Browsing by Subject "DNA nucleotidylexotransferase"

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Open Access
Induction of apoptosis by overexpression of the DNA-binding and DNA-PK-activating protein C1D
(1999) Rothbarth, K.; Spiess, E.; Juodka, B.; Yavuzer, U.; Nehls, P.; Stammer, H.; Werner, D.
Apoptosis is induced in various tumor cell lines by vector-dependent overexpression of the conserved gene C1D that encodes a DNA-binding and DNA-PK-activating protein. C1D is physiologically expressed in 50 human tissues tested, which points to its basic cellular function. The expression of this gene must be tightly regulated because elevated levels of C1D protein, e.g. those induced by transient vector-dependent expression, result in apoptotic cell death. Cells transfected with C1D-expressing constructs show terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling of DNA ends. Transfections with constructs in which C1D is expressed in fusion with the (enhanced) green fluorescent protein from A. victoria (EGFP) allow the transfected cells to be identified and the morphological changes induced to be traced. Starting from intense nuclear spots, green fluorescence reflecting C1D expression increases dramatically at 12-24 hours post-transfection. Expression of C1D-EGFP protein is accompanied by morphological changes typical of apoptotic cell death, e.g. cytoplasmic vacuolation, membrane blebbing and nuclear disintegration. Cell shrinkage and detachment from extracellular matrix are observed in monolayer cultures while suspension cells become progressively flattened. The facility to differentiate between transfected and non-transfected cells reveals that non-transfected cells co-cultured with transfected cells also show the morphological changes of apoptosis, which points to a bystander effect. C1D-dependent apoptosis is not induced in cells with non-functional p53. Accordingly, C1D-induced apoptosis is discussed in relation to its potential to activate DNA-PK, which has been considered to act as an upstream activator of p53.
Open Access
The role of bcl-2 family of genes during kindling
(Blackwell Publication, 2005) Akcali, K. C.; Sahiner, M.; Sahiner, T.
Purpose: Several experimental models of human temporal lobe epilepsy have shown that apoptotic death of neurons is an important part of this degenerative disease. However, the role of apoptotic regulators is not clear during the epileptogenesis. Therefore we investigated the expression pattern of bcl-2 family of genes during the formation of kindling model of epilepsy in rats. Methods: We examined the expression pattern of bax, bcl-2, bcl-xL, mtd, and bcl-w both at messenger RNA (mRNA) and protein level in the brain tissues during the formation of epilepsy with kindling model in adult rats, which has been the most acceptable form of experimental model of human epilepsy. We also assessed the onset of DNA fragmentation by using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. Results: Animals have started to have epileptic discharges after day 10 of kindling model. Recurrent subthreshold electrical stimuli induced not only epileptic foci but also the expression of box, an inducer of apoptosis, in this time period. Conversely, bcl-xL, which is an inhibitor of apoptosis, had an opposite pattern of expression both at mRNA and protein level during the formation of epilepsy. We did not observe DNA fragmentation by TUNEL staining. Conclusions: Our study shows differential expression of Bax and Bcl-x L at the CA1 region during the formation of hippocampal kindling model. The absence of DNA fragmentation during this period suggests that epileptic changes in neurons have the potential to induce DNA fragmentation by altering the expression levels of Bax and Bcl-xL.