Hyperproliferation and p53 Status of Lens Epithelial Cells Derived from αB-crystallin Knockout Mice

Abstract

␣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.␣〉-crystallin (␣B) 1 is a stress-inducible chaperone expressed in lens fiber cells and in the lens epithelium. It has also been detected at varying levels in numerous tissues, particularly heart, kidney, skeletal muscle, lung, and brain (1-3). However, the function of ␣B in these tissues remains elusive. A number of recent studies suggest a broad cellular function of ␣B associated with growth (4 -13). ␣A-and ␣B-crystallins have autokinase activity (4). ␣B expression increases in mitotically arrested NIH 3T3 fibroblasts, and in Chinese hamster ovary cells induced to express ␣B, the localization of ␣B changes from the cytoplasm to nucleus during interphase (5, 6). Nearly 20% of ␣B polypeptides isolated from the lens are phosphorylated in vivo, suggesting an association with signal transduction pathways (8, 12). Phosphorylated forms of ␣B have been detected in the centrosomes and midbodies of dividing cells (7). ␣B also accumulates in the developing central nervous syste...

Alpha‐crystallin expression affects microtubule assembly and prevents their aggregation

“…Until recently very little information was available on the effect of expression of ␣A-and ␣B-crystallin on the microtubule cytoskeleton. Our previous work with ␣A-/-and ␣B-/-lens epithelial cells had demonstrated that ␣A-and ␣B-crystallin have important but distinct effects on the integrity of the mitotic spindle microtubules (18,20). A loss of microtubules was observed in some of the anaphase spindles of ␣A-/-and ␣B-/-cells between the separating chromosomes (18,20).…”

“…Our previous work with ␣A-/-and ␣B-/-lens epithelial cells had demonstrated that ␣A-and ␣B-crystallin have important but distinct effects on the integrity of the mitotic spindle microtubules (18,20). A loss of microtubules was observed in some of the anaphase spindles of ␣A-/-and ␣B-/-cells between the separating chromosomes (18,20). The current work suggests that this phenotype may be the result of poor microtubule nucleation and/or destabilization of microtubules.…”

Inhibition of Lens Photodamage by UV-Absorbing Contact Lenses

“…30 Cells were also colabeled with a ␤-tubulin antibody and an Alexa-568 -labeled secondary antibody to allow visualization of the microtubule cytoskeleton. 31 Cells were examined by confocal microscopy. 31 Four random microscopic fields were examined under each condition.…”

Alpha-crystallin: an ATP-independent complete molecular chaperone toward sorbitol dehydrogenase

“…Moreover, mutation or lack of the aA gene induces cataract in mice and humans [13], while mutation or lack of the aB gene causes a desmin-related myopa-* Corresponding author. thy [14] or hyperproliferation and genomic instability, respectively [15,16]. A common feature of sHsps is a conserved C-terminal region of about 90 amino acids called the 'a-crystallin domain,' responsible for their molecular chaperone action.…”