Ren Q, Van Groeningen C J, Hardcastle A, Aherne G W, Geoffroy F, Allegra C J, Johnston P G, Grem J L
Developmental Therapeutics Department, National Cancer Institute, National Naval Medical Center, Bethesda, MD 20889-5105, USA.
Oncol Res. 1997;9(2):77-88.
To explore the determinants of cytotoxicity during prolonged exposure to pharmacologically relevant concentrations of 5-fluorouracil (FUra), we studied the effects of FUra at concentrations ranging from 0.1 to 1 microM in HCT 116 and HT 29 colon cancer cells grown in the presence of physiologic levels of leucovorin. A 5- and 7-day exposure to 1 microM FUra reduced cell growth to 46% and 20% of control in HT 29 cells and to 74% and 38% of control in HCT 116 cells. Concurrent exposure to thymidine (10 or 20 microM) or uridine (1 mM) provided partial protection against FUra toxicity in HT 29 cells, but did not protect HCT 116 cells. After a 24-h exposure to 1 microM [3H]FUra, free 5-fluoro-2'-deoxyuridine-5' -monophosphate (FdUMP) and FUDP. + FUTP levels were 0.7 and 144 pmol/10(6) cells in HT 29 cells, respectively, and 3.9 and 178 pmol/10(6) cells in HCT 116 cells. FdUMP and FUDP + FUTP pools increased by 5.7- and 2.0-fold in HT 29 cells and by 1.7- and 3.3-fold in HCT 116 cells over the next 48 h, but did not accumulate thereafter. After a 24-h exposure to 1 microM [3H]FUra, FUra-RNA levels were 158 and 280 fmol/microgram in HT 29 and HCT 116 cells, respectively; FUra-RNA levels increased over time, and reached 700 and 1156 fmol/microgram at day 5. Concurrent exposure to 1 mM uridine for 72 h did not diminish [3H]FUra-RNA incorporation. Upon removal of [3H]FUra following a 24-h exposure, FUra-RNA levels remained relatively stable with 57-78% retained at 120 h. A low level of [3H]FUra-DNA incorporation was detected in HT 29 cells. Thymidylate synthase (TS) catalytic activity in control cells was 2-fold higher in HCT 116 cells compared to HT 29 cells (47 vs. 23 pmol/min/mg). Total TS content increased 1.5- to 3-fold over control in both cell lines during FUra exposure, and ternary complex formation was evident for up to 96 h-dTTP pools were not depleted in FUra-treated cells, suggesting that residual TS catalytic activity was sufficient to maintain dTTP pools relative to demand. Surprisingly, the partial inhibition of TS was accompanied by a striking accumulation of immunoreactive "dUMP" pools in both lines; dUTP pools also increased 2-to 3-fold. In summary, the gradual and stable accumulation of FUra in RNA noted in both lines may account for the thymidine-insensitive component of FUra toxicity. Because dTTP pools were not appreciably diminished, the interference with nascent DNA chain elongation and induction of single-strand breaks in newly synthesized DNA in both cell lines may be due to misincorporation of deoxyuridine nucleotides.
为了探究在长时间暴露于药理学相关浓度的5-氟尿嘧啶（FUra）过程中细胞毒性的决定因素，我们研究了在生理水平的亚叶酸存在下，浓度范围为0.1至1微摩尔的FUra对HCT 116和HT 29结肠癌细胞的影响。在HT 29细胞中，1微摩尔FUra暴露5天和7天可使细胞生长分别降至对照的46%和20%；在HCT 116细胞中则降至对照的74%和38%。同时暴露于胸苷（10或20微摩尔）或尿苷（1毫摩尔）可部分保护HT 29细胞免受FUra毒性，但对HCT 116细胞无保护作用。在暴露于1微摩尔[³H]FUra 24小时后，HT 29细胞中游离的5-氟-2'-脱氧尿苷-5'-单磷酸（FdUMP）和FUDP + FUTP水平分别为0.7和144皮摩尔/10⁶细胞，在HCT 116细胞中分别为3.9和178皮摩尔/10⁶细胞。在接下来的48小时内，HT 29细胞中FdUMP和FUDP + FUTP池分别增加了5.7倍和2.0倍，HCT 116细胞中分别增加了1.7倍和3.3倍，但此后不再积累。在暴露于1微摩尔[³H]FUra 24小时后，HT 29和HCT 116细胞中的FUra-RNA水平分别为158和280飞摩尔/微克；FUra-RNA水平随时间增加，在第5天达到700和1156飞摩尔/微克。同时暴露于1毫摩尔尿苷72小时并未减少[³H]FUra-RNA的掺入。在暴露24小时后去除[³H]FUra，FUra-RNA水平保持相对稳定，在120小时时仍保留57 - 78%。在HT 29细胞中检测到低水平的[³H]FUra-DNA掺入。对照细胞中的胸苷酸合成酶（TS）催化活性在HCT 116细胞中比HT 29细胞高2倍（47对23皮摩尔/分钟/毫克）。在FUra暴露期间，两种细胞系中的总TS含量均比对照增加了1.5至3倍，并且三元复合物形成在长达96小时内都很明显——在FUra处理的细胞中dTTP池未耗尽，这表明残留的TS催化活性足以维持相对于需求的dTTP池。令人惊讶的是，TS的部分抑制伴随着两种细胞系中免疫反应性“dUMP”池的显著积累；dUTP池也增加了2至3倍。总之，两种细胞系中观察到的FUra在RNA中的逐渐和稳定积累可能解释了FUra毒性中对胸苷不敏感的成分。由于dTTP池没有明显减少，两种细胞系中对新生DNA链延伸的干扰以及新合成DNA中单链断裂的诱导可能是由于脱氧尿苷核苷酸的错误掺入。
