Influence of antineoplastic drug (5-fluorouracil) to the dNTP pool of a human salivary gland adenocarcinoma cell line (HSG)

抗肿瘤药物（5-氟尿嘧啶）对人唾液腺腺癌细胞系（HSG）dNTP库的影响

• 批准号: 08672158
• 负责人: NISHIDA Toshinobu
• 金额: $ 1.41万
• 依托单位: The University of Tokushima
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08672158/
• 关键词: HPLC; HSG cell line; deoxyribonucleosidetriphosphate; cell cycle; HSG ell ine; deoxyribonucleosidetriphesphete; ヌクレオチドプール; 増殖・分化; アポトーシス

Deoxyribonucleosidetriphosphate (dNTP) pools in a human salivary gland adenocarcinoma cell line (HSG) which characterization was relatively well elucidated, was measured on high pressure liquid chromatography (HPLC) system. HPLC analysis were performed on a Waters 510 pump with a LC spectrophotometer (using A254 nm). Chromatography on Partisyl-10 SAX (4.6x250 mm) was done and eluted with 0.4M ammonium phosphate (pH 2.65) at a flow rate of 2 ml/min. HSG cells were seeded 100 mm plastic Petri dishes and grown in 6 ml of Dulbecco Is modified Eagle Is medium supplemented with 10% newborn calf serum in the presence of 5% CO2 in an incubator. We investigated about the method of dNTPs extraction from HSG cells, periodate-methylamine degradation of rNTPs and partial purification and concentration by solid phase Sep-Pak cartridge column. From these results, 12% TCA treatment for preparation of cell extracts and GARRETT Is method for periodate-oxidation treatment were more effective than the other methods we investigated. And partial purification by Sep-Pak cartridge was very effective if a sample dose not include TCA.We prepared cell extract of HSG cells which were cultured over 62 hours, by addition of TCA and then dNTP pools were measured. Thus, the amounts of dNTP per 1x106 cells were dCTP,5.3 (]SY.+-。[) 2.9 pmol ; dATP,13.7 (]SY.+-。[) 5.9 pmol ; dTTP,26.3 (]SY.+-。[) 11.5 pmol ; dGTP,6.2 (]SY.+-。[) 4.4 pmol on several stages of cell cycles. These results suspect that dNTP pools change followed by cell cycle, but the details were must be invetigated in detail. Degradation of rNTP must performed carefully to reduce by-products, because rNTP pools (CTP,ATP,UTP,GTP were 11,83,36,21 nmol/106 cells, respectively) were about 3000 times larger than dNTP pools. Additionally, concentration of dNTPs were very low and crude cell extracted sample injected to HPLC system, reproducibility of HPLC analysis were much more improved.

用高效液相色谱（HPLC）系统测定了一个已比较明确的人涎腺腺癌细胞系（HSG）中的脱氧核苷三磷酸（dNTP）库。在具有LC分光光度计（使用λ 254 nm）的沃茨510泵上进行HPLC分析。在Partisyl-10 SAX（4.6 × 250 mm）上进行色谱分析，用0.4 M磷酸铵（pH 2.65）以2 ml/min的流速洗脱。将HSG细胞接种在100 mm塑料培养皿中，并在培养箱中添加10%新生小牛血清的6 ml Dulbecco Is改良Eagle Is培养基中，在5% CO2的存在下生长。本文研究了HSG细胞中dNTPs的提取方法、高碘酸-甲胺降解rNTPs的方法以及固相Sep-Pak柱的部分纯化和浓缩方法。从这些结果来看，12%TCA处理制备细胞提取物和加勒特Is方法用于高碘酸盐氧化处理比我们研究的其他方法更有效。对培养62小时以上的HSG细胞，加入TCA制备细胞提取液，测定dNTP池。因此，每1 × 106个细胞中dNTP的量为dCTP，5.3（μ S ±）。[] 2.9 pmol ; dATP，13.7（]SY.+-. [）5.9 pmol ; dTTP，26.3（]SY.+-. 11.5 pmol ; dGTP，6.2（±）SY. [）4.4 pmol对细胞周期的几个阶段。这些结果怀疑dNTP池随细胞周期变化而变化，但具体情况有待进一步研究。由于rNTP池（CTP、ATP、UTP、GTP分别为11、83、36、21 nmol/106 cells）比dNTP池大3000倍左右，因此降解rNTP时必须小心，以减少副产物。此外，dNTPs的浓度很低，细胞提取的粗样品进样到HPLC系统中，HPLC分析的重现性得到了很大的改善。