Mercer W E, Ullrich S J, Shields M T, Lin D, Alder H
Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
Ann N Y Acad Sci. 1992 Oct 28;660:209-18. doi: 10.1111/j.1749-6632.1992.tb21073.x.
In this study the effect of antisense oligomers targeted against the mRNA transcripts of p34cdc2 kinase on G1 progression into S-phase was examined. For this purpose, antisense, sense, or nonsense oligomers were introduced directly into the cytoplasm of T98G cells grown in monolayer cultures by glass-capillary microinjection. The microinjection of antisense oligomers (but not sense or nonsense oligomers) into growth-arrested cells before serum stimulation inhibited G1 progression into S-phase. This inhibition was correlated with a reduction in the steady-state levels of nuclear p34cdc2 protein. Microinjection of antisense oligomers into cells at 2 and 6 hours after serum stimulation also resulted in a marked inhibition in the ability of cells to enter S-phase. The inhibitory effect decreased when cells were microinjected at 12 hours after serum stimulation. When cells were microinjected at 18 and 24 hours after serum stimulation, only a slight inhibition was observed. As the antisense oligomers were introduced directly into the cytoplasm of cells at each of the time points examined, the observed differences in the inhibitory effects of the antisense oligomers at later times after serum stimulation cannot be explained by differences in uptake. An alternative explanation is that after a certain threshold level of nuclear p34cdc2 protein is reached in late G1 phase; no further increase is necessary, because the cells become committed to enter S-phase. In yeast, p34cdc2 appears to play an important role in the G1/S-phase transition at a control point in late G1 phase called START (reviewed by Lewin). In mammalian cells a control point that could be equivalent to START is the "restriction point" which is defined as the time after which inhibition of protein synthesis fails to block entry into S-phase (reviewed by Pardee). The effects observed with antisense oligomers to p34cdc2 kinase are strikingly similar to what is observed when low concentrations of the drug cycloheximide are added to these cells at different times after serum stimulation; entry into S-phase is significantly inhibited when cycloheximide is added up to 12 hours postimulation. Thus, the results reported in this study are in agreement with the idea that p34cdc2 kinase plays a role in the G1/S phase transition in mammalian cells. Finally, introduction of antisense oligomers directly into the cytoplasm of cells grown in monolayer cultures by glass-capillary microinjection appears to be a viable alternative to simply adding the oligomers to the culture medium.(ABSTRACT TRUNCATED AT 400 WORDS)
在本研究中，检测了针对p34cdc2激酶mRNA转录本的反义寡聚体对G1期向S期进展的影响。为此，通过玻璃毛细管显微注射将反义、正义或无义寡聚体直接导入单层培养的T98G细胞的细胞质中。在血清刺激前，向生长停滞的细胞显微注射反义寡聚体（而非正义或无义寡聚体）可抑制G1期向S期的进展。这种抑制与核p34cdc2蛋白稳态水平的降低相关。在血清刺激后2小时和6小时向细胞显微注射反义寡聚体也导致细胞进入S期的能力受到显著抑制。当在血清刺激后12小时对细胞进行显微注射时，抑制作用减弱。当在血清刺激后18小时和24小时对细胞进行显微注射时，仅观察到轻微抑制。由于在每个检测的时间点反义寡聚体都是直接导入细胞的细胞质中，因此血清刺激后较晚时间点反义寡聚体抑制作用的观察差异无法用摄取差异来解释。另一种解释是，在G1晚期达到一定阈值水平的核p34cdc2蛋白后，无需进一步增加，因为细胞已注定进入S期。在酵母中，p34cdc2似乎在G1/S期转换中于G1晚期的一个称为START的控制点发挥重要作用（Lewin综述）。在哺乳动物细胞中，一个可能等同于START的控制点是“限制点”，其定义为蛋白质合成抑制不再阻止进入S期后的时间（Pardee综述）。用针对p34cdc2激酶的反义寡聚体观察到的效应与在血清刺激后不同时间向这些细胞添加低浓度药物放线菌酮时观察到的效应惊人地相似；在刺激后长达12小时添加放线菌酮时，进入S期受到显著抑制。因此，本研究报告的结果与p34cdc2激酶在哺乳动物细胞G1/S期转换中起作用的观点一致。最后，通过玻璃毛细管显微注射将反义寡聚体直接导入单层培养细胞的细胞质中，似乎是简单地将寡聚体添加到培养基中的一种可行替代方法。（摘要截短至400字）
