TANUMA Sei-ichi UNIVERSITY OF TOKYO HOSPITAL,PROFESSOR, 薬学部, 教授 (10142449)
KAMII Yoshiyuki UNIVERSITY OF TOKYO HOSPITAL,ASSISTANT, 医学部・附属病院, 助手 (70177567)
TSUCHIDA Yoshiaki UNIVERSITY OF TOKYO HOSPITAL,PROFESSOR, 医学部・附属病院, 教授 (80010164)
OBANA Kazuko UNIVERSITY OF TOKYO HOSPITAL,ASSISTANT, 医学部・附属病院, 助手 (60272580)
|Budget Amount *help
¥7,900,000 (Direct Cost : ¥7,900,000)
Fiscal Year 1996 : ¥1,900,000 (Direct Cost : ¥1,900,000)
Fiscal Year 1995 : ¥6,000,000 (Direct Cost : ¥6,000,000)
During the research period in 1995, apoptosis induction was examined for each of 16 human neuroblastoma cell lines, using the following three apoptosis inducers, namely, actinomycin D,adriamycin, and camptothecin, . We have already found that the cleavage of DNA in apoptotic cells occurs in at least two steps, i.e., large fragmentation of DNA of 2-50 kilobase pairs and internucleosomal fragments of 180 basepairs. Depending upon the size of DNA fragmentation oberved in each tumor cell line, we found that 16 human neuroblastoma cell lines investigated were classified into the followign three categories ; (1) cell line in which even large fragmentation was not induced by any of the aforementioned three apoptotic inducers, (2) cell line in which only large fragmentation was induced followed by no further small fragmentaton, and (3) cell line in which small fragmentation into internucleosomal fragments with typical apoptotic DNA ladder formation was induced following large fragmentation.
ing the year 1996, our study was directed mainly toward identification of DNase lambda, a newly recognized key-endonuclease responsible for large fragmentation, by comparing the enzymatic activity between a pair of neruoblastoma cell lines ; one cell line (TGW) with positive DNase lambda activity and the oher one (GOTO) lacking it. We exmined whether large DNA fragmentation can be induced in vitro by autodigested nuclei of DNase lambda positive TGW cells, and confirmed the induction of large fragmentation with about 20 kb fragments.
Using this autodigestion system, fragmentation induction in vitro was further exmined in GOTO cells without DNase lambda, and in SK-N-SH cells with both DNase lambda and DNase gamma (a key-endonuclease for small, internucleosomal fragmentation). As has been expected, no induction was seen in GOTO cells, while in SK-N-SH cells small fragmentation in vitro was shown.
For further genetic analysis studies on DNase gamma in small DNA fragmentation b, three neuroblastoma cells with distinct biological characteristics (GOTO,TWGW,and SK-N-SH) were propagated and kept frozen. Less