Regulatory Mechanisms of Mitotic Catastrophe Induced by Doxorubicin

PURPOSE: Doxorubicin may act through DNA intercalation/binding, inhibition of topoisomerase II, free radical generation, or damage to cell membranes and induce apoptosis, senescence or mitotic catastrophe. Because low dose of doxorubicin induce senescence or mitotic cell death, we attempted to investigate the possible link between the drug-induced senescence and mitotic cell death as comparing with apoptotic cell death induced by high doses of doxorubicin in human hepatoma cells. And we examined the role of p21 in the prevention of doxorubicin-induced mitotic catastrophe (or multinucleation) to understand the regulatory mechanisms of mitotic catastrophe in human colorectal carcinoma cells. MATERIALS AND METHODS: We used human hepatoma (Huh-7) and human hepatocellular carcinoma cell lines (SNU-354, -398, -449, and -475) to distinguish the mode of low doses (LD) of doxorubicin-induced cell death from that of apoptosis induced by high doses (HD) of the same drug, morphologically and biochemically, and human colorectal carcinoma cell line HCT116 to understand the regulatory mechanisms of mitotic catastrophe induced by doxorubicin. We checked changes of several senescence markers, such as SA-beta-galactosidase activity and osteonectin, SM22, TGase II, and PAI-1 expressions by RT-PCR, and evaluated morphological and biochemical differences between mitotic catastrophe and apoptosis using electron or fluorescence microscophy and western blotting. To know the role of p21 in multinucleation formation we analyzed the cell cycle profiles using FACS, activities of Cdc2 or Cdk2. RESULTS: LD doxorubicin induced abnormal mitosis and then senescence-like phenotype (SLP) in Huh-7 human hepatoma cell lines. Subsequently, these cells were undergoing cell death through mitotic catastrophe which was caused by abnormal mitosis. Cellular morphologies and biochemical characters of mitotic cell death were different to HD doxorubicin-induced apoptosis. 1) Membrane morphology changed during the mitotic cell death was similar to those observed in necrosis. 2) Nuclear lamin B distribution differed in cells undergoing LD doxorubicin-induced mitotic cell death and HD doxorubicin-induced apoptosis. 3) Mitochondrial cytochrome c was released both in doxorubicin-induced apoptosis and mitotic cell death. 4) p38, JNK MAP kinases, and NF-kB were significantly activated during doxorubicin-induced apoptosis but not mitotic cell death. 5) Caspases were significantly activated during doxorubicin-induced apoptosis but not mitotic cell death In HCT116 human colorectal carcinoma cells, LD doxorubicin induced multinucleation in p21-/- cells, but not in p21+/+ cells. In p21-/- cells, abnormal mitosis caused by multi-polar spindle structures were observed and activities of Cdc2 and Cdk2 were not decreased. But in p21+/+ cells, LD doxorubicin induced G₁cell cycle accumulation and decrease of activities of Cdc2 and Cdk2. These multinucleation observed in LD doxorubicin-treated p21-/- cells was dramatically decreased by pre-treatment with Purvalanol A, an inhibitor of Cdc2 and Cdk2. But 500 nM doxorubicin induced G₂cell cycle arrest both in p21+/+ and p21-/- cells. G₂cell cycle arrest induced by 500 nM doxorubicin was caused from ATM/Chk signaling pathways. Although caffeine abrogated G₂arrest both in p21+/+ and p21-/- cells, multinucleation was accelerated only in p21-/- cells. CONCLUSION: In part I, LD doxorubicin induces SLP in Huh-7 human hepatoma cells, and then those cells with SLP subsequently died by abnormal mitosis or mitotic catastrophe. And two distinct modes of cell death by doxorubicin were due to differences in cellular morphologies and many biochemical characters. In part II, p21 played a key role in preventing multinucleation via G₁or G₂cell cycle arrest induced by 50 or 500 nM doxorubicin, respectively. Even though G₂arrest was abrogated by caffeine, multinucleation was prevented in p21+/+ cells because p21 induced again cell cycle arrest at G₁phase.

Author dc:creator

• 엄, 영우

Contributors dc:contributor

• 최, 경숙
• 대학원 의학과
• 200124438

• Doxorubicin
• Mitotic catastrophe
• Senescence-like phenotype
• Apoptosis
• p21
• Multinucleation

Language dc:language

en

Identifier

http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000000287
000000000287

OAI identifier oai:identifier

oai:repository.ajou.ac.kr:201003/2288