P27 Kip1 Degradation Research Articles

Expression of Skp2, a p27Kip1 ubiquitin ligase, in malignant lymphoma: correlation with p27Kip1 and proliferation index

Reduced levels of p27(Kip1) are frequent in human cancers and have been associated with poor prognosis. Skp2, a component of the Skp1-Cul1-F-box protein (SCF) ubiquitin ligase complex, has been implicated in p27(Kip1) degradation. Increased Skp2 levels are seen in some solid tumors and are associated with reduced p27(Kip1). We examined the expression of these proteins using single and double immunolabeling in a large series of lymphomas to determine if alterations in their relative levels are associated with changes in cell proliferation and lymphoma subgroups. We studied the expression of Skp2 in low-grade and aggressive B-cell lymphomas (n = 86) and compared them with p27(Kip1) and the proliferation index (PI). Fifteen hematopoietic cell lines and peripheral blood lymphocytes were studied by Western blot analysis. In reactive tonsils, Skp2 expression was limited to proliferating germinal center and interfollicular cells. Skp2 expression in small lymphocytic lymphomas (SLLs) and follicular lymphomas (FCLs) was low (mean percentage of positive tumor cells, less than 20%) and was inversely correlated (r = -0.67; P <.0001) with p27(Kip1) and positively correlated with the PI (r = 0.82; P <.005). By contrast, whereas most mantle cell lymphomas (MCLs) demonstrated low expression of p27(Kip1) and Skp2, a subset (n = 6) expressed high Skp2 (exceeding 20%) with a high PI (exceeding 50%). Skp2 expression was highest in diffuse large B-cell lymphomas (DLBCLs) (mean, 22%) and correlated with Ki-67 (r = 0.55; P <.005) but not with p27(Kip1). Cytoplasmic Skp2 was seen in a subset of aggressive lymphomas. Our data provide evidence for p27(Kip1) degradative function of Skp2 in low-grade lymphomas. The absence of this relationship in aggressive lymphomas suggests that other factors contribute to deregulation of p27(Kip1) expression in these tumors.

Effects of ErbB-2 overexpression on mitogenic signalling and cell cycle progression in human breast luminal epithelial cells.

Most breast cancers arise from luminal epithelial cells and 25-30% of these tumours overexpress the ErbB-2 receptor. Herein, a non-transformed, immortalized cell system was used to investigate the effects of ErbB-2 overexpression in luminal epithelial cells. The phenotypic consequence of ErbB-2 overexpression is a shortening of the G1 phase of the cell cycle and early S phase entry, which leads to hyperproliferation. We show that this effect was mediated through the up-regulation of cdk6 and cyclins D1 and E, and enhanced degradation and relocalization of p27(Kip1). These changes were effected predominantly through enhanced MAPK signalling, resulting in cdk2 hyperactivation. PI3K signalling also participated in cell cycle progression, since PI3K and MAPK coordinately regulated changes in cyclin D1 and cdk6 expression. Cdk4 activity was not required for cell cycle progression in these cells, and was constitutively inhibited through its association with p16(INK4A). MAPK-dependent induction of p21(Cip1) was also necessary for G1 phase progression, although its degradation by the proteasome was required for S phase entry. These data provide new insights into the complex molecular mechanisms underlying mitogenic cell cycle control in luminal epithelial cells, the cell type relevant to primary breast cancer, and show how ErbB-2 overexpression subverts this normal control.

Phosphatidylinositol 3-Kinase/Akt Pathway Regulates Tuberous Sclerosis Tumor Suppressor Complex by Phosphorylation of Tuberin

Normal cellular functions of hamartin and tuberin, encoded by the TSC1 and TSC2 tumor suppressor genes, are closely related to their direct interactions. However, the regulation of the hamartin-tuberin complex in the context of the physiologic role as tumor suppressor genes has not been documented. Here we show that insulin or insulin growth factor (IGF) 1 stimulates phosphorylation of tuberin, which is inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 but not by the mitogen-activated protein kinase inhibitor PD98059. Expression of constitutively active PI3K or active Akt, including Akt1 and Akt2, induces tuberin phosphorylation. We further demonstrate that Akt/PKB associates with hamartin-tuberin complexes, promoting phosphorylation of tuberin and increased degradation of hamartin-tuberin complexes. The ability to form complexes, however, is not blocked. Akt also inhibits tuberin-mediated degradation of p27(kip1), thereby promoting CDK2 activity and cellular proliferation. Our results indicate that tuberin is a direct physiological substrate of Akt and that phosphorylation of tuberin by PI3K/Akt is a major mechanism controlling hamartin-tuberin function.

Expression of p27 Kip1 in bladder cancers: immunohistochemical study and prognostic value in a series of 95 cases

Prognostic value of p27 Kip1 immunohistochemical expression was evaluated in a series of 95 bladder carcinomas. Low p27 Kip1 expression was correlated with higher tumor grade ( P=0.01) and stage ( P=0.009), associated with poor overall survival ( P=0.01) and, for superficial cancers, with disease-free survival ( P=0.05). Thirty-five cases exhibited a heterogeneous expression related in some instances to tumoral architecture. Seventeen cases showed a cytoplasmic reactivity related to low nuclear expression ( P=0.057). Loss of p27 Kip1 expression is a pejorative event in bladder tumors and inhibition of p27 Kip1 degradation could offer new therapeutic ways.

Modulation of caspase activation and p27 Kip1 degradation in the p53-induced apoptosis in IW32 erythroleukemia cells

To examine the p53-mediated biological activities and signalling pathways, we generated stable transfectants of the p53-null IW32 murine erythroleukemia cells expressing the temperature-sensitive p53 mutant DNA, tsp53 val135. Two clones with different levels of p53 protein expression were selected for further characterization. At permissive temperature, clone 1–5 cells differentiated along the erythroid pathway, and clone 3–2 cells that produced greater levels (3.5-fold) of p53 underwent apoptosis. Apoptosis of 3–2 cells was accompanied by mitochondrial cytochrome c release and caspase activation as well as by cleavage of caspase substrates. Bax protein was induced to a similar extent in these clones by wild-type p53; expression of p21 Cip1/Waf1 and p27 Kip1 proteins was also increased. However, significantly lesser extent of induction for both CDK inhibitors was detected in the apoptotic 3–2 clone. The general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD.fmk) blocked the p53-induced apoptosis in 3–2 cells, with a concomitant elevation of p27 Kip1, suggesting that p27 Kip1 protein underwent caspase-dependent proteolysis in the apoptotic 3–2 cells. Together these results linked a pathway involving cytochrome c release, caspase activation and p27 Kip1 degradation to the p53-induced apoptosis in IW32 erythroleukemia cells.

Xic1 degradation in Xenopus egg extracts is coupled to initiation of DNA replication.

CDK2 activity is regulated by phosphorylation/dephosphorylation, subcellular localization, cyclin levels, and cyclin dependent kinase inhibitors (CKIs). Using Xenopus egg extracts, we find that degradation of Xic1, a Xenopus p21(cip1)/p27(kip1) family member, is coupled to initiation of DNA replication. Xic1 turnover requires the formation of a prereplication complex (pre-RC). Additionally, downstream initiation factors including CDK2, Cdc7, and Cdc45, but not RPA or DNA polymerase alpha, are necessary for activating the degradation system. Xic1 degradation is attenuated following completion of DNA replication. Unlike degradation of p27(kip1) in mammalian cells, CDK2 activity is not directly involved in Xic1 degradation and interactions between Xic1 and CDK2/cyclin E are dispensable for Xic1 turnover. Interestingly, a C-terminal region (162-192) of Xic1 is essential and apparently sufficient for triggering Xic1 ubiquitination prior to degradation. These observations demonstrate that a direct link exists between DNA replication and CKI degradation.

Interaction and colocalization of PGP9.5 with JAB1 and p27(Kip1).

PGP9.5 (UCH-L1) is a member of the ubiquitin C-terminal hydrolase (UCH) family of proteins that is expressed in neuronal tissues. Our previous studies have shown that PGP9.5 was highly expressed in primary lung cancers and lung cancer cell lines. Additionally, the frequency of PGP9.5 over expression increases with tumor stage, indicating that PGP9.5 may play a role in lung cancer tumorigenesis. We used the yeast two-hybrid system to identify proteins that interact with PGP9.5. We show that PGP9.5 interacts with at least three proteins, one of which is JAB1, a Jun activation domain binding protein that can bind to p27(Kip1) and is involved in the cytoplasmic transportation of p27(Kip1) for its degradation. We also show that PGP9.5 is associated with JAB1 in vitro and in vivo; and that both proteins can be a part of a heteromeric complex containing p27(Kip1) in the nucleus in lung cancer cells. Furthermore, under serum-restimulation, nuclear translocation of both PGP9.5 and JAB1 coincides with a reduced level of p27(Kip1) in the nucleus. In contrast, when cells are contact inhibited, both PGP9.5 and JAB1 became more perinuclear and cytoplasmic in localization while p27(Kip1) was present only in the nucleus. Therefore, PGP9.5 may contribute to p27(Kip1) degradation via its interaction and nuclear translocation with JAB1.

Phosphorylation of p27Kip1 on Serine 10 Is Required for Its Binding to CRM1 and Nuclear Export

Phosphorylation of the cyclin-dependent kinase inhibitor p27(Kip1) has been thought to regulate its stability. Ser(10) is the major phosphorylation site of p27(Kip1), and phosphorylation of this residue affects protein stability. Phosphorylation of p27(Kip1) on Ser(10) has now been shown to be required for the binding of CRM1, a carrier protein for nuclear export. The p27(Kip1) protein was translocated from the nucleus to the cytoplasm at the G(0)-G(1) transition of the cell cycle, and this export was inhibited by leptomycin B, a specific inhibitor of CRM1-dependent nuclear export. The nuclear export and subsequent degradation of p27(Kip1) at the G(0)-G(1) transition were observed in cells lacking Skp2, the F-box protein component of an SCF ubiquitin ligase complex, indicating that these early events are independent of Skp2-mediated proteolysis. Substitution of Ser(10) with Ala (S10A) markedly reduced the extent of p27(Kip1) export, whereas substitution of Ser(10) with Asp (S10D) or Glu (S10E) promoted export. Co-immunoprecipitation analysis showed that CRM1 preferentially interacted with S10D and S10E but not with S10A, suggesting that the phosphorylation of p27(Kip1) on Ser(10) is required for its binding to CRM1 and for its subsequent nuclear export.

The COP9 Signalosome Inhibits p27kip1 Degradation and Impedes G1-S Phase Progression via Deneddylation of SCF Cul1

The COP9 signalosome (CSN) is a conserved protein complex with homologies to the lid subcomplex of the 26S proteasome. It promotes cleavage of the Nedd8 conjugate (deneddylation) from the cullin component of SCF ubiquitin ligases. We provide evidence that cullin neddylation and deneddylation is highly dynamic, that its equilibrium can be effectively modulated by CSN, and that neddylation allows Cul1 to form larger protein complexes. CSN2 integrates into the CSN complex via its C-terminal region and its N-terminal half region is necessary for direct interaction with Cul1. The polyclonal antibodies against CSN2 but not other CSN subunits cause accumulation of neddylated Cul1/Cul2 in HeLa cell extract, indicating that CSN2 is essential in cullin deneddylation. Further, CSN inhibits ubiquitination and degradation of the cyclin-dependent kinase inhibitor p27(kip1) in vitro. Microinjection of the CSN complex impeded the G1 cells from entering the S phase. Moreover, anti-CSN2 antibodies negate the CSN-dependent p27 stabilization and the G1/S blockage, suggesting that these functions require the deneddylation activity. We conclude that CSN inhibits SCF ubiquitin ligase activity in targeting p27 proteolysis and negatively regulates cell cycle at the G1 phase by promoting deneddylation of Cul1.

Molecular cloning and characterisation of p15 CDK-BP, a novel CDK-binding protein

The suc1/Cks proteins are well-conserved regulatory components of cyclin-dependent kinases 1 and 2 (CDK1/2). These small molecular mass proteins form a stable complex with CDK1/2 and are essential for normal regulation of CDKs during the cell division cycle and for degradation of p27 kip1. Despite the high degree of homology between the nine known CDKs, only CDK1, CDK2 and, to a lesser extent, CDK3 are able to bind to the suc1/Cks proteins. No additional suc1/Cks-related proteins interacting with other CDKs have been reported. We have purified, from starfish oocytes, a 15 kDa protein, p15 CDK-BP, which cross-reacts with anti-Cks antibodies (L. Azzi, L. Meijer, A.C. Ostvold, J. Lew, J.H. Wang, J. Biol. Chem. 269 (1994)). Following microsequencing of internal peptides and generation of corresponding oligonucleotides we cloned two cDNAs encoding two closely related proteins, p15A and p15B. The predicted protein sequences display distant but distinct homology with the Suc1/Cks proteins, including the genuine starfish Cks homologue protein, p9 CksMg. P15 transcripts are essentially expressed in oocytes. Recombinant p15B or native p15 CDK-BP bind a 34 kDa protein cross-reacting with anti-PSTAIRE antibodies, a feature characteristic of CDK-related proteins. In addition p15B interacts tightly with CDK4, CDK6, CDK8 and the yeast CDC28-related kinase Pho85, but not with CDK1, CDK2 or CDK7. P15 does not appear to alter the catalytic activity of the bound kinases.

Induction of Apoptosis in Human Leukemia Cells by the CDK1 Inhibitor

We have examined the effects of the CDK1 inhibitor CGP74514A on cell cycle- and apoptosis-related events in human leukemia cells. An 18-hr exposure to 5 mM CGP74514A induced mitochondrial damage (i.e., loss of Dym) and apoptosis in multiple human leukemia cell lines (e.g., U937, HL-60, KG-1, CCRF-CEM, Raji, and THP; range 30-95%). In U937 cells, CGP74514A- induced apoptosis (5 mM) became apparent within 4 hr and approached 100% by 24 hr. The pan- caspase inhibitor Boc-fmk and the caspase-8 inhibitor IETD-fmk opposed CGP74514A-induced caspase-9 activation and PARP degradation, but not cytochrome c or Smac/DIABLO release. CGP74514A-mediated apoptosis was substantially blocked by ectopic expression of full-length Bcl- 2, a loop-deleted mutant Bcl-2, and Bcl-xL. CGP74514A treatment (5 mM; 18 hr) resulted in increased p21CIP1 expression, p27KIP1 degradation, diminished E2F1 expression, and dephosphorylation of p34cdc2. It also induced early (i.e., within 2 hr) inhibition of CDK1 activity and dephosphorylation of pRb, followed by pRb degradation, but did not block pRb phosphorylation at CDK2- and CDK4- specific sites. These findings indicate that the selective CDK1 inhibitor, CGP74514A, induces complex changes in cell cycle-related proteins in human leukemia cells accompanied by extensive mitochondrial damage, caspase activation, and apoptosis.Key Words:Leukemia, CDK1 Inhibitor, Apoptosis, CGP74514A

The Cytoplasmic Shuttling and Subsequent Degradation of p27Kip1 Mediated by Jab1/CSN5 and the COP9 Signalosome Complex

The fifth component of the COP9 signalosome complex, Jab1/CSN5, directly binds to and induces specific down-regulation of the cyclin-dependent kinase inhibitor p27 (p27(Kip1)). Nuclear-cytoplasmic translocation plays an important role because leptomycin B (LMB), a chemical inhibitor of CRM1-dependent nuclear export, prevents p27 degradation mediated by Jab1/CSN5. Here we show that Jab1/CSN5 functions as an adaptor between p27 and CRM1 to induce nuclear export and subsequent degradation. Jab1/CSN5, but not p27, contains a typical leucine-rich nuclear export signal (NES) sequence conserved among different species, through which CRM1 bound to Jab1/CSN5 in an LMB-sensitive manner. Alteration of conserved leucine residues to alanine within Jab1/CSN5-NES abolished the interaction with CRM1 in vitro and impaired LMB-sensitive nuclear export and the ability to induce p27 breakdown in cultured cells. A Jab1/CSN5 truncation mutant lacking NES reversed p27 down-regulation induced by the full-length Jab1/CSN5, indicating that this mutant functions as a dominant negative (DN-Jab1). Introduction of DN-Jab1 into proliferating fibroblasts increased the level of p27 protein, thereby inducing growth arrest of the cells. Random mutagenesis analysis revealed that specific aspartic acid, leucine, and asparagine residues contained in the Jab1/CSN5-binding domain of p27 were required for interaction with Jab1/CSN5 and for down-regulation of p27. Glycerol gradient and cell fractionation experiments showed that at least two different forms of Jab1/CSN5-containing complexes existed within the cell. One is the conventional 450-kDa COP9 signalosome (CSN) complex located in the nucleus, and the other is much smaller (around 100-kDa), containing only a subset of CSN components (CSN4-8 but not CSN1-3), and mainly located in the cytoplasm. Treatment of cells with LMB greatly reduced the level of the smaller complex, suggesting that it originated from the CSN complex by nuclear export. Besides Jab1/CSN5, CSN3, -6, -7, and -8 were capable of inducing p27 down-regulation, when ectopically expressed. These results indicate that cytoplasmic shuttling regulated by Jab1/CSN5 and other CSN components may be a new pathway to control the intracellular abundance of the key cell cycle regulator.

Degradation of p27Kip1 at the G0-G1 Transition Mediated by a Skp2-independent Ubiquitination Pathway

Targeting of the cyclin-dependent kinase inhibitor p27(Kip1) for proteolysis has been thought to be mediated by Skp2, the F-box protein component of an SCF ubiquitin ligase complex. Degradation of p27(Kip1) at the G(0)-G(1) transition of the cell cycle has now been shown to proceed normally in Skp2(-/-) lymphocytes, whereas p27(Kip1) proteolysis during S-G(2) phases is impaired in these Skp2-deficient cells. Degradation of p27(Kip1) at the G(0)-G(1) transition was blocked by lactacystin, a specific proteasome inhibitor, suggesting that it is mediated by the ubiquitin-proteasome pathway. The first cell cycle of stimulated Skp2(-/-) lymphocytes appeared normal, but the second cycle was markedly inhibited, presumably as a result of p27(Kip1) accumulation during S-G(2) phases of the first cell cycle. Polyubiquitination of p27(Kip1) in the nucleus is dependent on Skp2 and phosphorylation of p27(Kip1) on threonine 187. However, polyubiquitination activity was also detected in the cytoplasm of Skp2(-/-) cells, even with a threonine 187 --> alanine mutant of p27(Kip1) as substrate. These results suggest that a polyubiquitination activity in the cytoplasm contributes to the early phase of p27(Kip1) degradation in a Skp2-independent manner, thereby promoting cell cycle progression from G(0) to G(1).

Transcriptional Up-regulation of p27Kip1 during Contact-Induced Growth Arrest in Vascular Endothelial Cells

By plating porcine aortic endothelial cells at two different densities and thereby inducing two different time courses of contact-induced growth arrest, the temporal correlation between p27Kip1 expression and cell cycle progression was investigated. When the quiescent cells were replated, they synchronously entered S phase with a peak at 20 h in both cases, while the cells plated at 25 and 80% of confluent densities exited the cell cycle by 96 and 48 h, respectively. Nuclear p27Kip1 disappeared when the cells reentered the cell cycle and then recovered when the cells exited the cell cycle. The change in p27Kip1 was associated with a concomitant change in Kip1 mRNA. The p27Kip1 degradation activity did not increase in the cells reentering the cell cycle, nor did it decrease in the cells exiting the cell cycle. The Kip1 mRNA stability decreased in the growing cells and increased in the cells exiting the cell cycle and at confluence. A nuclear run-on assay revealed a close correlation between the Kip1 transcriptional activity and the level of Kip1 mRNA. We conclude that the cell–cell contact up-regulated the Kip1 gene transcription and increased the Kip1 mRNA stability, which was related to the recovery of p27Kip1 protein during contact-induced growth arrest in endothelial cells.

Effect of Insulin on Cell Cycle Progression in MCF-7 Breast Cancer Cells: DIRECT AND POTENTIATING INFLUENCE

We recently demonstrated that in MCF-7 breast cancer cells, insulin promoted the phosphorylation and activation of geranylgeranyltransferase I (GGTI-I), increased the amounts of geranylgeranylated Rho-A and potentiated the transactivating activity of lysophosphatidic acid (LPA) (Chappell, J., Golovchenko, I., Wall, K., Stjernholm, R., Leitner, J., Goalstone, M., and Draznin, B. (2000) J. Biol. Chem. 275, 31792-31797). In the present study, we explored the mechanism of this potentiating effect of insulin on LPA. Insulin (10 nm) potentiated the ability of LPA to stimulate cell cycle progression and DNA synthesis in MCF-7 cells. The potentiating effect of insulin appears to involve increases in the expression of cyclin E and decreases in the expression of the cyclin-dependent kinase inhibitor p27Kip1. All potentiating effects of insulin were inhibited in the presence of an inhibitor of GGTase I, GGTI-286 (3 microm) or by an expression of a dominant negative mutant of Rho-A. In contrast to its potentiating action, a direct mitogenic effect of insulin in MCF-7 cells involves activation of phosphatidylinositol 3-kinase and increased expression of cyclin D1. We conclude that the ability of insulin to increase the cellular amounts of geranylgeranylated Rho-A results in potentiation of the LPA effect on cyclin E expression and degradation of p27Kip1 and cell cycle progression in MCF-7 breast cancer cells.

The activation of the phosphotyrosine phosphatase eta (r-PTP eta) is responsible for the somatostatin inhibition of PC Cl3 thyroid cell proliferation.

The aim of this study was the characterization of the intracellular effectors of the antiproliferative activity of somatostatin in PC Cl3 thyroid cells. Somatostatin inhibited PC Cl3 cell proliferation through the activation of a membrane phosphotyrosine phosphatase. Conversely, PC Cl3 cells stably expressing the v-mos oncogene (PC mos) were completely insensitive to the somatostatin antiproliferative effects since somatostatin was unable to stimulate a phosphotyrosine phosphatase activity. In PC mos cells basal phosphotyrosine phosphatase activity was also reduced, suggesting that the expression of a specific phosphotyrosine phosphatase was impaired in these transformed cells. We suggested that this phosphotyrosine phosphatase could be r-PTP eta whose expression was abolished in the PC mos cells. To directly prove the involvement of r-PTP eta in somatostatin's effect, we stably transfected this phosphatase in PC mos cells. This new cell line (PC mos/PTP eta) recovered somatostatin's ability to inhibit cell proliferation, showing dose-dependence and time course similar to those observed in PC Cl3 cells. Conversely, the transfection of a catalytically inactive mutant of r-PTP eta did not restore the antiproliferative effects of somatostatin. PC mos/PTP eta cells showed a high basal phosphotyrosine phosphatase activity which, similarly to PC Cl3 cells, was further increased after somatostatin treatment. The specificity of the role of r-PTP eta in somatostatin receptor signal transduction was demonstrated by measuring its specific activity after somatostatin treatment in an immunocomplex assay. Somatostatin highly increased r-PTP eta activity in PCCl3 and PC mos/PTP eta (+300%, P < 0.01) but not in PCmos cells. Conversely, no differences in somatostatin-stimulated SHP-2 activity, (approximately +50%, P < 0.05), were observed among all the cell lines. The activation of r-PTP eta by somatostatin caused, acting downstream of MAPK kinase, an inhibition of insulin-induced ERK1/2 activation with the subsequent blockade of the phosphorylation, ubiquitination, and proteasome degradation of the cyclin-dependent kinase inhibitor p27(kip1). Ultimately, high levels of p27(kip1) lead to cell proliferation arrest. In conclusion, somatostatin inhibition of PC Cl3 cell proliferation requires the activation of r-PTP eta which, through the inhibition of MAPK activity, causes the stabilization of the cell cycle inhibitor p27(kip1).

The p42/p44 Mitogen-activated Protein Kinase Activation Triggers p27Kip1 Degradation Independently of CDK2/Cyclin E in NIH 3T3 Cells

The p42/p44 mitogen-activated protein (MAP) kinase is stimulated by various mitogenic stimuli, and its sustained activation is necessary for cell cycle G(1) progression and G(1)/S transition. G(1) progression and G(1)/S transition also depend on sequential cyclin-dependent kinase (CDK) activation. Here, we demonstrate that MAP kinase inhibition leads to accumulation of the CDK inhibitor p27(Kip1) in NIH 3T3 cells. Blocking the proteasome-dependent degradation of p27(Kip1) impaired this accumulation, suggesting that MAP kinase does not act on p27(Kip1) protein synthesis. In the absence of extracellular signals (growth factors or cell adhesion), genetic activation of MAP kinase decreased the expression of p27(Kip1) as assessed by cotransfection experiments and by immunofluorescence detection. Importantly, MAP kinase activation also decreased the expression of a p27(Kip1) mutant, which cannot be phosphorylated by CDK2, suggesting that MAP kinase-dependent p27(Kip1) regulation is CDK2-independent. Accordingly, expression of dominant-negative CDK2 did not impair the down-regulation of p27(Kip1) induced by MAP kinase activation. These data demonstrate that the MAP kinase pathway regulates p27(Kip1) expression in fibroblasts essentially through a degradation mechanism, independently of p27(Kip1) phosphorylation by CDK2. This strengthens the role of this CDK inhibitor as a key effector of G(1) growth arrest, whose expression can be controlled by extracellular stimuli-dependent signaling pathways.

Dipyridamole inhibits PDGF-stimulated human peritoneal mesothelial cell proliferation

It has been proposed that proliferation of human peritoneal mesothelial cells (HPMCs) accompanied by collagen synthesis may contribute to the development of peritoneal fibrosis (PF) in patients of long-term continuous ambulatory peritoneal dialysis (CAPD). However, the precise molecular mechanism regulating HPMC proliferation has never been reported. Dipyridamole has been reported to have potential as an antiproliferative and antifibrotic agent. We investigated the mechanism and effect of dipyridamole in regulation of HPMC proliferation. HPMCs were cultured from human omentum by an enzyme digestion Cell proliferation was measured by the methyltetrazolium assay and intracellular cAMP was measured using an enzyme immunoassay kit. Cell-cycle distribution of HPMC was analyzed by flow cytometry. Extracellular signal-regulated protein kinase (p44/p42 ERK) activity and expressions of cell-cycle proteins (cyclin D(1), CDK4, pRB and p27(Kip1)) were determined by Western blotting. The addition of DP suppressed PDGF-stimulated HPMC proliferation by cell-cycle arrest at the G1 phase. The antimitogenic effect of dipyridamole was mediated through the cAMP pathway. PDGF (25 ng/mL) increased the ERK1/2 activity of HPMC within 15 minutes, which maximized at 30 minutes, and the pretreatment with dipyridamole (17 microg/mL) substantially reduced the ERK response to PDGF by approximately 78.5%. PDGF induced elevated protein levels of cyclin D(1), but the CDK4 protein level did not change. Dipyridamole and DBcAMP had no effect on the levels of cyclin D(1) and CDK4 in PDGF-stimulated HPMC. PDGF decreased p27(Kip1) and induced pRB phosphorylation of HPMC. In contrast, dipyridamole prevented PDGF-induced p27(Kip1) degradation and attenuated PDGF-stimulated pRB phosphorylation. Dipyridamole appears to inhibit PDGF-stimulated HPMC proliferation through attenuated ERK activity, preservation of p27(Kip1), and decreased pRB phosphorylation. Thus, dipyridamole may have therapeutic efficacy to prevent or alleviate PF.

Regulation of FRTL-5 thyroid cell growth by phosphatidylinositol (OH) 3 kinase-dependent Akt-mediated signaling.

Thyrotropin (TSH)-initiated cell cycle progression from G1 to S phase in FRTL-5 thyroid cells requires serum, insulin, or insulin-like growth factor 1 (IGF-1) and involves activation of 3-hydroxy-3-methylglutaryl-CoA reductase, geranylgeranylation of RhoA, p27Kip1 degradation, and activation of cyclin-dependent kinase (cdk) 2. In the present report, we show that the serine-threonine kinase Akt is an important mediator of insulin/IGF-1/serum effects on cell cycle progression in FRTL-5 thyroid cells. The phosphoinositol (OH) 3 kinase inhibitors, Wortmannin (WM) and Ly294002 (LY), block the ability of insulin/IGF-1 to reduce p27 expression, to induce expression of cyclins E, D1, and A as well as cdk 2 and 4, and to phosphorylate retinoblastoma protein. They also inhibit insulin/IGF-1-increased DNA synthesis and cell cycle entrance (S+G2/M). Insulin/IGF-1 rapidly induced activation of Aktl in a PI3 kinase-dependent manner, and increased Aktl RNA levels. Most importantly, FRTL-5 cells transfected with a constitutively active form of Aktl have higher basal rates of DNA synthesis and no longer require exogenous insulin/IGF-1 or serum for TSH-induced growth. In sum, Aktl appears to have an important role in insulin/IGF-1 regulation of FRTL-5 thyroid cell growth and cell cycle progression.

IFNα 2b induces apoptosis and proteasome-mediated degradation of p27Kip1 in a human lung cancer cell line

IFNs are a family of cytokines involved in antiviral defense, cell growth regulation and immune activation. IFNs either inhibit cell proliferation or control apoptosis depending on factors such as cell type and state of cell differentiation. It is important to determine how IFN-induced gene products interact with other cellular proteins to produce these responses. We have investigated the effect of IFNalpha 2b on a human small cell lung carcinoma (SCLC) cell line H82. We have found that IFNalpha efficiently induces apoptosis in H82 cells. The induction of apoptosis by IFNalpha 2b is accompanied by decreased levels of c-myc and Cdk2. We have also observed that in H82 cells IFNalpha induces downregulation of p27 and this is in contrast to the upregulation of p27 observed in other cell types where IFNs induce cell cycle arrest. IFNalpha-induced downregulation of p27 is due to protein destabilization and can be prevented by the proteasome inhibitor LLnL. The data suggest that in H82 cells, IFNalpha 2b induces degradation of p27Kip1 independently of CDK2 kinase activity and through a ubiquitin or ubiquitin-related pathway and that the degradation of p27Kip1 could be a molecular event of importance for IFN-induced apoptosis in cancer cells.