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Cultured mouse embryonic stem cells are a heterogeneous population with diverse differentiation potential. In particular, the subpopulation marked by Zscan4 expression has high stem cell potency and shares with 2 cell stage preimplantation embryos both genetic and epigenetic mechanisms that orchestrate zygotic genome activation. Although embryonic de novo genome activation is known to rely on metabolites, a more extensive metabolic characterization is missing. Here we analyze the Zscan4(+) mouse stem cell metabolic phenotype associated with pluripotency maintenance and cell reprogramming. We show that Zscan4(+) cells have an oxidative and adaptable metabolism, which, on one hand, fuels a high bioenergetic demand and, on the other hand, provides intermediate metabolites for epigenetic reprogramming. Our findings enhance our understanding of the metastable Zscan4(+) stem cell state with potential applications in regenerative medicine.

Plant extracts are an important source of bioactive compounds for many drug discovery programs. Given the chemical complexity, however, the identification and characterization of bioactive compounds in crude plant extracts still remain a big challenge. Moreover, complex mixture of phytochemicals generally combine the advantage of targeting multiple molecular pathways so that a desired biological response may be due to a mixture of different bioactive components. Ethno-pharmacology history and available information about the plant to be studied may help to set specific bio-assays that can predict the reputed therapeutic activity and facilitate the isolation of the bioactives responsible for that biological activity. In a collaborative effort, we have analyzed the bioactivities of Cayenne pepper (Capsicum) a spicy staple of many foods, largely used in folk medicine for its extensive curative properties. Used either topically or internally, Capsicum may help support healthy bone, joint and muscle function by reducing inflammation. It is commonly used as a general stimulant and to heal ulcers, improve circulation and boost immune system. To elucidate the mechanism of action of Capsicum at cellular level, we have studied the effects of the variety Capsicum annuum fiesta on immortalized human keratinocytes. We have analyzed cell viability, intracellular ROS levels, stress granules formation and cell cycle distribution demonstrating that C. annuum fiesta exerts a protective role reducing intracellular ROS levels in a dose-dependent manner and stimulating cell viability at low concentrations without affecting cell proliferation. Another area of interest in our lab involves the characterization of Uncaria tomentosa extract (UT-ex) that has been widely used in folk medicine to treat a variety of health conditions including skin burns and cancer. Cell viability, intracellular ROS levels and cell cycle distribution were evaluated following treatment of immortalized and transformed keratinocytes cells with UT-ex. All cell lines tested showed dose-dependent susceptibility to Uncaria cytotoxicity, untransformed HaCaT keratinocytes being, however, less sensitive than squamous carcinoma cells. Interestingly, we found that following UT-ex treatment PARP1 cleavage occurred concomitantly with the reduction of the cleaved form of Y-box binding protein 1 (YB-1), a nuclear prosurvival factor involved in DNA damage repair. Taken together, our observations suggest that Uncaria induced cell death can be ascribed to its ability to simultaneously induce oxidative DNA damage and antagonize the mechanism of DNA repair. Our data support the use of Uncaria extract as adjuvant therapy for treatment of precancerous and early forms of squamous cell carcinomas.

The prototype cold-shock Y-box binding protein 1 (YB-1) is a multifunctional protein that regulates a variety of fundamental biological processes including cell proliferation and migration, DNA damage, matrix protein synthesis and chemotaxis. The plethora of functions assigned to YB-1 is strictly dependent on its subcellular localization. In resting cells, YB-1 localizes to cytoplasm where it is a component of messenger ribonucleoprotein particles. Under stress conditions, YB-1 contributes to the formation of stress granules (SGs), cytoplasmic foci where untranslated messenger RNAs (mRNAs) are sorted or processed for reinitiation, degradation, or packaging into ribonucleoprotein particles (mRNPs). Following DNA damage, YB-1 translocates to the nucleus and participates in DNA repair thereby enhancing cell survival. Recent data show that YB-1 can also be secreted and YB-1-derived polypeptides are found in plasma of patients with sepsis and malignancies. Here we show that in response to oxidative insults, YB-1 assembly in SGs is associated with an enhancement of YB-1 protein secretion. An enriched fraction of extracellular YB-1 (exYB-1) significantly inhibited proliferation of receiving cells and such inhibition was associated to a G2/M cell cycle arrest, induction of p21WAF and reduction of &Delta, Np63&alpha, protein level. All together, these data show that acute oxidative stress causes sustained release of YB-1 as a paracrine/autocrine signal that stimulate cell cycle arrest.

Uncaria tomentosa (Willd.) DC. (Rubiacee), also known as uña de gato, is a plant that grows wild in the upper Amazon region of Peru and has been widely used in folk medicine to treat several health conditions including cancer. We have produced an aqueous extract from Uncaria tomentosa (UT-ex) and analyzed its effects on squamous carcinoma cells and immortalized HaCaT keratinocytes. Squamous cell carcinoma (SCC) is an uncontrolled growth of abnormal cells arising in the skin's squamous layer of epidermis. When detected at an early stage, SCCs are almost curable, however, if left untreated, they can penetrate the underlying tissue and become disfiguring. We have evaluated cell proliferation, apoptosis and the level of reactive oxygen species following UT-ex treatment. UT-ex affected cell cycle progression and reduced cell viability in a dose and time-dependent manner. From a mechanistic point of view, this delay in cell growth coincided with the increase of reactive oxygen species (ROS). Furthermore, PARP1 cleavage was associated to the reduction of Y-box binding protein 1 (YB-1) 36kDa, a nuclear prosurvival factor involved in DNA damage repair. These data indicate that UT-ex-induced cell death can be ascribed, at least in part, to its ability both to induce oxidative DNA damage and antagonize the mechanism of DNA repair relying upon YB-1 activity. They also show that non metastatic SCCs are more susceptible to UT-ex treatment than untransformed keratinocytes supporting the use of UT-ex for the treatment of precancerous and early forms of squamous cell carcinomas. Preliminary chemical investigation of UT-ex revealed the presence of hydrophilic low-medium molecular weight metabolites with anticancer potential towards squamous carcinoma cells.

Cutaneous squamous cell carcinomas (SCCs) typically lack somatic oncogene-activating mutations and most of them contain p53 mutations. However, the presence of p53 mutations in skin premalignant lesions suggests that these represent early events during tumor progression and additional alterations may be required for SCC development. SCC cells frequently express high levels of ΔNp63α and Y-box binding 1 (YB-1 or YBX1) oncoproteins. Here, we show that knockdown of YB-1 in spontaneously immortalized HaCaT and non-metastatic SCC011 cells led to a dramatic decrease of ΔNp63α, cell detachment and death. In highly metastatic SCC022 cells, instead, YB-1 silencing induces PI3K/AKT signaling hyperactivation which counteracts the effect of YB-1 depletion and promotes cell survival. In summary, our results unveil a functional cross-talk between YB-1, ΔNp63α and the PI3K/AKT pathway critically governing survival of squamous carcinoma cells.

Poly(ADP-ribose)polymerase 1 (PARP-1) inhibitors are thought as breakthrough for cancer treatment in solid tumors such as breast cancer through their effects on PARP's enzymatic activity. Our previous findings showed that the hydrophilic PARP inhibitor PJ34 enhances the sensitivity of p53 proficient MCF7 breast carcinoma cells to topotecan, a DNA Topoisomerase I (TOP 1) inhibitor. In the present study, we combine the classical TOP 1 poison camptothecin or its water-soluble derivative topotecan with PJ34 to investigate the potentiation of chemotherapeutic efficiency in MCF7 (p53(WT)), MDA-MB231 (p53(mut)) breast carcinoma cells and SCC022 (p53(null)) squamous carcinoma cells. We show that, following TPT-PJ34 combined treatment, MCF7 cells exhibit apoptotic death while MDA-MB231 and SCC022 cells are more resistant to these agents. Specifically, in MCF7, (i) PJ34 in combination with TPT causes a G2/M cell cycle arrest followed by massive apoptosis; (ii) PJ34 addition reverts TPT-dependent PARP-1 automodification and triggers caspase-dependent PARP-1 proteolysis; (iii) TPT, used as a single agent, stimulates p53 expression while in combination with PJ34 increases p53, TAp63α and TAp63γ protein levels with a concomitant reduction of MDM2 protein. The identification of p63 proteins as new players involved in the cancer cell response to TPT-PJ34 is relevant for a better understanding of the PARP1-dependent signaling of DNA damage. Furthermore, our data indicate that, in response to TPT-PJ34 combined chemotherapy, a functional cooperation between p53 and TAp63 proteins may occur and be essential to trigger apoptotic cell death.

Y-box binding protein 1 (YBX-1 or YB-1) is an oncoprotein that promotes replicative immortality, tumor cell invasion and metastasis. The increase in the abundance of YB-1 in the cell or YB-1 translocation from the cytoplasm to the nucleus is characteristic of malignant cell growth. We have previously reported that ΔNp63α, a transcription factor that is known to play a pivotal role in keratinocyte proliferation and differentiation, promotes YB-1 nuclear accumulation. Here, we show that YB-1 is highly expressed in proliferating keratinocytes and is down-regulated during keratinocyte differentiation. ΔNp63α reduces YB-1 protein turnover and leads to accumulation of ubiquitin-conjugated YB-1 into the nucleus. Reduction of YB-1 protein level, following treatment with a DNA-damaging agent, is inhibited by ΔNp63α suggesting that YB-1 and ΔNp63α interplay can support keratinocyte proliferation and protect cells from apoptosis under genotoxic stress.

The Y-box binding protein 1 (YB-1) belongs to the cold-shock domain protein superfamily, one of the most evolutionarily conserved nucleic acid-binding proteins currently known. YB-1 performs a wide variety of cellular functions, including transcriptional and translational regulation, DNA repair, drug resistance, and stress responses to extracellular signals. Inasmuch as the level of YB-1 drastically increases in tumor cells, this protein is considered to be one of the most indicative markers of malignant tumors. Here, we present evidence that ΔNp63α, the predominant p63 protein isoform in squamous epithelia and YB-1, can physically interact. Into the nucleus, ΔNp63α and YB-1 cooperate in PI3KCA gene promoter activation. Moreover, ΔNp63α promotes YB-1 nuclear accumulation thereby reducing the amount of YB-1 bound to its target transcripts such as that encoding the SNAIL1 protein. Accordingly, ΔNp63α enforced expression was associated with a reduction of the level of SNAIL1, a potent inducer of epithelial to mesenchymal transition. Furthermore, ΔNp63α depletion causes morphological change and enhanced formation of actin stress fibers in squamous cancer cells. Mechanistic studies indicate that ΔNp63α affects cell movement and can reverse the increase of cell motility induced by YB-1 overexpression. These data thus suggest that ΔNp63α provides inhibitory signals for cell motility. Deficiency of ΔNp63α gene expression promotes cell mobilization, at least partially, through a YB-1-dependent mechanism.

The p63 protein is a member of the p53 gene family [1].The TP63 gene encodes isoforms that contain (TA) or lack (∆N) a transactivation domain. ∆Np63α, a critical pro-proliferative factor and a marker of epidermal stemness, is the most commonly expressed p63 protein and is essential for morphogenesis of organs/tissues developing by epithelial-mesenchimal interactions such as the epidermis, teeth, hair and glands [2]. We have recently shown that ∆Np63α is a molecular partner of the Y-box binding protein 1 (YB-1). YB-1, a marker of malignant tumor, is a nucleic acid binding protein with pleiotropic functions, such as alternative splicing, regulation of transcription and translation. Although YB-1 is predominantly cytoplasmic, it is highly expressed in the nuclear compartment of proliferating keratinocytes and squamous carcinoma cells. Here we show that ∆Np63α induces the nuclear accumulation of post-translationally modified forms of YB-1, sumoylation and ubiquitination are both involved in this phenomenon. During keratinocyte differentiation, YB-1 silencing restrains cell proliferation. In proliferating HaCaT and squamous carcinoma cells, YB-1 knockdown causes dramatic cell death and detachment. Our observations suggest that ∆Np63α and YB-1 functional interaction is critically associated with the proliferation and survival of normal and transformed keratinocytes.

Cutaneous squamous cell carcinomas (SCCs) typically lack somatic oncogene-activating mutations and most of them contain p53 mutations. However, the presence of p53 mutations in skin premalignant lesions suggests that these represent early events during tumor progression and additional alterations may be required for SCC development. SCC cells frequently express high levels of ΔNp63α and Y-box binding 1 (YB-1 or YBX1) oncoproteins. Here, we show that knockdown of YB-1 in spontaneously immortalized HaCaT and non-metastatic SCC011 cells led to a dramatic decrease of ΔNp63α, cell detachment and death. In highly metastatic SCC022 cells, instead, YB-1 silencing induces PI3K/AKT signaling hyperactivation which counteracts the effect of YB-1 depletion and promotes cell survival. In summary, our results unveil a functional cross-talk between YB-1, ΔNp63α and the PI3K/AKT pathway critically governing survival of squamous carcinoma cells.

Stearoyl-Coenzyme A desaturase 1 (SCD1) belongs to the fatty acid family of desaturases. In lactating ruminants, the SCD1 protein is highly expressed in the mammary gland and is relevant for the fatty acid composition of milk and dairy products. Bovine mammary epithelial cells (BME-UV1), cultured in vitro, have been proposed as a model to reproduce the biology of the mammary gland. The present study was designed to investigate the responsiveness of bovine SCD1 promoter to serum, insulin, oleic acid, and NFY transcription factor in BME-UV1 cells. A luciferase-based reporter assay was used to monitor the transcriptional activity of the SCD1 promoter region in BME-UV1 cells treated or not with insulin and/or oleic acid. The level of endogenous SCD1 mRNA was evaluated by Real time PCR. Insulin (20 ng/mL) induced a 2.0 to 2.5-fold increase of SCD1 promoter activity. Additionally, the effect of insulin was inhibited by oleic acid, serum components, and NFY enforced expression. Serum and NFY showed no synergistic or additive effect on SCD1 promoter activity suggesting that they repress SCD1 transcription through the same responsive element.

Depending on their genetic background (p53 wt versus p53 null ), carcinoma cells are more or less sensitive to drug-induced cell cycle arrest and/or apoptosis. Among the members of the p53 family, p63 is characterized by two N-terminal isoforms, TAp63 and ΔNp63. TAp63 isoform has p53-like functions, while ΔNp63 acts as a dominant negative inhibitor of p53. We have previously published that TAp63 is involved in poly(ADP-ribose)polymerase-1 (PARP-1) signaling of DNA damage deriving from DNA topoisomerase I (TOP I) inhibition in carcinoma cells. In the present study, we treated MCF7 breast carcinoma cells (p53 + /ΔNp63 − ) or SCC022 (p53 − /ΔNp63 + ) squamous carcinoma cells with the TOP I inhibitor topotecan (TPT) and the PJ34 PARP inhibitor, to compare their effects in the two different cell contexts. In MCF7 cells, we found that PJ34 addition reverts TPT-dependent PARP-1 auto-modification and triggers caspase-dependent PARP-1 proteolysis. Moreover, TPT as single agent stimulates p53 ser15 phosphorylation, p53 PARylation and occupancy of the p21WAF promoter by p53 resulting in an increase of p21WAF expression. Interestingly, PJ34 in combination with TPT enhances p53 occupancy at the BAX promoter and is associated with increased BAX protein level. In SCC022 cells, instead, TPT + PJ34 combined treatment reduces the level of the anti-apoptotic ΔNp63α protein without inducing apoptosis. Remarkably, in such cells, either exogenous p53 or TAp63 can rescue the apoptotic program in response to the treatment. All together our results suggest that in cancer cells PARP inhibitor(s) can operate in the choice between growth arrest and apoptosis by modulating p53 family-dependent signal

Congenital Dyserythropoietic Anemia type II is an autosomal recessive disorder characterized by unique abnormalities in the differentiation of cells of the erythroid lineage. The vast majority of CDA II cases result from mutations in the SEC23B gene. To date, 53 different causative mutations have been reported in 86 unrelated cases (from the CDA II European Registry), 47 of them Italian. We have now identified SEC23B mutations in 23 additional patients, 17 Italians and 6 non-Italian Europeans. The relative allelic frequency of the mutations was then reassessed in a total of 64 Italian and 45 non-Italian unrelated patients. Two mutations, E109K and R14W, account for over one-half of the cases of CDA II in Italy. Whereas the relative frequency of E109K is similar in Italy and in the rest of Europe (and is also prevalent in Moroccan Jews), the relative frequency of R14W is significantly higher in Italy (26.3% vs. 10.7%). By haplotype analysis we demonstrated that both are founder mutations in the Italian population. By using the DMLE+ program our estimate for the age of the E109K mutation in Italian population is ≈2,200 years; whereas for the R14W mutation it is ≈3,000 years. We hypothesize that E109K may have originated in the Middle East and may have spread in the heyday of the Roman Empire. Instead, R14W may have originated in Southern Italy. The relatively high frequency of the R14W mutation may account for the known increased prevalence of CDA II in Italy. Am. J. Hematol. 86:727–732, 2011. © 2011 Wiley-Liss, Inc.

Abstract 2098 Congenital dyserythropoietic anemias (CDAs) designate a group of genetic disorders in which ineffective erythropoiesis is the predominant mechanism of anemia marked by distinct morphologic abnormalities of the majority of erythroblasts in the bone marrow. CDA type II (CDAII) is the most common type of CDA. It is characterized by a recessive model of inheritance, mild to moderate anemia, jaundice, and splenomegaly (Fukuda MN, Glycobiology 1990; Fukuda MN, Clin Haematol 1993), by the presence of bi- and multinucleated erythroblasts in bone marrow, with nuclei of equal size and DNA content, suggesting a cytokinesis disturbance (Schwarz K and Iolascon A. et al., Nat Genet. 2009). The specific hallmark of diagnosis is the presence of the more abundant protein of membrane red cell, band 3, in a hypoglycosilated state; this is thinner and migrated slightly faster than in controls on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Anselstetter V et al., Br J Haematol 1977). The causative gene of CDAII, SEC23B, is a member of the SEC23 subfamily of the SEC23/SEC24 family, which is involved in vesicle trafficking. The encoded protein has similarity to yeast Sec23p (66.4%) component of COPII, the coat protein complex responsible for vesicle budding from the ER. The function of this gene product has been implicated in cargo selection and concentration. The SEC23B gene spans approximately 54 kb on human chromosome 20p11.23 and codifies for a protein of 767 aminoacids divided in five functional domains: zink finger, trunk, β-sheet, helical and gelsolin domain. Although most of the SEC23B mutations are sporadic events, 4 mutations (R14W, E109K, R497C, I318T) accounted for more than 50% of mutated alleles. The aim of this study is the in vitro characterization of the R14W mutation, the most frequent variant in Italy, particularly in South of Italy (Russo R et al., Am J Hematol. in press). We used the human erythroleukemia cell line, HEL, as in vitro model because it is more similar to mature red cell. By using in silico tool ESyPred3D Web Server 1.0, we predicted that this aminoacidic substitution alters the zink finger domain 3D structure, when compared to wild type protein. This tool implements homology modeling approach followed by a final analysis with MODELLER release 4 in order to build a 3D model of the submitted protein (Lambert et al, 2002). However, when we transfected the SEC23B-R14W we observed a strong reduction of gene expression in the mutant when compared to SEC23B-wt construct by qRT-PCR. These results have been also confirmed at the protein level. In fact the protein expression of SEC23B-R14W showed a reduction comparable to gene expression respect to SEC23B-wt construct. Immunofluorescence analyses by confocal microscopy, were used for the investigation of the cellular localization of SEC23B-R1W protein and, interestingly, the localization of mutant protein was not changed when compared to that wt. Our data allow us to hypothesize that the mutation R14W gives rise to anomalous protein product quantity, but the protein function would be like not altered. Our findings demonstrated that the most frequent mutation found in Italy, SEC23B-R14W, results in a reduced half-life of the mutated mRNA, without altering the cellular localization in HEL cell line. SEC23B belongs to a multiproteic compelx that assembles with the others complex proteins in accordance with a specific structure. Each structure establishes a cargo selectivity. Further studies are necessary in order to understand what is the role of SEC23B in selectivity of the cargo in erythroid cells and how its disruption could determines the appearance of the principal pathological phenotype in CDAII patients, for example the hypoglycosilation of band 3. Disclosures: No relevant conflicts of interest to declare.

Abstract 1005 The Congenital Dyserythropoietic Anemia type II (CDA II) is an autosomal recessive disorder characterized by an impaired differentiation-proliferation pathway of the erythroid lineage. The vast majority of CDA II cases are associated with mutations in the SEC23B gene, codifying a component of the cytoplasmic coat protein II complex, involved in the anterograde transport of correctly folded protein from the endoplasmic reticulum towards the Golgi (Fromme JC et al, Trends Cell Biol 2008). To date, 86 unrelated cases from CDA II International Registry and 53 different causative mutations have been described (Schwarz K et al, Nat Gen 2009; Bianchi P et al, Hum Mut 2009; Iolascon A et al, Haematologica 2010; Fermo E et al, Blood Cells Mol Dis 2010; Russo R et al, in submission [humu-2010-0373]). This set of patients is composed of 45 cases of Italian origin, and 39 without Italian origin (WIO). Two cases are of unknown origin. Our aim is to characterize the allelic distribution of SEC23B gene mutations in Italian country, in order to define a possible founder effect for the most frequent variants. In this study we included 17 Italian and 6 WIO patients still unpublished, with an overall count of 62 Italian and 45 WIO patients, respectively. Written consent was obtained from all patients in accordance with the Declaration of Helsinki. Genomic DNA preparation, mutational search, oligonucleotide primers design and direct sequencing were performed as previously described (Iolascon A et al, Haematologica 2010). Overall we found 30 different causative mutations in Italian patients (13 missense, 8 nonsense, 4 frameshift, 4 splice site mutations and one amino acid deletion) and 32 in WIO patients (19 missense, 8 nonsense, 3 frameshift and 2 splice site mutations). The most representative variants in Italian cohort are E109K and R14W substitutions (27.2%), confirming our previous data (Iolascon A et al, Haematologica 2010). When we analyzed the allelic frequency of 10 mutations (E109K, R14W, R701C, R497C, R217X, R79X, A524V, R264X, R339X, R535X) common to both cohort, we found that R14W variant showed an higher recurrence in Italian CDA II patients when compared to WIO patients (27.2% vs 10.7%). Moreover, E109K substitution showed almost the same allelic frequency between both groups, with only a little prevalence in Italian cohort (27.2% in Italian patients vs 25.0% in those WIO). This observation lead us to investigate whether their frequencies are due to a possible mutational hot spot (i.e. arisen independently) or, perhaps, to a possible founder effect for both mutations, E109K and R14W. So, we performed an haplotype analysis in a case-control study. Here, we focused our attention on E109K mutation. Using the genotype data of CEU population from HapMap Project web site (http://hapmap.ncbi.nlm.nih.gov/) we downloaded a list of 9370 single nucleotide polymorphisms (SNPs) within a region of 1685 kb upstream, 2656 kb downstream of the SEC23B gene and throughout the gene. From this list, we selected 12 tag-SNPs covering a region of about 1.2 Mb upstream and downstream of the E109K mutation. Tag-SNPs were identified using Haploview software (Barrett JC et al, Bioinformatics 2005), with a threshold of r2 > 0.8 and with a minor allele frequency > 0.35. From 27% of patients with E109K mutation, genotyping was performed by direct sequencing on 16 unrelated patients, 14 homozygotes and 2 heterozygotes for E109K variant. As healthy controls we downloaded genotypes information of 174 unrelated Caucasian subjects (CEU population) available from HapMart. Haplotype analysis, performed using Haploview software, showed that the haplotype CATAGT (composed of 6 SNPs spanning in a 28.8 kb region upstream and downstream of the mutation) is common to the majority of E109K-patients (96.9%); instead, the same haplotype was found only in a smaller percentage (3%) in healthy controls. Additional genetics analyses are ongoing to estimate the age of E109K mutation using a (i) marker by marker approach or (ii) an intra-allelic model to assess the linkage disequilibrium across the marker set, implemented in DMLE+2.2 software. Since the R14W mutation showed an allelic frequency in Italy similar to those observed for E109K, we are testing if also R14W may occur on a common genetic background. Disclosures: No relevant conflicts of interest to declare.