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Sodium orthovanadate stimulation of DNA synthesis in nakano mouse lens epithelial cells in serum‐free medium
Author(s) -
Jones Trevor R.,
Reid Ted W.
Publication year - 1984
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/jcp.1041210125
Subject(s) - vanadate , sodium orthovanadate , thymidine , incubation , dna synthesis , stimulation , insulin , biology , cell growth , microbiology and biotechnology , medicine , biochemistry , endocrinology , dna , chemistry , phosphorylation , phosphatase
Abstract Quiescent cultured Nakano mouse lens cells incubated for 40 hours with sodium orthovanadate incorporated 3 H‐thymidine at an accelerated rate; the greatest response occurred at 20 μM vanadate, whereas by 2 μM an incorporation rate equivalent to unstimulated cells was noted. Microscopic examination of the cells revealed that those exposed to concentrations of vanadate greater than 100 μM had lysed by the end of the 40‐hour incubation. Reduction in vanadate exposure time to 1 hour caused the cells to incorporate the greatest amount of 3 H‐thymidine at a vanadate concentration of 200 μM to 500 μM. Half‐maximum incorporation of 3 H‐thymidine (after a 40‐hour incubation) was induced by a 2‐hour incubation with 20 μM vanadate. Studies with insulin showed that while 20 ng/ml insulin alone did not increase 3 H‐thymidine incorporation, 20 ng/ml insulin in combination with 20 μM vanadate resulted in a significant increase in 3 H‐thymidine uptake over cells exposed to only vanadate. Insulin alone will increase cell number and insulin with vanadate are synergistic in the stimulation of DNA synthesis, but the two together show no further increase in cell number over that produced by insulin alone. Thus, vanadate can increase progression from G1/G0 to S‐phase, but cannot stimulate cells to divide. Studies designed to detect DNA damage and repair rather than S‐phase DNA synthesis demonstrated that vanadate was not causing increased 3 H‐thymidine uptake by damaging DNA. Cell counts revealed that vanadate, while able to induce DNA synthesis, does not induce mitosis. Autoradiography and equilibrium sedimentation experiments demonstrated that gene amplification was not occurring. A known vanadate exchange inhibitor blocked the ability of vanadate to increase 3 H‐thymidine incorporation which is consistent with the idea that cellular internalization of vanadate is required for this effect to be seen. 86 Rb + uptake experiments demonstrate that the vanadate concentration inducing 50% inhibition of (Na + , K + )ATPase is nearly two orders of magnitude more concentrated that vanadate concentrations shown capable of inducing 3 H‐thymidine uptake. This strongly suggests that (Na + , K + )ATPase inhibition is not the central mechanism by which DNA synthesis is stimulated by vanadate.