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J. Biol. Chem., Vol. 278, Issue 38, 36876-36886, September 19, 2003

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Hyperproliferation and p53 Status of Lens Epithelial Cells Derived from B-crystallin Knockout Mice^*

Fang Bai , Jing Hua Xi , Eric F. Wawrousek , Timothy P. Fleming ¶ and Usha P. Andley  || **

From the Department of Ophthalmology and Visual Sciences, ^||Department of Biochemistry and Molecular Biophysics, and ^¶Department of Surgery, Washington University School of Medicine, St. Louis, Missouri 63110 and the NEI, National Institutes of Health, Bethesda, Maryland 20892

B-Crystallin, a major protein of lens fiber cells, is a stress-induced chaperone expressed at low levels in the lens epithelium and numerous other tissues, and its expression is enhanced in certain pathological conditions. However, the function of B in these tissues is not known. Lenses of B-/- mice develop degeneration of specific skeletal muscles but do not develop cataracts. Recent work in our laboratory indicates that primary cultures of B-/- lens epithelial cells demonstrate genomic instability and undergo hyperproliferation at a frequency 4 orders of magnitude greater than that predicted by spontaneous immortalization of rodent cells. We now demonstrate that the hyperproliferative B-/- lens epithelial cells undergo phenotypic changes that include the appearance of the p53 protein as shown by immunoblot analysis. Sequence analysis showed a lack of mutations in the p53 coding region of hyperproliferative B-/- cells. However, the reentry of hyperproliferative B-/- cells into S phase and mitosis after DNA damage by -irradiation were consistent with impaired p53 checkpoint function in these cells. The results demonstrate that expression of functionally impaired p53 is one of the factors that promote immortalization of lens epithelial cells derived from B-/- mice. Fluorescence in situ hybridization using probes prepared from centromere-specific mouse P1 clones of chromosomes 1 and 9 demonstrated that the hyperproliferative B-/- cells were 30% diploid and 70% tetraploid, whereas wild type cells were 83% diploid. Further evidence of genomic instability was obtained when the hyperproliferative B-/- cells were labeled with anti--tubulin antibodies. Examination of the hyperproliferative B-/- mitotic profiles revealed the presence of cells that failed to round up for mitosis, or arrested in cytokinesis, and binucleated cells in which nuclear division had occurred without cell division. These results suggest that the stress protein and molecular chaperone B-crystallin protects cells from acquiring impaired p53 protein and genomic instability.

Received for publication, April 16, 2003 , and in revised form, June 6, 2003.

^* This work was supported by National Institutes of Health Grant R01EY05681 (to U. P. A.) and by National Institutes of Health Core Grant EY02687 and a grant from Research to Prevent Blindness, Inc. to the Department of Ophthalmology and Visual Sciences, Washington University School of Medicine. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** Recipient of the Lew R. Wasserman award from Research to Prevent Blindness, Inc. To whom correspondence should be addressed: Dept. of Ophthalmology and Visual Sciences, Washington University School of Medicine, 660 S. Euclid Ave., Campus Box 8096, St. Louis, MO 63110. Fax: 314-362-3638; E-mail: andley{at}vision.wustl.edu.

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