抗叶酸抗代谢物对细胞周期的影响：对细胞毒性和细胞生长抑制的意义。

Cell cycle effects of antifolate antimetabolites: implications for cytotoxicity and cytostasis.

作者信息

Tonkinson J L, Marder P, Andis S L, Schultz R M, Gossett L S, Shih C, Mendelsohn L G

机构信息

Lilly Research Laboratories, Lilly Corporate Center, Eli Lilly and Co., Indianapolis, IN 46285, USA.

出版信息

Cancer Chemother Pharmacol. 1997;39(6):521-31. doi: 10.1007/s002800050608.

DOI:10.1007/s002800050608

PMID:9118464

Abstract

PURPOSE

Cell cycle-related events in CCRF-CEM lymphocytic leukemia cells were examined subsequent to inhibition of thymidylate synthase (TS) or GAR formyltransferase (GARFT) and prior to cell death or stasis.

METHODS

Cell populations were treated with the GARFT inhibitors 6R-5, 10-dideazatetrahydrofolate (Lometrexol) or LY309887, the TS inhibitor ZD1694, or the multitargeted antifolate LY231514. DNA content, nucleoside precursor incorporation and proliferating cell nuclear antigen (PCNA) expression as functions of drug treatment were assessed by multiparameter flow cytometry. Cellular respiration was measured by MTT analysis and apoptosis was detected by extraction of DNA fragments.

RESULTS

Cell populations treated for up to 96h with lometrexol or LY309887 did not replicate and maintained a cell cycle distribution with distinct G1, S and G2/M regions. The number of S phase cells in treated populations was slightly elevated relative to control as measured by DNA content and PCNA. However, these cells were unable to incorporate 5-bromodeoxyuridine (BrdU). Throughout treatment, cells incubated with GARFT inhibitors maintained intact membranes and respired at a level comparable to untreated cells. In contrast, ZD1694 as well as LY231514, induced synchronization of the treatment population at the G1/S interface within 12h of drug addition. This was followed by synchronous entry of the population into S phase. After 24 h of treatment, more than 90% of the cells were capable of incorporating BrdU and stained positive for PCNA. DNA fragmentation occurred in cells treated with ZD1694 or LY231514 but not in those treated with GARFT inhibitors. In addition, the viable cells remaining after 24-48 h of treatment with ZD1694 or LY231514 were respiring at twice the level of untreated cells.

CONCLUSION

These results demonstrate that the distinct endpoints of GARFT and TS inhibition are preceded by distinct cell cycle and metabolic alterations.

摘要

目的

在胸苷酸合成酶（TS）或甘氨酰胺核糖核苷酸甲酰基转移酶（GARFT）受到抑制后且细胞死亡或停滞之前，检测CCRF-CEM淋巴细胞白血病细胞中与细胞周期相关的事件。

方法

用GARFT抑制剂6R-5,10-二去氮四氢叶酸（洛美曲索）或LY309887、TS抑制剂ZD1694或多靶点抗叶酸药LY231514处理细胞群体。通过多参数流式细胞术评估DNA含量、核苷前体掺入情况以及增殖细胞核抗原（PCNA）表达随药物处理的变化。通过MTT分析测量细胞呼吸，并通过提取DNA片段检测细胞凋亡。

结果

用洛美曲索或LY309887处理长达96小时的细胞群体未进行复制，并维持具有明显G1、S和G2/M区域的细胞周期分布。通过DNA含量和PCNA测量，处理群体中S期细胞的数量相对于对照略有增加。然而，这些细胞无法掺入5-溴脱氧尿苷（BrdU）。在整个处理过程中，用GARFT抑制剂孵育的细胞保持完整的细胞膜，并以与未处理细胞相当的水平进行呼吸。相比之下，ZD1694以及LY231514在添加药物后12小时内诱导处理群体在G1/S界面同步化。随后群体同步进入S期。处理24小时后，超过90%的细胞能够掺入BrdU并PCNA染色呈阳性。ZD1694或LY231514处理的细胞发生DNA片段化，但GARFT抑制剂处理的细胞未发生。此外，用ZD1694或LY231514处理24-48小时后剩余的活细胞呼吸水平是未处理细胞的两倍。