Nuclear degeneration in the developing lens and its regulation by TNFalpha.
DNA fragmentation in lens fibre cell nuclei undergoing programmed degeneration was identified by terminal deoxynucleotidyl transferase (TdT)-mediated biotin-dUTP nick end labelling (TUNEL). Lens epithelial cells in culture were induced to differentiate into lens fibre-like clumps of cells (lentoids) by insulin and it was shown that the TUNEL method was also an effective means of labelling degenerating nuclei in lentoid cells in lens epithelial cell cultures. Using immuno-fluorescence and confocal microscopy, it was shown that TNFalpha and TNF receptor (TNFR1, and TNFR2) immunoreactivity was present in sections of chick embryo lenses. TNFalpha immunoreactivity was associated with the lens epithelium and lens fibres. TNFR1 immunoreactivity was present in lens epithelial cells, cortical lens fibres, and lens fibre cell nuclei, while TNFR2 immunoreactivity had a similar distribution to that of TNFR1, but was not associated with nuclei. Similar patterns of TNFalpha, TNFR1, and TNFR2 immunoreactivity were observed in lens epithelial cell cultures. When added to lens epithelial cell cultures, TNFalpha, at concentrations of 50 to 100 ng ml-1, and agonistic antibodies to both TNFR1 and TNFR2 significantly (P<0.05) enhanced the number of degenerating (TUNEL-positive) nuclei. On the other hand, a neutralising antibody to TNFalpha significantly (P<0. 05) reduced the number of TUNEL-positive nuclei. These results demonstrate that TUNEL is an effective means of labelling degenerating lens fibre nuclei during lens fibre and lentoid differentiation, and suggest a potential role for TNFalpha-like factors and their receptors in the degeneration of lens fibre cell nuclei during lens differentiation. We further suggest that the nuclear degeneration of lens fibre cells is analogous to the nuclear events that occur during apoptosis, and that in lens cells the nuclear degeneration is uncoupled from the plasma membrane events of apoptosis that normally lead to cell death.
Wride
M A
MA
Department of Physiology, University of Alberta, Edmonton, Alberta, T6G 2H7, Canada.
Sanders
E J
EJ
D000818:Animals; D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis; D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism*; D002478:Cells, Cultured; D002642:Chick Embryo; D053938:DNA Fragmentation; D004253:DNA Nucleotidylexotransferase / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D007908:Lens, Crystalline / Q000196:embryology* / Q000378:metabolism; D018613:Microscopy, Confocal; D011419:Propidium / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D047990:TNF Receptor-Associated Factor 1; D047992:TNF Receptor-Associated Factor 2; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
D017304:Annexin A5; D011506:Proteins; D047990:TNF Receptor-Associated Factor 1; D047992:TNF Receptor-Associated Factor 2; D014409:Tumor Necrosis Factor-alpha; D011419:Propidium; D004253:DNA Nucleotidylexotransferase