p27Kip1 - p(RhoB)lematic in lung cancer.

Lung cancer is the leading cause of cancer mortality worldwide, with adenocarcinomas of the non-small cell lung carcinoma (NSCLC) subtype accounting for the majority of cases. Therefore, an urgent need exists for a more detailed dissection of the molecular events driving NSCLC development and the identification of clinically relevant biomarkers. Even though originally identified as a tumour suppressor, recent studies associate the cytoplasmically (mis)localised CDK inhibitor p27Kip1 (p27) with unfavourable responses to chemotherapy and poor outcomes in NSCLC, supporting the hypothesis that the protein can execute oncogenic activities. In a recent issue of The Journal of Pathology, Calvayrac and coworkers uncover a novel molecular mechanism that can explain this oncogenic role of p27. They demonstrate that cytoplasmic p27 binds and inhibits the small GTPase RhoB and thereby relieves a selection pressure for RhoB loss that is frequently observed in NSCLC. This is supported not only by studies with genetically modified mice, but also through identification of a cohort of human lung cancer patients with cytoplasmic p27 and continued RhoB expression, where this signature correlates with decreased survival. This not only establishes a potentially useful biomarker, but also provides yet another facet of the complex roles p27 undertakes in tumourigenesis. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Cold-inducible RNA-binding protein (CIRP) induces translation of the cell-cycle inhibitor p27Kip1.

The CDK inhibitor p27Kip1 plays a central role in controlling cell proliferation and cell-cycle exit. p27Kip1 protein levels oscillate during cell-cycle progression and are regulated by mitogen or anti-proliferative signaling. The abundance of the protein is frequently determined by post-transcriptional mechanisms including ubiquitin-mediated proteolysis and translational control. Here, we report that the cold-inducible RNA-binding protein (CIRP) selectively binds to the 5' untranslated region of the p27Kip1 mRNA. CIRP is induced, modified and relocalized in response to various stress stimuli and can regulate cell survival and cell proliferation particularly during stress. Binding of CIRP to the 5'UTR of the p27Kip1 mRNA significantly enhanced reporter translation. In cells exposed to mild hypothermia, the induction of CIRP correlated with increased translation of a p27Kip1 5'UTR reporter and with the accumulation of p27Kip1 protein. shRNA-mediated CIRP knockdown could prevent the induction of translation. We found that p27Kip1 is central for the decreased proliferation at lower temperature, since p27Kip1 KO mouse embryonic fibroblasts (MEFs) hardly increased their doubling time in hypothermic conditions, whereas wild-type MEFs significantly delayed proliferation in response to cold stress. This suggests that the CIRP-dependent p27Kip1 upregulation during mild hypothermia contributes to the cold shock-induced inhibition of cell proliferation.

FLT3 and FLT3-ITD phosphorylate and inactivate the cyclin-dependent kinase inhibitor p27Kip1 in acute myeloid leukemia.

P27 Kip1 (p27) can prevent cell proliferation by inactivating cyclin-dependent kinases. This function is impaired upon phosphorylation of p27 at tyrosine residue 88. We observed that FLT3 and FLT3-ITD can directly bind and selectively phosphorylate p27 on this residue. Inhibition of FLT3-ITD in cell lines strongly reduced p27 tyrosine 88 phosphorylation and resulted in increased p27 levels and cell cycle arrest. Subsequent analysis revealed the presence of tyrosine 88 phosphorylated p27 in primary patient samples. Inhibition of FLT3 kinase activity with AC220 significantly reduced p27 tyrosine 88 phosphorylation in cells isolated from FLT3 wild type expressing acute myeloid leukemia (AML) patients. In FLT3-ITD positive AML patients, p27 tyrosine 88 phosphorylation was reduced in 5 out of 9 subjects, but, surprisingly, was increased in 4 patients. This indicated that other tyrosine kinases such as Src family kinases might contribute to p27 tyrosine 88 phosphorylation in FLT3-ITD positive AML cells. In fact, incubation with the Src family kinase inhibitor dasatinib could decrease p27 tyrosine 88 phosphorylation in these patient samples, indicating that p27 phosphorylated on tyrosine 88 may be a therapeutic marker for the treatment of AML patients with tyrosine kinase inhibitors.

CDK4 T172 phosphorylation is central in a CDK7-dependent bidirectional CDK4/CDK2 interplay mediated by p21 phosphorylation at the restriction point.

Cell cycle progression, including genome duplication, is orchestrated by cyclin-dependent kinases (CDKs). CDK activation depends on phosphorylation of their T-loop by a CDK-activating kinase (CAK). In animals, the only known CAK for CDK2 and CDK1 is cyclin H-CDK7, which is constitutively active. Therefore, the critical activation step is dephosphorylation of inhibitory sites by Cdc25 phosphatases rather than unrestricted T-loop phosphorylation. Homologous CDK4 and CDK6 bound to cyclins D are master integrators of mitogenic/oncogenic signaling cascades by initiating the inactivation of the central oncosuppressor pRb and cell cycle commitment at the restriction point. Unlike the situation in CDK1 and CDK2 cyclin complexes, and in contrast to the weak but constitutive T177 phosphorylation of CDK6, we have identified the T-loop phosphorylation at T172 as the highly regulated step determining CDK4 activity. Whether both CDK4 and CDK6 phosphorylations are catalyzed by CDK7 remains unclear. To answer this question, we took a chemical-genetics approach by using analogue-sensitive CDK7(as/as) mutant HCT116 cells, in which CDK7 can be specifically inhibited by bulky adenine analogs. Intriguingly, CDK7 inhibition prevented activating phosphorylations of CDK4/6, but for CDK4 this was at least partly dependent on its binding to p21 (cip1) . In response to CDK7 inhibition, p21-binding to CDK4 increased concomitantly with disappearance of the most abundant phosphorylation of p21, which we localized at S130 and found to be catalyzed by both CDK4 and CDK2. The S130A mutation of p21 prevented the activating CDK4 phosphorylation, and inhibition of CDK4/6 and CDK2 impaired phosphorylations of both p21 and p21-bound CDK4. Therefore, specific CDK7 inhibition revealed the following: a crucial but partly indirect CDK7 involvement in phosphorylation/activation of CDK4 and CDK6; existence of CDK4-activating kinase(s) other than CDK7; and novel CDK7-dependent positive feedbacks mediated by p21 phosphorylation by CDK4 and CDK2 to sustain CDK4 activation, pRb inactivation, and restriction point passage.

Novel antibodies directed against the human erythropoietin receptor: creating a basis for clinical implementation.

Recombinant human erythropoietin (rHuEPO) is an effective treatment for anaemia but concerns that it causes disease progression in cancer patients by activation of EPO receptors (EPOR) in tumour tissue have been controversial and have restricted its clinical use. Initial clinical studies were flawed because they used polyclonal antibodies, later shown to lack specificity for EPOR. Moreover, multiple isoforms of EPOR caused by differential splicing have been reported in cancer cell lines at the mRNA level but investigations of these variants and their potential impact on tumour progression, have been hampered by lack of suitable antibodies. The EpoCan consortium seeks to promote improved pathological testing of EPOR, leading to safer clinical use of rHuEPO, by producing well characterized EPOR antibodies. Using novel genetic and traditional peptide immunization protocols, we have produced mouse and rat monoclonal antibodies, and show that several of these specifically recognize EPOR by Western blot, immunoprecipitation, immunofluorescence, flow cytometry and immunohistochemistry in cell lines and clinical material. Widespread availability of these antibodies should enable the research community to gain a better understanding of the role of EPOR in cancer, and eventually to distinguish patients who can be treated safely by rHuEPO from those at increased risk from treatment.

Nogo-A couples with Apg-1 through interaction and co-ordinate expression under hypoxic and oxidative stress.

Nogo-A is the largest isoform of the Nogo/RTN4 (reticulon 4) proteins and has been characterized as a major myelin-associated inhibitor of regenerative nerve growth in the adult CNS (central nervous system). Apart from the myelin sheath, Nogo-A is expressed at high levels in principal neurons of the CNS. The specificity of Nogo-A resides in its central domain, NiG. We identified Apg-1, a member of the stress-induced Hsp110 (heat-shock protein of 110 kDa) family, as a novel interactor of NiG/Nogo-A. The interaction is selective because Apg-1 interacts with Nogo-A/RTN4-A, but not with RTN1-A, the closest paralogue of Nogo-A. Conversely, Nogo-A binds to Apg-1, but not to Apg-2 or Hsp105, two other members of the Hsp110 family. We characterized the Nogo-A-Apg-1 interaction by affinity precipitation, co-immunoprecipitation and proximity ligation assay, using primary hippocampal neurons derived from Nogo-deficient mice. Under conditions of hypoxic and oxidative stress we found that Nogo-A and Apg-1 were tightly co-regulated in hippocampal neurons. Although both proteins were up-regulated under hypoxic conditions, their expression levels were reduced upon the addition of hydrogen peroxide. Taken together, we suggest that Nogo-A is closely involved in the neuronal response to hypoxic and oxidative stress, an observation that may be of relevance not only in stroke-induced ischaemia, but also in neuroblastoma formation.

Complement C7 and clusterin form a complex in circulation.

Introduction: The complement system is part of innate immunity and is comprised of an intricate network of proteins that are vital for host defense and host homeostasis. A distinct mechanism by which complement defends against invading pathogens is through the membrane attack complex (MAC), a lytic structure that forms on target surfaces. The MAC is made up of several complement components, and one indispensable component of the MAC is C7. The role of C7 in MAC assembly is well documented, however, inherent characteristics of C7 are yet to be investigated. Methods: To shed light on the molecular characteristics of C7, we examined the properties of serum-purified C7 acquired using polyclonal and novel monoclonal antibodies. The properties of serum­purified C7 were investigated through a series of proteolytic analyses, encompassing Western blot and mass spectrometry. The nature of C7 protein-protein interactions were further examined by a novel enzyme-linked immunosorbent assay (ELISA), as well as size­exclusion chromatography. Results: Protein analyses showcased an association between C7 and clusterin, an inhibitory complement regulator. The distinct association between C7 and clusterin was also demonstrated in serum-purified clusterin. Further assessment revealed that a complex between C7 and clusterin (C7-CLU) was detected. The C7-CLU complex was also identified in healthy serum and plasma donors, highlighting the presence of the complex in circulation. Discussion: Clusterin is known to dissociate the MAC structure by binding to polymerized C9, nevertheless, here we show clusterin binding to the native form of a terminal complement protein in vivo. The presented data reveal that C7 exhibits characteristics beyond that of MAC assembly, instigating further investigation of the effector role that the C7-CLU complex plays in the complement cascade.

PIAS1 is increased in human prostate cancer and enhances proliferation through inhibition of p21.

Prostate cancer development and progression are associated with alterations in expression and function of elements of cytokine networks, some of which can activate multiple signaling pathways. Protein inhibitor of activated signal transducers and activators of transcription (PIAS)1, a regulator of cytokine signaling, may be implicated in the modulation of cellular events during carcinogenesis. This study was designed to investigate the functional significance of PIAS1 in models of human prostate cancer. We demonstrate for the first time that PIAS1 protein expression is significantly higher in malignant areas of clinical prostate cancer specimens than in normal tissues, thus suggesting a growth-promoting role for PIAS1. Expression of PIAS1 was observed in the majority of tested prostate cancer cell lines. In addition, we investigated the mechanism by which PIAS1 might promote prostate cancer and found that down-regulation of PIAS1 leads to decreased proliferation and colony formation ability of prostate cancer cell lines. This decrease correlates with cell cycle arrest in the G0/G1 phase, which is mediated by increased expression of p21(CIP1/WAF1). Furthermore, PIAS1 overexpression positively influences cell cycle progression and thereby stimulates proliferation, which can be mechanistically explained by a decrease in the levels of cellular p21. Taken together, our data reveal an important new role for PIAS1 in the regulation of cell proliferation in prostate cancer.

EpoR Activation Stimulates Erythroid Precursor Proliferation by Inducing Phosphorylation of Tyrosine-88 of the CDK-Inhibitor p27Kip1.

Erythrocyte biogenesis needs to be tightly regulated to secure oxygen transport and control plasma viscosity. The cytokine erythropoietin (Epo) governs erythropoiesis by promoting cell proliferation, differentiation, and survival of erythroid precursor cells. Erythroid differentiation is associated with an accumulation of the cyclin-dependent kinase inhibitor p27Kip1, but the regulation and role of p27 during erythroid proliferation remain largely unknown. We observed that p27 can bind to the erythropoietin receptor (EpoR). Activation of EpoR leads to immediate Jak2-dependent p27 phosphorylation of tyrosine residue 88 (Y88). This modification is known to impair its CDK-inhibitory activity and convert the inhibitor into an activator and assembly factor of CDK4,6. To investigate the physiological role of p27-Y88 phosphorylation in erythropoiesis, we analyzed p27Y88F/Y88F knock-in mice, where tyrosine-88 was mutated to phenylalanine. We observed lower red blood cell counts, lower hematocrit levels, and a reduced capacity for colony outgrowth of CFU-Es (colony-forming unit-erythroid), indicating impaired cell proliferation of early erythroid progenitors. Compensatory mechanisms of reduced p27 and increased Epo expression protect from stronger dysregulation of erythropoiesis. These observations suggest that p27-Y88 phosphorylation by EpoR pathway activation plays an important role in the stimulation of erythroid progenitor proliferation during the early stages of erythropoiesis.

Inability to phosphorylate Y88 of p27Kip1 enforces reduced p27 protein levels and accelerates leukemia progression.

The cyclin-dependent kinase (CDK) inhibitor p27Kip1 regulates cell proliferation. Phosphorylation of tyrosine residue 88 (Y88) converts the inhibitor into an assembly factor and activator of CDKs, since Y88-phosphorylation restores activity to cyclin E,A/CDK2 and enables assembly of active cyclin D/CDK4,6. To investigate the physiological significance of p27 tyrosine phosphorylation, we have generated a knock-in mouse model where Y88 was replaced by phenylalanine (p27-Y88F). Young p27-Y88F mice developed a moderately reduced body weight, indicative for robust CDK inhibition by p27-Y88F. When transformed with v-ABL or BCR::ABL1p190, primary p27-Y88F cells are refractory to initial transformation as evidenced by a diminished outgrowth of progenitor B-cell colonies. This indicates that p27-Y88 phosphorylation contributes to v-ABL and BCR::ABL1p190 induced transformation. Surprisingly, p27-Y88F mice succumbed to premature v-ABL induced leukemia/lymphoma compared to p27 wild type animals. This was accompanied by a robust reduction of p27-Y88F levels in v-ABL transformed cells. Reduced p27-Y88F levels seem to be required for efficient cell proliferation and may subsequently support accelerated leukemia progression. The potent downregulation p27-Y88F levels in all leukemia-derived cells could uncover a novel mechanism in human oncogenesis, where reduced p27 levels are frequently observed.

Stimulation of c-Jun/AP-1-Activity by the Cell Cycle Inhibitor p57Kip2.

p57 is a member of the Cip/Kip family of cell cycle inhibitors which restrict the eukaryotic cell cycle by binding to and inhibiting cyclin/CDK complexes. They are considered as tumor suppressors and inactivating genomic mutations of p57 are associated with human overgrowth disorders. Increasing evidence suggests that p57 controls additional cellular processes beyond cell cycle control such as apoptosis, cell migration or transcription. Here we report that p57 can stimulate AP-1 promotor activity. While transactivation by c-Jun is strongly activated by p57, it did not enhance c-Fos induced transcription. This indicates that c-Jun is the target of p57 in the canonical AP-1 heterodimeric transcription factor. We could detect endogenous p57/c-Jun containing complexes in cells by co-immunoprecipitation. The strong stimulation of c-Jun activity is not the consequence of activating phosphorylation in the transactivation domain (TAD) of c-Jun, but rather due to negative interference with c-Jun repressors and positive interference with c-Jun activators. In contrast to full-length p57, the amino- and carboxy-terminal domains of p57 are insufficient for a significant activation of c-Jun induced transcription. When expressed in presence of full length p57, the p57 C-terminus abrogated and the N-terminus enhanced c-Jun activation. This indicates that the C-terminus may bind and sequester a putative activator of c-Jun, whereas the N-terminus may sequester a c-Jun repressor. Interestingly, the p57 aminoterminus is sufficient for binding to the two c-Jun repressors HDAC1 and HDAC3. These data are consistent with a model of c-Jun activation where p57 is a part of large nuclear remodeling/transcription complexes. p57 might stimulate transcription by inhibiting transcription repressor proteins like HDACs via its N-terminus and/or attracting transcription activators through its C-terminus. These data suggest that in addition to its role as a CDK inhibitor and tumor suppressor, p57 may also exert tumor promoting functions by activation of the proto-oncoprotein c-Jun.

The CDK inhibitor p57Kip2 enhances the activity of the transcriptional coactivator FHL2.

The eukaryotic cell cycle is negatively regulated by cyclin-dependent kinase inhibitors (CKIs). p57Kip2 is a member of the Cip/Kip family of CKIs and frequently inactivated by genomic mutations associated with human overgrowth disorders. There is increasing evidence for p57 to control cellular processes in addition to cell cycle and CDK regulation including transcription, apoptosis, migration or development. In order to obtain molecular insights to unknown functions of p57, we performed a protein interaction screen. We identified the transcription regulator four-and-a-half LIM-only protein 2 (FHL2) as a novel p57-binding protein. Co-immunoprecipitation and reporter gene assays were used to elucidate the physiological and functional relevance of p57/FHL2 interaction. We found in cancer cells that endogenous p57 and FHL2 are in a complex. We observed a substantial induction of established FHL2-regulated gene promoters by p57 in reporter gene experiments and detected strong induction of the intrinsic transactivation activity of FHL2. Treatment of cells with histone deacetylase (HDAC) inhibitors and binding of exogenous FHL2 to HDACs indicated repression of FHL2 transcription activity by HDACs. In the presence of the HDAC inhibitor sodium butyrate activation of FHL2 by p57 is abrogated suggesting that p57 shares a common pathway with HDAC inhibitors. p57 competes with HDACs for FHL2 binding which might partly explain the mechanism of FHL2 activation by p57. These results suggest a novel function of p57 in transcription regulation.

RanGAP1*SUMO1 is phosphorylated at the onset of mitosis and remains associated with RanBP2 upon NPC disassembly.

The RanGTPase activating protein RanGAP1 has essential functions in both nucleocytoplasmic transport and mitosis. In interphase, a significant fraction of vertebrate SUMO1-modified RanGAP1 forms a stable complex with the nucleoporin RanBP2/Nup358 at nuclear pore complexes. RanBP2 not only acts in the RanGTPase cycle but also is a SUMO1 E3 ligase. Here, we show that RanGAP1 is phosphorylated on residues T409, S428, and S442. Phosphorylation occurs before nuclear envelope breakdown and is maintained throughout mitosis. Nocodazole arrest leads to quantitative phosphorylation. The M-phase kinase cyclin B/Cdk1 phosphorylates RanGAP1 efficiently in vitro, and T409 phosphorylation correlates with nuclear accumulation of cyclin B1 in vivo. We find that phosphorylated RanGAP1 remains associated with RanBP2/Nup358 and the SUMO E2-conjugating enzyme Ubc9 in mitosis, hence mitotic phosphorylation may have functional consequences for the RanGTPase cycle and/or for RanBP2-dependent sumoylation.

p27 binds cyclin-CDK complexes through a sequential mechanism involving binding-induced protein folding.

p27 controls cell proliferation by binding and regulating nuclear cyclin-dependent kinases (CDKs). In addition, p27 interacts with other nuclear and cytoplasmic targets and has diverse biological functions. We seek to understand how the structural and dynamic properties of p27 mediate its several functions. We show that, despite showing disorder before binding its targets, p27 has nascent secondary structure that may have a function in molecular recognition. Binding to Cdk2-cyclin A is accompanied by p27 folding, and kinetic data suggest a sequential mechanism that is initiated by binding to cyclin A. p27 regulates CDK-cyclin complexes involved directly in cell cycle control and does not interact with other closely related CDKs. We show that p27-cyclin interactions are an important determinant of this specificity and propose that the homologous cell cycle regulators p21 and p57 function by a similar sequential, folding-on-binding mechanism.

Oncostatin M induces growth arrest by inhibition of Skp2, Cks1, and cyclin A expression and induced p21 expression.

Oncostatin M has been characterized as a potent growth inhibitor for various tumor cells. Oncostatin M-treated glioblastoma cells cease proliferation and instigate astrocytal differentiation. The oncostatin M-induced cell cycle arrest in G(1) phase is characterized by increased level of the cyclin-dependent kinase (CDK) inhibitory proteins p21(Cip1/Waf1/Sdi1) and p27(Kip1). Induction of p21 protein corresponds to increased mRNA level, whereas p27 accumulates due to increased stability of the protein. Interestingly, stabilization of p27(Kip1) occurs even in S phase, showing that p27 stabilization is a direct consequence of oncostatin M signaling and not a result of the cell cycle arrest. Degradation of p27 in late G(1) and S phase is initiated by the ubiquitin ligase complex SCF-Skp2/Cks1. Oncostatin M inhibits expression of two components of this E3 ligase complex (Skp2 and Cks1). Although combined overexpression of Skp2 and Cks1 rescues p27 degradation in S phase, it can not override p27 accumulation in G(1) phase and cell cycle arrest by oncostatin M. In addition to increasing Cdk inhibitor level, oncostatin M also impairs cyclin A expression. Cyclin A mRNA and protein level decline shortly after oncostatin M addition. The accumulation of two CDK inhibitor proteins and the repression of cyclin A expression may explain the broad and potent antiproliferative effect of the cytokine.

CRM1/Ran-mediated nuclear export of p27(Kip1) involves a nuclear export signal and links p27 export and proteolysis.

We show that p27 localization is cell cycle regulated and we suggest that active CRM1/RanGTP-mediated nuclear export of p27 may be linked to cytoplasmic p27 proteolysis in early G1. p27 is nuclear in G0 and early G1 and appears transiently in the cytoplasm at the G1/S transition. Association of p27 with the exportin CRM1 was minimal in G0 and increased markedly during G1-to-S phase progression. Proteasome inhibition in mid-G1 did not impair nuclear import of p27, but led to accumulation of p27 in the cytoplasm, suggesting that export precedes degradation for at least part of the cellular p27 pool. p27-CRM1 binding and nuclear export were inhibited by S10A mutation but not by T187A mutation. A putative nuclear export sequence in p27 is identified whose mutation reduced p27-CRM1 interaction, nuclear export, and p27 degradation. Leptomycin B (LMB) did not inhibit p27-CRM1 binding, nor did it prevent p27 export in vitro or in heterokaryon assays. Prebinding of CRM1 to the HIV-1 Rev nuclear export sequence did not inhibit p27-CRM1 interaction, suggesting that p27 binds CRM1 at a non-LMB-sensitive motif. LMB increased total cellular p27 and may do so indirectly, through effects on other p27 regulatory proteins. These data suggest a model in which p27 undergoes active, CRM1-dependent nuclear export and cytoplasmic degradation in early G1. This would permit the incremental activation of cyclin E-Cdk2 leading to cyclin E-Cdk2-mediated T187 phosphorylation and p27 proteolysis in late G1 and S phase.

Erythropoietin drives breast cancer progression by activation of its receptor EPOR.

Breast cancer is a leading cause of cancer-related deaths. Anemia is common in breast cancer patients and can be treated with blood transfusions or with recombinant erythropoietin (EPO) to stimulate red blood cell production. Clinical studies have indicated decreased survival in some groups of cancer patients treated with EPO. Numerous tumor cells express the EPO receptor (EPOR), posing a risk that EPO treatment would enhance tumor growth, but the mechanisms involved in breast tumor progression are poorly understood.Here, we have examined the functional role of the EPO-EPOR axis in pre-clinical models of breast cancer. EPO induced the activation of PI3K/AKT and MAPK pathways in human breast cancer cell lines. EPOR knockdown abrogated human tumor cell growth, induced apoptosis through Bim, reduced invasiveness, and caused downregulation of MYC expression. EPO-induced MYC expression is mediated through the PI3K/AKT and MAPK pathways, and overexpression of MYC partially rescued loss of cell proliferation caused by EPOR downregulation. In a xenotransplantation model, designed to simulate recombinant EPO therapy in breast cancer patients, knockdown of EPOR markedly reduced tumor growth.Thus, our experiments in vitro and in vivo demonstrate that functional EPOR signaling is essential for the tumor-promoting effects of EPO and underline the importance of the EPO-EPOR axis in breast tumor progression.

Local statin therapy differentially interferes with smooth muscle and endothelial cell proliferation and reduces neointima on a drug-eluting stent platform.

OBJECTIVE: Therapeutic strategies to provide local inhibition of mitogen mediated proliferation and migration of human coronary artery smooth muscle cells (CASMC) by means of drug-eluting stents have been shown to enable effective limitation of neointimal hyperplasia. However, currently available drug-eluting stents utilize compounds that may also adversely affect endothelial regrowth, thus possibly precipitating subsequent cardiac events. Accordingly, identification of compounds that differentially inhibit smooth muscle and endothelial cell migration and proliferation could be of substantial clinical usefulness. METHODS AND RESULTS: In addition to lipid lowering, statins are known to display auxiliary pleiotropic activities. The purpose of this study was to evaluate the effect of local administration of cerivastatin on proliferation, migration and cytotoxicity of CASMC as well as coronary artery endothelial cells (CAEC) and to evaluate the effect of cerivastatin-coated stents on the inhibition of neointima formation as well as endothelial regrowth within the stented vessel. Cerivastatin displayed a differential effect on CASMC as compared to CAEC with regard to proliferation and migration; both were more profoundly inhibited in CASMC. Appreciable cytotoxicity and pro-apoptotic effects were low in both cell lines at therapeutic concentrations. Cerivastatin-elution led to significant inhibition of neointima formation in the rat carotid stent model, endothelial coverage of in-stent vascular tissue was similar with control and cerivastatin-eluting stents. CONCLUSIONS: As proof of principle, our study provides evidence that local application of a HMG-CoA reductase inhibitor on a drug-eluting stent platform can efficiently limit neointima formation. Consequently, these compounds warrant further clinical evaluation to confirm this finding. Our data further suggest that the anti-restenotic effect of local statin administration might be associated with a more protective interaction with the endothelium than that observed with compounds currently employed on drug-eluting stents.

SUMO-1 and p53.

Abundance and activity of the tumor suppressor p53 are regulated by many different posttranslational modifications. These include phosphorylation, acetylation, ribosylation, O-glycosylation or ubiquitination. Three years ago, covalent modification with the ubiquitin- related protein SUMO-1 has been added to this list. SUMO-1 resembles ubiquitin both in structure and in the mechanism of attachment, and is reversibly attached to a large number of proteins. Molecular consequences of this dynamic modification vary between targets and include alterations in protein/protein or protein/DNA interactions, changes in localization, enzymatic activity, or stability. A role of SUMOylation in modulating p53 transcriptional activity has been reported, but is still an issue of controversy. Here we will briefly summarize the pathway of SUMOylation and discuss possible implications for p53 function.