Will C L, Dolnick B J
Department of Experimental Therapeutics, Roswell Park Memorial Institute, Buffalo, New York 14263.
J Biol Chem. 1989 Dec 15;264(35):21413-21.
This laboratory previously reported that 5-fluorouracil (FUra) increases dihydrofolate reductase (DHFR) precursor mRNA (pre-mRNA) levels relative to DHFR mRNA levels in a methotrexate-resistant KB cell line; these data suggested that incorporation of FUra into RNA may, in part, lead to cell death through the inhibition of mRNA processing (Will, C. L., and Dolnick, B.J. (1987) J. Biol. Chem. 262, 5433-5436). Utilizing a methotrexate-resistant KB cell line designated 1BT, we now report the kinetic basis for altered levels of DHFR RNA observed in FUra-treated cells. Long-term exposure to FUra had no effect on the steady-state level of DHFR pre-mRNA containing intron V or I. However, steady-state levels of total DHFR mRNA decreased 2.0-fold on a per cell basis in cells exposed to 1.0 microM FUra. No significant change in the half-life of total DHFR mRNA or pre-mRNA was observed in cells exposed to FUra (t1/2 = approximately 11.5 h and 50 min, respectively). Nuclear/cytoplasmic RNA labeling experiments demonstrated that the rate of nuclear DHFR RNA conversion to cytoplasmic DHFR mRNA decreased approximately 1.8-fold in FUra-treated cells. These results provide further evidence the FUra may inhibit processing of mRNA precursors and/or affect the stability of nuclear DHFR mRNA.
本实验室先前报道，在一种耐甲氨蝶呤的KB细胞系中，相对于二氢叶酸还原酶（DHFR）mRNA水平，5-氟尿嘧啶（FUra）可增加DHFR前体mRNA（前体mRNA）水平；这些数据表明，FUra掺入RNA可能部分通过抑制mRNA加工导致细胞死亡（威尔，C.L.，和多尔尼克，B.J.（1987年）《生物化学杂志》262，5433 - 5436）。利用一种名为1BT的耐甲氨蝶呤KB细胞系，我们现在报告在FUra处理的细胞中观察到的DHFR RNA水平改变的动力学基础。长期暴露于FUra对含有内含子V或I的DHFR前体mRNA的稳态水平没有影响。然而，在暴露于1.0 microM FUra的细胞中，总DHFR mRNA的稳态水平在每个细胞基础上下降了2.0倍。在暴露于FUra的细胞中，未观察到总DHFR mRNA或前体mRNA半衰期的显著变化（半衰期分别约为11.5小时和50分钟）。核/细胞质RNA标记实验表明，在FUra处理的细胞中，核DHFR RNA转化为细胞质DHFR mRNA的速率下降了约1.8倍。这些结果提供了进一步的证据，表明FUra可能抑制mRNA前体的加工和/或影响核DHFR mRNA的稳定性。
