Porsin B, Formento J-L, Filipski E, Etienne M-C, Francoual M, Renée N, Magné N, Lévi F, Milano G
Oncopharmacology Unit, Centre Antoine Lacassagne, Nice, France.
Eur J Cancer. 2003 Apr;39(6):822-8. doi: 10.1016/s0959-8049(02)00598-1.
Dihydropyrimidine dehydrogenase (DPD) is the rate-limiting enzyme of 5-fluorouracil (FU) catabolism. The relevance of the measurement of DPD activity for identifying DPD-deficient patients is lessened by circadian variability in DPD activity. Our purpose was to determine whether or not DPD mRNA is sustained by a circadian rhythm. Synchronised mice (male B6D2F1) were sacrificed at 3, 7, 11, 15, 19 or 23 Hours After Light Onset (HALO; eight mice per time-point). Liver DPD activity was determined by a radio-enzymatic assay and liver DPD expression by a reverse transcriptase-polymerase chain reaction (RT-PCR) enzyme-linked immunosorbent assay (ELISA) method. Mice synchronisation was controlled by leucocyte and neutrophil counts. Individual DPD activity ranged from 555 to 1575 pmol/min/mg prot; mean DPD activity was highest at 3 HALO (mean+/-standard error of the mean (S.E.M.); 1105+/-70) and lowest at 15 HALO (889+/-71). Individual liver DPD expression varied from 761 to 3481 units (DPD/beta actin ratio); the mean was lowest at 3 HALO (1406+/-112) and highest at 15 HALO (2067+/-214). Cosinor analysis indicated that respective double amplitudes of DPD activity and expression were 21 and 30% of the 24-h mean. The acrophases for activity and expression were 6:40 and 14:10 HALO, respectively, meaning that maximum activity occurred 16 h after the maximum observed expression. These results, revealing the existence of a circadian rhythm in DPD expression, should stimulate further studies to enhance our understanding of the molecular mechanisms involved in the circadian regulation of the DPD enzyme.
二氢嘧啶脱氢酶（DPD）是5-氟尿嘧啶（FU）分解代谢的限速酶。DPD活性的昼夜变化降低了通过测量DPD活性来识别DPD缺陷患者的相关性。我们的目的是确定DPD mRNA是否受昼夜节律维持。在光照开始后3、7、11、15、19或23小时（HALO；每个时间点8只小鼠）处死同步化的小鼠（雄性B6D2F1）。通过放射酶法测定肝脏DPD活性，并通过逆转录聚合酶链反应（RT-PCR）酶联免疫吸附测定（ELISA）方法测定肝脏DPD表达。通过白细胞和中性粒细胞计数来控制小鼠同步化。个体DPD活性范围为555至1575 pmol/分钟/毫克蛋白；平均DPD活性在3 HALO时最高（平均值±平均值标准误差（S.E.M.）；1105±70），在15 HALO时最低（889±71）。个体肝脏DPD表达从761至3481单位（DPD/β肌动蛋白比值）不等；平均值在3 HALO时最低（1406±112），在15 HALO时最高（2067±214）。余弦分析表明，DPD活性和表达各自的双振幅分别为24小时平均值的21%和30%。活性和表达的峰相位分别为6:40和14:10 HALO，这意味着最大活性出现在最大观察到的表达之后16小时。这些结果揭示了DPD表达中存在昼夜节律，应促使进一步研究以加深我们对参与DPD酶昼夜调节的分子机制的理解。
