DFF45/ICAD restores cisplatin-induced nuclear fragmentation but not DNA cleavage in DFF45-deficient neuroblastoma cells.
We have previously defined a homozygously deleted region at chromosome 1p36.2-p36.3 in human neuroblastoma cell lines, NB-1 and NB-C201, and identified six genes including DFF45/ICAD within this region. In this study, we found that NB-C201 cells are much more resistant to various genotoxic stresses such as cisplatin (CDDP) than CHP134 and SH-SY5Y cells that do not have the homozygous deletion. To examine a role(s) of DFF45 in the regulation of apoptosis in response to CDDP, we have established stably DFF45-expressing NB-C201 cell clones (DFF45-1 and DFF45-3) and a control cell clone (NB-C201-C) using a retrovirus-mediated gene transfer. In contrast to NB-C201-C cells, DFF45-3 cells displayed apoptotic nuclear fragmentation in response to CDDP. Although CDDP-induced proteolytic cleavage of procaspase-3 and DFF45 in DFF45-3 cells, we could not detect a typical apoptotic DNA fragmentation. Additionally, deletion analysis revealed that C-terminal region of DFF45 is required for inducing nuclear fragmentation. Unexpectedly, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays demonstrated that DFF45 has undetectable effect on CDDP sensitivity of NB-C201 cells. Taken together, our present results suggest that DFF45/DFF40 system may be sufficient for CDDP-induced nuclear fragmentation but not DNA cleavage.
Takahashi
M
M
Division of Biochemistry, Chiba Cancer Center Research Institute, Chiba, Japan.
Ozaki
T
T
Takahashi
A
A
Miyauchi
M
M
Ono
S
S
Takada
N
N
Koda
T
T
Todo
S
S
Kamijo
T
T
Nakagawara
A
A
D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000235:genetics; D051017:Apoptosis Regulatory Proteins / Q000235:genetics* / Q000378:metabolism; D015152:Blotting, Northern; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000187:drug effects / Q000235:genetics; D002470:Cell Survival / Q000187:drug effects; D002945:Cisplatin / Q000494:pharmacology*; D053837:DNA Cleavage / Q000187:drug effects; D053938:DNA Fragmentation / Q000187:drug effects*; D017353:Gene Deletion; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D009447:Neuroblastoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D013997:Time Factors; D014162:Transfection
D000970:Antineoplastic Agents; D051017:Apoptosis Regulatory Proteins; C109664:caspase-activated DNase inhibitor; D002945:Cisplatin