Mivechi N F, Dewey W C, Feuerstein B G, Deen D F, Marton L J
Radiat Res. 1986 Dec;108(3):269-81.
Alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, was used to study the effect of polyamine depletion on delayed heat sensitization in Chinese hamster ovary cells (CHO). The cells were treated with 1 or 10 mM DFMO for 8 or 48 h and then given a single heat treatment (43 degrees C, 90 min) at intervals up to 150 h after DFMO addition. Cellular survival, DNA polymerase activity, and polyamine levels were measured. Delayed heat sensitization for cell lethality began 50-55 h (about two cell divisions) after addition of 10 or 1 mM of DFMO for 8 or 48 h, respectively; i.e., cell survival of heated control cells was about 10(-1), but decreased to 10(-4)-10(-5) in heated DFMO-treated cells by 100 h. During this same interval, delayed heat sensitization also was observed for loss of DNA polymerase beta activity (from 20% in cells heated without DFMO treatment to 7% in heated DFMO-treated cells), but none was observed for DNA polymerase alpha activity. Delayed heat sensitization disappeared at 120-130 h after DFMO addition, with survival of heated DFMO-treated cells returning to that for heated control cells. The onset of delayed heat sensitization occurred 30-40 h after intracellular levels of putrescine and spermidine were depleted by more than 95%; however, spermine levels were not lowered, and in some cases even increased. Levels of putrescine and spermidine increased 5-10 h before delayed heat sensitization disappeared. While putrescine reached 25% of control, spermidine exceeded control levels during this time. Furthermore, delayed heat sensitization could be reversed by adding 10(-3) M putrescine or 5 X 10(-5) M spermidine 85-95 h after DFMO addition; in both cases spermidine increased 5-10 h before the decrease in heat sensitization. Finally, neither delayed heat sensitization nor depletion of spermidine was observed in nondividing plateau-phase cells treated with DFMO, although putrescine was depleted. These results lead to the hypothesis that DFMO-induced heat sensitization which occurs after inhibition of the synthesis of putrescine is secondary to the depletion of spermidine in some critical compartment of the cell or to a biochemical alteration. This depletion or biochemical alteration apparently occurs as the cells divide about two times after the intracellular levels of soluble spermidine have been depleted.
α-二氟甲基鸟氨酸（DFMO）是鸟氨酸脱羧酶的不可逆抑制剂，用于研究多胺耗竭对中国仓鼠卵巢细胞（CHO）延迟热致敏的影响。将细胞用1或10 mM DFMO处理8或48小时，然后在添加DFMO后长达150小时的间隔内进行单次热处理（43℃，90分钟）。测量细胞存活率、DNA聚合酶活性和多胺水平。分别在添加10或1 mM DFMO 8或48小时后，细胞致死性的延迟热致敏在50 - 55小时（约两个细胞分裂周期）开始；即，加热对照细胞的存活率约为10⁻¹，但在加热的DFMO处理细胞中到100小时时降至10⁻⁴ - 10⁻⁵。在相同间隔期间，DNA聚合酶β活性丧失也观察到延迟热致敏（从未用DFMO处理加热的细胞中的20%降至加热的DFMO处理细胞中的7%），但DNA聚合酶α活性未观察到延迟热致敏。DFMO添加后120 - 130小时延迟热致敏消失，加热的DFMO处理细胞的存活率恢复到加热对照细胞的水平。延迟热致敏的开始发生在腐胺和亚精胺的细胞内水平耗尽超过95%后30 - 40小时；然而，精胺水平未降低，在某些情况下甚至升高。腐胺和亚精胺水平在延迟热致敏消失前5 - 10小时增加。虽然腐胺达到对照的25%，但在此期间亚精胺超过对照水平。此外，在添加DFMO后85 - 95小时添加10⁻³ M腐胺或5×10⁻⁵ M亚精胺可逆转延迟热致敏；在这两种情况下，亚精胺在热致敏降低前5 - 10小时增加。最后，在用DFMO处理的非分裂平台期细胞中未观察到延迟热致敏和亚精胺耗竭，尽管腐胺被耗尽。这些结果导致这样的假设，即DFMO诱导的热致敏在抑制腐胺合成后发生，是细胞某些关键区室中亚精胺耗竭或生化改变的继发结果。这种耗竭或生化改变显然发生在可溶性亚精胺的细胞内水平耗尽后细胞分裂约两次时。
