Hyposmotic Stress Induces Cell Growth Arrest via Proteasome Activation and Cyclin/Cyclin-dependent Kinase Degradation

Abstract

Ordered cell cycle progression requires the expression and activation of several cyclins and cyclin-dependent kinases (Cdks). Hyperosmotic stress causes growth arrest possibly via proteasome-mediated degradation of cyclin D1. We studied the effect of hyposmotic conditions on three colonic (Caco2, HRT18, HT29) and two pancreatic (AsPC-1 and PaCa-2) cell lines. Hyposmosis caused reversible cell growth arrest of the five cell lines in a cell cycle-independent fashion, although some cell lines accumulated at the G 1 /S interface. Growth arrest was followed by apoptosis or by formation of multinucleated giant cells, which is consistent with cell cycle catastrophe. Hyposmosis dramatically decreased Cdc2, Cdk2, Cdk4, cyclin B1, and cyclin D3 expression in a time-dependent fashion, in association with an overall decrease in cellular protein synthesis. However, some protein levels remained unaltered, including cyclin E and keratin 8. Selective proteasome inhibition prevented Cdk and cyclin degradation and reversed hyposmotic stress-induced growth arrest, whereas calpain and lysosome enzyme inhibitors had no measurable effect on cell cycle protein degradation. Therefore, hyposmotic stress inhibits cell growth and, depending on the cell type, causes cell cycle catastrophe with or without apoptosis. The growth arrest is due to decreased protein synthesis and proteasome activation, with subsequent degradation of several cyclins and Cdks.The cell cycle involves a meticulously ordered series of events that control defined cell cycle stage check points and ultimate cell division. These events are tightly regulated by the expression and degradation, activation and inactivation, and subcellular localization of cyclins and cyclin-dependent kinases (Cdks) 1 (1-3). Cyclins associate with, and activate, Cdks and are periodically synthesized then degraded during cell cycle progression, whereas cellular Cdk levels tend to remain in excess throughout the normal cell cycle (1-5). In mammalian cells, Cdk4 and Cdk6 associate with D-type cyclins and regulate G 1 cell cycle phase progression. Cdk2 associates with the Eand A-type cyclins, and the respective complexes control G 1 /S transition and S phase progression, respectively (1-5). Cdc2 (also termed Cdk1) and the B-type cyclin form a cytoplasmic complex at the G 2 /M phase checkpoint, which translocates to the nucleus just prior to nuclear envelope breakdown during prophase. Abnormal sequestering of cyclin B1/Cdc2 complexes in the cytoplasm leads to perturbation of cell cycle progression (6 -8).Most mammalian cells have developed compensatory mechanisms to respond to changes in the osmolarity of the surrounding medium, which allow them to re-establish homeostasis of osmotically disturbed aspects of cell structure and function (9, 10). These mechanisms are necessary, because osmotic stress may severely compromise eukaryotic cell function due to the importance of maintaining homeostasis of inorganic ions as a prerequisite for normal progression of metabolic processes. Disruption of such me...

Enhancement of the p27Kip1‐mediated antiproliferative effect of trastuzumab (Herceptin) on HER2‐overexpressing tumor cells

“…73,74 Moreover, CQ up to 100 M has no effect on cyclin or Cdk expression levels. 75 We did not find any significant cytotoxic effects or changes in levels of Cdk2 or cyclin E induced by CQ at the concentration used. The major effect of CQ is due to the increase in Ab-mediated internalization of HER2 and does not represent a synergism between 2 signaling pathways induced by CQ and HCT, respectively.…”

P38SAPK2 phosphorylates cyclin D3 at Thr-283 and targets it for proteasomal degradation

“…On the other hand, it has been reported that cyclic AMP inhibits translation of cyclin D3 in T lymphocytes at the level of elongation by inducing eEF2-phosphorylation and thereby inhibiting its activity (Gutzkow et al, 2003). Moreover, it is known that cyclin D3 is degraded via proteasome when cells are subjected to tumor necrosis factor (Hu et al, 2002) or under hyposmotic stress (Tao et al, 2002). Our results indicating that cyclin D3 degradation might be induced in cells under different conditions of hyperosmotic stress (NaCl, sodium arsenite or cadmium sulfate) in a proteasome-depending manner are in agreement with these reports.…”

“…Whereas the factors involved in cyclin D3 transcription are largely unknown, recent reports indicate that cyclin D3 levels might be regulated by degradation via proteasome after hyposmotic stress (Tao et al, 2002) or in cells treated with tumor necrosis factor-a (Hu et al, 2002). It has also been shown that the formation of ternary complexes cyclin D3-cdk4 and p21 Cip1 or p27 Kip1 stabilizes the D1 and D3 cyclins (Bagui et al, 2003).…”

Escherichia coli STb toxin induces apoptosis in intestinal epithelial cell lines

“…Aside from enterotoxins, pore-forming toxins such as Staphylococcus aureus -toxin [35] and PorB of Neisseria gonorrhoeae [36] also induce intrinsic apoptosis. HRT-18 cells were chosen as they are extensively used in apoptosis studies [37,38]. IEC-18 cells, on the other hand, were chosen since in vivo studies conducted on STb are often undertaken using a rat model as an alternative to the pig.…”