The biochemical role of polyADP-ribosylation on the internucleosomal DNA fragmentation associated with apoptosis was investigated in HL 60 human premyelocytic leukemia cells. An increase in the activity of poly(ADP-ribose)polymerase preceded DNA fragmentation during the UV-induced apoptotic process. The DNA fragmentation was prevented by inhibitors of poly(ADP-ribose)polymerase, suggesting a requirement of polyADP-ribosylation for DNA fragmentation. There was no significant increase of intracellular $Ca^{2+}$ level and $Ca^{2+}/Mg^{2+}$-dependent nuclease activity during UV-induced apoptosis. Intracellular level of $NAD^+$ did not changed significantly. It was found that UV light and chemotherapeutic drugs including adriamycin, mitomycin C and cisplatin increased polyADP-ribosylation of nuclear proteins, particularly histone H1. A poly(ADP-ribose)polymerase inhibitor, 3-aminobenzamide, prevented both internucleosomal DNA fragmentation and histone H1 polyADP-ribosylation in cells treated with these apoptosis inducers. These results suggest a correlation between the polyADP-ribosylation of histone H1 and internucleosomal DNA fragmentation in the apoptotic processes induced by DNA damaging agents. Our suggestion is further supported by the finding that a xeroderma pigmentosum cell line, GM04312B, which is defective in introducing nicks at the site of DNA damage, failed to induce DNA fragmentation as well as histone H1 polyADP-ribosylation after UV irradiation. To investigate the possible role of histone H1 polyADP-ribosylation, the micrococcal nuclease-susceptibility of chromatin was measured. Nuclease-susceptibility of chromatin was increased during UV-induced apoptosis, and the cotreatment of 3-aminobenzamide reduced both polyADP-ribosylation of histone H1 and nuclease-susceptibility of chromatin.
A23187, colchicine and cycloheximide which do not induce DNA damage and PARP activation also provoked apoptosis in HL 60 cells. Apoptotic processes induced by these agents, however, was not prevented by 3-aminobenzamide. The activation of PARP and histone H1 polyADP-ribosylation were not also detected during these apoptotic processes. During colchicine-induced apoptosis, significant increase of $Mg^{2+}$-dependent nuclease activity was observed. These results suggest that the apoptosis-inducing mechanisms of various agents were diverse. However, among this diversity, DNA damaging agents have some common steps including PARP activation and histone H1 polyADP-ribosylation along their apoptotic pathways.
자외선, adriamycin, mitomycin C, cisplatin 등의 DNA 손상 인자들은 인간의 골수암 세포주 (HL 60)에서 apoptosis 기작에 의한 세포의 죽음을 초래하였다. 이 과정중에 핵내 효소인 poly(ADP-ribose)polymerase가 활성화됨이 관찰되었고, 이 효소의 억제제 3-aminobenzamide를 처리하였을 경우에는 apoptosis 특이적인 internucleosoml DNA fragmentation이 억제되었다. DNA 손상 물질에 의한 apoptosis 과정에 있어서 세포내 칼슘 농도의 변화와 nuclease의 활성도 변화는 관찰되지 않았고 세포내의 NAD+ 함량 역시 의미 있는 변화를 보이지 않았다.
자외선에 의한 apoptosis 과정 중에서 분자량 33 kDa과 110 kDa의 두 단백질이 주요하게 polyADP-ribosylation됨이 관찰되었고 이중 33 kDa 단백질은 histone H1임이 확인 되었다. 자외선을 조사 받았을 경우 DNA에 incision을 도입할 수 없는 xeroderma pigmentosum 세포주 (GM04312B)를 이용한 실험은 자외선에 의한 histone H1의 polyADP-ribosylation과 apoptosis 특이적인 internucleosomal DNA fragmentation이 동시에 억제됨을 보임으로써 이 둘간의 상호연관성을 제시하였다. 또한 자외선 처리에 의해 histone H1이 polyADP-ribosylation될 경우 chromatin의 internucleosomal region이 nuclease의 공격을 받기 쉽게 된다는 것이 micrococcal nuclease를 이용한 실험에서 관찰되었다.
A23187, colchicine, cycloheximde 등 DNA를 손상시키지 않는 물질들도 HL 60 세포주에서 apoptosis를 유도하였으나 그 기작은 DNA 손상 인자에 의한 apoptosis 유도 기작과 상당히 다름이 관찰되었다. Poly(ADP-ribose)polymerase 의 활성화와 histone H1의 polyADP-ribosylation은 관찰되지 않았으나 마그네슘 의존성 nuclease의 활성도가 증가하였다. 이상의 결과로 보아 apoptosis의 생화학적 기작은 그 유도물질에 따라 다양함을 알 수 있다. 그러나 DNA 손상인자들은 그 apoptosis 유도기작내에 poly(ADP-ribose)polymerase의 활성화 와 histone H1의 polyADP-ribosylation등 몇가지의 공통된 과정을 지니고 있다고 생각되어진다.
