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Simple Summary Renal cell carcinoma (RCC) is often late diagnosed at an advanced stage, worsening the prognosis of the patients. Thus, an early marker is desirable. This paper presents an innovative combined approach useful to identify, for the first time, the presence of LOX-1 protein within the urine of clear cell RCC patients. The LOX-1 protein is related to metabolic disorder-associated carcinogenesis and is shown to be quantitatively correlated to tumor grade and stage. The analysis of volatile compounds released by urine shows the diagnostic potentialities of volatilome and indicates that at least one volatile compound is correlated with both LOX-1 and cancer. In this work, we propose the potential use of a noninvasive approach that enables an early, routine ccRCC diagnosis and leads to a better management of the patients. Abstract Renal cell carcinoma (RCC) represents around 3% of all cancers, within which clear cell RCC (ccRCC) are the most common type (70–75%). The RCC disease regularly progresses asymptomatically and upon presentation is recurrently metastatic, therefore, an early method of detection is necessary. The identification of one or more specific biomarkers measurable in biofluids (i.e., urine) by combined approaches could surely be appropriate for this kind of cancer, especially due to easy obtainability by noninvasive method. OLR1 is a metabolic gene that encodes for the Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), implicated in inflammation, atherosclerosis, ROS, and metabolic disorder-associated carcinogenesis. Specifically, LOX-1 is clearly involved in tumor insurgence and progression of different human cancers. This work reports for the first time the presence of LOX-1 protein in ccRCC urine and its peculiar distribution in tumoral tissues. The urine samples headspace has also been analyzed for the presence of the volatile compounds (VOCs) by SPME-GC/MS and gas sensor array. In particular, it was found by GC/MS analysis that 2-Cyclohexen-1-one,3-methyl-6-(1-methylethyl)- correlates with LOX-1 concentration in urine. The combined approach of VOCs analysis and protein quantification could lead to promising results in terms of diagnostic and prognostic potential for ccRCC tumors.

Renal cell carcinoma (RCC) represents around 3% of all cancers, within which clear cell RCC (ccRCC) are the most common type (70–75%). The RCC disease regularly progresses asymptomatically and upon presentation is recurrently metastatic, so an early method of detection is necessary. The identification of one or more spe-cific biomarkers measurable in biofluids (i.e urine) by combined approaches could surely be appropriate for this kind of cancer, especially due to easy obtainability by non invasive method. OLR1 is a metabolic gene that encodes for the Lectin-like oxidized low-density lipoprotein re-ceptor-1 (LOX-1), implicated in inflammation, atherosclerosis, ROS and metabolic disor-der-associated carcinogenesis. Specifically, LOX-1 is clearly involved in tumor insurgence and progression of different human cancers. This work reports for the first time the presence of LOX-1 protein in ccRCC urine and its peculiar distribution in tumoral tissues. In parallel, urine samples headspace has been analyzed for the presence of the volatile compounds (VOCs) by SPME-GC/MS and gas sensor array. In particular, it was found by GC/MS analysis that 2-Cyclohexen-1-one,3-methyl-6-(1-methylethyl)- correlates with LOX-1 concentration in urine. Thus, the combined approach of VOCs analysis and protein quantification could led to promis-ing results in terms of diagnostic and prognostic potential for ccRCC tumor.

Osteoarthritis (OA) is the most common joint disease characterized by destruction of articular cartilage. OA-induced cartilage degeneration causes inflammation, oxidative stress and the hypertrophic shift of quiescent chondrocytes. Clusterin (CLU) is a ubiquitous glycoprotein implicated in many cellular processes and its upregulation has been recently reported in OA cartilage. However, the specific role of CLU in OA cartilage injury has not been investigated yet. We analyzed CLU expression in human articular cartilage in vivo and in cartilage-derived chondrocytes in vitro. CLU knockdown in OA chondrocytes was also performed and its effect on proliferation, hypertrophic phenotype, apoptosis, inflammation and oxidative stress was investigated. CLU expression was upregulated in human OA cartilage and in cultured OA cartilage-derived chondrocytes compared with control group. CLU knockdown reduced cell proliferation and increased hypertrophic phenotype as well as apoptotic death. CLU-silenced OA chondrocytes showed higher MMP13 and COL10A1 as well as greater TNF-α, Nox4 and ROS levels. Our results indicate a possible cytoprotective role of CLU in OA chondrocytes promoting cell survival by its anti-apoptotic, anti-inflammatory and antioxidant properties and counteracting the hypertrophic phenotypic shift. Further studies are needed to deepen the role of CLU in order to identify a new potential therapeutic target for OA.

Recurrence and metastasis are the primary causes of mortality in patients with colorectal cancer (CRC), and therefore effective tools to reduce morbidity and mortality of CRC patients are necessary. LOX-1, the ox-LDL receptor, is strongly involved in inflammation, obesity, and atherosclerosis, and several studies have assessed its role in the carcinogenesis process linking ROS, metabolic disorders and cancer. We have already demonstrated in vitro that LOX-1 expression correlates to the aggressiveness of human colon cancer and its downregulation weakens the tumoral phenotype, indicating its potential function as a biomarker and a target in CRC therapy. Here we further investigate in vivo the role of LOX-1 in colon tumorigenesis by xenografting procedures, injecting nude mice both subcutaneously and intravenously with human high grade metastatic colorectal cancer cells, DLD-1, in which LOX-1 expression has been downregulated by shRNA (LOX-1RNAi cells). Histopathological and immunohistochemical evaluations have been performed on xenograft tumors. The experiments have been complemented by the analysis of the volatile compounds (VOCs) collected from the cages of injected mice and analyzed by gas-chromatography and gas sensors. After intravenous injection of LOX-1RNAi cells, we found that LOX-1 silencing influences both the engraftment of the tumor and the metastasis development, acting by angiogenesis. For the first time, we have observed that LOX-1 inhibition significantly prevents metastasis formation in injected mice and, at the same time, induces a downregulation of VEGF-A165, HIF-1α, and β-catenin whose expression is involved in cell migration and metastasis, and a variation of histone H4 acetylation pattern suggesting also a role of LOX-1 in regulating gene transcription. The analysis of the volatile compounds (VOCs) collected from the cages of injected mice has evidenced a specific profile in those xenograft mice in which metastasis originates. These findings underline the role of LOX-1 as a potential target for inhibition of tumor progression and metastasis, enhancing current therapeutic strategies against colorectal cancer.

Background Targeting new molecular pathways leading to Osteoporosis (OP) and Osteoarthritis (OA) is a hot topic for drug discovery. Clusterin (CLU) is a glycoprotein involved in inflammation, proliferation, cell death, neoplastic disease, Alzheimer disease and aging. The present study focuses on the expression and the role of CLU in influencing the decrease of muscle mass and fiber senescence in OP-OA condition. Methods Vastus lateralis muscle biopsies were collected from 20 women with OP undergoing surgery for fragility hip fracture and 20 women undergoing arthroplasty for hip osteoarthritis. Results We found an overexpression of CLU in degenerated fibers in OP closely correlated with interleukin 6 (IL6) and histone H4 acetylation level. Conversely, in OA muscle tissues we observed a weak expression of CLU but no nuclear histone H4 acetylation. Ex vivo studies on isolated human myoblasts confirmed CLU overexpression in OP as compared to OA (p

// Sabina Pucci 1, * , Maria Jose Zonetti 1 , Tommaso Fisco 1 , Chiara Polidoro 1 , Gianfranco Bocchinfuso 2 , Antonio Palleschi 2 , Giuseppe Novelli 1 , Luigi G. Spagnoli 1 , Paola Mazzarelli 1, * 1 Department of Biomedicine and Prevention, Tor Vergata University of Rome, Rome, Italy 2 Department of Chemical Sciences and Technologies, Tor Vergata University of Rome, Rome, Italy * These authors have contributed equally to this work Correspondence to: Luigi G. Spagnoli, e-mail: spagnoli@alleanzacontroilcancro.it Sabina Pucci, e-mail: sabina.pucci@uniroma2.it Keywords: breast cancer, carnitine palmitoyl transferase-1A, HDAC inhibitor, tumor metabolism, tumor specific target Received: July 28, 2015 Accepted: January 01, 2016 Published: January 21, 2016 ABSTRACT Transcriptional mechanisms epigenetically-regulated in tumoral tissues point out new targets for anti-cancer therapies. Carnitine palmitoyl transferase I (CPT1) is the rate-limiting enzyme in the transport of long-chain fatty acids for β-oxidation. Here we identified the tumor specific nuclear CPT1A as a product of the transcript variant 2, that doesn’t retain the classical transferase activity and is strongly involved in the epigenetic regulation of cancer pro-survival, cell death escaping and tumor invasion pathways. The knockdown of CPT1A variant 2 by small interfering RNAs (siRNAs), was sufficient to induce apoptosis in MCF-7, SK-BR3 and MDA-MB-231 breast cancer cells. The cell death triggered by CPT1A silencing correlated with reduction of HDAC activity and histone hyperacetylation. Docking experiments and molecular dynamics simulations confirmed an high binding affinity of the variant 2 for HDAC1. The CPT1A silenced cells showed an up-regulated transcription of pro-apoptotic genes (BAD, CASP9, COL18A1) and down-modulation of invasion and metastasis related-genes (TIMP-1, PDGF-A, SERPINB2). These findings provide evidence of the CPT1 variant 2 involvement in breast cancer survival, cell death escape and invasion. Thus, we propose nuclear CPT1A as a striking tumor specific target for anticancer therapeutics, more selective and effective as compared with the well-known HDAC inhibitors.

The identification of new predictive biomarkers and therapeutic target for tailored therapy in breast cancer onset and progression is an interesting challenge. OLR-1 gene encodes the cell membrane receptor LOX-1 (lectin-like oxidized low-density lipoprotein receptor). We have recently identified a novel alternative OLR-1 isoform, LOX-1Δ4, whose expression and functions are still not clarified. In the present paper, we demonstrated that LOX-1 is overexpressed in 70% of human breast cancer (n = 47) and positively correlated to the tumor stage and grade (p

Myotonic Dystrophy type 1 (DM1) is a multisystemic disease, autosomal dominant, caused by a CTG repeat expansion in DMPK gene. We assessed the appropriateness of patient-specific induced pluripotent stem cell-derived cardiomyocytes (CMs) as a model to recapitulate some aspects of the pathogenetic mechanism involving cardiac manifestations in DM1 patients. Once obtained in vitro, CMs have been characterized for their morphology and their functionality. CMs DM1 show intranuclear foci and transcript markers abnormally spliced respect to WT ones, as well as several irregularities in nuclear morphology, probably caused by an unbalanced lamin A/C ratio. Electrophysiological characterization evidences an abnormal profile only in CMs DM1 such that the administration of antiarrythmic drugs to these cells highlights even more the functional defect linked to the disease. Finally, Atomic Force Measurements reveal differences in the biomechanical behaviour of CMs DM1, in terms of frequencies and synchronicity of the beats. Altogether the complex phenotype described in this work, strongly reproduces some aspects of the human DM1 cardiac phenotype. Therefore, the present study provides an in vitro model suggesting novel insights into the mechanisms leading to the development of arrhythmogenesis and dilatative cardiomyopathy to consider when approaching to DM1 patients, especially for the risk assessment of sudden cardiac death (SCD). These data could be also useful in identifying novel biomarkers effective in clinical settings and patient-tailored therapies.

Niemann-Pick type C disease (NPCD) is an autosomal recessive storage disorder, characterized by abnormal sequestration of unesterified cholesterol within the late endo-lysosomal compartment of cells. In the central nervous system, hypoxic insults could result in low-density lipoprotein (LDL) oxidation and Lectin-like oxidized LDL receptor-1 (LOX-1) induction, leading to a pathological hippocampal response, namely, ischemic long-term potentiation (i-LTP). These events may correlate with the progressive neural loss observed in NPCD. To test these hypotheses, hippocampal slices from Wild Type (WT) and NPC1−/− mice were prepared, and field potential in the CA1 region was analyzed during transient oxygen/glucose deprivation (OGD). Moreover, LOX-1 expression was evaluated by RT-qPCR, immunocytochemical, and Western blot analyses before and after an anoxic episode. Our results demonstrate the development of a precocious i-LTP in NPC1−/− mice during OGD application. We also observed a higher expression of LOX-1 transcript and protein in NPC1−/− mice with respect to WT mice; after anoxic damage to LOX-1 expression, a further increase in both NPC1−/− and WT mice was observed, although the protein expression seems to be delayed, suggesting a different kinetic of induction. These data clearly suggest an elevated susceptibility to neurodegeneration in NPC1−/− mice due to oxidative stress. The observed up-regulation of LOX-1 in the hippocampus of NPC1−/− mice may also open a new scenario in which new biomarkers can be identified.

Alternative splicing (AS) is a process in which precursor messenger RNA (pre-mRNA) splicing sites are differentially selected to diversify the protein isoform population. Changes in AS patterns have an essential role in normal development, differentiation and response to physiological stimuli. It is documented that AS can generate both “risk” and “protective” splice variants that can contribute to the pathogenesis of several diseases including atherosclerosis. The main endothelial receptor for oxidized low-density lipoprotein (ox-LDLs) is LOX-1 receptor protein encoded by the OLR1 gene. When OLR1 undergoes AS events, it generates three variants: OLR1, OLR1D4 and LOXIN. The latter lacks exon 5 and two-thirds of the functional domain. Literature data demonstrate a protective role of LOXIN in pathologies correlated with LOX-1 overexpression such as atherosclerosis and tumors. In this review, we summarize recent developments in understanding of OLR1 AS while also highlighting data warranting further investigation of this process as a novel therapeutic target.

The identification of predictive biomarkers for neoadjuvant chemoradiation therapy (CRT) is a current clinical need. The heterodimer Ku70/80 plays a critical role in DNA repair and cell death induction after damage. The aberrant expression and localization of these proteins fail to control DNA repair and apoptosis. sClusterin is the Ku70 partner that sterically inhibits Bax-dependent cell death after damage in some pathologic conditions. This study sought to evaluate the molecular relevance of Ku70-Ku80-Clu as a molecular cluster predicting the response to neoadjuvant CRT in patients with locally advanced rectal cancer (LARC).Patients enrolled in this study underwent preoperative CRT followed by surgical excision. A retrospective study based on individual response, evaluated by computed tomography and diffusion-weighted magnetic resonance imaging, identified responder (56%) and no-responder patients (44%). Ku70/80 and Clu expression were observed in biopsy specimens obtained before and after treatment with neoadjuvant CRT from the same LARC patients. In vitro studies before and after irradiation were also performed on radioresistant (SW480) and radiosensitive (SW620) colorectal cancer cell lines, mimicking sensitive or resistant tumor behavior.We found a conventional nuclear localization of Ku70/80 in pretherapeutic tumor biopsies of responder patients, in agreement with their role in DNA repair and regulating apoptosis. By contrast, in the no-responder population we observed an unconventional overexpression of Ku70 in the cytoplasm (P.001). In this context we also overexpression of sClu in the cytoplasm, which accorded with its role in stabilizing of Bax-Ku70 complex, inhibiting Bax-dependent apoptosis. Strikingly, Ku80 in these tumor tissues was lost (P.005). In vitro testing of colon cancer cells finally confirmed the results observed in tumor biopsy specimens, proving that Ku70/80-Clu deregulation is extensively involved in the resistance mechanism.These results strongly suggest a potential role of these proteins as a new prognostic tool to predict the response to chemoradiation in LARC.

The identification of new biomarkers and targets for tailored therapy in human colorectal cancer (CRC) onset and progression is an interesting challenge. CRC tissue produces an excess of ox-LDL, suggesting a close correlation between lipid dysfunction and malignant transformation. Lectin-like oxidized LDL receptor-1 (LOX-1) is involved in several mechanisms closely linked to tumorigenesis. Here we report a tumor specific LOX-1 overexpression in human colon cancers: LOX-1 results strongly increased in the 72% of carcinomas (P

The up‐regulation of lectin‐like oxidized low‐density lipoprotein receptor‐1 (LOX‐1), encoded by the OLR1 gene, plays a fundamental role in the pathogenesis of atherosclerosis. Moreover, OLR1 polymorphisms were associated with increased susceptibility to acute myocardial infarction (AMI) and coronary artery diseases (CAD). In these pathologies, the identification of therapeutic approaches that can inhibit or reduce LOX‐1 overexpression is crucial. Predictive analysis showed a putative hsa‐miR‐24 binding site in the 3′UTR of OLR1, ‘naturally’ mutated by the presence of the rs1050286 single nucleotide polymorphism (SNP). Luciferase assays revealed that miR‐24 targets OLR1 3′UTR‐G, but not 3′UTR‐A (P < 0.0005). The functional relevance of miR‐24 in regulating the expression of OLR1 was established by overexpressing miR‐24 in human cell lines heterozygous (A/G, HeLa) and homozygous (A/A, HepG2) for rs1050286 SNP. Accordingly, HeLa (A/G), but not HepG2 (A/A), showed a significant down‐regulation of OLR1 both at RNA and protein level. Our results indicate that rs1050286 SNP significantly affects miR‐24 binding affinity to the 3′UTR of OLR1, causing a more efficient post‐transcriptional gene repression in the presence of the G allele. On this basis, we considered that OLR1 rs1050286 SNP may contribute to modify OLR1 susceptibility to AMI and CAD, so ORL1 SNPs screening could help to stratify patients risk.

The influence of the microenvironment through the various steps of cancer progression is signed by different cytokines and growth factors, that could directly affect cell proliferation and survival, either in cancer and stromal cells. In colon cancer progression, the cooperation between hypoxia, IL-6 and VEGF-A165 could regulate the DNA repair capacity of the cell, whose impairment is the first step of colon cancer development. This cooperation redirects the activity of proteins involved in the metabolic shift and cell death, affecting the cell fate. The pathways triggered by micro environmental factors could modulate cancer-related gene transcription, affecting also small non coding mRNA, microRNAs. MicroRNAs have emerged as key post-transcriptional regulators of gene expression, directly involved in human cancers. The present review will focus first on the intertwined connection between cancer microenvironment and aberrant expression of microRNAs which contribute to carcinogenesis. In particular, the epigenetic mechanisms triggered by tissue microenvironment will be discussed, in view of the recent identification of miRNAs able to directly or indirectly modulate the epigenetic machinery (epi-miRNAs) and that are involved in the epithelial to mesenchimal transition and metastases development.

The identification of useful markers for early diagnosis of human colon cancer is a major goal still in progress. Clusterin is a pleiotropic protein with a broad range of functions. It has recently drawn much attention because of its association with cancer promotion and metastasis. It is involved in prosurvival and apoptosis processes that are carried out by two different isoforms. Secreted clusterin isoform (sCLU) is cytoprotective and its prosurvival function is the basis of the current phase I/II clinical trials against prostate, lung, and breast cancers. We have already shown that in colorectal cancer (CRC) there is an increased expression of sCLU. In this report, we investigated whether sCLU is released in the blood and stool of colon cancer patients in order to study sCLU as a potential diagnostic molecular marker for colon cancer screening.The quantitative expression of sCLU was determined by dot blot immunodosage in the serum and stool of CRC patients (n=63) and age-matched controls without clinical history of neoplasia, CRC, or systemic or bowel inflammatory disease (n=50). Unpaired t-tests and Mann-Whitney U-tests were used for continuous variables. The diagnostic performance of clusterin was appraised by means of receiver operating characteristic (ROC) curves.We found a significant increase of sCLU in the serum and stool of CRC patients (P=0.0002 and P0.000, respectively) as compared with controls. ROC curves provided cutoff points showing a trade-off between sensitivity and specificity. With a cutoff point of 88.5 microg/ml, sCLU in blood showed a 55.6% sensitivity and 100% specificity, and with a cutoff point of 34.6 microg/g, the stool test reached 66.7% sensitivity and 84% specificity in discriminating between nonneoplastic and colorectal neoplastic lesions. Human cancer xenografts in nude mice indicated a positive correlation between increasing serum clusterin level and tumor size.This study highlights the potential of clusterin detection in stool to be a valuable tool to improve the effectiveness and efficiency of large-scale clinical cancer screening.

Activation of pro-survival pathways and apoptotic cell death escape are considered hallmarks of oncogenic cell transformation. Tissue microenvironment strongly influences tumorigenesis, redirecting some pathways versus a persisting pro-survival state. Here, we report evidence on the role of interleukin 6 (IL-6) in affecting pro-survival pathways in colon cancer progression, modulating the expression and the molecular interactions among the pro-apoptotic factor Bax, the DNA repair proteins Ku70/86 and Clusterin isoforms. In human colorectal carcinomas (n = 50) at different stages of disease, we found an increased IL-6 production, the loss of Ku86 and Clusterin 50-55 kDa pro-apoptotic isoform. Conversely, we observed the overexpression of Bax and the 40 kDa prosurvival sClusterin (sCLU) isoform. Bax co-localized with Ku70 that was found atypically expressed in the cytoplasm of advanced stage colon cancers (Dukes'C-D; n = 22). IL-6 treatment of a colon cancer cell line, Caco-2, modulated the expression of genes involved in tumor invasion and apoptosis, as observed by microarrays. In particular, IL-6 downmodulated Bax expression at mRNA level. Concomitantly, IL-6 exposure influenced Bax also at protein level acting on the Bax-Ku70-sCLU physical interactions in the cytoplasm, by affecting the Ku70 acetylation and phosphorylation state, thus leading to the inhibition of Bax pro-apoptotic activity. In addition, we found that IL-6 treatment induced a significant downregulation of Ku86 and a strong increase of sCLU, confirming tumor biopsies data. In contrast Somatostatin treatment of Caco-2 cells was able to restore apoptosis, demonstrating that Ku70-Bax-CLU interactions could be dynamically modulated. Hence, IL-6 could favor tumor expansion, promoting cell survival and apoptosis escape throughout the different stages of tumor evolution. Uncovering the molecular mechanisms of action of these factors may offer strategies for selectively manipulate the cancer cells sensitivity to therapy.

Objectives— Clusterin is a heterodimeric glycoprotein which is implicated in several biological processes. The nuclear (n-CLU) and cytoplasmic secreted (s-CLU) isoforms have recently been described, but their role is still unclear. The aim of this study is to investigate the expression of clusterin and its isoforms during proliferative arrest and apoptosis of vascular smooth muscle cells (SMCs). Methods and Results— Clusterin expression was evaluated by immunohistochemistry and Western blotting in human arteries and rat aortas. In human diffuse myointimal thickening, clusterin was detected in cell cytoplasm and extracellular space, whereas it was practically absent in the media. In rat aortas 15 days after ballooning, intimal cells (IT cells) overexpressed s-CLU and n-CLU, the latter mainly in the inner neointima; clusterin expression decreased at 60 days. In vitro, IT cells maintained high clusterin expression and its antisense markedly reduced proliferation and increased apoptosis. Western blotting showed that all- trans retinoic acid-induced proliferative arrest and increased α-smooth muscle actin expression did associate to s-CLU and B-myb reduction, whereas bax-related apoptosis was associated to a shift from the s-CLU to n-CLU isoform. Conclusions— Clusterin overexpression characterized neointimal SMCs; s-CLU expression decreased in IT cells during all- trans retinoic acid-induced proliferative arrest and redifferentiation, whereas n-CLU overexpression was characteristic of apoptosis.

Clusterin is a ubiquitous secretory heterodimeric disulfide-linked glycoprotein, which is implicated in several physiological processes, including immune regulation, cell adhesion and morphological transformation, lipid transportation, tissue remodelling, membrane recycling and cell-cell interactions. A large number of studies have focused their interest on clusterin gene products as mediators of cell cycle progression and cell death induction, although data on the different isoforms and their role in the different cell processes are still obscure. Recently, an increased clusterin expression in breast cancer has been reported. In order to elucidate the role of clusterin in tumor progression and whether one of its isoforms is preferentially expressed in tumorigenesis, we examined its presence throughout the different steps of human colon carcinoma, one of the best-characterized models of human tumor progression. The immunohistochemical observation of 30 bioptic and surgical colon specimens demonstrated a cell compartment clusterin translocation from the nucleus to the cytoplasm directly related to tumor progression. In fact, a nuclear localization found in healthy colonic mucosa is consistent with the involvement of the proapoptotic nuclear form in the regulation of cell cycle progression and in cell death induction. The progression towards high-grade and metastatic carcinoma leads to cytoplasmic clusterin distribution. Protein extracts from freshly isolated cells of the same patients confirm in high-grade carcinomas with metastatic nodes the complete loss of the proapoptotic nuclear form and a cytoplasmic overexpression of the highly glycosylated form. Data obtained from in vitro experiments confirm that this form is released in the extracellular space and corresponded to the fully glycosylated one. These data suggest that the controversial data on clusterin function in tumors may be related to the pattern shift of its isoform production. As the secreted form of clusterin is correlated to cell matrix formation, cell membrane remodeling and cell-cell adhesion, the overexpression of this form in highly aggressive tumors and metastatic nodes could be a potential new prognostic and predictive marker for colon carcinoma aggressiveness.

Background Acute myocardial infarction (AMI), is related to a diffuse active inflammation of the coronary tree associated with rupture of one of the multiple vulnerable plaques. The presence of soluble mediators of inflammation with their synergic or antagonistic actions coordinates the physiological response determining the plaque fate and the fatal event. The present study focus on the cytokines network operating in human coronary plaques of patients died from AMI and controls, pointing out that coronaries of AMI patients produce PTX3 protein twice as that of controls and express high level of PTX3 mRNA. Results The presence of CX3CR1 polymorphisms is significantly correlated with the incidence and the outcome of acute myocardial infarction inducing in the whole coronary tree a strong recruitment of Th1 polarized inflammation that is directly correlated to PTX3 expression. Conclusions Moreover we found a positive correlation between the expression of PTX3 in the plaque and the content of macrophage cells showing a M2 polarization indicating the possible role of this chemokine as mediator of immune response that would orchestrate plaque evolution and inflammatory cell type activation.

The loss of IL-2 production is the main defect accounting for age-related immunodeficiencies. We have investigated the molecular mechanisms involved in the decrease of IL-2 production in CD4 F T cells from aging mice. Our results demonstrate that the stability of IL-2 mRNA increases in T cells from young mice, whereas it declines in T cells from old mice with the time of stimulation, suggesting the existence of different mechanisms of post-transcriptional regulation in young and old mice. We found that the IL-2 mRNA level in T cells from young but not from old mice increased up to 6- to 10-fold by addition of cycloheximide (CHX) while the stability of IL-2 mRNA is not affected. We then looked for IL-2 inducible inhibitory factors in T cells from young and old mice and demonstrated the presence of Nil-2-a, a zinc finger protein which negatively controls IL-2 gene transcription in human cells. This protein could be detected in T cells from both young and old mice, yet, in the presence of CHX, its binding activity was reduced by 75% in T cells from young but not from old mice. These findings show that Nil-2-a accounts for the negative control of IL-2 production in the mouse and explain the reduced IL-2 production in aging.

CD4+ cells from young (3 months) and old (19 months) mice were stimulated by plate-bound anti-CD3 monoclonal antibody (mAb) alone or also by soluble anti-CD28 mAb. Supernatants were analysed by enzyme-linked immunosorbent assay (ELISA) to determine cytokine concentrations. Total RNA was extracted from cells, reverse transcribed and the cDNA amplified by polymerase chain reaction (PCR) to evaluate the amount of specific mRNA. The results indicate that anti-CD3 alone is not sufficient to induce interleukin-2 (IL-2) production in CD4+ cells from both young and old mice. However, anti-CD28, together with anti-CD3 mAb, induces a much higher production of IL-2 in CD4+ cells from young as compared with old mice. Conversely, interferon-gamma (IFN-gamma) production is also induced by anti-CD3 alone and is higher in CD4+ cells from old as compared with young mice. Upon addition of anti-CD28 mAb, IFN-gamma production increases in both groups, but it remains much higher in old than in young mice. Also the production of IL-4 and IL-10 is induced by anti-CD3 mAb but it is increased by the addition of anti-CD28 mAb. CD4+ cells from old mice produce more IL-4 and IL-10 as compared with cells from young mice. The amounts of cytokine specific mRNA in CD4+ cells from young and old mice parallel the cytokine levels in culture supernatants. Results on the mRNA turnover indicate that when CD4+ cells are stimulated by anti-CD3 or costimulated also by anti-CD28 mAb, the IFN-gamma, IL-4 and IL-10 specific mRNAs are more stable in old than in young mice, suggesting that mRNA stability has a relevant role in the different patterns of cytokine production.

Fractalkine is a proinflammatory chemokine that participates in atherosclerotic process mediating the interactions of vascular cells and leukocytes and selective recruitment of Th1 lymphocytes, through interaction with CX3CR1 receptor. The polymorphism of the fractalkine receptor 280M-containing haplotype, which codifies for a receptor with minor expression and with a reduced binding capability, represents a novel protective factor of atherosclerotic disease. We investigated the association among CX3CR1 genotype, the inflammatory infiltrate subpopulations recruited in the plaque, and the in situ expression of fractalkine and its receptor, in patients who died of myocardial infarction (AMI) compared with subjects who died of noncardiac causes. Patients with nonlethal AMI (AMI survivors) were also investigated to correlate the CX3CR1 polymorphisms and the incidence of lethal AMI. A strong T cells infiltrate was found in infarct related artery (IRA) plaques of AMI patients presenting the V249 T280 haplotype (84%). Conversely, a decreased T cell recruitment was associated with I249T280 haplotype in the controls (64%). The significant higher presence of the variant allele I249 in homo- and heterozygosis, found in controls (91%) and in AMI survivors (94%), with respect to the patients who died of AMI (48%), showed the relevance of this polymorphism both in the onset and outcome of acute myocardial infarction. The presence of CX3CR1 polymorphisms could influence the incidence and the outcome of acute myocardial infarction, altering the inflammation of the whole coronary tree by the impaired recruitment of Th1 polarized subpopulation in the coronary plaque.

International audience; Background: In prostate cancer the secreted form of clusterin (sCLU) has been described as an anti-apoptotic protein whose expression is increased after therapeutic intervention, whereas, the nuclear protein form nCLU was reported to have pro-apoptotic properties.Methodology: In order to provide new therapeutic approaches targeting CLU, we developed a strategy based on exon skipping by using a lentiviral construct to preferentially induce the nuclear spliced form of the protein. The molecular construct was transduced in LNCaP cells for testing the modulation of sensitivity of the transduced cells to pro-apoptotic stress.Results and Conclusions: We showed an increase of nCLU/sCLU expression ratio in the prostate cancer cell line “LNCaP” after lentiviral vector-U7 nCLU transduction. Moreover, we showed a significant inhibition of cell proliferation in nCLU-U7 LNCaP cells after treatment with cisplatin and after exposure to ionizing radiation compared to control cells. Finally, we showed that nCLU-U7 LNCaP cells exposure to UV-C significantly reduced an increase of cell death compared to control. Finally, we showed that modulating nCLU expression had profound impact on Ku70/Bax interaction as well as Rad17 expression which could be a key mechanism in sensitizing cells to cell death. In conclusion, this is the first report showing that increasing of nCLU/sCLU expression ratio by using an “on demand alternative splicing” strategy successfully increased sensitivity to radiotherapy and chemotherapy of prostate cancer cells.

B-myb and c-myb expression is high in neuroblastoma cells and declines during retinoic acid-induced differentiation. We show here that B-myb down-regulation during retinoic acid-induced differentiation of LAN-5 neuroblastoma cells occurs at the transcriptional level. In addition, we measured B-myb and c-myb messenger RNA half-lives, and found that, unlike c-myb, B-myb messenger RNA was remarkably stable (>10 h). Inhibition of protein synthesis by treatment with cycloheximide increased B-myb messenger RNA levels, suggesting that one or more labile proteins act as repressors of B-myb transcription. In the same cell line, blocking protein synthesis decreased the level of c-myb mRNA under both normal and differentiative conditions. Thus, B-myb and c-myb undergo similar transcriptional regulation, but there are specific differences in the stability of their messenger RNAs and in the mechanisms through which their transcription is controlled. These differences could reflect different functional roles played by c-myb and B-myb in neuroblastoma cells.

In our previous studies aiming at the design of appropriate strategies to accelerate the recovery of the immune system after irradiation, we found that recombinant murine (rmu) IL-3 treatment induces differentiation and growth of thymocytes and splenic T and B lymphocytes in mice exposed to X-rays (200-500 cGy). These studies were extended to investigate the effects of recombinant human (rhu) IL-11. Results indicate that rhulL-11 is able to restore thymus and spleen cell numbers as well as T and B cell mitotic responsiveness in mice exposed to 200 cGy but not to 300 cGy. However, recovery of thymus and spleen cell numbers and functions could be accelerated also in mice exposed to higher doses if rhulL-11 was given with rmulL-3. Recovery was complete as soon as 7 days after irradiation. A large dose range of both cytokines was explored and the synergistic effect of the two cytokines was evident when a relatively small dose of rhulL-11 was injected with graded doses of rmulL-3. The recovery of the immune system in irradiated mice injected with these cytokines was independent from Bcl-2 expression, suggesting that elimination of damaged cells by apoptosis is unaffected by hematopoietic cytokines.

Review on XRCC5 (X-ray repair complementing defective repair in Chinese hamster cells 5 (double-strand-break rejoining)), with data on DNA, on the protein encoded, and where the gene is implicated.

Cell transformation is strictly linked to important metabolic changes which are instrumental for initial survival of cancer cells and subsequent spreading of disease. Early (i.e., anerobic glycolysis) and late metabolic changes (i.e., fatty acid metabolism) are required for progression and clinical emergence of cancer. Besides well-known tumor suppressors and oncogenes, several metabolic genes have been found implicated in this multistep process, among which are fatty acid synthase (FASN) and carnitine palmitoyl transferase I (CPT I). An intriguing link between these metabolic shifts and a change in the balance between nuclear and secreted forms of CLU (nCLU/sCLU) has been suggested. The shifting balance between CLU forms during tumor progression, by affecting the fate of the cell, seems to be strongly influenced by the metabolic shift occurring in the different steps of tumor progression.

In no other field has the function of clusterin (CLU) been more controversial than in cancer genetics. After more than 20 years of research, there is still uncertainty with regard to the role of CLU in human cancers. Some investigators believe CLU to be an oncogene, others-an inhibitor of tumorigenesis. However, owing to the recent efforts of several laboratories, the role of CLU in important cellular processes like proliferation, apoptosis, differentiation, and transformation is beginning to emerge. The "enigmatic" CLU is becoming less so. In this chapter, we will review the work of research teams interested in understanding how CLU is regulated by oncogenic signaling. We will discuss how and under what circumstances oncogenes and epigenetic factors modify CLU expression, with important consequences for mammalian tumorigenesis.

Cancer cells need to interact synergistically with their surrounding microenvironment to form a neoplasm and to progress further to colonize distant organs. The microenvironment can exert profound epigenetic effects on cells through cell-derived interactions between cells, or through cell-derived factors deposited into the microenvironment. Tumor progression implies immune-escaping and triggers several processes that synergistically induce a cooperation among transformed and stromal cells, that compete for space and resources such as oxygen and nutrients. Therefore, the extra cellular milieu and tissue microenvironment heterotypic interactions cooperate to promote tumor growth, angiogenesis, and cancer cell motility, through elevated secretion of pleiotropic cytokines and soluble factors. Clusterin (CLU), widely viewed as an enigmatic protein represents one of the numerous cellular factors sharing the intracellular information with the microenvironment and it has also a systemic diffusion, tightly joining the "In and the Out" of the cell with a still debated variety of antagonistic functions. The multiplicity of names for CLU is an indication of the complexity of the problem and could reflect, on one hand its multifunctionality, or alternatively could mask a commonality of function. The posited role for CLU, further supported as a cytoprotective prosurvival chaperone-like molecule, seems compelling, in contrast its tumor suppressor function, as a guide of the guardians of the genome (DNA-repair proteins Ku70/80, Bax cell death inducer), could really reflect the balanced expression of its different forms, most certainly depending on the intra- and extracellular microenvironment cross talk. The complicated balance of cytokines network and the regulation of CLU forms production in cancer and stromal cells undoubtedly represent a potential link among adaptative responses, genomic stability, and bystander effect after oxidative stresses and damage. This review focuses on the tumor-microenvironment interactions strictly involved in controlling local cancer growth, invasion, and distant metastases that play a decisive role in the regulation of CLU different forms expression and release. In addition, we focus on the pleiotropic action of the extracellular form of this protein, sCLU, that may play a crucial role in redirecting stromal changes, altering intercellular communications binding cell surface receptors and contributing to influence the secretion of chemokines in paracrine and autocrine fashion. Further elucidation of CLU functions inside and outside ("in and out") of cancer cell are warranted for a deeper understanding of the interplay between tumor and stroma, suggesting new therapeutic cotargeting strategies.

The transition from normal to malignant phenotype implies the activation of some pathways that underlie the aberrant clone expansion. In some way, the conventional function of proteins involved in DNA repair, cell death/growth induction, vascularization, and metabolism is inhibited or shifted toward other pathways by soluble mediators that orchestrate such change depending on the microenvironment conditions. The adenoma-carcinoma sequence of the colon represents one of the most well studied and characterized models of human tumor progression. In this section, we focus our attention on defined pathways that underlie the initiation, promotion, and progression of colon cancer, conferring aggressiveness to the neoplastic cells. Clusterin (CLU) is a pleiotropic protein with a broad range of functions. It has recently drawn much attention because of its association with cancer promotion and metastasis. It is involved in prosurvival and apoptosis processes that are carried out by two different forms. sCLU is cytoprotective and its prosurvival function is the basis of the current Phase I/II clinical trials. In colorectal cancer an increase of sCLU expression occurs, whereas the nuclear proapoptotic form is downregulated. Several controversial data have been published on colon cancer discussing its role as tumor suppressor or prosurvival factor in colon cancer. Here, we report the dynamic interaction of the different forms of CLU with their partners DNA-repair protein Ku70 and proapoptotic factor Bax during colon cancer progression, which seems to be a crucial point for the neoplastic cell fate. We also highlight that the appearance and the progressive increase of the sCLU in colorectal tumors correlate to a significant increase of CLU in serum and stool of patients. On the basis of results obtained by CLU immuno-dosage in blood and stool of colon cancer patients, we report that sCLU could represent a diagnostic molecular marker for colon cancer screening.

Cell transformation is strictly linked to important metabolic changes which are instrumental for initial survival of cancer cells and subsequent spreading of disease. Early (i.e., anerobic glycolysis) and late metabolic changes (i.e., fatty acid metabolism) are required for progression and clinical emergence of cancer. Besides well-known tumor suppressors and oncogenes, several metabolic genes have been found implicated in this multistep process, among which are fatty acid synthase (FASN) and carnitine palmitoyl transferase I (CPT I). An intriguing link between these metabolic shifts and a change in the balance between nuclear and secreted forms of CLU (nCLU/sCLU) has been suggested. The shifting balance between CLU forms during tumor progression, by affecting the fate of the cell, seems to be strongly influenced by the metabolic shift occurring in the different steps of tumor progression.

Growth factors and their respective receptors are key regulators in development and homeostasis of the nervous system, and changes in the function, expression, or downstream signaling of growth factors are involved in many neuropathological disorders. Recently, research has yielded a rich harvest of information about molecules and gene, and currently the assumption "a gene-a protein", where each gene encodes the structure of a single protein, is becoming a paradox. In the past years, the discovery of synergic or antagonistic proteins deriving from the same gene is a novelty upsetting. In some way, the conventional function of proteins involved in DNA repair, cell death/growth induction, vascularization, and metabolism is inhibited or shifted toward other pathways by soluble mediators that orchestrate such change depending on the microenvironment conditions. In this chapter, we focus on the antithetic properties that proteins could exert, depending on the microenvironment that orchestrates the complex networks among proteins and their respective partners.

Carnitine palmitoyl transferase I (CPT1) catalyzes the transport of long-chain fatty acids into mitochondria for beta-oxidation. A link between CPT1 and apoptosis has been suggested on the basis of several experimental data. Nevertheless, results are contradictory about the effective role of CPT1 in cell survival control and cancer development. Conversely, Fatty acid synthase (FAS) enzyme, required for the synthesis of fatty acids, is found over-expressed in tumors and inhibition of FAS triggers apoptosis in human cancer cells. We have studied the tumor-specific modulation of CPT1 and FAS in human colorectal cancer (n = 11) and breast carcinomas (n = 24). CPT1 was significantly decreased in the cytoplasm of tumoral samples (p < or = 0.04), whereas FAS was increased (p < or = 0.04). A striking CPT1 nuclear localization was evident in the tumors (p < or = 0.04). In the nuclear environment the protein would modulate the levels of acetyl/acyl-CoA implicated in the regulation of gene transcription. At this purpose, we performed in vitro experiments using epithelial neoplastic (MCF-7, Caco-2, HepG2 cells) and non neoplastic cell lines (MCF-12F) confirming a nuclear localization of CPT1 protein exclusively in neoplastic cells. Moreover histone deacetylase (HDAC) activity showed significantly higher levels in nuclear extracts from neoplastic than from control cells. HDAC1 and CPT1 proteins coimmunoprecipitated in nuclear extracts from MCF-7 cells. The treatment with HDAC inhibitors such as trichostatin A and butyrate significantly decreased nuclear expression of CPT1 and its bond to HDAC1. We also identified the existence of CPT1A mRNA transcript variant 2 in MCF-7, beside to the classic isoform 1. The peculiar localization of CPT1 in the nuclei of human carcinomas and the disclosed functional link between nuclear CPT1 and HDAC1 propose a new role of CPT1 in the histonic acetylation level of tumors.

Acute myocardial infarction (AMI) associated with unfavorable prognosis is likely to be the consequence of a diffuse active chronic inflammatory process that destabilizes the whole coronary tree and myocardium, suggesting a possible common causal agent underlying both conditions. The main objective of this study was to investigate whether Chlamydia pneumoniae (CP) infection occurred beyond the coronary plaques, namely in the myocardium of individuals who died of AMI. The presence of CP cell wall antigen (OMP-2) and CP-HSP60 was investigated in the myocardium and coronary plaques of 10 AMI and 10 age-matched control patients by immunohistochemistry, electron microscopy, and molecular biology. OMP-2 antigens were found in the unaffected myocardium of 9 of 10 AMI patients. Conversely, only 1 of 10 control patients exhibited a positive staining for CP. Moreover, OMP-2 and CP-HSP60 were detected in the whole coronary tree. CP presence was strongly associated with a T-cell inflammatory infiltrate. Our results suggest that CP may underlie both coronary and myocardial vulnerabilities in patients who died of AMI and corroborate the notion that CP may act by reducing cardiac reserves, thus worsening the ischemic burden of myocardium.

Given the increasing incidence of colorectal cancer (CRC), performing new and cost-effective stool tests is of particular importance for early diagnosis and treatment. In the present review, we describe the main characteristics, and the performance of the most recently developed stool tests in the screening setting of colorectal tumoral diseases. Most of the studies reported high sensitivity both for adenomas and CRC diagnosis; less than half studies reported also high specificity with respect to stage and localization of the tumor. However, the performance of every single test was extremely variable, reaching >95% specificity for most of DNA-based markers, although lacking sensitivity even in case of invasive CRC. A new potential stool marker of colon cancer is clusterin, a protein of particular interest for its high sensitivity and positive predictive value in patients with highly aggressive CRC.

Purpose: In order to characterize the biological effects and molecular mechanism underlying indocyanine-green (ICG)-mediated photo-oxidative cell damage, human cultured retinal pigmented epithelium (RPE) cells preloaded with ICG were exposed to 810-nm laser irradiation. Cell viability and death induction were examined, as well as the modulation of proteins involved in cell death and DNA repair. Methods: ARPE-19 cells preloaded with 100 µM ICG were irradiated using continuous and micropulsed 810-nm laser for the dye photoactivation, and cell viability and apoptosis were evaluated. The expression and subcellular localization of Bax, Ku70, Ku80 and clusterin/ApoJ were analyzed by immunocytochemistry and Western blot. Results: ICG photoactivation induced apoptosis in RPE cells. The micropulsed laser irradiation induced a higher percentage of cell killing as compared to continuous wave. Cell killing was inhibited by sodium azide, suggesting the involvement of reactive oxygen species in the laser-induced cell damage. Bax was strongly induced after 4 and up to 24 h of treatment. The nuclear proapoptotic isoform of clusterin/ApoJ was selectively upregulated after 24 h of treatment. The DNA repair machinery was upregulated after 4 and up to 24 h. Conclusion: These data elucidate some molecular mechanisms involved in cell death induced by ICG photosensitization. The increase and relocalization of Bax into the mitochondria and the upregulation and translocation of the proapoptotic isoform of clusterin/ApoJ in the nucleus demonstrated the involvement of these proteins in the photo-oxidative cell death pathway. These data point out new molecular targets and suggest potential applications in the therapy of the retinal diseases that could benefit by selective RPE treatment.

The Ubiquitination Factor E4A gene (UBE4A) encodes for a U-box-type ubiquitin ligase, originally described as an E4 ubiquitination factor. UBE4A is a mammalian homolog of Saccharomyces cerevisiae Ufd2. The UBE4A gene has been mapped on the human chromosome region 11q23.3, a critical region involved in some specific cancers such as neuroblastoma. Northern blots analysis on foetal and adult human tissues revealed a single band of approximately 7.5 kb transcript most abundant in the heart, skeletal muscle and kidney. We generated a polyclonal antibody to UBE4A and performed immunoblot and immunohistochemical analysis. The UBE4A protein appeared as a single band of approximately 125 kDa. UBE4A was present in the skeletal muscle, kidney and liver; a faint band was visible in peripheral blood leukocytes and spleen. We did not reveal expression of UBE4A in whole brain, colon, lung and heart. At the cellular level, UBE4A results predominantly expressed in the nucleus and the cytoplasm of cortical neurons and liver and in the nucleus of tubular kidney cells. In the liver, the nucleus of similar cells appeared to be unstained or stained at different levels suggesting that UBE4A may have a cell cycle dependent expression or a role of in cell cycle control. In conclusion, our results show that UBE4A is expressed in different tissues in a pattern that seems to be dependent from cell type and cell cycle and that UBE4A might have a specific role in different biochemical processes other than ubiquitination, including growth or differentiation.

Background Multinodular goitre (MNG) is a common disorder characterised by an enlargement of the thyroid, occurring as a compensatory response to hormonogenesis impairment. The incidence of MNG is dependent on sex (female:male ratio 5:1) and several reports have documented a genetic basis for the disease. Last year we mapped a MNG locus to chromosome Xp22 in a region containing the peroxiredoxin IV (Prx-IV) gene. Since Prx-IV is involved in the removal of H2O2 in thyroid cells, we hypothesize that mutations in Prx-IV gene are involved in pathogenesis of MNG. Methods Four individuals (2 affected, 2 unrelated unaffected) were sequenced using automated methods. All individuals were originated from the original three-generation Italian family described in previous studies. A Southern blot analysis using a Prx-IV full-length cDNA as a probe was performed in order to exclude genomic rearrangements and/or intronic mutations. In addition a RT-PCR of PRX-IV was performed in order to investigate expression alterations. Results No causative mutations were found. Two adjacent nucleotide substitutions were detected within introns 1 and 4. These changes were also detected in unaffected individuals, suggesting that they were innocuous polymorphisms. No gross genomic rearrangements and/or restriction fragment alterations were observed on Southern analysis. Finally, using RT-PCR from tissue-specific RNA, no differences of PRX-IV expression-levels were detected between affected and unaffected samples. Conclusions Based on sequence and genomic analysis, Prx-IV is very unlikely to be the MNG2 gene.

The Ku70/80 heterodimer is the regulatory subunit of the DNA-dependent protein kinase (DNA-PK) and its DNA-binding activity mediates DNA double-strand breaks repair. Although Ku80 was recently proposed as a caretaker gene involved in the control of genome integrity, no data are available on Ku70/80 DNA-binding activity in human tumors. Heterodimer DNA-binding activity and protein expression were assayed by electrophoretic-mobility-shift-assay (EMSA) and Western blot analysis, in nuclear and cytoplasmic extracts from eight breast, seven bladder primary tumors and three metastatic nodes from breast cancers. Corresponding normal tissues of the same patients were used as controls. Ten out of 15 tumors showed nuclear Ku-binding activity 3-10 times higher than in the normal tissues, irrespective of bladder or breast origin. Conversely, in 5/15 primary tumors and in all the metastatic nodes analysed, nuclear Ku-activity was 1.5-4.5-fold lower than in the corresponding normal tissues. Cytoplasmic heterodimer activity significantly differed between tumor and normal tissues, displaying a 2-10-fold increase in neoplastic tissues. Three different patterns combining both Ku expression and activity with tumor characteristics were identified. In low aggressive breast tumors p70/p80 proteins were expressed in tumor but not in normal tissues. The heterodimer binding-activity matched the protein levels. In non-invasive bladder carcinomas no significant differences in protein expression between tumor and the corresponding normal tissues were found, however heterodimer binding-activity was increased in tumor samples. In breast and bladder tumors, at the advanced stage and in node metastases, the binding activity was strongly reduced in tumor biopsies, however no differences were demonstrated between normal and tumor protein levels. Our results suggest a different modulation of Ku70/80 DNA-binding activity in human neoplastic tissues, possibly related to tumor progression. Findings provide further data on tissue-specific protein expression and post-translational regulation of heterodimer activity.

Previous studies on DNA repair in ageing have demonstrated increased frequencies of single and double strand breaks in lymphocytes from elderly subjects and, as a consequence, decreased efficiency in DNA replication. We have investigated the relationship between cell proliferation and the nuclear expression of ku protein in a human population of 43 subjects of different ages. Ku is an heterodimeric protein composed of two subunits of 70 and 80 kDa, which is involved in the early steps of DNA damage recognition. In the present study, PBL from subjects of different ages were PHA-activated to evaluate the stimulation index and the production of Th1- and Th2-type cytokines. Moreover, nuclear extracts were obtained from activated lymphocytes to evaluate by a gel retardation assay the presence and the functional activity of the heterodimer ku 70/80. Our results indicate that ageing affects the mitotic responsiveness and cytokine production to a significant extent, but only marginally the expression of ku 70/80. These findings suggest that the age-related impairment in DNA repair mechanisms are only in part related to the reduced expression of ku protein able to recognize DNA damage.

Detectable levels of MAX messenger RNA were found in a set of human neuroblastoma tumors and established cell lines. MAX mRNA levels were independent of tumor stage and N-myc genomic amplification. By contrast, N-myc mRNA transcripts were detectable only in tumors with amplification of N-myc gene and in cell lines. Analysis by reverse transcriptase polymerase chain reaction and hybridization to specific oligodeoxynucleotide probes revealed approximately equal amounts of two MAX transcripts in all cases analyzed. Immunoprecipitations with a specific antibody to MAX detected two proteins of M(r) 21,000 and 22,000 in approximately equal amounts in all neuroblastoma lines regardless of N-myc amplification and/or expression. On the other hand, protein binding to the myc DNA consensus sequence correlated with N-myc expression in neuroblastoma cells. Thus, N-myc expression might be a limiting factor in the formation of the N-myc-MAX heterodimer in neuroblastomas.