Induction of apoptosis by overexpression of the DNA-binding and DNA-PK-activating protein C1D

Apoptosis is induced in various tumor cell lines by vector-dependent overex pression of the conserved gene C1D that encodes a DNA-binding and DNA-PK-ac tivating protein. C1D is physiologically expressed in 50 human tissues test ed, which points to its basic cellular function. The expression of this gen e must be tightly regulated because elevated levels of C1D protein, e.g. th ose induced by transient vector-dependent expression, result in apoptotic c ell death. Cells transfected with C1D-expressing constructs show terminal d eoxynucleotidyl transferase-mediated dUTP nick end-labeling of DNA ends. Tr ansfections with constructs in which C1D is expressed in fusion with the (e nhanced) green fluorescent protein from A. victoria (EGFP) allow the transf ected cells to be identified and the morphological changes induced to be tr aced. Starting from intense nuclear spots, green fluorescence reflecting C1 D expression increases dramatically at 12-24 hours post-transfection, Expre ssion of C1D-EGFP protein is accompanied by morphological changes typical o f apoptotic cell death, e.g. cytoplasmic vacuolation, membrane blebbing and nuclear disintegration. Cell shrinkage and detachment from extracellular m atrix are observed in monolayer cultures while suspension cells become prog ressively flattened. The facility to differentiate between transfected and nontransfected cells reveals that non-transfected cells cocultured with tra nsfected cells also show the morphological changes of apoptosis, which poin ts to a bystander effect. C1D-dependent apoptosis is not induced in cells with non-functional p53, Ac cordingly, C1D-induced apoptosis is discussed in relation to its potential to activate DNA-PK, which has been considered to act as an upstream activat or of p53.