Lens epithelial cells derived from αB‐crystallin knockout mice demonstrate hyperproliferation and genomic instability

Abstract

alphaB-crystallin is a member of the small heat shock protein family and can act as a molecular chaperone preventing the in vitro aggregation of other proteins denatured by heat or other stress conditions. Expression of alphaB-crystallin increases in cells exposed to stress and enhanced in tumors of neuroectodermal origin and in many neurodegenerative diseases. In the present study, we examined the properties of lens epithelial cells derived from mice in which the alphaB-crystallin gene had been knocked out. Primary rodent cells immortalize spontaneously in tissue culture with a frequency of 10(-5) to 10(-6). Primary lens epithelial cells derived from alphaB-crystallin-/- mice produced hyperproliferative clones at a frequency of 7.6 x 10(-2), four orders of magnitude greater than predicted by spontaneous immortalization (1). Hyperproliferative alphaB-crystallin-/- cells were shown to be truly immortal since they have been passaged for more than 100 population doublings without any diminution in growth potential. In striking contrast to the wild-type cells, which were diploid, the alphaB-crystallin-/- cultures had a high proportion of tetraploid and higher ploidy cells, indicating that the loss of alphaB-crystallin is associated with an increase in genomic instability. Further evidence of genomic instability of alphaB-crystallin-/- cells was observed when primary cultures were infected with Ad12-SV40 hybrid virus. In striking contrast to wild-type cells, alphaB-crystallin-/- cells expressing SV40 T antigen exhibited a widespread cytocidal response 2 to 3 days after attaining confluence, indicating that SV40 T antigen enhanced the intrinsic genomic instability of alphaB-crystallin-/- lens epithelial cells. These observations suggest that the widely distributed molecular chaperone alphaB-crystallin may play an important nuclear role in maintaining genomic integrity.

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

“…Our previous work with lens epithelial cells cultured from ␣B knockout mice showed that deletion of the ␣B gene permits the emergence of lens epithelial cells with vastly increased proliferative ability (19). In the present work we elucidated mechanistically how ␣B interacts with proteins involved in cell cycle regulation.…”

“…Lenses were obtained from 6 -12-week-old mice. To obtain primary lens epithelial cells, capsule/epithelial explants of wild type (129Sv) and ␣BϪ/Ϫ mouse lenses were dissected and cultured in 20% fetal bovine serum-minimal essential medium in 35-mm tissue culture plates as described previously (19,20), and cultured for 1-2 weeks until growth of cells was detectable. Cells were passaged using trypsin-EDTA, and plated in 35-mm plates.…”

“…Attachment efficiency determined 2 h after plating was found to be 85%, and was the same for wild type and ␣BϪ/Ϫ cells. Cell numbers were measured with a Coulter counter (19). Hyperproliferative ␣BϪ/Ϫ cells were plated at low densities to examine clonal growth (19).…”

“…Because HSPB2 is not expressed in the lens (18), its deletion is not expected to contribute to lens phenotypes. Lens epithelial cells derived form ␣BϪ/Ϫ mice demonstrate hyperproliferation and genomic instability (19). In contrast, cells derived from mice lacking ␣A-crystallin (␣A), a protein normally associated with ␣B in the lens, have a diminished growth potential (20).…”

The lens epithelium: Focus on the expression and function of the α-crystallin chaperones

“…Primary cultures of αB knockout lens epithelial cells demonstrate genomic instability and undergo hyperproliferation at frequency 4 orders of magnitude greater than that predicted by spontaneous immortalization of rodent cells (Andley et al, 2001). Mechanistic studies demonstrate an upregulation of functionally impaired p53 protein .…”