Jullien M, Harel L, Golde A, Villaudy J, Pugnet P
Exp Cell Res. 1984 Jun;152(2):390-401. doi: 10.1016/0014-4827(84)90640-2.
Our results showed that the expression of the src gene in chick embryo fibroblasts (CEF) released the density-dependent inhibition (DDI) of phosphate metabolism (phosphate uptake and phosphorylation of small organic compounds). With increasing cell density, phosphate metabolism decreased by 58% in normal CEF and, in contrast, increased by 20% in Rous sarcoma virus (RSV)-transformed CEF. The same change in the DDI was observed in CEF infected by NY68 (a ts mutant for transformation of RSV) and maintained at the permissive temperature (37 degrees C) instead of the restrictive temperature (41.5 degrees C) for the expression of transformation. An interesting feature was that the release of the DDI of phosphate metabolism was an early event in the process of transformation, since it was almost concomitant with the stimulation of the pp60 src kinase activity following the shift from 41.5 to 37 degrees C of NY68 CEF. The phosphorylation of small organic compounds (Po) was more strongly increased by the change in temperature than was 32Pi accumulation. Furthermore, the percentage increases of Po and adenosine triphosphate (ATP) labelling with 32P were similar, suggesting that the expression of src gene enhanced ATP synthesis. In glucose-free medium, the stimulation of Po-labelling was still observed but was decreased. Therefore the activation of glycolytic activity is not an absolute requirement, but is necessary for the maximum effect of transformation on the release of DDI of phosphate metabolism. Oligomycin added in complete medium did not prevent the increase in Po-labelling. From these results, we assumed that ATP turnover was stimulated as a consequence of enhanced ATP degradation. We verified that the stimulation of Po phosphorylation was not a consequence of increased ATP utilization for RNA or protein synthesis. The stimulation of Po labelling was specifically abolished by quercetin. This drug inhibited the transformed cells more strongly than the non-transformed cells.
我们的结果表明，src基因在鸡胚成纤维细胞（CEF）中的表达解除了磷酸盐代谢（磷酸盐摄取和小有机化合物的磷酸化）的密度依赖性抑制（DDI）。随着细胞密度的增加，正常CEF中的磷酸盐代谢下降了58%，相比之下，劳氏肉瘤病毒（RSV）转化的CEF中的磷酸盐代谢增加了20%。在被NY68（RSV转化的温度敏感突变体）感染并维持在允许温度（37摄氏度）而非限制温度（41.5摄氏度）以进行转化表达的CEF中，观察到了DDI的相同变化。一个有趣的特征是，磷酸盐代谢DDI的解除是转化过程中的一个早期事件，因为它几乎与NY68 CEF从41.5摄氏度转变到37摄氏度后pp60 src激酶活性的刺激同时发生。温度变化对小有机化合物（Po）磷酸化的增加作用比对32Pi积累的增加作用更强。此外，用32P标记的Po和三磷酸腺苷（ATP）的增加百分比相似，这表明src基因的表达增强了ATP合成。在无葡萄糖培养基中，仍可观察到对Po标记的刺激，但有所降低。因此，糖酵解活性的激活不是绝对必需的，但对于转化对磷酸盐代谢DDI解除的最大效应是必要的。在完全培养基中添加寡霉素并不能阻止Po标记的增加。从这些结果中，我们推测ATP周转是由于ATP降解增强而受到刺激。我们证实，Po磷酸化的刺激不是RNA或蛋白质合成中ATP利用增加的结果。槲皮素特异性地消除了对Po标记的刺激。这种药物对转化细胞的抑制作用比对未转化细胞的抑制作用更强。
