Purchase this article with an account.
A.J. Maniotis, K. Valyi–Nagy, J. Moses, J. Karavitis, R. Folberg; DNA Sequestration Increases Among More Invasive Uveal Melanoma Cells Because of Changes in the Cytoskeleton and Higher Order Chromatin Structure . Invest. Ophthalmol. Vis. Sci. 2004;45(13):1067.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: We wanted to determine if DNA sequestration and exposure varies in human cells as a function of a cell's normal or invasive capabilities, its higher order chromatin structure, its cytoskeleton, and its place in the cell cycle. Methods: Using a lysed cell model system, we grew uveal melanoma cells on coverslips, treated the cells with Triton–X100, incubated cells with nucleases, and labeled the preparations with DNA–binding dyes. A modified smear preparation was used without using Triton by placing cells on glass slides and allowing them to air–dry, and treating them with restriction enzymes identically as described with the lysed model system. Pharmacological agents were used to interfere with actin, microtubules, intermediate filaments respectively. Results: The nuclei of poorly invasive uveal melanoma cells (OCM–1a) and cells from fibrocystic disease of the breast (MCF–10a) were rapidly digested with Alu and MSP I, but highly invasive uveal melanoma cells (MUM2B) and breast cancer cells (MB231) resisted digestion. Identical differential results were obtained using the smear preparation technique. Only 3 oncogenes were required in order to increase the extent of DNA sequestration in normal fibroblasts. Interference with microtubules, actin and intermediate filaments altered the sensitivity of chromatin to nucleases. Differential sensitivity of microsurgically–removed mitotic chromosomes indicated that differential stability persisted into mitosis: chromosomes removed from more invasive cells were also more resistant to restriction enzymes. Conclusions: Alu and MSP I sequences are more sequestered in the nuclei of highly invasive cells than in poorly invasive cells or normal cells, and this relationship also is present in the genomes of mitotic cells. The trend in increasing sequestration among more highly invasive cells was also dependent upon actin, microtubules, intermediate filaments, and disulfide–rich proteins associated specifically with chromatin. These data suggest a new role for microtubules and intermediate filaments that might be described in terms of them providing the cell with a structural system that exposes Alu and MSP I sequences.
This PDF is available to Subscribers Only