CELLULAR RAS ACTIVITY IS REQUIRED FOR PASSAGE THROUGH MULTIPLE POINTS OF THE G(0)/G(1) PHASE IN BALB/C 3T3 CELLS
CELLULAR RAS ACTIVITY IS REQUIRED FOR PASSAGE THROUGH MULTIPLE POINTS OF THE G(0)/G(1) PHASE IN BALB/C 3T3 CELLS
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DOI：
10.1128/mcb.14.8.5441
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发表时间：
1994-08-01
影响因子：
5.3
通讯作者：
STACEY, DW
中科院分区：
文献类型：
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作者：
DOBROWOLSKI, S;HARTER, M;STACEY, DW
显微注射实验表明在G晚期需要细胞ras活性(1)。在这项研究中，我们使用了两种不同的方法来确定在细胞周期的G(0)/G(1)期早期对细胞ras活性的额外需求。静止的BALB/c细胞在血清刺激前注射抗ras抗体。因此，在细胞周期中需要ras功能的最早阶段，细胞的进展将受到抑制。或者，与先前的研究一样，在静止细胞中加入血清数小时后注射抗ras，细胞在G(1)晚期受到抑制。然后用已知在g中期起作用的化学细胞周期抑制剂处理注射的培养物(1)。在血清刺激之前注射抗ras的细胞被保留在化学抑制剂执行点之前的ras需求点，而在血清刺激后3至5小时注射的细胞被保留在化学抑制剂执行点下游的ras需求点。为了证实这些结果，在加入血清之前或几小时后，向静止的BALB/c细胞注射抗ras抗体。然而，在这种情况下，第二次注射致癌ras或腺病毒E1A蛋白来克服抗ras抗体的抑制作用。在血清添加前注射的细胞在Ras需求的早期明显受到抑制，因为它们比在血清添加后注射抗Ras抗体的细胞进入S期的时间长5或6小时。
Microinjection experiments demonstrated a requirement for cellular ras activity late in G(1). In this study, we used two separate methods to identify an additional requirement for cellular ras activity early in the G(0)/G(1), phase of the cell cycle. Quiescent BALB/c cells were injected with anti-ras antibody prior to stimulation with serum. The cells would therefore be inhibited in progression through the cell cycle at the earliest point requiring ras function. Alternatively, cells were inhibited in late G(1) as in previous studies by injecting anti-ras several hours after serum addition to quiescent cells. The injected cultures were then treated with chemical cell cycle inhibitors known to function in mid-G(1). Cells injected with anti-ras prior to serum stimulation were retained at a point of ras requirement prior to the execution point of the chemical inhibitor, while cells injected 3 to 5 h after serum stimulation were retained at a point of ras requirement downstream of the execution point of the chemical inhibitor. To confirm these results, quiescent BALB/c cells were injected with anti-ras antibody prior to or several hours following serum addition. In this case, however, second injections of oncogenic ras or adenoviral E1A protein were performed to overcome the inhibitory effects of the anti-ras antibody. Cells injected prior to serum addition were clearly inhibited at an early point of Ras requirement since they required 5 or 6 h longer to enter S phase than cells injected with anti-ras antibody after serum addition.