Karn J, Watson J V, Lowe A D, Green S M, Vedeckis W
MRC Laboratory of Molecular Biology, Medical Research Council Centre, Cambridge, UK.
Oncogene. 1989 Jun;4(6):773-87.
Early passage murine fibroblasts infected with retroviral vectors carrying human c-myc 'minigenes' express high levels of c-myc and have a dramatically shortened G1-phase of the cell cycle. Cells infected with viruses where c-myc is expressed from the viral LTR (MSN-4 virus) express more c-myc protein than cells infected with viruses where c-myc is expressed from the SV40 early promoter (NSM-7 virus). Populations of cells were infected with high titre viruses, selected for drug-resistance, pulse labelled with bromodeoxyuridine (BrdUrd) and chased in BrdUrd free media. This allows accurate, simultaneous, measurement of the rate of exit of unlabelled cells from G1 and progression of BrdUrd-labelled cells through S-phase. The length of the G1-phase in cell populations infected with the MSN-4 virus is 4.65 h, a reduction of nearly 30% compared to the G1-phase length of 6.50 h seen in cells infected with the VSN-2 control virus. Cells infected with NSM-7 virus show an intermediate phenotype and have a G1-phase of 5.25 h. The lengths of the S-phase (4.50 to 4.75 h) and G2 + M phases (2.75 h) were not significantly altered by exogenous c-myc expression. When chases are performed in growth-factor free media, the G1-phase of infected and non-infected cells is extended by approximately 2 h. Cells infected with the c-myc viruses continue to cycle more rapidly than uninfected cells. Growth factor-deprived cells, restimulated with serum, show similar alterations of the cell cycle kinetics. MSN-4 and NSM-7 infected cells, expressing high levels of c-myc, enter S-phase 2 to 4 h earlier, but less synchronously, than control cells, and sustain subsequent rounds of DNA synthesis, while control cells do not. However, cells carrying activated c-myc genes have nearly-normal morphologies and are not tumourgenic in syngenic mice. These results demonstrate that c-myc levels are rate limiting for events in G1, and the length of G1 varies proportionally with the level of exogenous c-myc expression.
用携带人c-myc“小基因”的逆转录病毒载体感染的早期传代小鼠成纤维细胞，表达高水平的c-myc，并且细胞周期的G1期显著缩短。用病毒LTR（MSN-4病毒）表达c-myc的病毒感染的细胞，比用SV40早期启动子（NSM-7病毒）表达c-myc的病毒感染的细胞表达更多的c-myc蛋白。用高滴度病毒感染细胞群体，选择抗药的细胞，用溴脱氧尿苷（BrdUrd）进行脉冲标记，然后在不含BrdUrd的培养基中追踪。这使得能够准确、同时测量未标记细胞从G1期退出的速率以及BrdUrd标记的细胞通过S期的进程。用MSN-4病毒感染的细胞群体中G1期的长度为4.65小时，与用VSN-2对照病毒感染的细胞中6.50小时的G1期长度相比，减少了近30%。用NSM-7病毒感染的细胞表现出中间表型，G1期为5.25小时。S期（4.50至4.75小时）和G2+M期（2.75小时）的长度并未因外源性c-myc表达而发生显著改变。当在无生长因子的培养基中进行追踪时，感染和未感染细胞的G1期延长约2小时。用c-myc病毒感染的细胞继续比未感染的细胞循环更快。用血清重新刺激生长因子剥夺的细胞，显示出类似的细胞周期动力学改变。表达高水平c-myc的MSN-4和NSM-7感染细胞比对照细胞提前2至4小时进入S期，但同步性较差，并维持随后的几轮DNA合成，而对照细胞则不然。然而，携带活化c-myc基因的细胞具有近乎正常的形态，并且在同基因小鼠中不具有致瘤性。这些结果表明，c-myc水平是G1期事件的速率限制因素，并且G1期的长度与外源性c-myc表达水平成比例变化。
