Genetic and Epigenetic Control of CDKN1C Expression: Importance in Cell Commitment and Differentiation, Tissue Homeostasis and Human Diseases.

The CDKN1C gene encodes the p57Kip2 protein which has been identified as the third member of the CIP/Kip family, also including p27Kip1 and p21Cip1. In analogy with these proteins, p57Kip2 is able to bind tightly and inhibit cyclin/cyclin-dependent kinase complexes and, in turn, modulate cell division cycle progression. For a long time, the main function of p57Kip2 has been associated only to correct embryogenesis, since CDKN1C-ablated mice are not vital. Accordingly, it has been demonstrated that CDKN1C alterations cause three human hereditary syndromes, characterized by altered growth rate. Subsequently, the p57Kip2 role in several cell phenotypes has been clearly assessed as well as its down-regulation in human cancers. CDKN1C lies in a genetic locus, 11p15.5, characterized by a remarkable regional imprinting that results in the transcription of only the maternal allele. The control of CDKN1C transcription is also linked to additional mechanisms, including DNA methylation and specific histone methylation/acetylation. Finally, long non-coding RNAs and miRNAs appear to play important roles in controlling p57Kip2 levels. This review mostly represents an appraisal of the available data regarding the control of CDKN1C gene expression. In addition, the structure and function of p57Kip2 protein are briefly described and correlated to human physiology and diseases.

Iron overload enhances human mesenchymal stromal cell growth and hampers matrix calcification.

BACKGROUND: Iron overload syndromes include a wide range of diseases frequently associated with increased morbidity and mortality. Several organs are affected in patients with iron overload including liver, heart, joints, endocrine glands, and pancreas. Moreover, severe bone and hemopoietic tissue alterations are observed. Because of the role of bone marrow mesenchymal stromal cells (BM-MSCs) in bone turnover and hematopoiesis, iron effects on primary BM-MSCs cultures were evaluated. METHODS: Primary human BM-MSCs cultures were prepared and the effects of iron on their proliferation and differentiation were characterized by biochemical analyses and functional approaches. RESULTS: Addition of iron to the culture medium strongly increased BM-MSCs proliferation and induced their accelerated S phase entry. Iron enters BM-MSCs through both transferrin-dependent and transferrin-independent mechanisms, inducing the accumulation of cyclins E and A, the decrease of p27(Kip1), and the activation of MAPK pathway. Conversely, neither apoptotic signs nor up-regulation of reactive oxygen species were observed. Iron inhibited both differentiation of BM-MSCs into osteoblasts and in vitro matrix calcification. These effects result from the merging of inhibitory activities on BM-MSCs osteoblastic commitment and on the ordered matrix calcification process. CONCLUSIONS: We demonstrated that BM-MSCs are a target of iron overload. Iron accelerates BM-MSCs proliferation and affects BM-MSCs osteoblastic commitment, hampering matrix calcification. GENERAL SIGNIFICANCE: Our study reports, for the first time, that iron, at concentration found in overloaded patient sera, stimulates the growth of BM-MSCs, the BM multipotent stromal cell component. Moreover, iron modulates the physiological differentiation of these cells, affecting bone turnover and remodeling.

Resveratrol: from basic studies to bedside.

Plants produce a remarkable amount of low molecular mass natural products endowed with a large array of pivotal biological activities. Among these molecules, resveratrol (3,5,4'-trihydroxystilbene) has been identified as an important modulator of cell phenotype with a complex and pleiotropic mode of action. Extensive literature regarding its activity, mainly employing cellular models, suggests that this polyphenol controls cell proliferation, induces differentiation, and activates apoptosis and autophagy. The compound also modulates angiogenesis and inflammation. Similarly, studies on implanted cancers and chemical-induced tumors confirm the potential chemotherapeutical interest of the compound. Likewise, several reports clearly demonstrated, in animal models, that the compound might positively affect the development and evolution of chronic diseases including type 2 diabetes, obesity, coronary heart disease, metabolic syndrome, and neurogenerative pathologies. Finally, a number of investigations stated that the toxicity of the molecule is scarce. Despite these promising observations, few clinical trials have yet been performed to evaluate the effectiveness of the molecule both in prevention and treatment of human chronic disease. Preliminary findings therefore suggest the need for more extensive clinical investigations.

Transferrin-immune complex disease: a potentially overlooked gammopathy mediated by IgM and IgG.

The combination of marked hypersideremia, hypertransferrinemia, and monoclonal gammopathy of underdetermined significance (MGUS) should alert clinicians to the possible presence of an anti-transferrin immunoglobulin, an uncommon acquired disorder also defined as transferrin-immune complex disease (TICD). The authors have previously described a case of TICD with 100% transferrin saturation and liver iron overload. However, the findings in the few cases so far reported are heterogeneous, and the presence of high transferrin saturation and liver iron overload is not universal. In this article, the authors have described the identification of two additional patients with anti-transferrin monoclonal gammopathy, hypersideremia, and hypertransferrinemia, but with incomplete transferrin saturation and no hepatic iron overload. The autoantibodies were purified by using transferrin as affinity bait and characterized. One subject showed a high-titer monoclonal anti-transferrin IgM with a κ-type light chain. This finding is the first observation of IgM autoantibodies against transferrin. The other patient developed the disease after pregnancy. In this study, monoclonal antibody was an IgG mounting a κ-type light chain with altered molecular weight. These results highlight that transferrin might induce the development of a monoclonal immune response of different classes and specificity. The identification, in a single hematologic center, of three different subjects with anti-transferrin monoclonal gammopathy suggests that the disease probably represents a still underdiagnosed condition. From a clinical standpoint, these patients must be followed up both as MGUS and as hemochromatosis.

p57Kip2 is a downstream effector of BCR-ABL kinase inhibitors in chronic myelogenous leukemia cells.

Chronic myelogenous leukemia (CML) is characterized by the expression of BCR-ABL tyrosine kinase, which results in increased cell proliferation and inhibition of apoptosis. In this study, we show that BCR-ABL-positive CML cell lines treated with imatinib (STI571) undergo G1 cell cycle arrest associated with the accumulation of p57(Kip)², a cyclin-dependent kinase inhibitor (CKI). Interestingly, p57(Kip)² increase precedes the reported STI571-dependent upregulation of p27(Kip)¹. A number of complementary approaches allow the demonstration that p57(Kip)² buildup is due to the transcriptional activation of CDKN1C, the p57(Kip)²-encoding gene, while neither p57(Kip)² half-life elongation nor its cell relocalization were observed. We also identified a heretofore undescribed pattern of p57(Kip)² phosphorylated isoforms which, however, did not change in response to STI571 cell treatment. The imatinib-dependent p57(Kip)² upregulation occurs only in STI571-responsive cells, while the CKI accumulation was not evidenced in an imatinib-resistant clone. Nilotinib and dasatinib (second-generation BCR-ABL inhibitors), at concentrations comparable to those used in therapy, increase the CKI but do not affect p27(Kip)¹ level. Finally, CD34(+) cells from CML patients display a clear imatinib-dependent p57(Kip)² upregulation, which was not observed in CD34(+) cells from control subjects. In conclusion, our study points to p57(Kip)² as a novel and precocious effector of BCR-ABL targeting drugs.

Cystatin B and its EPM1 mutants are polymeric and aggregate prone in vivo.

Progressive myoclonus epilepsy type 1 (EPM1) is a neurodegenerative disease correlating with mutations of the cystatin B gene. Cystatin B is described as a monomeric protein with antiprotease function. This work shows that, in vivo, cystatin B has a polymeric structure, highly resistant to SDS, urea, boiling and sensitive to reducing agents and alkaline pH. Hydrogen peroxide increases the polymeric structure of the protein. Mass spectrometry analysis shows that the only component of the polymers is cystatin B. EPM1 mutants of cystatin B transfected in cultured cells are also polymeric. The banding pattern generated by a cysteine-minus mutant is different from that of the wild-type protein as it contains only monomers, dimers and some very high MW bands while misses components of MW intermediate between 25 and 250 kDa. Overexpression of wild-type or EPM1 mutants of cystatin B in neuroblastoma cells generates cytoplasmic aggregates. The cysteine-minus mutant is less prone to the formation of inclusion bodies. We conclude that cystatin B in vivo has a polymeric structure sensitive to the redox environment and that overexpression of the protein generates aggregates. This work describes a protein with a physiological role characterized by highly stable polymers prone to aggregate formation in vivo.

p27Kip1 and human cancers: A reappraisal of a still enigmatic protein.

p27Kip1 is a cell cycle regulator firstly identified as a cyclin-dependent kinase inhibitor. For a long time, its function has been associated to cell cycle progression inhibition at G1/S boundary in response to antiproliferative stimuli. The picture resulted complicated by the discovery that p27Kip1 is an intrinsically unstructured protein, with numerous CDK-dependent and -independent functions and involvement in many cellular processes, such as cytoskeleton dynamics and cell motility control, apoptosis and autophagy activation. Depending on the cell context, these activities might turn to be oncogenic and stimulate cancer progression and metastatization. Nevertheless, p27Kip1 role in cancer biology suppression was underscored by myriad data reporting its down-regulation and/or cytoplasmic relocalization in different tumors, while usually no genetic alterations were found in human cancers, making the protein a non-canonical oncosuppressor. Recently, mostly due to advances in genomic analyses, CDKN1B, p27Kip1 encoding gene, has been found mutated in several cancers, thus leading to a profound reappraisal of CDKN1B role in tumorigenesis. This review summarizes the main p27Kip1 features, with major emphasis to its role in cancer biology and to the importance of CDKN1B mutations in tumor development.

Tyrosine kinase inhibitors and mesenchymal stromal cells: effects on self-renewal, commitment and functions.

The hope of selectively targeting cancer cells by therapy and eradicating definitively malignancies is based on the identification of pathways or metabolisms that clearly distinguish "normal" from "transformed" phenotypes. Some tyrosine kinase activities, specifically unregulated and potently activated in malignant cells, might represent important targets of therapy. Consequently, tyrosine kinase inhibitors (TKIs) might be thought as the "vanguard" of molecularly targeted therapy for human neoplasias. Imatinib and the successive generations of inhibitors of Bcr-Abl1 kinase, represent the major successful examples of TKI use in cancer treatment. Other tyrosine kinases have been selected as targets of therapy, but the efficacy of their inhibition, although evident, is less definite. Two major negative effects exist in this therapeutic strategy and are linked to the specificity of the drugs and to the role of the targeted kinase in non-malignant cells. In this review, we will discuss the data available on the TKIs effects on the metabolism and functions of mesenchymal stromal cells (MSCs). MSCs are widely distributed in human tissues and play key physiological roles; nevertheless, they might be responsible for important pathologies. At present, bone marrow (BM) MSCs have been studied in greater detail, for both embryological origins and functions. The available data are evocative of an unexpected degree of complexity and heterogeneity of BM-MSCs. It is conceivable that this grade of intricacy occurs also in MSCs of other organs. Therefore, in perspective, the negative effects of TKIs on MSCs might represent a critical problem in long-term cancer therapies based on such inhibitors.

Histone Deacetylase Inhibitors Increase p27(Kip1) by Affecting Its Ubiquitin-Dependent Degradation through Skp2 Downregulation.

Histone deacetylase inhibitors (HDACIs) represent an intriguing class of pharmacologically active compounds. Currently, some HDACIs are FDA approved for cancer therapy and many others are in clinical trials, showing important clinical activities at well tolerated doses. HDACIs also interfere with the aging process and are involved in the control of inflammation and oxidative stress. In vitro, HDACIs induce different cellular responses including growth arrest, differentiation, and apoptosis. Here, we evaluated the effects of HDACIs on p27(Kip1), a key cyclin-dependent kinase inhibitor (CKI). We observed that HDACI-dependent antiproliferative activity is associated with p27(Kip1) accumulation due to a reduced protein degradation. p27(Kip1) removal requires a preliminary ubiquitination step due to the Skp2-SCF E3 ligase complex. We demonstrated that HDACIs increase p27(Kip1) stability through downregulation of Skp2 protein levels. Skp2 decline is only partially due to a reduced Skp2 gene expression. Conversely, the protein decrease is more profound and enduring compared to the changes of Skp2 transcript. This argues for HDACIs effects on Skp2 protein posttranslational modifications and/or on its removal. In summary, we demonstrate that HDACIs increase p27(Kip1) by hampering its nuclear ubiquitination/degradation. The findings might be of relevance in the phenotypic effects of these compounds, including their anticancer and aging-modulating activities.

Resveratrol mimics insulin activity in the adipogenic commitment of human bone marrow mesenchymal stromal cells.

Bone marrow mesenchymal stromal cells (BM-MSCs) are multipotent cells capable of differentiating toward osteoblatic and adipocytic phenotypes. BM-MSCs play several key roles including bone remodeling, establishment of hematopoietic niche and immune tolerance induction. Here, we investigated the effect of resveratrol (RSV), a therapeutically promising natural polyphenol, on the commitment of human BM-MSCs primary cultures. Cell differentiation was evaluated by means of morphological analysis, specific staining and expression of osteogenic and adipocytic master genes (Runx-2, PPARγ). To maintain BM-MSC multipotency, all experiments were performed on cells at very early passages. At any concentration RSV, added to standard medium, did not affect the phenotype of confluent BM-MSCs, while, when added to osteogenic or adipogenic medium, 1 µM RSV enhances the differentiation toward osteoblasts or adipocytes, respectively. Conversely, the addition of higher RSV concentration (25 µM) to both differentiation media resulted exclusively in BM-MSCs adipogenesis. Surprisingly, the analysis of RSV molecular effects demonstrated that the compound completely substitutes insulin, a key component of adipogenic medium. We also observed that RSV treatment is associated to enhanced phosphorylation of CREB, a critical effector of insulin adipogenic activity. Finally, our observations contribute to the mechanistic elucidation of the well-known RSV positive effect on insulin sensitivity and type 2 diabetes mellitus.

P27Kip1 serine 10 phosphorylation determines its metabolism and interaction with cyclin-dependent kinases.

p27Kip1 is a critical modulator of cell proliferation by controlling assembly, localization and activity of cyclin-dependent kinase (CDK). p27Kip1 also plays important roles in malignant transformation, modulating cell movement and interaction with the extracellular matrix. A critical p27Kip1 feature is the lack of a stable tertiary structure that enhances its "adaptability" to different interactors and explains the heterogeneity of its function. The absence of a well-defined folding underlines the importance of p27Kip1 post-translational modifications that might highly impact the protein functions. Here, we characterize the metabolism and CDK interaction of phosphoserine10-p27Kip1 (pS10- p27Kip1), the major phosphoisoform of p27Kip1. By an experimental strategy based on specific immunoprecipitation and bidimensional electrophoresis, we established that pS10-p27Kip1 is mainly bound to cyclin E/CDK2 rather than to cyclin A/CDK2. pS10- p27Kip1 is more stable than non-modified p27Kip1, since it is not (or scarcely) phosphorylated on T187, the post-translational modification required for p27Kip1 removal in the nucleus. pS10-p27Kip1 does not bind CDK1. The lack of this interaction might represent a mechanism for facilitating CDK1 activation and allowing mitosis completion. In conclusion, we suggest that nuclear p27Kip1 follows 2 almost independent pathways operating at different rates. One pathway involves threonine-187 and tyrosine phosphorylations and drives the protein toward its Skp2-dependent removal. The other involves serine-10 phosphorylation and results in the elongation of p27Kip1 half-life and specific CDK interactions. Thus, pS10-p27Kip1, due to its stability, might be thought as a major responsible for the p27Kip1-dependent arrest of cells in G1/G0 phase.

Resveratrol and cancer treatment: is hormesis a yet unsolved matter?

Plants produce many low molecular mass natural compounds endowed with biological activity. Among them, resveratrol (3,5,4'-trihydroxystilbene) has been demonstrated to be able to affect a plethora of pivotal cellular molecular processes, including transduction pathways and gene expression. These activities result, in turn, in several different cell phenotypes. Particularly, frequent effects of resveratrol treatment appear to be the reduction of growth and the activation of programmed cell death. Accordingly, a number of trials are currently under development to evaluate the possibility of using resveratrol in cancer therapy, both as single agent or in association with other anticancer compounds. However, some reports suggest that, at low concentrations, not only resveratrol does not inhibit the proliferation and/or the survival of cells but, conversely, it induces proliferation and/or protects cells against toxic agents. On the basis of these biphasic effects, it has been proposed that resveratrol belongs to the so-called hormetic compounds. Hormesis is an expression employed by toxicologists to describe a U-shaped (or J-shaped) dose response characterized by a beneficial effect at low doses and a toxic (or inhibitory) activity at high dose. In this review, we will reappraise data that might suggest or disprove that resveratrol is endowed with clear hormetic properties.

p57(Kip2) and cancer: time for a critical appraisal.

p57(Kip2) is a cyclin-dependent kinase inhibitor belonging to the Cip/Kip family, which also includes p21(Cip1) and p27(Kip1). So far, p57(Kip2) is the least-studied Cip/Kip protein, and for a long time its relevance has been related mainly to its unique role in embryogenesis. Moreover, genetic and molecular studies on animal models and patients with Beckwith-Wiedemann syndrome have shown that alterations in CDKN1C (the p57(Kip2) encoding gene) have functional relevance in the pathogenesis of this disease. Recently, a number of investigations have identified and characterized heretofore unexpected roles for p57(Kip2). The protein appears to be critically involved in initial steps of cell and tissue differentiation, and particularly in neuronal development and erythropoiesis. Intriguingly, p27(Kip1), the Cip/Kip member that is most homologous to p57(Kip2), is primarily involved in the process of cell cycle exit. p57(Kip2) also plays a critical role in controlling cytoskeletal organization and cell migration through its interaction with LIMK-1. Furthermore, p57(Kip2) appears to modulate genome expression. Finally, accumulating evidence indicates that p57(Kip2) protein is frequently downregulated in different types of human epithelial and nonepithelial cancers as a consequence of genetic and epigenetic events. In summary, the emerging picture is that several aspects of p57(Kip2)'s functions are only poorly clarified. This review represents an appraisal of the data available on the p57(Kip2) gene and protein structure, and its role in human physiology and pathology. We particularly focus our attention on p57(Kip2) changes in cancers and pharmacological approaches for modulating p57(Kip2) levels.

The tyrosine kinase inhibitor dasatinib induces a marked adipogenic differentiation of human multipotent mesenchymal stromal cells.

BACKGROUND: The introduction of specific BCR-ABL inhibitors in chronic myelogenous leukemia therapy has entirely mutated the prognosis of this hematologic cancer from being a fatal disorder to becoming a chronic disease. Due to the probable long lasting treatment with tyrosine-kinase inhibitors (TKIs), the knowledge of their effects on normal cells is of pivotal importance. DESIGN AND METHODS: We investigated the effects of dasatinib treatment on human bone marrow-derived mesenchymal stromal cells (MSCs). RESULTS: Our findings demonstrate, for the first time, that dasatinib induces MSCs adipocytic differentiation. Particularly, when the TKI is added to the medium inducing osteogenic differentiation, a high MSCs percentage acquires adipocytic morphology and overexpresses adipocytic specific genes, including PPARγ, CEBPα, LPL and SREBP1c. Dasatinib also inhibits the activity of alkaline phosphatase, an osteogenic marker, and remarkably reduces matrix mineralization. The increase of PPARγ is also confirmed at protein level. The component of osteogenic medium required for dasatinib-induced adipogenesis is dexamethasone. Intriguingly, the increase of adipocytic markers is also observed in MSCs treated with dasatinib alone. The TKI effect is phenotype-specific, since fibroblasts do not undergo adipocytic differentiation or PPARγ increase. CONCLUSIONS: Our data demonstrate that dasatinib treatment affects bone marrow MSCs commitment and suggest that TKIs therapy might modify normal phenotypes with potential significant negative consequences.

Targeting p27Kip1 protein: its relevance in the therapy of human cancer.

INTRODUCTION: Cell division cycle progression is achieved by a sequential and stringently concerted activation of a family of serine-threonine kinases, namely the cyclin-dependent kinases (CDKs). p27(Kip1) is a pivotal CDK inhibitor and a tight modulator of CDK-dependent phenotypes. Thus, p27(Kip1) plays a fundamental role in key cellular processes such as proliferation, differentiation, apoptosis, substrate adhesion and motility. Intriguingly, when p27(Kip1) is localized in the nucleus, it acts as an antiproliferative protein, while, in the cytosol, p27(Kip1) promotes cytoskeleton remodeling and might positively influence metastatization. Downregulation of p27(Kip1) nuclear level or its cytosolic mislocalization are consistently correlated with poor prognosis of numerous types of human epithelial and non-epithelial cancers. AREAS COVERED: This review illustrates the basic structural features of p27(Kip1) protein, its metabolism and alterations in human malignancies, along with describing anticancer strategies based on targeting p27(Kip1). EXPERT OPINION: Given the role of p27(Kip1) in the control of cell proliferation and its decreased level observed in malignancies with poor outcome, drugs able to handle the protein levels and localization might represent an important goal for novel specific and effective anticancer strategies. Although no convincing proofs have been reported, putative negative consequences of p27(Kip1) targeting might be also conceivable.