Experimental cell research
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Brefeldin A (BFA) has recently been shown to induce apoptosis in human tumor cells in a p53-independent fashion. In this study, BFA-induced apoptosis was analyzed in the human Jurkat T-cell line. Apoptosis occurred 8 h after treatment with BFA and at concentrations as low as 10 ng/ml and increased with the duration of BFA exposure. ⋯ Z-Phe-Ala-FMK, a cysteine protease inhibitor lacking aspartate at the P1 position, did not have any impact on BFA-induced apoptosis. Furthermore, Jurkat cells transfected with the proto-oncoprotein Bcl-2, which is able to block various apoptotic conditions, showed remarkable resistance to the apoptotic effect of BFA. Thus, the data indicate that BFA-induced apoptosis requires caspase(s) activation, primarily the activation of caspase-3, and is inhibited by overexpression of Bcl-2.
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The calmodulin-dependent protein kinase-II (CaMK-II) inhibitor KN-93 has been shown to reversibly arrest mouse and human cells in the G1 phase of the cell cycle [Tombes, R. M., Westin, E., Grant. S., and Krystal, G. (1995) Cell Growth Differ. 6, 1073-1070; Rasmussen, G., and Rasmussen, C. (1995) Biochem. ⋯ All of these phenomena were unaffected by KN-93's inactive analog, KN-92, and were reversible upon KN-93 washout. The kinetics of recovery from cell cycle arrest were similar to those reported for other G1 phase blockers. These results suggest a mechanism by which G1 Ca2+ signals could be linked via calmodulin-dependent phosphorylations to the cell cycle-controlling machinery through cyclins and cdk inhibitors.
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In previous studies (W. Jiang, S. M. ⋯ Thus, even though overexpression of cyclin D1 can induce the expression of cyclin E and phosphorylated CDK2, premature activation of cyclin E-CDK2 kinase activity in quiescent cells or during progression through G1 appears to be blocked by CDKIs. Nevertheless, the R6ccnD1 cells have a shorter G1 phase than the control cells presumably due to the high levels of both cyclin D1 and cyclin E. Taken together, these results indicate that overexpression of cyclin D, which is frequently seen in human tumors, can have complex effects on the expression of other genes that control cell cycle progression.
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The mechanism by which cells take up retinol from retinol-binding protein (RBP) and the role of the RBP-transthyretin (TTR) complex remain unclear. Here we report on retinol uptake through the RBP-TTR complex by primary cultured rat hepatocytes (parenchymal cells, PC) and nonparenchymal cells (NPC) following incubation with [3H]retinol-RBP or the [3H]retinol-RBP-TTR complex under several conditions. The cellular accumulation of retinol was time and temperature dependent in both PC and NPC. ⋯ From these results we postulate that TTR may take part as a positive regulator in the delivery of RBP-bound retinol from plasma, possibly by a membrane receptor, and that retinol uptake takes place preferentially from the RBP-TTR complex into both PC and NPC. The uptake of [3H]retinol (2 microM) by PC was saturated, whereas uptake by NPC was not. These results indicate that the physiological importance of TTR in retinol delivery may be especially important to vitamin A-storing stellate (Ito) cells in the NPC fraction.
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Cell cycle analysis indicates that retinoic acid (RA) inhibition of MCF-7 cell growth occurs through induction of G1 arrest with a concomitant reduction in the proportion of cells in S and G2 + M phases. RA did not affect cyclins D1, A, and E and cyclin-dependent kinase 2 (CDK2) expression, but significantly reduced cyclin D3 and CDK4 expression after 24 h. RA also inhibited cyclin B1 and CDC2 expression, possibly responsible for the reduction of the proportion of cells in G2 + M and S phases. ⋯ These observations demonstrate a marked effect of RA on some of the key cell cycle regulatory proteins in MCF-7 cells. Cyclin D3 and CDK4 are likely the early targets of RA, followed by reduced pRB expression and phosphorylation, as well as by the inhibition of the E2F1 transcription factor which controls progression from G1 to S phase. Most of these events precede the observed reduction in MCF-7 cell growth, which begins at Day 3 of RA treatment.