Assays Based on the Role of Dystroglycan and Alpha-Dystroglycan Proteolysis in Tumor Cell Growth
PATENT SUMMARY: The present invention relates to methods and compositions for the diagnosis and treatment of cells lacking normal growth arresting characteristics. This characteristic is referred to as "tumorigenicity," which means the properties of a cell normally associated with tumor forming properties, especially, growth arresting properties, normal cell arrest, and appearance in the 3D-BM assay. Normal, non-tumorigenic cells will be polarized and, in the case of mammary epithelial cells, form acini with regulated growth properties. In the case of tumorigenic cells, the cells are disorganized and sometimes invasive, and exhibit abnormal growth.
It has been found that many tumor cells lack normal cell surface Éø-dystroglycan and thereby lack dystroglycan function. Re-establishment of dystroglycan function has been achieved in one cell line by transfection and over-expression of the dystroglycan gene. By re-establishing dystroglycan function, the once tumorigenic cells reverted to non-tumorigenic cells which polarized and arrested growth in the presence of basement membrane proteins, demonstrating that dystroglycan functions as a tumor suppressor. Loss of a tumor suppressor function, like that of dystroglycan, facilitates the development of tumors, therefore, cells lacking a tumor suppressor are said to have a higher "potential tumorigenicity". In some cases, loss of a single tumor suppressor, like dystroglycan, can indicate a tumorigenic state, and in other cases additional changes to the cell are required before it becomes capable of forming tumors. For the purpose of this application, either case is described as a higher potential tumorigenicity.
Most importantly, it has been found that dystroglycan can be lost from the cell surface by proteolytic shedding; some tumors cells shed a fragment of Éø-dystroglycan into the surrounding medium. These forms of Éø-dystroglycan are distinguishable because normal Éø-dystroglycan has a molecular weight of 180 kD, while the shed fragment has a molecular weight (Mr) of 120-130 kD (FIGS. 1A). As is known in the field, the term "Mr" refers to relative mobility on electrophoretic gels. This shedding is inhibited by the presence of metaloproteinase inhibitors (FIG. 1B and C). The present assays may be carried out on tissue samples, the cells themselves, or on the surrounding medium. In vivo, the surrounding medium will comprise the blood and its serum.
Using the above information, one can measure the potential tumorigenicity of cells by assaying for the presence of a fragment of Éø-dystroglycan in medium, particularly fragments having an Mr of 120-130 kD. Identifying the presence of the Éø-dystroglycan fragment indicates a higher potential tumorigenicity.
Using the above information, one can also measure the potential tumorigenicity of cells by assaying to determine the ratio of the total amount of Éø-dystroglycan present in a cell sample relative to the amount of É¿-dystroglycan present in the sample. A ratio showing a deficiency of Éø-dystroglycan relative to É¿-dystroglycan indicates Éø-dystroglycan shedding. A correlation between tumorigenicity and the loss of Éø-dystroglycan through proteolysis has been shown. Treatment of the tumorigenic cells with a metaloprotease inhibitor, at concentrations that inhibit dystroglycan shedding, reverses the tumorigenic phenotype (FIG. 3). Furthermore, treatment of cells with a genetic construct for Éø-dystroglycan also reverses the tumorigenic phenotype.
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