Abstract
The intracellular concentration of ionized calcium is involved in regulating mitosis. However, little is known about intracellular levels of calcium during G1. We have demonstrated in vascular smooth muscle cells a mid-G1 decrease in ionized calcium concentration followed by a 2-fold rise at the G1/S interface (44 nM +/- 0.6 nM versus 98 nM +/- 1.1 nM, p < 0.01). The elevation of intracellular calcium is preceded by an increase in c-myb mRNA levels and is abolished with antisense but not missense c-myb oligonucleotides. Furthermore, cells stably transfected with c-myb show a similar 2-fold augmentation in intracellular calcium concentrations, as compared with untransfected cells, which is also abolished by antisense c-myb oligonucleotides. The c-myb-induced rise in intracellular calcium is dependent upon the presence of extracellular calcium and is not suppressed by L type calcium channel blockers. We conclude that c-myb induces an elevation in intracellular calcium levels of vascular smooth muscle cells at the G1/S interface which provides a novel role for this proto-oncogene as well as a potentially important control point for cell cycle regulation.
Highlights
From the $Department of Biology, Massachusetts Institute of Technology, CambridgeMassachusetts 02139, the §Department of Medicine
Flow cytometric analysis of cellular DNA shows partial cell cycle synchronization and reveals that increased intracellular calcium levels at 24 h occur as the cell population enters S phase (Fig. 1, bottom).Identical experiments using Indo-1 have beencarried out with primary rat aortic smooth muscle cells (SMC) isolated as described previously [12] with similar changes in intracellular levels of ionized calcium
The results show that intracellular calcium levelsdecrease as fected cells to 25 WM antisense c-myb oligonucleotide or anti- cells transit from Goto G1, increase for a prolonged period of sense 2-base pair mismatch c-myb oligonucleotide.The treat- time to the startingconcentrations ascells enter S phase, and ment of growth-arrested LTR-MYB with antisense c-myb undergo a subsequent prolonged drop to levels typical ofG1 oligonucleotide,but not2-base pair mismatch oligonucleotide, at later points in the cell cycle
Summary
SDS-gel electrophoresis on an 8%polyof intracellular calcium concentration in c-myb-transfected clones (LTR-MYB and SV40"YB) show a substantial increase in Fura-2 ratios in growth-arrestedas well as predominantly late GIclones as compared withuntransfected control acrylamide gel of "C-labeled molecular weight markers (first lane), cells at the same stageof the cell cycle (Fig. 5). The same results as described abovewere generated with threeadditional c-myb-transfected clones (Fura-2 ratios for LTR-MYB andSV40-MYB clones average 1.37 & 0.16 as compared with designated ratios for untransfected nifedipine (added in the dark), a specific blocker of L type calcium channels, hadno effect upon proto-oncogene-dependent elevations of intracellular calcium (Fura-2 ratios to nifedipine-free controls: 0.98 & 0.1 (no nifedipine); 0.94 f 0.1 (1 p~ nifedipine); 1.02 f 0.15 (3 p M nifedipine), p = not significant).
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