Schaefer E L, Seidenfeld J
Department of Pharmacology, Northwestern University Medical School, Chicago, Illinois 60611.
J Cell Physiol. 1987 Dec;133(3):546-52. doi: 10.1002/jcp.1041330316.
Numerous reports have shown that polyamines are required for cell proliferation. A current model for regulating commitment to DNA replication in cultured fibroblasts stimulated from quiescence by serum addition postulates sequential action by specific growth factors. To temporally localize polyamine-dependent steps within this defined sequence, mouse Balb/c-3T3 fibroblasts were partially depleted of polyamines by treatment with DL-alpha-difluoromethylornithine (DFMO), next rendered quiescent by serum deprivation, then stimulated by 10% serum with or without exogenous putrescine (Pu). Depletion of polyamines was verified by HPLC, and entry of cells into S phase was monitored by autoradiography. After 24 h of incubation with [3H]-thymidine, polyamine-depleted cells had labeling indices similar to quiescent cells if they were serum-stimulated without Pu, but progressed to S phase to the same degree as control cultures if polyamines were restored by adding Pu at the time of serum stimulation. These observations suggested that commitment of quiescent cells to DNA replication may require polyamines. To determine if polyamine-dependent steps occur during the pre-commitment period (up to 12 h after serum stimulation) or only in traverse of G1 (12 h to 24 h, post-commitment), polyamine-depleted quiescent cells were serum-stimulated for 12 h without Pu, then returned to low serum with Pu. Labeling indices of these cultures remained nearly as low as those of unstimulated cells. Reducing serum concentration from 10% to 0.5% at 12 h after stimulation did not effect labeling indices of control cells not depleted of polyamines by DFMO. These results supported the postulated requirement for polyamines during pre-commitment events. However, polyamine-deficient quiescent cells serum-stimulated without Pu for periods longer than 24 h had labeling indices at 36 and 48 h significantly greater than at 24 h. This suggested that polyamine depletion may decrease the rate at which quiescent cells commit to DNA replication, rather than producing an absolute blockade during the pre-commitment period.
大量报告表明，多胺是细胞增殖所必需的。目前关于调控静止的培养成纤维细胞在添加血清后进入DNA复制过程的模型假定特定生长因子会依次发挥作用。为了在这个确定的序列中对多胺依赖步骤进行时间定位，用DL-α-二氟甲基鸟氨酸（DFMO）处理使小鼠Balb/c-3T3成纤维细胞中的多胺部分耗竭，接着通过血清剥夺使其进入静止状态，然后用含或不含外源性腐胺（Pu）的10%血清进行刺激。通过高效液相色谱法（HPLC）验证多胺的耗竭情况，并通过放射自显影监测细胞进入S期的情况。在用[³H]-胸腺嘧啶核苷孵育24小时后，如果在无Pu的情况下进行血清刺激，多胺耗竭的细胞的标记指数与静止细胞相似，但如果在血清刺激时添加Pu使多胺得以恢复，它们进入S期的程度与对照培养物相同。这些观察结果表明，静止细胞进入DNA复制过程可能需要多胺。为了确定多胺依赖步骤是发生在预先决定期（血清刺激后长达12小时）还是仅发生在G1期的进程中（决定后12小时至24小时），将多胺耗竭的静止细胞在无Pu的情况下用血清刺激12小时，然后在有Pu的情况下恢复到低血清状态。这些培养物的标记指数几乎与未刺激细胞的标记指数一样低。在刺激后12小时将血清浓度从10%降至0.5%对未被DFMO耗竭多胺的对照细胞的标记指数没有影响。这些结果支持了预先决定期事件中对多胺的假定需求。然而，在无Pu的情况下用血清刺激超过24小时的多胺缺乏的静止细胞在36小时和48小时的标记指数明显高于24小时时的标记指数。这表明多胺耗竭可能会降低静止细胞进入DNA复制过程的速率，而不是在预先决定期产生绝对的阻滞。
