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(HPLC)系统测定了特性较好的人涎腺腺癌细胞系(HSG)的脱氧核糖核苷三磷酸(DNTP)池。高效液相色谱法在Waters 510色谱柱上进行，使用LC分光光度计(使用A254 nm)。色谱柱为Partisyl-10 SAX(4.6×250 mm)，流动相为0.4M磷酸二铵(pH 2.65)，流速为2ml/min。将HSG细胞接种于100 mm塑料培养皿中，在6mlDulbecco is改良的Eagle is培养液中添加10%的新生小牛血清，在5%CO2的培养箱中培养。研究了从HSG细胞中提取dNTPs的方法、rNTPs的高碘酸甲胺降解以及固相Sep-Pak柱的部分纯化和浓缩。从这些结果来看，12%TCA法制备细胞提取液和Garrett is法处理高碘酸氧化法的效果要好于我们研究的其他方法。当样品中不含TCA时，用Sep-Pak柱进行部分纯化是非常有效的。我们通过加入TCA制备了培养超过62小时的HSG细胞提取液，然后测定了dNTP池。因此，每1×106个细胞的dNTP数量为dCTP，5.3(SY+-)。[)2.9pmol；dATP，13.7(SY+-)。[)5.9pmol；dTTP，26.3([Sy.+-].[)11.5pmol；dGTP，6.2([Sy.+-].[)4.4pmol.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。RNTP的降解必须仔细进行，以减少副产物，因为rNTP池(CTP、ATP、UTP和GTP分别为11、83、36、21nmol/106个细胞)大约是dNTP池的3000倍。此外，dNTPs的浓度很低，粗细胞提取的样品进入高效液相系统后，高效液相分析的重复性得到了很大的提高。

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.