PHOSPHATE UPTAKE AND CONTROL OF FIBROBLAST GROWTH
PHOSPHATE UPTAKE AND CONTROL OF FIBROBLAST GROWTH
复制标题
DOI：
10.1002/jcp.1040920114
复制
发表时间：
1977-01-01
影响因子：
5.6
通讯作者：
CUNNINGHAM, DD
中科院分区：
文献类型：
--
作者：
BARSH, GS;GREENBERG, DB;CUNNINGHAM, DD
成纤维细胞样细胞的生长到静止伴随着磷酸盐摄取速率的大幅下降。由于3T3细胞[瑞士小鼠]可以通过降低培养液中的磷酸盐浓度而被阻止在细胞周期的G1(或GO)期，因此研究了在生长到静止期间观察到的磷酸盐摄取量下降可能是抑制DNA合成和细胞分裂的关键事件。通过改变培养基中的磷浓度来控制吸收磷的速率。动力学实验表明，生长和静止细胞的磷吸收部分是由简单扩散和载体介导的吸收所解释的。磷在生长细胞中的扩散是在静止细胞中的2.5倍。不同初始密度培养的3T3细胞对磷的吸收与细胞密度呈负相关，表明细胞对磷的吸收与生长速度有关，而不是简单地随培养时间的延长而下降。对磷酸盐的测量表明，静止细胞吸收磷酸盐的速度降低不仅仅是因为培养液中磷酸盐的减少。为了检查先前描述的运输抑制剂的释放可能解释当细胞生长到静止时观察到的磷酸盐吸收下降的可能性，从生长和非生长的培养物中移除培养基，并测试其支持磷酸盐吸收的能力。生长期培养基比静止期培养基磷吸收速率更高，表明运输抑制物正在释放。缓蚀剂的释放量与介质中磷酸盐的浓度成正比。为了直接确定当细胞生长到静止时，磷吸收的下降是否是DNA合成下降的关键事件，生长中的培养物在细胞附着后立即切换到低磷的培养基中。这将磷酸盐的吸收速度降低到低于在通常浓度的磷酸盐中生长的静止细胞的水平。这是对3T3、多瘤病毒转化的3T3、人类二倍体包皮和次级鸡胚胎细胞进行的。DNA合成和细胞数量的测量表明，这种降低的磷酸盐吸收速率对细胞生长几乎没有影响，直接表明在生长到融合过程中观察到的磷酸盐吸收的下降并不是导致DNA合成的下降。对细胞内磷酸盐池大小的测量也表明，细胞内磷酸盐吸收的变化与细胞内磷酸盐池大小的变化并不直接平行，而且在生长到静止期间，细胞内磷酸盐池的大小并不调节DNA合成或细胞分裂。
Growth to quiescence of fibroblast-like cells is accompanied by a large decrease in the rate of phosphate uptake. Since 3T3 cells [Swiss mouse] can be arrested in the G1 (or Go) phase of the cell cycle by lowering the concentration of phosphate in the medium, the possibility that the decline in phosphate uptake observed during growth to quiescence might be a key event in the inhibition of DNA synthesis and cell division was examined. The rate of phosphate uptake was controlled by varying the phosphate concentration in the medium. Kinetic experiments showed that phosphate uptake in both growing and quiescent cells was partly accounted for by simple diffusion and carrier-mediated uptake. Diffusion of phosphate into the growing cells was 2.5-fold greater than in the quiescent cells. When phosphate uptake was measured in 3T3 cells plated at different initial densities, there was an inverse relationship between phosphate uptake and cell density showing that phosphate uptake was correlated with growth rate and did not decline simply as a consequence of time in culture. Measurements of phosphate demonstrated that the lowered rate of phosphate uptake by quiescent cells was not due merely to a reduction of phosphate in the medium. To check the possibility that release of a previously described transport inhibitor might account for the decline in phosphate uptake observed as cells grow to quiescence, media was removed from growing and non-growing cultures and its ability to support phosphate uptake was tested. The medium from growing cultures supported a higher rate of phosphate uptake than the medium from the quiescent cultures did, indicating that a transport inhibitor was being released. The amount of inhibitor released was proportional to the concentration of phosphate in the medium. To directly determine if the decline in phosphate uptake was a key event in the decline in DNA synthesis as cells grew to quiescence, growing cultures were switched to a medium with low phosphate immediately after cell attachment. This lowered the rate of phosphate uptake to a level below that of quiescent cells grown in the usual concentration of phosphate. This was done for 3T3, Polyoma virus-transformed 3T3, human diploid foreskin and secondary chick embryo cells. Measurements of DNA synthesis and cell number showed that this lowered rate of phosphate uptake had virtually no effect on cell growth, directly demonstrating that the decline in phosphate uptake observed during growth to confluency was not causing the decline in DNA synthesis. Measurements of intracellular phosphate pool size also showed that changes in phosphate uptake were not directly paralleled by changes in intracellular phosphate pool size and that intracellular phosphate pool size was not regulating DNA synthesis or cell division during growth to quiescence.