LINC01605 Is a Novel Target of Mutant p53 in Breast and Ovarian Cancer Cell Lines.

TP53 is the most frequently mutated gene in human cancers. Most TP53 genomic alterations are missense mutations, which cause a loss of its tumour suppressor functions while providing mutant p53 (mut_p53) with oncogenic features (gain-of-function). Loss of p53 tumour suppressor functions alters the transcription of both protein-coding and non-protein-coding genes. Gain-of-function of mut_p53 triggers modification in gene expression as well; however, the impact of mut_p53 on the transcription of the non-protein-coding genes and whether these non-protein-coding genes affect oncogenic properties of cancer cell lines are not fully explored. In this study, we suggested that LINC01605 (also known as lincDUSP) is a long non-coding RNA regulated by mut_p53 and proved that mut_p53 directly regulates LINC01605 by binding to an enhancer region downstream of the LINC01605 locus. We also showed that the loss or downregulation of LINC01605 impairs cell migration in a breast cancer cell line. Eventually, by performing a combined analysis of RNA-seq data generated in mut_TP53-silenced and LINC01605 knockout cells, we showed that LINC01605 and mut_p53 share common gene pathways. Overall, our findings underline the importance of ncRNAs in the mut_p53 network in breast and ovarian cancer cell lines and in particular the importance of LINC01605 in mut_p53 pro-migratory pathways.

Exploring the Role of Fallopian Ciliated Cells in the Pathogenesis of High-Grade Serous Ovarian Cancer.

High-grade serous epithelial ovarian cancer (HGSOC) is the fifth leading cause of cancer death in women and the first among gynecological malignancies. Despite an initial response to standard chemotherapy, most HGSOC patients relapse. To improve treatment options, we must continue investigating tumor biology. Tumor characteristics (e.g., risk factors and epidemiology) are valuable clues to accomplish this task. The two most frequent risk factors for HGSOC are the lifetime number of ovulations, which is associated with increased oxidative stress in the pelvic area caused by ovulation fluid, and a positive family history due to genetic factors. In the attempt to identify novel genetic factors (i.e., genes) associated with HGSOC, we observed that several genes in linkage with HGSOC are expressed in the ciliated cells of the fallopian tube. This finding made us hypothesize that ciliated cells, despite not being the cell of origin for HGSOC, may take part in HGSOC tumor initiation. Specifically, malfunction of the ciliary beat impairs the laminar fluid flow above the fallopian tube epithelia, thus likely reducing the clearance of oxidative stress caused by follicular fluid. Herein, we review the up-to-date findings dealing with HGSOC predisposition with the hypothesis that fallopian ciliated cells take part in HGSOC onset. Finally, we review the up-to-date literature concerning genes that are located in genomic loci associated with epithelial ovarian cancer (EOC) predisposition that are expressed by the fallopian ciliated cells.

Critical lysine residues within the overlooked N-terminal domain of human APE1 regulate its biological functions.

Apurinic/apyrimidinic endonuclease 1 (APE1), an essential protein in mammals, is involved in base excision DNA repair (BER) and in regulation of gene expression, acting as a redox co-activator of several transcription factors. Recent findings highlight a novel role for APE1 in RNA metabolism, which is modulated by nucleophosmin (NPM1). The results reported in this article show that five lysine residues (K24, K25, K27, K31 and K32), located in the APE1 N-terminal unstructured domain, are involved in the interaction of APE1 with both RNA and NPM1, thus supporting a competitive binding mechanism. Data from kinetic experiments demonstrate that the APE1 N-terminal domain also serves as a device for fine regulation of protein catalytic activity on abasic DNA. Interestingly, some of these critical lysine residues undergo acetylation in vivo. These results suggest that protein-protein interactions and/or post-translational modifications involving APE1 N-terminal domain may play important in vivo roles, in better coordinating and fine-tuning protein BER activity and function on RNA metabolism.

High-performance metabolic marker assessment in breast cancer tissue by mass spectrometry.

BACKGROUND: The identification of reliable markers for diagnosis of breast cancer has been thoroughly addressed by metabolic profiling using nuclear magnetic resonance (NMR) spectroscopy or imaging. Several clear diagnostic indicators have emerged using either in vitro analysis of tissue extracts, ex vivo analysis of biopsies or in vivo direct spectral observations. Most of the breast cancer characteristic metabolites could be assayed by mass spectrometry (MS) to exploit the superior sensitivity of this technique and therefore reduce the traumatic impact of current biopsy procedures. METHODS: Following extraction, aqueous metabolite mixtures were obtained that were submitted to liquid-chromatography, electrospray-ionization, mass spectrometry (LC/ESI-MS) analysis to estimate the content of choline (Cho) and its phosphorylated derivatives, phosphocholine (PCho) and glycerophosphocholine (GPCho). The determinations were performed using 10 samples from breast tissue biopsies, surgical specimens and one single sample of a hepatic metastasis. In addition, some measurements were also repeated using high-resolution ¹H NMR spectroscopy to complement the mass spectrometry results. RESULTS: The contents of Cho, PCho and GPCho in breast tissue extracts were estimated by LC/ESI-MS based on standard compound calibration curves. Sharply increased ratios of phosphorylated-to-unphosphorylated metabolites, PCho/ Cho and (PCho+GPCho)/Cho, were observed in all tumor samples, although without discrimination between benign and malignant lesions, contrary to samples from healthy individuals and from those with fibrocystic disease. CONCLUSIONS: The assessment of breast cancer markers by LC/ESI-MS is feasible and diagnostically valuable. In addition to high sensitivity, the approach also shows a resolution advantage for assaying choline derivatives compared to NMR, and could complement the latter.

A proteomic approach to the bilirubin-induced toxicity in neuronal cells reveals a protective function of DJ-1 protein.

Unconjugated bilirubin (UCB) is a powerful antioxidant and a modulator of cell growth through the interaction with several signal transduction pathways. Although newborns develop a physiological jaundice, in case of severe hyperbilirubinemia UCB may become neurotoxic causing severe long-term neuronal damages, also known as bilirubin encephalopathy. To investigate the mechanisms of UCB-induced neuronal toxicity, we used the human neuroblastoma cell line SH-SY5Y as an in vitro model system. We verified that UCB caused cell death, in part due to oxidative stress, which leads to DNA damage and cell growth reduction. The mechanisms of cytotoxicity and cell adaptation to UCB were studied through a proteomic approach that identified differentially expressed proteins involved in cell proliferation, intracellular trafficking, protein degradation and oxidative stress response. In particular, the results indicated that cells exposed to UCB undertake an adaptive response that involves DJ-1, a multifunctional neuroprotective protein, crucial for cellular oxidative stress homeostasis. This study sheds light on the mechanisms of bilirubin-induced neurotoxicity and might help to design a strategy to prevent or ameliorate the neuronal damages leading to bilirubin encephalopathy.

Genome-wide analysis and proteomic studies reveal APE1/Ref-1 multifunctional role in mammalian cells.

Apurinic apyrimidinic endonuclease/redox effector factor 1 (APE1/Ref-1) protects cells from oxidative stress by acting as a central enzyme in base excision repair pathways of DNA lesions and through its independent activity as a redox transcriptional co-activator. Dysregulation of this protein has been associated with cancer development. At present, contrasting data have been published regarding the biological relevance of the two functions as well as the molecular mechanisms involved. Here, we combined both mRNA expression profiling and proteomic analysis to determine the molecular changes associated with APE1 loss-of-expression induced by siRNA technology. This approach identified a role of APE1 in cell growth, apoptosis, intracellular redox state, mitochondrial function, and cytoskeletal structure. Overall, our data show that APE1 acts as a hub in coordinating different and vital functions in mammalian cells, highlighting the molecular determinants of the multifunctional nature of APE1 protein.

Sleeping beauty genetic screen identifies miR-23b::BTBD7 gene interaction as crucial for colorectal cancer metastasis.

BACKGROUND: Metastatic colorectal cancer (CRC) remains a deadly disease. Identifying locally advanced CRC patients with high risk of developing metastasis and improving outcome of metastatic CRC patients require discovering master regulators of metastasis. In this context, the non-coding part of the human genome is still largely unexplored. METHODS: To interrogate the non-coding part of the human genome and disclose regulators of CRC metastasis, we combined a transposon-based forward genetic screen with a novel in vitro assay, which forces cells to grow deprived of cell-substrate and cell-cell contacts (i.e. forced single cell suspension assay - fSCS). FINDINGS: We proved that fSCS selects CRC cells with mesenchymal and pro-metastatic traits. Moreover, we found that the transposon insertions conferred CRC cells resistance to fSCS and thus metastatic advantage. Among the retrieved transposon insertions, we demonstrated that the one located in the 3'UTR of BTBD7 disrupts miR-23b::BTBD7 interaction and contributes to pro-metastatic traits. In addition, miR-23b and BTBD7 correlate with CRC metastasis both in preclinical experiments and in clinical samples. INTERPRETATION: fSCS is a simple and scalable in vitro assay to investigate pro-metastatic traits and transposon-based genetic screens can interrogate the non-coding part of the human genome (e.g. miRNA::target interactions). Finally, both Btbd7 and miR-23b represent promising prognostic biomarkers and therapeutic targets in CRC. FUND: This work was supported by Marie Curie Actions (CIG n. 303877) and Friuli Venezia Giulia region (Grant Agreement n°245574), Italian Association for Cancer Research (AIRC, MFAG n°13589), Italian Ministry of Health (GR-2010-2319387 and PE-2016-02361040) and 5x1000 to CRO Aviano.

Bilirubin-induced cell toxicity involves PTEN activation through an APE1/Ref-1-dependent pathway.

Unconjugated bilirubin (UCB) is the major degradation product of the heme catabolism. A growing body of evidences suggests that UCB plays major biological effects by inhibiting cell proliferation in cancer cell lines and eliciting cell toxicity particularly in neurons and glial cells. Early molecular events responsible for bilirubin-induced cytotoxicity remain poorly understood. Using HeLa cells and mouse embryonic fibroblasts, we found that UCB at a concentration of free pigment (Bf) of 80 nM induced oxidative stress, promoting a significant increase in intracellular reactive oxygen species (ROS) and a decreased cell survival (by the MTT test). The ROS increase activated the antioxidant cell response through APE1/Ref-1, a master redox regulator in eukaryotic cells. Activation of APE1/Ref-1 was followed by a concomitant activation of Egr-1 transcription factor and by an upregulation of PTEN tumor suppressor, an Egr-1 target gene, leading to inhibition of cell growth. Blocking ROS generation with N-acetylcysteine pretreatment, restored cell survival, limited the upregulation of PTEN in response to UCB, and prevented the inhibition of cell proliferation. HeLa cells transfected with mutants of the PTEN promoter or silenced with APE1/Ref-1 small interference RNA confirmed that UCB modulates a signaling pathway involving APE1/Ref-1, Egr-1, and PTEN. These findings describe a new molecular pathway involved in the cytotoxic effects of UCB.

BNC2 is a putative tumor suppressor gene in high-grade serous ovarian carcinoma and impacts cell survival after oxidative stress.

Rs3814113 is the single-nucleotide polymorphism (SNP) showing the strongest association with high-grade serous ovarian carcinoma (HGSOC) incidence and is located in an intergenic region about 44 kb downstream of basonuclin 2 (BNC2) gene. Lifetime number of ovulations is associated with increased risk to develop HGSOC, probably because of cell damage of extrauterine Müllerian epithelium by ovulation-induced oxidative stress. However, the impact of low-penetrance HGSOC risk alleles (e.g. rs3814113) on the damage induced by oxidative stress remains unclear. Therefore, the purpose of this study was to investigate whether rs3814113 genetic interval regulates BNC2 expression and whether BNC2 expression levels impact on cell survival after oxidative stress. To do this, we analyzed gene expression levels of BNC2 first in HGSOC data sets and then in an isogenic cell line that we engineered to carry a 5 kb deletion around rs3814113. Finally, we silenced BNC2 and measured surviving cells after hydrogen peroxide (H2O2) treatment to simulate oxidative stress after ovulation. In this paper, we describe that BNC2 expression levels are reduced in HGSOC samples compared with control samples, and that BNC2 expression levels decrease following oxidative stress and ovulation in vitro and in vivo, respectively. Moreover, deletion of 5 kb surrounding rs3814113 decreases BNC2 expression levels in an isogenic cell line, and silencing of BNC2 expression levels increases cell survival after H2O2 treatment. Altogether, our findings suggest that the intergenic region located around rs3814113 regulates BNC2 expression, which in turn affects cell survival after oxidative stress response. Indeed, HGSOC samples present lower BNC2 expression levels that probably, in the initial phases of oncogenic transformation, conferred resistance to oxidative stress and ultimately reduced the clearance of cells with oxidative-induced damages.

Effects of histone acetylation on sodium iodide symporter promoter and expression of thyroid-specific transcription factors.

Inhibitors of histone deacetylases (HDACs) activate the sodium iodide symporter (NIS) expression in thyroid tumor cells. In this study, mechanisms accounting for these effects were investigated. Various human thyroid tumor cell lines (ARO, BCPAP, FRO, TPC-1) were treated with the HDAC inhibitors Na butyrate (NaB) and tricostatin A (TSA), and the effects on the expression of NIS and several thyroid-specific transcription factors together with the activity of NIS promoter were evaluated. TSA and NaB increased NIS mRNA levels in all cell lines. Among thyroid-specific transcription factors, only expression of PAX8 in ARO cells was increased. Down-regulation of thyroid-specific transcription factor-1 expression was observed in BCPAP and TPC-1 cell lines. Thyroid-specific transcription factor-2 mRNA was reduced in FRO, BCPAP, and TPC-1 cells. Histone acetylation had no significant effects on HEX expression. Altogether, these data indicate that the increase of NIS expression is not mediated by modification of expression of thyroid-specific transcription factors. Accordingly, in transfection experiments performed in the HeLa cell line (which does not express thyroid-specific transcription factors), treatment with TSA and NaB increased NIS promoter activity. Stimulation of NIS promoter activity was also obtained by overexpressing histone acetylating proteins pCAF and p300 in HeLa cells. Conversely, overexpression of the HDAC 1 enzyme inhibited basal activity of the NIS promoter. Effects of TSA and NaB on NIS expression were also evaluated in nonthyroid cell lines MCF-7, Hep-G2, and SAOS-2. In all cell lines TSA and NaB greatly increased NIS mRNA levels. We concluded that control of NIS expression by inhibition of HDAC appears not to be mediated by cell-specific mechanisms, suggesting it as a potential strategy to induce radioiodine sensitivity in different human tumors.

Extracellular nucleotides activate Runx2 in the osteoblast-like HOBIT cell line: a possible molecular link between mechanical stress and osteoblasts' response.

Dynamic mechanical loading increases bone density and strength and promotes osteoblast proliferation, differentiation and matrix production, by acting at the gene expression level. Molecular mechanisms through which mechanical forces are conversed into biochemical signalling in bone are still poorly understood. A growing body of evidence point to extracellular nucleotides (i.e., ATP and UTP) as soluble factors released in response to mechanical stimulation in different cell systems. Runx2, a fundamental transcription factor involved in controlling osteoblasts differentiation, has been recently identified as a target of mechanical signals in osteoblastic cells. We tested the hypothesis that these extracellular nucleotides could be able to activate Runx2 in the human osteoblastic HOBIT cell line. We found that ATP and UTP treatments, as well as hypotonic stress, promote a significant stimulation of Runx2 DNA-binding activity via a mechanism involving PKC and distinct mitogen-activated protein kinase cascades. In fact, by using the specific inhibitors SB203580 (specific for p38 MAPK) and PD98059 (specific for ERK-1/2 MAPK), we found that ERK-1/2, but not p38, play a major role in Runx2 activation. On the contrary, another important transcription factor, i.e., Egr-1, that we previously demonstrated being activated by extracellular released nucleotides in this osteoblastic cell line, demonstrated to be susceptible to both ERK-1/2 and p38 kinases. These data suggest a possible differential involvement of these two transcription factors in response to extracellularly released nucleotides. The biological relevance of our data is strengthened by the finding that a target gene of Runx2, i.e., Galectin-3, is up-regulated by ATP stimulation of HOBIT cells with a comparable kinetic of that found for Runx2. Since it is known that osteocytes are the primary mechanosensory cells of the bone, we hypothesize that they may signal mechanical loading to osteoblasts through release of extracellular nucleotides. Altogether, these data suggest a molecular mechanism explaining the purinoreceptors-mediated activation of specific gene expression in osteoblasts and could be of help in setting up new pharmacological strategies for the intervention in bone loss pathologies.

Overoxidation of peroxiredoxins as an immediate and sensitive marker of oxidative stress in HepG2 cells and its application to the redox effects induced by ischemia/reperfusion in human liver.

Oxidative stress is a major pathogenetic event occurring in several liver disorders and is a major cause of liver damage due to Ischemia/Reperfusion (I/R) during liver transplantation. While several markers of chronic oxidative stress are well known, early protein targets of oxidative injury are not well defined. In order to identify these proteins, we used a differential proteomics approach to HepG2 human liver cells treated for 10 min with 500 microM H(2)O(2). This dose was sufficient to induce a slight decrease of total GSH and total protein thiol content without affecting cell viability. By performing Differential Proteomic analysis, by means of two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry, we identified four proteins which resulted sensitive to H(2)O(2) treatment. The main changes were due to post-translational modifications of native polypeptides. Three of these proteins belong to the Peroxiredoxin family of hydroperoxide scavengers, namely PrxI, PrxII and PrxVI, that showed changes in their pI as result of overoxidation. Mass mapping experiments demonstrated the specific modification of peroxiredoxins active site thiol into sulphinic and/or sulphonic acid, thus explaining the increase in negative charge measured for these proteins. The oxidation kinetic of all peroxiredoxins was extremely rapid and sensitive, occurring at H(2)O(2) doses unable to affect the common markers of cellular oxidative stress. Recovery experiments demonstrated a quite different behaviour between 1-Cys and 2-Cys containing Prxs as their retroreduction features is concerned, thus suggesting a functional difference between different class of Prxs. The in vivo relevance of our study is demonstrated by the finding that overoxidation of PrxI occurs during I/R upon liver transplantation and is dependent on the time of warm ischemia. Our present data could be of relevance in setting up more standardized procedures to preserve organs for transplantations.

Control of phosphatase and tensin homolog (PTEN) gene expression in normal and neoplastic thyroid cells.

The lipid phosphatase, phosphatase and tensin homolog (PTEN), is a key element in controlling cell growth and survival and has a well established role as tumor suppressor protein in many neoplasia. Several data indicate that silencing of PTEN gene expression may be relevant in follicular thyroid cell transformation. Thus, in the present study regulation of PTEN gene expression in thyroid cells was investigated. Cotransfection experiments indicated that in normal FRTL-5 rat thyroid cells, PTEN promoter activity was increased by overexpression of the transcription factor early growth response protein-1 (Egr-1). Moreover, Western blot experiments indicated that when Egr-1 expression was up-regulated by treating FRTL-5 cells with H2O2, an increase in PTEN expression was also observed. TSH induced opposite modifications on PTEN and Egr-1 protein levels. Moreover, acute or chronic TSH stimulation determined distinct effects. In fact, acute TSH stimulation (30 and 60 min) induced a decrease in PTEN, but an increase in Egr-1 protein levels. These effects were cAMP dependent; in fact, they were mimicked by forskolin. A chronic TSH treatment (5 d) stimulated PTEN protein expression, whereas Egr-1 protein was down-regulated. In contrast to normal thyroid cells, when the thyroid tumor cell lines ARO and BCPAP were exposed to H2O2, neither Egr-1 nor PTEN protein levels were increased. Acute stimulation of ARO and BCPAP cells with forskolin increased Egr-1, but not PTEN, protein levels. Therefore, thyroid tumor cell lines show alteration of PTEN gene expression regulation. RT-PCR experiments performed on human thyroid tumors showed that the absence of Egr-1 mRNA is always paralleled by the absence of PTEN mRNA. Thus, modification of the Egr-1-dependent mechanisms may play a role in the silencing of PTEN gene expression occurring during thyroid cell transformation.

The importance of redox state in liver damage.

Oxidative stress is a major pathogenetic event occurring in several liver disorders ranging from metabolic to proliferative ones, and is a major cause of liver damage due to Ischemia/Reperfusion (I/R) during liver transplantation. The main sources of ROS are represented by mitochondria and cytocrome P450 enzymes in the hepatocyte, by Kupffer cells and by neutrophils. Cells are provided with efficient molecular strategies to strictly control the intracellular ROS level and to maintain the balance between oxidant and antioxidant molecules. A cellular oxidative stress condition is determined by an imbalance between the generation of ROS and the antioxidant defense capacity of the cell and can affect major cellular components including lipids, proteins and DNA. Proteins are very important signposts of cellular redox status and through their structure/function modulation, ROS can also influence gene expression profile by affecting intracellular signal transduction pathways. While several enzymatic (such as superoxide dismutase, catalase, glutathione peroxidase) and non enzymatic (such as 4-hydroxynonenal, decrease of glutathione, vitamin E, vitamin C, malondialdehyde) markers of chronic oxidative stress in liver are well known, early protein targets of oxidative injury are yet not well defined. Identification of these markers will enable early detection of liver diseases and will allow monitoring the degree of liver damage, the response to pharmacological therapies and the development of new therapeutic approaches. In the new era of molecular medicine, new proteomics methodologies promise to establish a relationship between pathological hallmarks of disease and protein structural and functional abnormalities in liver disease, thus allowing a better understanding and a more rational therapy on these disorders.

Osteoblastic cell secretome: a novel role for progranulin during risedronate treatment.

It is well established that osteoblasts, the key cells involved in bone formation during development and in adult life, secrete a number of glycoproteins harboring autocrine and paracrine functions. Thus, investigating the osteoblastic secretome could yield important information for the pathophysiology of bone. In the present study, we characterized for the first time the secretome of human Hobit osteoblastic cells. We discovered that the secretome comprised 89 protein species including the powerful growth factor progranulin. Recombinant human progranulin (6nM) induced phosphorylation of mitogen-activated protein kinase in both Hobit and osteocytic cells and induced cell proliferation and survival. Notably, risedronate, a nitrogen-containing bisphosphonate widely used in the treatment of osteoporosis, induced the expression and secretion of progranulin in the Hobit secretome. In addition, our proteomic study of the Hobit secretome revealed that risedronate induced the expression of ERp57, HSP60 and HSC70, three proteins already shown to be associated with the prevention of bone loss in osteoporosis. Collectively, our findings unveil novel targets of risedronate-evoked biological effects on osteoblast-like cells and further our understanding of the mechanisms of action of this currently used compound.

Specific inhibition of the redox activity of ape1/ref-1 by e3330 blocks tnf-α-induced activation of IL-8 production in liver cancer cell lines.

APE1/Ref-1 is a main regulator of cellular response to oxidative stress via DNA-repair function and co-activating activity on the NF-κB transcription factor. APE1 is central in controlling the oxidative stress-based inflammatory processes through modulation of cytokines expression and its overexpression is responsible for the onset of chemoresistance in different tumors including hepatic cancer. We examined the functional role of APE1 overexpression during hepatic cell damage related to fatty acid accumulation and the role of the redox function of APE1 in the inflammatory process. HepG2 cells were stably transfected with functional and non-functional APE1 encoding plasmids and the protective effect of APE1 overexpression toward genotoxic compounds or FAs accumulation, was tested. JHH6 cells were stimulated with TNF-α in the presence or absence of E3330, an APE1 redox inhibitor. IL-8 promoter activity was assessed by a luciferase reporter assay, gene expression by Real-Time PCR and cytokines (IL-6, IL-8, IL-12) levels measured by ELISA. APE1 over-expression did not prevent cytotoxicity induced by lipid accumulation. E3330 treatment prevented the functional activation of NF-κB via the alteration of APE1 subcellular trafficking and reduced IL-6 and IL-8 expression induced by TNF-α and FAs accumulation through blockage of the redox-mediated activation of NF-κB. APE1 overexpression observed in hepatic cancer cells may reflect an adaptive response to cell damage and may be responsible for further cell resistance to chemotherapy and for the onset of inflammatory response. The efficacy of the inhibition of APE1 redox activity in blocking TNF-α and FAs induced inflammatory response opens new perspectives for treatment of inflammatory-based liver diseases.

APE1/Ref-1 interacts with NPM1 within nucleoli and plays a role in the rRNA quality control process.

APE1/Ref-1 (hereafter, APE1), a DNA repair enzyme and a transcriptional coactivator, is a vital protein in mammals. Its role in controlling cell growth and the molecular mechanisms that fine-tune its different cellular functions are still not known. By an unbiased proteomic approach, we have identified and characterized several novel APE1 partners which, unexpectedly, include a number of proteins involved in ribosome biogenesis and RNA processing. In particular, a novel interaction between nucleophosmin (NPM1) and APE1 was characterized. We observed that the 33 N-terminal residues of APE1 are required for stable interaction with the NPM1 oligomerization domain. As a consequence of the interaction with NPM1 and RNA, APE1 is localized within the nucleolus and this localization depends on cell cycle and active rRNA transcription. NPM1 stimulates APE1 endonuclease activity on abasic double-stranded DNA (dsDNA) but decreases APE1 endonuclease activity on abasic single-stranded RNA (ssRNA) by masking the N-terminal region of APE1 required for stable RNA binding. In APE1-knocked-down cells, pre-rRNA synthesis and rRNA processing were not affected but inability to remove 8-hydroxyguanine-containing rRNA upon oxidative stress, impaired translation, lower intracellular protein content, and decreased cell growth rate were found. Our data demonstrate that APE1 affects cell growth by directly acting on RNA quality control mechanisms, thus affecting gene expression through posttranscriptional mechanisms.

Subcellular localization of APE1/Ref-1 in human hepatocellular carcinoma: possible prognostic significance.

APE1/Ref-1, normally localized in the nucleus, is a regulator of the cellular response to oxidative stress. Cytoplasmic localization has been observed in several tumors and correlates with a poor prognosis. Because no data are available on liver tumors, we investigated APE1/Ref-1 subcellular localization and its correlation with survival in 47 consecutive patients undergoing hepatocellular carcinoma (HCC) resection. APE1/Ref-1 expression was determined by immunohistochemistry in HCC and surrounding liver cirrhosis (SLC) and compared with normal liver tissue. Survival probability was evaluated using Kaplan-Meier curves (log-rank test) and Cox regression. Cytoplasmic expression of APE1/Ref-1 was significantly higher in HCC than in SLC (P = 0.00001); normal liver showed only nuclear reactivity. Patients with poorly differentiated HCC showed a cytoplasmic expression three times higher than those with well-differentiated HCC (P = 0.03). Cytoplasmic localization was associated with a median survival time shorter than those with negative cytoplasmic reactivity (0.44 compared with 1.64 years, P = 0.003), and multivariable analysis confirmed that cytoplasmic APE1/Ref-1 localization is a predictor of survival. Cytoplasmic expression of APE1/Ref-1 is increased in HCC and is associated with a lower degree of differentiation and a shorter survival time, pointing to the use of the cytoplasmic localization of APE1/Ref-1 as a prognostic marker for HCC.

Proteomic analysis of liver tissues subjected to early ischemia/reperfusion injury during human orthotopic liver transplantation.

Knowledge of early molecular events occurring upon ischemia/reperfusion (I/R) during liver transplantation (LT) is of great importance to improve the therapeutic intervention of surgical treatment. However, nowadays, few data are available on early protein targets of I/R injury. To identify these proteins, we used a differential proteomics approach in the characterization human liver biopsies during I/R upon LT. Analyses were performed on nine donor livers during LT. By using 2-DE and MALDI-TOF MS, we identified 36 proteins which resulted significantly altered upon I/R injury. The majority of these proteins are functionally involved in lipid and energy metabolism, in different metabolic pathways, in redox signalling and in oxidative-stress response. Our data represent the first global approach in the study of I/R injury in liver.