Dickson R B, Aitken S, Lippman M E
Methods Enzymol. 1987;146:329-40. doi: 10.1016/s0076-6879(87)46034-5.
In summary we have presented data on [3H]dThd incorporation into DNA for a positive and negative growth modulator. These data clearly do not correspond to those based on net DNA synthesis (ortho[32P]phosphate incorporation into purified DNA) and previous knowledge of the effects of these hormones on cell number and cellular DNA accumulation. The paradox was resolved by directly analyzing effects of hormones on de novo pyrimidine biosynthesis. E2 stimulated both de novo and scavenger pathways in the first wave of DNA synthesis and only de novo in the second (and subsequent) waves. In contrast, tam progressively inhibited de novo pyrimidine biosynthesis. These hormonal effects on intracellulr pyrimidine pool sizes rendered [3H]dThd incorporation data by itself uninterpretable. [3H]dThd is a useful measure of DNA synthesis only if verified by independent measures of net DNA synthesis for the same time course and treatment conditions. In addition it may prove beneficial in various experimental systems to develop direct assays of de novo pyrimidine biosynthesis to assess mitogen effects. The experiments presented may also prove to be useful in evaluating the effects of mitogens and antimitogens on cells synchronized in the cell cycle by any of a variety of means.
总之，我们已经给出了关于[3H]胸苷掺入DNA的相关数据，这些数据来自一种正性和一种负性生长调节剂。这些数据显然与基于净DNA合成（将正磷酸[32P]掺入纯化的DNA）以及这些激素对细胞数量和细胞DNA积累影响的先前认知不符。通过直接分析激素对从头嘧啶生物合成的影响，这个矛盾得以解决。在第一轮DNA合成中，雌激素刺激了从头合成途径和补救途径，而在第二轮（及后续轮次）DNA合成中仅刺激了从头合成途径。相比之下，他莫昔芬逐渐抑制了从头嘧啶生物合成。这些激素对细胞内嘧啶池大小的影响使得仅靠[3H]胸苷掺入数据本身难以解释。只有在相同时间进程和处理条件下通过净DNA合成的独立测量方法进行验证时，[3H]胸苷才是DNA合成的有用指标。此外，在各种实验系统中，开发直接检测从头嘧啶生物合成的方法以评估促有丝分裂原的作用可能会被证明是有益的。所展示的实验在评估促有丝分裂原和抗有丝分裂原对通过多种方式之一同步于细胞周期的细胞的影响时，也可能会被证明是有用的。
