p27Kip1, an Intrinsically Unstructured Protein with Scaffold Properties

The Cyclin-dependent kinase (CDK) regulator p27Kip1 is a gatekeeper of G1/S transition. It also regulates G2/M progression and cytokinesis completion, via CDK-dependent or -independent mechanisms. Recently, other important p27Kip1 functions have been described, including the regulation of cell motility and migration, the control of cell differentiation program and the activation of apoptosis/autophagy. Several factors modulate p27Kip1 activities, including its level, cellular localization and post-translational modifications. As a matter of fact, the protein is phosphorylated, ubiquitinated, SUMOylated, O-linked N-acetylglicosylated and acetylated on different residues. p27Kip1 belongs to the family of the intrinsically unstructured proteins and thus it is endowed with a large flexibility and numerous interactors, only partially identified. In this review, we look at p27Kip1 properties and ascribe part of its heterogeneous functions to the ability to act as an anchor or scaffold capable to participate in the construction of different platforms for modulating cell response to extracellular signals and allowing adaptation to environmental changes

An Unanticipated Modulation of Cyclin-Dependent Kinase Inhibitors: The Role of Long Non-Coding RNAs

It is now definitively established that a large part of the human genome is transcribed. However, only a scarce percentage of the transcriptome (about 1.2%) consists of RNAs that are translated into proteins, while the large majority of transcripts include a variety of RNA families with different dimensions and functions. Within this heterogeneous RNA world, a significant fraction consists of sequences with a length of more than 200 bases that form the so-called long non-coding RNA family. The functions of long non-coding RNAs range from the regulation of gene transcription to the changes in DNA topology and nucleosome modification and structural organization, to paraspeckle formation and cellular organelles maturation. This review is focused on the role of long non-coding RNAs as regulators of cyclin-dependent kinase inhibitors' (CDKIs) levels and activities. Cyclin-dependent kinases are enzymes necessary for the tuned progression of the cell division cycle. The control of their activity takes place at various levels. Among these, interaction with CDKIs is a vital mechanism. Through CDKI modulation, long non-coding RNAs implement control over cellular physiology and are associated with numerous pathologies. However, although there are robust data in the literature, the role of long non-coding RNAs in the modulation of CDKIs appears to still be underestimated, as well as their importance in cell proliferation control

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 p27Kip1, 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

A cancer-associated CDKN1B mutation induces p27 phosphorylation on a novel residue: a new mechanism for tumor suppressor loss-of-function

CDKN1B haploinsufficiency promotes the development of several human cancers. The gene encodes p27Kip1 , a protein playing pivotal roles in the control of growth, differentiation, cytoskeleton dynamics and cytokinesis. CDKN1B haploinsufficiency has been associated with chromosomal or gene aberrations. However, very few data exist on the mechanisms by which CDKN1B missense mutations facilitate carcinogenesis. Here, we report a functional study on a cancer-associated germinal p27Kip1 variant, namely glycine9->arginine-p27Kip1 (G9R-p27Kip1 ) identified in a parathyroid adenoma. We unexpectedly found that G9R-p27Kip1 lacks the major tumor suppressor activities of p27Kip1 including its antiproliferative and pro-apoptotic functions. In addition, G9R-p27Kip1 transfection in cell lines induces the formation of more numerous and larger spheres when compared to wild type p27Kip1 -transfected cells. We demonstrated that the mutation creates a consensus sequence for basophilic kinases causing a massive phosphorylation of G9R-p27Kip1 on S12, a residue normally never found modified in p27Kip1 . The novel S12 phosphorylation appears responsible for the loss of function of G9R-p27Kip1 since S12AG9R-p27Kip1 recovers most of the p27Kip1 tumor suppressor activities. In addition, the expression of the phosphomimetic S12D-p27Kip1 recapitulates G9R-p27Kip1 properties. Mechanistically, S12 phosphorylation enhances the nuclear localization of the mutant protein, and also reduces its CDK2/CDK1 inhibition activity. To our knowledge, this is the first reported case of quantitative phosphorylation of a p27Kip1 variant on a physiologically unmodified residue associated with the loss of several tumor suppressor activities. In addition, our findings demonstrate that haploinsufficiency might be due to unpredictable post-translational modifications due to generation of novel consensus sequences by cancer-associated missense mutations

Caspase 3 and 8 deficiency in human neuroblastoma

An altered apoptotic response represents a pivotal feature of cancer and is involved in cancerogenesis and resistance to chemotherapy. So far, however, only a few studies have been devoted to survey caspase content in malignant cell lines and primary tumor specimens. In this report, we investigated the expression of two pivotal caspases, 3 and 8, in 63 neuroblastoma specimens by three complementary techniques (i.e., reverse transcriptase polymerase chain reaction, immunoblotting, and immunohistochemistry). We confirmed the frequent absence of caspase 8 expression. Moreover and most important, we demonstrated, for the first time to our knowledge, that a significant percentage of neuroblastomas lack caspase 3 mRNA and protein. Both caspase alterations do not show any correlation with tumor stage and MYCN status. Immunohistochemistry showed a large number of caspase-negative cell islets also present in positive samples. Our findings suggest that the absence of caspases might play an important role in neuroblastoma development and resistance to apoptosisbased treatments

Hypocalcemia: A key biomarker in hospitalized COVID-19 patients

BACKGROUND: At the end of 2019 a new respiratory syndrome emerged in China named Coronavirus disease 2019 (COVID-19). Considering the severity of the disease in adult subjects with one or more chronic pathologies, it was mandatory to find simple and effective biomarkers for negative prognosis of the disease easily available at the admission to the hospital. MATERIAL AND METHODS: To identify possible parameters showing association with the outcome in COVID-19 patients with previous chronic diseases, blood biochemical profiles of 511 patients, enrolled from March to June 2020, were retrospectively evaluated. The pathologies taken into consideration were diabetes, arterial hypertension, chronic kidney disease, cardiovascular diseases, chronic obstructive pulmonary disease, obesity, and cancer. All the data were collected upon admission to the emergency room (ER) during the indicated period. RESULTS AND DISCUSSION: In accord with previous studies, we observed that serum and ionized calcium were prevalently altered in our cohort. We determined that hypocalcemia was a major parameter associated with mechanical ventilation and poor prognosis, correlating also with the presence of comorbidities such as cardiovascular diseases, chronic kidney disease, and cancer. In addition, we found a positive correlation between hypocalcemia and clinical complications during hospitalizations, strengthening the relevance of calcium concentration as a useful prognostic biomarker in COVID-19 patients

Effects of Magnetic Stimulation on Dental Implant Osseointegration: A Scoping Review

This PRISMA-ScR driven scoping review aims to evaluate the influence of magnetic field stimu-lation on dental implant osseointegration. Seven databases were screened adopting ad-hoc strings. All clinical and preclinical studies analyzing the effects of magnetic fields on dental im-plant osseointegration were included. From 3124 initial items, on the basis of the eligibility cri-teria, 33 articles, regarding both Pulsed ElectroMagnetic Fields (PEMF) and Static magnetic Fields from permanent Magnets (SFM) were finally included and critically analyzed. In vitro studies showed a positive effect of PEMF, but contrasting effects of SFM on bone cell proliferation, whereas cell adhesion and osteogenic differentiation were induced by both types of stimulation. In vivo studies showed an increased bone-to-implant contact rate in different animal models and clinical studies revealed positive effects on implant stability, under magnetic stimulation. In conclusion, although positive effects of magnetic exposure on osteogenesis activity and os-seointegration emerged, this scoping review highlighted the need for further preclinical and clinical studies. More standardized designs, accurate choice of stimulation parameters, adequate methods of evaluation of the outcomes, greater sample size and longer follow-ups are needed to clearly assess the effect of magnetic fields on dental implant osseointegration

Von Hippel-Lindau–dependent polycythemia is endemic on the island of Ischia: identification of a novel cluster

AbstractChuvash polycythemia (MIM 263400) is an autosomal recessive disorder characterized by a high hemoglobin level, relatively high serum erythropoietin, and early death. It results from a Von Hippel-Lindau (VHL) gene mutation (C598T) that causes increased HIF-1α activity and erythrocyte production in the face of normoxia. This polycythemia is endemic in Chuvashia, whereas its worldwide frequency is very low. We investigated the incidence of the Chuvash-type VHL mutation in Campania (South Italy) and identified 14 affected subjects (5 families). Twelve live on the island of Ischia (Bay of Naples). From analysis of the mutated allele, we found that the disease was more frequent on Ischia (0.070) than in Chuvashia (0.057). The haplotype of all patients matched that identified in the Chuvash cluster, thereby supporting the single-founder hypothesis. We also found that nonaffected heterozygotes had increased HIF-1α activity, which might confer a biochemical advantage for mutation maintenance. In conclusion, we have identified the first large cluster of Chuvash erythrocytosis outside Chuvashia, which suggests that this familial polycythemia might be endemic in other regions of the world

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

Molecular analysis of Fanconi anemia: the experience of the Bone Marrow Failure Study Group of the Italian Association of Pediatric Onco-Hematology

Fanconi anemia is an inherited disease characterized by congenital malformations, pancytopenia, cancer predisposition, and sensitivity to cross-linking agents. The molecular diagnosis of Fanconi anemia is relatively complex for several aspects including genetic heterogeneity with mutations in at least 16 different genes. In this paper, we report the mutations identified in 100 unrelated probands enrolled into the National Network of the Italian Association of Pediatric Hematoly and Oncology. In approximately half of these cases, mutational screening was carried out after retroviral complementation analyses or protein analysis. In the other half, the analysis was performed on the most frequently mutated genes or using a next generation sequencing approach. We identified 108 distinct variants of the FANCA, FANCG, FANCC, FANCD2, and FANCB genes in 85, 9, 3, 2, and 1 families, respectively. Despite the relatively high number of private mutations, 45 of which are novel Fanconi anemia alleles, 26% of the FANCA alleles are due to 5 distinct mutations. Most of the mutations are large genomic deletions and nonsense or frameshift mutations, although we identified a series of missense mutations, whose pathogenetic role was not always certain. The molecular diagnosis of Fanconi anemia is still a tiered procedure that requires identifying candidate genes to avoid useless sequencing. Introduction of next generation sequencing strategies will greatly improve the diagnostic process, allowing a rapid analysis of all the genes