Presence of DNase gamma-like endonuclease in nuclei of neuronal differentiated PC12 cells.
DNase gamma, which cleaves chromosomal DNA into nucleosomal units (DNA ladder formation), has been suggested to be the critical component of apoptotic machinery. Using rat pheochromocytoma PC12 cells, which are differentiated to sympathetic neurons by nerve growth factor (NGF), we investigated whether DNase gamma-like enzyme is present in neuronal cells and is involved in neuronal cell death. The nuclear auto-digestion assay for DNase catalyzing internucleosomal DNA cleavage revealed that nuclei from neuronal differentiated PC12 cells contain acidic and neutral endonucleases, while nuclei from undifferentiated PC12 cells have only acidic endonuclease. The DNA ladder formation observed in isolated nuclei from neuronal differentiated PC12 cells at neutral pH requires both Ca(2+) and Mg(2+), and is sensitive to Zn(2+). The molecular mass of the neutral endonuclease present in neuronal differentiated PC12 cell nuclei is 32000 as determined by activity gel analysis (zymography). The properties of the neuronal endonuclease present in neuronal differentiated PC12 cell nuclei were similar to those of purified DNase gamma from rat thymocytes and splenocytes. Interestingly, in neuronal differentiated PC12 cells, internucleosomal DNA fragmentation is observed following NGF deprivation, whereas undifferentiated PC12 cells fail to exhibit DNA ladder formation during cell death by serum starvation. These results suggest that the DNase gamma-like endonuclease present in neuronal differentiated PC12 cell nuclei is involved in internucleosomal DNA fragmentation during apoptosis, induced by NGF deprivation.
Nishimura
K
K
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Science University of Tokyo, Shinjuku-ku, Tokyo, Japan.
Tanuma
S
S