S366000, C435S371000
Reexamination Certificate
active
06344324
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the fields of molecular biology and toxicology. More specifically, the present invention relates to a molecularly engineered cell line that functions as a reporter system for the detection of cellular injury.
2. Description of the Related Art
Exposure of cells to genotoxic agents results in the induction of a number of damage response genes. In bacteria, damage to DNA or inhibition of its replication invokes the well-characterized SOS response with the induction of approximately 20 different genes (1). In yeast, an even larger number of genes are involved in the cellular injury response, and the identity of some of those responsible for executing cell cycle arrest and detecting and repairing DNA damage are now known (2,3). Thus, it is not surprising that the expression of quite a variety of genes is altered in response to cellular injury in mammalian cells (4). These genes are of interest, in part, because of the possibility that changes in their expression can be used as a surrogate for the early detection of cellular injury and quantification of its extent.
Among the injury-inducible mammalian genes, GADD153 may be as a candidate for early detection of cellular injury because of the large magnitude of the increase in its mRNA. GADD153 was originally cloned by hybridization subtraction of mRNA from CHO cells before and after UV irradiation (5). It is one of 5 “growth arrest and DNA damage” genes found to be coordinately induced by either growth arrest or DNA damage (6). GADD153 is highly conserved in mammalian species; hamster GADD153 shares 78% nucleotide sequence identity with the human exons (7) and >85% with the mouse exons (8). Although the mechanism responsible for the activation of GADD153 expression after DNA damage is still unclear, current evidence suggests that the magnitude of the increase is proportional to the extent of cellular injury with maximal GADD153 promoter activity occurring under circumstances of severe toxicity to the cell (9-13).
Current in vitro detection of cellular injury is normally accomplished by measuring the number of cells that survive exposure to the drug. A variety of assays exist, but all of them require a period of several days to weeks of cell growth. The detection of cellular injury in vivo is much more problematic, and is generally measured as gross change in the size of a tumor mass in the living host. Current screening using tumor models for the evaluation of candidate compounds based on tumor growth delay or survival is costly, labor-intensive, relatively insensitive and requires days to weeks of monitoring.
In experimental systems there are several ways to monitor the GADD153 promoter activity as a surrogate for cellular injury detection. These include linking GADD153 promoter fragment to the coding sequences of the chloramphenicol acetyltransferase gene (10), the beta-galactosidase gene (14), and firefly (15) or bacterial luciferase gene (11, 16). The ability of those reporter enzymes to catalyze substrates reflects the activity of GADD153 promoter. However, such enzyme-related assays require additional substrates or cofactors, are inconvenient and of limited use with living tissue.
Hence, the prior art is deficient in the lack of an effective reporter system that can readily quantifies cellular injury and be adapted for high throughput in vitro and in vivo screening. The present invention fulfills this long-standing need and desire in the art.
SUMMARY OF THE INVENTION
The present invention consists of a molecularly engineered cell line that functions as a reporter system for the detection of cellular injury. To produce this cell line a chimeric gene containing the GADD153 promoter linked to the coding region of an enhanced green fluorescent protein (EGFP) gene was stably integrated into the genome of UMSCC10b head and neck carcinoma cells. The molecularly engineered cell line is identified as the T10b45 cell line. Activation of the GADD153 promoter within the T10b45 cells grown in vitro or in vivo by agents or environmental factors injurious to the cell that act via a wide variety of different mechanisms can be quantified using flow cytometric measurement of EGFP expression following the treatment. Moreover, the reporter system is able to discriminate the relative potency of drug-DNA adducts, making it feasible to utilize it to categorize analogs which produce similar types of DNA adducts.
The successful use of the GADD153 promoter to create the T10b45 reporter system validates the concept that injury-responsive promoters can be used to develop reporter systems. It would be obvious to someone skilled in the art that clones of either the UMSCC10b cells, or other types of cells, could be constructed using other injury-responsive promoters capable of reporting on the activation of specific intracellular pathways or specific types of cellular injury.
The present invention features a method to use GADD153-driven EGFP expression to monitor cellular injury induced by a variety of genotoxic agents such as DNA cross-linking and methylating agents, oxygen free radicals, DNA intercalators, UV and &ggr;-radiation, and hypoxia.
The present invention also features a method to use GADD153-driven EGFP expression to monitor cellular injury in vivo.
In yet another embodiment of the present invention, there is provided a method to use GADD153-driven EGFP expression to categorize the relative potency of adducts produced by related platinum-containing drugs.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.
REFERENCES:
Ramiro et al. Enhanced green fluorescent protein as an efficient reporter gene for retroviral transduction of human multipotent lymphoid precursors. Human gene therapy. vol. 9, 1103-1109, 1998.*
Luethy et al. Activation of the gadd 153 promoter by genotoxic agents: A rapid and specific response to DNA damage. Cancer Research. vol. 52, pp. 5-10, 1992.
Gately Dennis P.
Howell Stephen B.
Lin Xinjian
Adler Benjamin Aaron
Crouch Deborah
Research Development Foundation
LandOfFree
Quantification of cellular injury using expression of a... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Quantification of cellular injury using expression of a..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Quantification of cellular injury using expression of a... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2942606