ISSN:
0021-9541
Keywords:
Life and Medical Sciences
;
Cell & Developmental Biology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Medicine
Notes:
Previous studies have shown that growth to quiescence of fibroblast-like cells is accompanied by a large decrease in the rate of phosphate uptake. Since 3T3 cells can be arrested in the G1 (or G0) phase of the cell cycle by lowering the concentration of phosphate in the medium, we examined 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.The experimental approach consisted of controlling the rate of phosphate uptake 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 as well as carrier-mediated uptake. In fact, 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, we found 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, we removed media from growing and non-growing cultures and tested its ability to support phosphate uptake. We found that 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. In addition, we found that 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, we switched growing cultures 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. In addition, measurements of intracellular phosphate pool size 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.
Additional Material:
9 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/jcp.1040920114
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