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

用高压液相色谱法（HPLC）测定了一株人唾液腺腺癌细胞系（HSG）中的脱氧核糖核苷磷酸（dNTP）池，并对其性质进行了较好的研究。HPLC分析采用Waters 510泵，LC分光光度计（A254 nm）。在Partisyl-10 SAX （4.6x250 mm）上进行层析，以0.4M磷酸铵（pH 2.65）洗脱，流速为2ml /min。将HSG细胞接种于100 mm的塑料培养皿中，并在培养箱中培养6ml添加10%新生牛犊血清的Dulbecco Is改良Eagle Is培养基。研究了从HSG细胞中提取dNTPs的方法、高周酸甲胺降解rNTPs的方法以及固相Sep-Pak柱部分纯化和浓缩的方法。从这些结果来看，12% TCA处理制备细胞提取物和GARRETT Is方法进行高碘酸氧化处理比我们研究的其他方法更有效。如果样品剂量不含TCA， Sep-Pak滤筒部分纯化是非常有效的。我们将培养62小时以上的HSG细胞提取液，加入TCA，然后测量dNTP池。因此，每1x106个细胞的dNTP量为dCTP，5.3 （]SY.+-）。[) 2.9 pmol；[j] .中国生物医学工程学报，2013,27(1):1 - 2。[) 5.9 pmol；[j] .浙江大学学报（自然科学版），2016,26(3):591 - 591。[) 11.5 pmol；dGTP,6.2 [j]；[) 4.4 pmol在细胞周期的几个阶段。这些结果怀疑dNTP池随着细胞周期的变化而变化，但细节必须详细研究。由于rNTP池（CTP，ATP，UTP，GTP分别为11,83,36,21 nmol/106 cells）比dNTP池大约3000倍，因此rNTP的降解必须谨慎进行以减少副产物。此外，由于dNTPs的浓度很低，并且将粗细胞提取样品注射到HPLC系统中，使得HPLC分析的重现性大大提高。

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.