Hydroxyurea inhibition of DNA synthesis in ascites tumor.
Hydroxyurea inhibition of DNA synthesis in ascites tumor.
复制标题
羟基脲抑制腹水肿瘤中的 DNA 合成。
DOI：
--
复制
发表时间：
1965
影响因子：
11.1
通讯作者：
C. Barnum
中科院分区：
文献类型：
--
作者：
J. Yarbro;B. Kennedy;C. Barnum
最近已证明，羟基脲在某些恶性肿瘤的治疗中具有价值。Frenkel等3报道了用羟基脲处理的大鼠骨髓对氚化胞苷酸转化为脱氧胞苷酸的干扰。这是本报告的目的，以描述一个调查的影响，羟基脲对P32纳入核酸的小鼠腹水肿瘤。材料与方法腹水瘤，最初是一种淋巴肉瘤，在我们的实验室已经使用了几年，4被用作这些研究的组织。将0.1 ml荷瘤小鼠腹水接种到C3 H小鼠腹腔内后，肿瘤迅速生长，在第6至第8天产生2-4 ml腹水，其中含有约50%的细胞。处死动物，合并几个肿瘤以获得所需体积。加入含有P32作为正磷酸盐的溶液，使得最终浓度为50 μ c/ml。加入P32的肿瘤细胞可以在冰浴中保持数小时，当随后在370 ℃温育时，没有显著的同位素掺入核酸中，并且没有合成能力的损失。将体积为0.4 ml的肿瘤液转移到含有0.1 ml盐水的试管中，其中含有和不含所研究的抑制剂。然后将制备物在370 ° C下温育所需的时间长度，同时轻轻摇动。通过将试管转移到冰浴中并加入2.5ml 5%三氯乙酸终止反应。将制备物在Potter-Elvehjem管中均质化并以600 g离心10 min，随后将一部分上清液转移至灰化管中用于随后的酸溶性磷评价。将酸不溶性残余物在冷的5%三氯乙酸中洗涤5次，并在缓冲至pH 7.8的10%NaCl中的沸水浴中提取核酸。用两个体积的95%乙醇从提取物中沉淀核酸，用0.1 N NaOH水解RNA，用三氯乙酸再沉淀DNA，并通过前面描述的方法水解。4将所有样品（酸溶性、RNA和DNA）湿灰化，稀释至体积，用端窗盖革管计数，并分析磷含量。每个样品的活性计算为每分钟每微克磷每毫升的计数，然后将DNA和RNA的活性除以酸溶性前体库活性，得到DNA和RNA的相对比活性。在评价抑制剂的作用时，将处理样品的相对比活性与未处理对照样品的相对比活性进行比较，称为比较比活性，以对照的百分比表示。结果。-表1显示了羟基脲浓度与P32掺入DNA和RNA的关系。几乎90%的DNA抑制
Hydroxyurea has recently been shown to be of value in the treatment of certain malignancies.', 2 Frenkel et al.3 have reported interference in the conversion of tritiated cytidylic acid into deoxycytidylic acid by bone marrow of rats treated with hydroxyurea. It is the purpose of this report to describe an investigation of the effect of hydroxyurea on the incorporation of P32 into the nucleic acids of a mouse ascites tumor. Materials and Methods.-An ascites tumor, originally a lymphosarcoma, which has been in use in our laboratory for several years,4 was used as the tissue for these studies. Following intraperitoneal inoculation of 0.1 ml of ascitic fluid from a tumor-bearing mouse into C3H mice, the tumor grows rapidly, yielding 2-4 ml of ascitic fluid containing about 50 per cent cells on the sixth to the eighth day. The animals were sacrificed and several tumors pooled to obtain the desired volume. A solution containing P32 as orthophosphate was added, such that the final concentration was 50 ,uc/ml. Tumor cells, with P32 added, could be maintained for several hours in an ice bath with no significant incorporation of isotope into nucleic acid and without loss of synthetic capacity when subsequently incubated at 370C. The tumor fluid was transferred in volumes of 0.4 ml into tubes containing 0.1 ml of saline with and without the inhibitor under study. The preparation was then incubated for the desired length of time at 370 with gentle shaking. The reaction was stopped by transferrring the tubes to an ice bath and adding 2.5 ml of 5 per cent trichloroacetic acid. The preparations were homogenized in Potter-Elvehjem tubes and centrifuged for 10 min at 600 g, following which a portion of the supernate was transferred to an ashing tube for subsequent evaluation of the acid-soluble phosphorus. The acid-insoluble residue was washed five times in cold 5 per cent trichloroacetic acid, and the nucleic acids extracted in a boiling water bath in 10 per cent NaCl buffered to pH 7.8. The nucleic acids were precipitated from the extract with two volumes of 95 per cent ethanol, the RNA was hydrolyzed with 0.1 N NaOH, and the DNA reprecipitated with trichloroacetic acid and hydrolyzed by methods previously described.4 All samples-acid-soluble, RNA, and DNA-were wet-ashed, diluted to volume, counted with an end-window Geiger tube, and analyzed for phosphorus content. The activity of each sample was calculated as counts per minute per microgram of phosphorus per milliliter, and the activity of the DNA and RNA was then divided by the acid-soluble precursor pool activity to give the relative specific activity of DNA and RNA. In evaluating the effect of the inhibitor the relative specific activity of a treated sample is compared to that in untreated controls and is referred to as the comparative specific activity, expressed as per cent of control. Results.-Table 1 indicates the relationship of concentration of hydroxyurea to incorporation of P32 into DNA and RNA. Almost 90 per cent inhibition of DNA