p57Kip2 is a cyclin-dependent kinase inhibitor belonging to the Cip/Kip family, which also includes p21Cip1 and p27Kip1. So far, p57Kip2 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 p57Kip2 encoding gene) have functional relevance in the pathogenesis of this disease. Recently, a number of investigations have identified and characterized heretofore unexpected roles for p57Kip2. The protein appears to be critically involved in initial steps of cell and tissue differentiation, and particularly in neuronal development and erythropoiesis. Intriguingly, p27Kip1, the Cip/Kip member that is most homologous to p57Kip2, is primarily involved in the process of cell cycle exit. p57Kip2 also plays a critical role in controlling cytoskeletal organization and cell migration through its interaction with LIMK-1. Furthermore, p57Kip2 appears to modulate genome expression. Finally, accumulating evidence indicates that p57Kip2 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 p57Kip2's functions are only poorly clarified. This review represents an appraisal of the data available on the p57Kip2 gene and protein structure, and its role in human physiology and pathology. We particularly focus our attention on p57Kip2 changes in cancers and pharmacological approaches for modulating p57Kip2 levels. Mol Cancer Res; 9(10); 1269–84. ©2011 AACR.

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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.

All-trans-retinoic acid (ATRA), the most biologically active metabolite of vitamin A, controls cell proliferation, apoptosis, and differentiation depending on the cellular context. These activities point to ATRA as a candidate for cancer therapy. A pivotal effect of the molecule is the modulation of p27 Kip1 , a cyclin-dependent kinase (CDK) inhibitor (CDKI). Here, we investigate the mechanisms by which ATRA regulates p27 Kip1 level in LAN-5, a neuroblastoma cell line. When added to the cells, ATRA causes a rapid nuclear increase of p27 Kip1 , which clearly precedes growth arrest. The early buildup is not due to impairment of the CDKI degradation, in contrast to previous observations. Particularly, we did not detect the downregulation of Skp2 and Cks1, two proteins involved in the nuclear ubiquitin-dependent p27Kip1 removal. Moreover, the morphogen does not impair the CDKI nuclear export and does not cause CDK2 relocalization. The characterization of CDKI isoforms by two-dimensional PAGE/immunoblotting showed that ATRA induces an early nuclear up-regulation of monophosphorylated p27 Kip1 . Immunologic studies established that this isoform corresponds to p27Kip1 phosphorylated on S10. The buildup of phospho(S10)p27Kip1 precedes the CDKI accumulation and increases its half-life. Finally, ATRA-treated nuclear LAN-5 extracts showed an enhanced capability of phosphorylating p27Kip1 on S10, thus explaining the nuclear up-regulation of the isoform. In conclusion, our data suggest a novel mechanism of ATRA antiproliferative activity, in which the morphogen rapidly up-regulates a nuclear kinase activity that phosphorylates p27 Kip1 on S10. In turn, this event causes the stabilization of p27Kip1 and its accumulation in the nuclear compartment. (Cancer Res 2006; 66(8): 4240-8)

© F e r r a t a S t o r t i F o u n d a t i o napproximately 80% of FA patients worldwide, will not be successful in at least 20%. Secondly, there is a wide spectrum of mutations, mainly private, spanning the entire genes, including intragenic deletions, as frequently observed in FANCA (http://www.rockefeller.edu/fanconi/). Since mutational screening would largely benefit from a preliminary knowledge of the candidate gene, complementation by cell fusion or by viral transduction, as well as protein analyses, have been established as screening strategies to identify the putative gene that is defective. 6,7 Thirdly, a false negative or inconclusive chromosomal breakage test might occur in patients who develop hematopoietic mosaicism due to reversion of the cellular FA phenotype. 8,9 This phenomenon arises if a spontaneous genetic event reconstitutes normal protein activity of one allele. Since it is unlikely that the same somatic event will also occur in primary skin fibroblasts, these cells are often used for mutational screening.In this paper, we report the molecular data of 100 FA families enrolled into the National Network of the Marrow Failure Study Group of the Italian Association of Pediatric Hematology and Oncology. This cohort consists of 76 new families, as well as 24 families that have been described in previous reports. 10,11 Methods Patients, cell lines, and DNA samplesPatients with a positive chromosomal breakage test were included in this study for molecular screening of FA genes by the National Network of the Marrow Failure Study Group of the Italian Association of Pediatric Hematology and Oncology. All the subjects or their legal guardians gave written informed consent to the investigation, according to the Declaration of Helsinki. Protocols were approved by the ethics review boards of the institutions that enrolled the patients. DNA was extracted from peripheral blood, lymphoblastoid cell lines and/or primary fibroblasts. Complementation and Western blot analysesLymphoblast cell lines, peripheral blood T lymphocytes or primary fibroblasts were transduced with retroviral expressing the cDNAs for FANCA, FANCC or FANCG as previously reported. 6 Complementation was considered to occur when the viability of the transduced cells increased by more than 20% that of controls at at least three different mitomycin C concentrations. Western blot analysis of FANCD2 using a monoclonal antibody (Santa Cruz, CA, USA; diluted 1:500) was performed as previously described. 10 Sequencing analysis and multiplex ligation-dependent probe amplificationThe coding exons of the FA genes and their flanking regions were sequenced using a set of oligonucleotides (the primer sequences are available upon request) according to standard procedures. 10 Six samples were first analyzed using the Ion PGM TM system for next generation sequencing of the 16 FA genes according to the manufacturer's protocols (Life Technologies). Variants with minor allele frequency less than 1% were then confirmed by Sanger sequencing as above. N...

Histone deacetylase inhibitors (HDACIs) represent a new class of targeted anticancer agents. Here, we evaluate the effects of butyrate (BuA) and other HDACIs on p57(Kip2), a cyclin-dependent kinase inhibitor (cki). We observed that inhibitors of class I/II histone deacetylases (HDACs), but not of class III HDACs, induce a remarkable accumulation of p57(Kip2) in several cells. The cki upregulation is associated with an increased gene expression that was not prevented by cycloheximide, indicating that HDACIs affect directly p57(Kip2) transcription. The characterization of p57(Kip2) promoter indicates that the first 165 bp are mostly involved in the BuA effects. Chromatin immunoprecipitation studies demonstrated that the BuA treatment causes the recruitment of Sp1 transcription factor. The Sp1 importance was confirmed by the reduction of BuA effects by mithramycin A (an Sp1 antagonist) and, most stringently, by Sp1 downregulation due to Sp1 siRNA. Moreover, both the treatments reduce the p57(Kip2) transcription in untreated cells, suggesting that Sp1 is required for the constitutive cki expression. Studies employing plasmids containing parts of the 165 bp of p57(Kip2) promoter indicate that the promoter region between -87 and -113 bp, which includes two putative Sp1 consensus sequences, plays a critical role in the response to HDACIs. Since this p57(Kip2) promoter region also embraces the consensus sequence for the transcriptional repressor chicken ovalbumin upstream promoter transcription factor-interacting protein 2 (CTIP2), we evaluated whether this factor is involved into the BuA effect. When CTIP2 was downregulated by a specific siRNA, we observed the enhancement of BuA activity on p57(Kip2) expression suggesting that CTIP2 might also be involved in HDACIs effects.

Objective: Provide evidence of HPV, C. trachomatis, and HSV infection in the oral cavity from patients with different types of stomatological lesions. Materials and Methods: Oral swabs samples were collected from a total of 318 patients. The infectious agents were analyzed using the PCR technique. HPV genotyping and HSV type were studied using the RFLP method. Results: We studied 137 benign lesions (B), 96 potentially malignant disorders (PMD) and 85 oral squamous cell carcinomas (OSCC). The prevalence of HPV was 34%. The most frequently genotypes detected were 6 low risk and 16 high risk. The prevalence of C. trachomatis was 16% and HSV 3%. Co-infections were detected mostly in benign lesions as following: HPV-C. trachomatis in 4%, C. trachomatis-HSV in 1.8% and HPV-HSV in 0.3%. Conclusion: This report is the first contribution to the identification and genotype characterization of HPV in a scenario little studied in our area, and it also contributes to improving our understanding on sexually transmitted infectious agents and their associations with the oral cavity. Besides, we detect the presence of C. trachomatis and HSV and co-infection with HPV in the oral cavity, which they should be taken into account for diagnostic and treatment purposes.

Fanconi anemia (FA) is an autosomal recessive disorder characterized by genomic instability, bone marrow failure, congenital malformations, and cancer predisposition. FA is a genetically heterogeneous disease with at least seven genes so far identified. The role of FA proteins is unknown although they interact in a common functional pathway. Here, we report six novel FANCA sequence changes and review all the mutations identified in Italy. Except for two missense substitutions, all are expected to cause a premature termination of the FANCA protein at various sites throughout the molecule. The premature terminations are due to nonsense and splice site mutations, as well as small insertions and deletions, and large genomic rearrangements. The expected truncated proteins were not detectable on Western blot analyses. The FANCA-S858R variant is instead expressed at lower level than that seen in normal cell lines and is associated with a non-ubiquinated FANCD2 protein, strongly suggesting that the amino acid substitution is a disease-causing mutation. The spectrum of FA mutations is widely in agreement with the heterogeneous ethnic origin of the Italian population.