Retention of lens specificity in long-term cultures of diploid rabbit lens epithelial cells

Abstract

Abstract. Rabbit lens epithelial cells from newborn animals exhibited limited growth when cultured under standard conditions. Cell lines were generated when explants from individual lenses were cultured in medium supplemented with conditioned medium or untreated rabbit serum. All lines exhibited a stable epithelial morphology. One line, N/ N1003A. was examined extensively with respect to its growth, ploidy, and maintenance of lens‐specific functions. Cells at population‐doubling level (pdl) 120 exhibited a normal chromosomal banding pattern, were diploid, were non‐tumorigenic in vivo, did not grow in suspension culture, and did not exhibit sustained growth in medium supplemented with low concentrations of serum. The shape of the growth curves and the final density for cells at pdl 24 and 181 exposed to various concentrations of serum were identical. The cells showed no diminution in growth as a function of in vitro age. The cells retained lens‐specific functions. Proteins were isolated from cells at pdl 40 and 170, and were separated on polyacrylamide gels. Western immunoblot analysis using antiserum to α‐crystallin, a tissue‐specific protein found in lens epithelial cells in vivo, indicated the presence of α‐A‐ and α‐B‐crystallin polypeptides. The cells also contained the transcription factors required for activating the murine α‐A‐crystallin gene promoter. which is known to function with precise tissue specificity. When an expression vector including the bacterial chloramphenicol acetyltransferase (CAT) gene controlled by the α‐A‐crystallin gene promoter was introduced into the lens epithelial cells, the CAT gene was expressed. This did not occur when this construct was introduced into cultured rabbit kidney epithelial cells. Cultured lens epithelial cells provide a stable system for investigating the factors that regulate growth, differentiation, and crystallin gene expression in lens epithelial cells.

Okadaic acid‐induced lens epithelial cell apoptosis requires inhibition of phosphatase‐1 and is associated with induction of gene expression including p53 and bax

“…The rabbit lens epithelial cells (RLECs), N/ N1003A, was a gift from Dr John Reddan [55]. N/N1003A cells were grown in Eagle's minimal essential medium (MEM; Sigma M0643) containing 10% rabbit serum (Sigma R4505).…”

Mafs, Prox1, and Pax6 Can Regulate Chicken βB1-Crystallin Gene Expression

“…Previous in situ hybridization studies showed that MafB mRNA was present in the embryonic lens (8,46). Expression of MafB in the adult lens was determined by Western immunoblotting of extracts obtained from microdissected adult lens fiber cells, lens epithelial cells, and extracts of two lens-derived cell lines, ␣TN4-1 (43) and N/N 1003A (42). MafB protein was detected in microdissected lens epithelial cells as well as in the established lens epithelial cell lines, but not in the fiber cells ( Fig.…”

“…Cell Culture and Transfection-N/N 1003A cells (a rabbit lens epithelium derived cell line) (42) were transfected following the LipofectAMINE Plus reagent protocol provided by Invitrogen. Generally 6 ϫ 10 5 cells were plated for each 60-mm dish at least 8 h before transfection, each dish received 2.5 g of promoter/chloramphenicol acetyltransferase (CAT) plasmid, 0.5 g of pCMV/␤GAL, and 0.5 g of various expression plasmids.…”

Human Bcl-2 activates ERK signaling pathway to regulate activating protein-1, lens epithelium-derived growth factor and downstream genes

“…RLECs, N/N1003A (a kind gift from Dr John Reddan) (Reddan et al, 1986), were grown in Eagle's minimum essential medium containing 10% rabbit serum as described previously (Mao et al, 2001). The medium was prepared in ion-exchanged double-distilled water to give an osmolarity of 30075 mosmol supplemented with 26 mM NaHCO 3 and 50 mg/ ml gentamycin sulfate.…”