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Multiple Sclerosis (MS) is a chronic disease of the central nervous system, the etiology of which, although not completely known, involves inflammation and autoimmunity. In the present study we aimed at identifying molecular markers of apoptosis, cellular stress and DNA damage in isolated peripheral blood mononuclear cells (PBMCs) of MS patients. The analysis was carried on 19 relapsing-remitting untreated MS patients and 13 healthy individuals. We investigated the emergency-driven synthesis of poly(ADP-ribose) (PAR), the expression level of the constitutive enzyme poly(ADP-ribose) polymerase-1 (PARP-1) and the DNA damage-induced phosphorylation of histone H2AX. PAR accumulation, PARP-1 and phosphorylated H2AX (γH2AX) were detected by immunofluorescence experiments on PBMCs isolated from 19 patients and 13 healthy volunteers. Our results show for the first time a net increased amount in PAR and γH2AX in MS patients compared to healthy individuals. Patients were further subdivided in three groups, according to the neuroimaging (MRI)-based classification of disease phase. Remarkably, we found a positive correlation between the level of γH2AX and MS aggressiveness. In addition, apoptosis in PBMCs was monitored by flow cytometry of both phosphatidylserine exposure (revealed by Annexin V-FITC labeling) and membrane permeability to propidium iodide. Our observations provide the evidence that the number of apoptotic cells was significantly higher in patients compared to healthy individuals, thus suggesting that apoptosis could affect MS lymphocyte function.

We have developed a cellular system constituted of human telomerase immortalized fibroblasts that gradually underwent neoplastic transformation during propagation in culture. We exploited this cellular system to investigate gene and miRNA transcriptional programs in cells at different stages of propagation, representing five different phases along the road to transformation, from non-transformed cells up to tumorigenic and metastatic ones. Here we show that gene and miRNA expression profiles were both able to divide cells according to their transformation phase. We identified more than 1,700 genes whose expression was highly modulated in cells at at least one propagation stage and we found that the number of modulated genes progressively increased at successive stages of transformation. These genes identified processes significantly deregulated in tumorigenic cells, such as cell differentiation, cell movement and extracellular matrix remodeling, cell cycle and apoptosis, together with upregulation of several cancer testis antigens. Alterations in cell cycle, apoptosis, and cancer testis antigen expression were particular hallmarks of metastatic cells. A parallel deregulation of a panel of 43 miRNAs strictly connected to the p53 and c-Myc pathways and with oncogenic/oncosuppressive functions was also found. Our results indicate that cen3tel cells can be a useful model for human fibroblast neoplastic transformation, which appears characterized by complex and peculiar alterations involving both genetic and epigenetic reprogramming, whose elucidation could provide useful insights into regulatory networks underlying cancerogenesis.

In this work, we show that vertical, high aspect-ratio (HAR) photonic crystals (PhCs), consisting of periodic arrays of 5 µm wide gaps with depth of 50 µm separated by 3 µm thick silicon walls, fabricated by electrochemical micromachining, can be used as three-dimensional microincubators, allowing cell lines to be selectively grown into the gaps. Silicon micromachined dice incorporating regions with different surface profiles, namely flat silicon and deeply etched PhC, were used as microincubators for culturing adherent cell lines with different morphology and adhesion properties. We extensively investigated and compared the proliferative behavior on HAR PhCs of eight human cell models, with different origins, such as the epithelial (SW613-B3; HeLa; SW480; HCT116; HT29) and the mesenchymal (MRC-5V1; CF; HT1080). We also verified the contribution of cell sedimentation into the silicon gaps. Fluorescence microscopy analysis highlights that only cell lines that exhibit, in the tested culture condition, the behavior typical of the mesenchymal phenotype are able to penetrate into the gaps of the PhC, extending their body deeply in the narrow gaps between adjacent silicon walls, and to grow adherent to the vertical surfaces of silicon. Results reported in this work, confirmed in various experiments, strongly support our statement that such three-dimensional microstructures have selection capabilities with regard to the cell lines that can actively populate the narrow gaps. Cells with a mesenchymal phenotype could be exploited in the next future as bioreceptors, in combination with HAR PhC optical transducers, e.g., for label-free optical detection of cellular activities involving changes in cell adhesion and/or morphology (e.g., apoptosis) in a three-dimensional microenvironment.

Cancer still represents a major health problem worldwide, which urges the development of more effective strategies. Resistance to chemotherapy, a major obstacle for cancer eradication, is mainly related to an intrinsic failure to activate the apoptotic pathways. However, a protective effect of autophagy toward cancer cells has been recently observed, thus adding further complexity to the development of an effective approach counteracting cancer cell growth and improving the response to therapy., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Sertoli cells play an essential role in the development of a functional testis. ROS (reactive oxygen species) are normally produced by the developing testicular cells and may be dangerous to spermatogenesis. The aim of this study was to investigate the developmental expression of genes involved in antioxidant defense as well as in the DNA damage response in rat Sertoli cells. As revealed by quantitative RT-PCR analysis, the expression pattern of the antioxidant enzymes GST (glutathione-S-transferase), CAT (catalase) and SOD (superoxide dismutase) showed a progressive decrease from birth to puberty. The expression level of the oncosuppressor p53 revealed a net reduction as well. We next focused on PARP-1 [poly(ADP-ribose) polymerase-1], a 'guardian of the genome' that combats stress conditions. At both the mRNA and protein level, PARP-1 expression was low at the early stage of development and increased later on. Maximal PARP-1 expression was preceded by a rise in the transcript level for MTs (metallothioneins), which provide zinc to zinc-dependent enzymes and proteins, including PARP-1. Our results showed an increased expression of PARP-1 during Sertoli cell development, together with a decrease in the expression of antioxidant enzymes. In conclusion, a role of PARP-1 in protecting the testicular differentiation is suggested.

Drug resistance of cancer cells is often correlated with apoptosis evasion; however, an active involvement of autophagy in this scenario has been recently proposed, based on the evidence that autophagy could exert a protective role toward the activation of apoptosis in cancer cells. In this review, we briefly review the basic features of apoptosis, and we describe in details the molecular patterns of autophagy, with a special emphasis on its still controversial physiological function(s). The crucial factors governing the cross talk between autophagy and apoptosis will be illustrated.

Tumors are generally composed of different cell types. In recent years, it has been shown that in many types of cancers a subset of cells show peculiar characteristics, such as the ability to induce tumors when engrafted into host animals, self-renew and being immortal, and give rise to a differentiated progeny. These cells have been defined as cancer stem cells (CSCs) or tumor initiating cells. CSCs can be isolated both from tumor specimens and established cancer cell lines on the basis of their ability to exclude fluorescent dyes, express specific cell surface markers or grow in particular culture conditions. A key feature of CSCs is their resistance to chemotherapeutic agents, which could contribute to the remaining of residual cancer cells after therapeutic treatments. It has been shown that CSC-like cells can be isolated after drug treatment of cancer cell lines; in this review, we will describe the strategies so far applied to identify and isolate CSCs. Furthermore, we will discuss the possible use of these selected populations to investigate CSC biology and develop new anticancer drugs.

Colon carcinoma represents a major problem in oncology, since this type of cancer responds poorly to conventional chemotherapy. Many groups are actively involved in the search of new experimental strategies to bypass this problem. We investigated the effects of 2-methoxyestradiol (2-ME), which derives from the NADPH-dependent cytochrome P450 metabolism of 17β-estradiol. This compound has raised much interest in the past few decades for its inhibitory effects on the growth of cancer cells of different origin; however, little is known about its use on colon carcinoma-derived cell lines. In the present study, we investigated the effects of 2-ME on cell proliferation and cell cycle of two human colon carcinoma cell lines, namely HCT116 and SW613-B3. Our results showed a net anti-proliferative effect of 2-ME on both cell lines, which is accompanied by cell cycle arrest; moreover, we demonstrated that 2-ME is able to induce apoptosis as well as autophagy. This body of evidence points out that 2-ME could be considered as a promising tool against colon carcinoma., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Poly(ADP-ribosylation) consists in the conversion of β-NAD(+) into ADP-ribose, which is then bound to acceptor proteins and further used to form polymers of variable length and structure. The correct turnover of poly(ADP-ribose) is ensured by the concerted action of poly(ADP-ribose) polymerase (PARP) and poly(ADP-ribose) glycohydrolase (PARG) enzymes, which are responsible for polymer synthesis and degradation, respectively. Despite the positive role of poly(ADP-ribosylation) in sensing and repairing DNA damage, generated also by ROS, PARP over-activation could allow NAD depletion and consequent necrosis, thus leading to an inflammatory condition in many diseases. In this respect, inhibition of PARP enzymes could exert a protective role towards a number of pathological conditions; i.e. the combined treatment of tumors with PARP inhibitors/anticancer agents proved to have a beneficial effect in cancer therapy. Thus, pharmacological inactivation of poly(ADP-ribosylation) could represent a novel therapeutic strategy to limit cellular injury and to attenuate the inflammatory processes that characterize many disorders., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The cell cycle inhibitor p21(CDKN1A) has been shown to participate in nucleotide excision repair by interacting with PCNA. Here we have investigated whether p21 plays a role in base excision repair (BER), by analyzing p21 interactions with BER factors, and by assessing the response of p21(-/-) human fibroblasts to DNA damage induced by alkylating agents. Absence of p21 protein resulted in a higher sensitivity to alkylation-induced DNA damage, as indicated by reduced clonogenic efficiency, defective DNA repair (assessed by the comet test), and by persistence of histone H2AX phosphorylation. To elucidate the mechanisms at the basis of the function of p21 in BER, we focused on its interaction with poly(ADP-ribose) polymerase-1 (PARP-1), an important player in this repair process. p21 was found to bind the automodification/DNA binding domain of PARP-1, although some interaction occurred also with the catalytic domain after DNA damage. This association was necessary to regulate PARP-1 activity since poly(ADP-ribosylation) induced by DNA damage was higher in p21(-/-) human fibroblasts than in parental p21(+/+) cells, and in primary fibroblasts after p21 knock-down by RNA interference. Concomitantly, recruitment of PARP-1 and PCNA to damaged DNA was greater in p21(-/-) than in p21(+/+) fibroblasts. This accumulation resulted in persistent interaction of PARP-1 with BER factors, such as XRCC1 and DNA polymerase beta, suggesting that prolonged association reduced the DNA repair efficiency. These results indicate that p21 regulates the interaction between PARP-1 and BER factors, to promote efficient DNA repair., (Copyright 2010 Elsevier B.V. All rights reserved.)

Among cell cycle regulatory proteins that are activated following DNA damage, the cyclin-dependent kinase inhibitor p21(CDKN1A) plays essential roles in the DNA damage response, by inducing cell cycle arrest, direct inhibition of DNA replication, as well as by regulating fundamental processes, like apoptosis and transcription. These functions are performed through the ability of p21 to interact with a number of proteins involved in these processes. Despite an initial controversy, during the last years several lines of evidence have also indicated that p21 may be directly involved in DNA repair. In particular, the participation of p21 in nucleotide excision repair (NER), base excision repair (BER), and DNA translesion synthesis (TLS), has been suggested to occur thanks to its interaction with proliferating cell nuclear antigen (PCNA), a crucial protein involved in several aspects of DNA metabolism, and cell-cycle regulation. In this review, the multiple roles of p21 in the DNA damage response, including regulation of cell cycle, apoptosis and gene transcription, are discussed together with the most recent findings supporting the direct participation of p21 protein in DNA repair processes. In particular, spatio-temporal dynamics of p21 recruitment to sites of DNA damage will be considered together with several lines of evidence indicating a regulatory role for p21. In addition, the relevance of post-translational regulation in the fate (e.g. degradation) of p21 protein after cell exposure to DNA damaging agents will be analyzed. Both sets of evidence will be discussed in terms of the overall DNA damage response., (2010 Elsevier B.V. All rights reserved.)

Drugs that are able to modulate the microtubule dynamics either by inhibiting tubulin polymerization or by blocking microtubule disassembly are of great interest in anti-cancer therapy; a number of them are currently applied in clinical development. Tubulin polymerization inhibitors, including arylthioindoles, are characterized by the presence of an indole nucleus and have been obtained from natural sources or prepared by semi-synthesis. We characterized the effect of 5-bromo-3-[(3,4,5-trimetoxyphenyl)thio]-1H-indole (RS 2518) on the metabolism of human cell lines derived from solid tumors. We found that this new compound impairs cell adhesion, arrests the cells in the G(2)/M cell cycle phase and inhibits cell proliferation, thus leading to apoptosis. The described effects of RS 2518 on cancer cells have led to its selection as a lead compound for further studies. Some analogues have been developed and tested on a panel of cancer cell lines.

Apoptosis, the best known form of programmed cell death, is tightly regulated by a number of sensors, signal transducers and effectors. Apoptosis is mainly active during embryonic development, when deletion of redundant cellular material is required for the correct morphogenesis of tissues and organs; moreover, it is essential for the maintenance of tissue homeostasis during cell life. Cells also activate apoptosis when they suffer from various insults, such as damage to DNA or to other cellular components, or impairment of basic processes, such as DNA replication and DNA repair. Removal of damaged cells is fundamental in maintaining the health of organisms. In addition, apoptosis induction following DNA damage is exploited to kill cancer cells. In this chapter we will review the main features of developmental and induced apoptosis.

New arylthioindoles along with the corresponding ketone and methylene compounds were potent tubulin assembly inhibitors. As growth inhibitors of MCF-7 cells, sulfur derivatives were superior or sometimes equivalent to the ketones, while methylene derivatives were substantially less effective. Esters 24, 27-29, 36, 39, and 41 showed approximately 50% of inhibition on human HeLa and HCT116/chr3 cells at 0.5 microM, and these compounds inhibited the growth of HEK, M14, and U937 cells with IC(50)'s in the 78-220 nM range. While murine macrophage J744.1 cell growth was significantly less affected (20% at higher concentrations), four other nontransformed cell lines remained sensitive to these esters. The effect of drug treatment on cell morphology was examined by time-lapse microscopy. In a protocol set up to evaluate toxicity on the Saccharomyces cerevisiae BY4741 wild type strain, compounds 24 and 54 strongly reduced cell growth, and 29, 36, and 39 also showed significant inhibition.

Drug resistance of cancer cells is often correlated with the evasion of apoptosis, thus a major goal in cancer research is to search for compounds able to counteract cancer by promoting apoptosis. A variety of compounds with anticancer activity are characterised by the presence of the pyrazole as core nucleus. We synthesised a panel of pyrrolyl-pyrazole-carboxamides and we focused on the new compound RS 2780 (N-2-phenylethyl 1-(4-chlorophenyl)-3-methyl-5-pyrrolylpyrazole-4-carboxamide). The biological effects of RS 2780 on cell proliferation and viability were first evaluated on human HeLa cancer cells. As revealed by cell growth and viability experiments, a 24-h treatment of HeLa cells with increasing concentrations of RS 2780 (ranging from 0.1 to 100 microM) proved to inhibit cell proliferation and to affect cell viability. Notably, the new compound was effective also on colon carcinoma SW613-B3 cells, which are extremely resistant to most drugs, while it does not alter the proliferation of normal fibroblasts. We observed that RS 2780 interferes with the structural and functional properties of mitochondria, leading to the activation of the mitochondria-dependent apoptotic pathway. Apoptosis occurrence was supported by a number of morphological and biochemical hallmarks, including chromatin condensation, internucleosomal DNA fragmentation, PARP-1 cleavage and caspase activation. In conclusion, our results demonstrate for the first time the antiproliferative properties of the new compound RS 2780 on HeLa and SW613-B3 cancer cells and show that its effects on mitochondria lead to apoptosis.

During apoptosis, apoptosis-inducing factor (AIF) is released from the mitochondrial intermembrane space to the cytosol and to the nucleus. We analyzed AIF in HeLa cells driven to apoptosis by either etoposide or actinomycin D, and we observed changes in the structure and function of mitochondria as well as the translocation of cytochrome c and AIF from mitochondria to the nucleus in early apoptosis. In cells with fragmented chromatin (i.e., in late apoptosis), the immunolabeling for AIF appeared to be distinct from chromatin, being mainly confined to mitochondria.

Poly(ADP-ribose) polymerase-1 (PARP-1) uses NAD(+) as a substrate to form ADP-ribose. During apoptosis, caspases cleave PARP-1 to avoid excessive NAD consumption. Because PARP-1 is a key regulator of the activity of DNases involved in caspase-dependent apoptosis, its cleavage is required to promote DNA degradation. To explore the situation in caspase-independent cell death, we investigated the effect of PARP-1 on the acid endonuclease leukocyte elastase inhibitor (LEI)-derived DNase II (L-DNase II). We found for the first time an association between PARP-1 and LEI/L-DNase II. Unexpectedly, we observed that LEI influenced the automodification of PARP-1.

A large complex of proteins, called CENPs, are associated with centromeric DNA. Some of them exhibit a cell cycle-related expression (e.g., CENP-E and -F) and are required for the transition from interphase to mitosis, whereas constitutive proteins (e.g., CENP-A, -B, -C, -G, and -H) reside permanently at the centromere and are essential for the correct kinetochore assembly. Poly(ADP-ribose) polymerase-1 (PARP-1), which plays an active role in many basic processes, was described as a possible regulator of CENPs. By multicolor immunofluorescence we therefore analyzed the distribution of PARP-1 and its interaction with CENP-B, -E, and -F during mitosis and apoptosis.

Vacuolar-H(+)-ATPase plays a critical role in the cellular balance of protons, thus regulating intracellular pH and contributing to apoptosis resistance, drug resistance, and invasive and metastatic behavior of cells. NiK-12192, a vacuolar-H(+)-ATPase inhibitor, caused a reduction in the volume and/or acidity of lysosomes, a polarization of alphavbeta5 integrin distribution, and a number of floating live cells, whereas signs of apoptosis appeared only after 72 h of treatment. In conclusion, NiK-12192, by affecting vacuolar- H(+)-ATPase activity (and intracellular pH), causes a modification of structures crucial for cell adhesion and induces cell death, likely by a modality involving an anoikis-mediated apoptosis.

Poly(ADP-ribose) polymerase-1 (PARP-1) is an important regulator of apoptosis. Its over-activation at the onset of apoptosis can inhibit the action of apoptotic endonucleases like caspase-activated DNase and DNAS1L3. Therefore, controlled PARP-1 proteolysis during caspase-dependent apoptosis is considered essential to promote DNA degradation. Yet, little is known about the interplay of PARP-1 and endonucleases that operate during caspase-independent cell death. Here we show that in the long-term cultured HeLa cells which undergo caspase-independent death, PARP-1 co-immunoprecipitates with leukocyte elastase inhibitor-derived DNase II (L-DNase II), an acid DNase implicated in this death pathway and activated by serine proteases. Our results indicate that, despite having putative poly(ADP-ribose)-acceptor sites, LEI/L-DNase II is neither significantly poly(ADP-ribosyl)ated nor inhibited by PARP-1 during caspase-independent apoptosis. Unexpectedly, caspase-independent apoptosis induced by hexa-methylene amiloride, LEI/L-DNase II can activate PARP-1 and promote its auto-poly(ADP-ribosyl)ation, thus inhibiting PARP-1 activity. Moreover, overexpression of LEI blocks the pro-survival effect of PARP-1 in this model of cell death. Our results provide the original evidence for a new mechanism of PARP-1 activity regulation in the caspase-independent death pathway involving LEI/L-DNase II.

Photosensitization of tumor cells after incubation with Rose Bengal acetate (RB-Ac) induces multiple organelle photodamage followed by apoptotic cell death. We used immunocytochemical techniques in multicolor fluorescence microscopy to elucidate whether this occurs through the simultaneous activation of different apoptotic pathways, in HeLa cells. We detected in situ the activated forms of caspases 9 and 3, and the translocation from the mitochondria to the nucleus of the apoptosis inducing factor; DNA electrophoretic techniques were also used to assess the occurrence of nuclear DNA cleavage into either high- or low-molecular-weight fragments. Both the caspase-dependent and caspase-independent apoptotic pathways are activated. The genomic DNA is degraded into high molecular weight molecules only, without the formation of oligonucleosome-sized fragments. The ability of RB-Ac to induce the simultaneous release of apoptogenic signals from different photodamaged organelles makes it an especially powerful cytotoxic agent.

Buthionine sulfoximine (BSO) is a well-known inhibitor of glutathione synthesis, producing slow glutathione (GSH) depletion and oxidative stress; some "responder" cells avoid BSO-induced death by trans-activating the prosurvival protein Bcl-2. Here we show that BSO activates a noncanonical, inhibitory NF-kappaB- and p65-independent NF-kappaB pathway via a multistep process leading to the up-regulation of Bcl-2. The slow BSO-induced GSH depletion allows separation of two redox-related phases, namely, early thiol disequilibrium and late frank oxidative stress; each phase contributes to the progressive activation of a p50-p50 homodimer. The early phase, coinciding with substantial thiol depletion, produces a cytosolic preparative complex, consisting of p50 and its interactor Bcl-3 linked by interprotein disulfide bridges. The late phase, coinciding with reactive oxygen species production, is responsible, probably via p38 activation, for nuclear targeting of the complex and trans-activation of Bcl-2.

In the apoptotic program of controlled cell dismantling, the most characteristic nuclear changes involve chromatin, which condenses and often collapses against the nuclear envelope in the form of crescents. A severe reorganization also occurs in ribonucleoprotein (RNP)-containing structures which are involved in the synthesis and processing of transcripts: already during early apoptosis, the nucleoplasmic RNPs (namely, perichromatin fibrils, perichromatin granules, and interchromatin granules) coalesce in the interchromatin space where they associate with segregated nucleolar components, to ectopically form fibro-granular heterogeneous clusters. This was found to occur in cell systems in vivo and in cultured cell lines, after different apoptogenic stimuli. These RNP aggregates we have called heterogeneous ectopic RNP-derived structures (HERDS) move from the nucleus to the cytoplasm, and may be found in apoptotic bodies, in late apoptosis. Immunolabeling experiments demonstrated that several other proteins which are normally located inside the nucleus also move into the cytoplasm, during apoptosis, independently from HERDS. Apoptotic cells have been suggested to be a powerful source of nuclear auto-antigens, which are produced by the partial proteolytic or nucleolytic cleavage of a wide variety of nuclear substrates. In the presence of defective phagocytosis (or when massive apoptosis overwhelms the clearance capability of the tissue scavenger cells), the disposal of apoptotic cells becomes insufficient and unphagocytosed late apoptotic cells may accumulate in the tissue where they may be engulfed by antigen-presenting cells (such as dendritic cells); an autoimmune response may thus be elicited, by which apoptosis-derived auto-antigens are recognized and presented to the immune system.

In mammalian cells, the H2AX histone is rapidly phosphorylated upon the induction of DNA double strand breaks and promotes their repair, which is required for preserving genomic integrity. Etoposide is an inhibitor of DNA topoisomerase II, which causes DNA breaks and induces H2AX phosphorylation. To elucidate whether H2AX may affect cellular sensitivity to etoposide, we studied the response to this agent in immortalized embryonic fibroblasts derived from H2AX knockout mice. Clonogenic assays in cells treated with the drug revealed a greater sensitivity of H2AX null cells compared to wild-type cells, possibly due to the persistence of a higher number of DNA breaks, as detected with the comet assay. In both cell lines, etoposide induced micronuclei formation and nuclear fragmentation; however, in H2AX deficient cells nuclear fragmentation was observed at a lower drug concentration. Flow cytometric analysis showed that etoposide induced a G2/M cell cycle arrest in both cell lines, which occurred at lower drug concentrations in H2AX deficient cells. G2/M arrest was paralleled by an accumulation of cyclin A and cyclin B1, suggesting that treated cells are not able to complete cell cycle correctly and undergo cell death. Taken together, our observations suggest that H2AX takes part to the cellular response to etoposide and confirm its role in the maintenance of genome stability.

The cell-cycle inhibitor p21(CDKN1A) has been suggested to directly participate in DNA repair, thanks to the interaction with PCNA. Yet, its role has remained unclear. Among proteins interacting with both p21 and PCNA, the histone acetyltransferase (HAT) p300 has been shown to participate in DNA repair. Here we report evidence indicating that p21 protein localizes and interacts with both p300 and PCNA at UV-induced DNA damage sites. The interaction between p300 and PCNA is regulated in vivo by p21. Indeed, loss of p21, or its inability to bind PCNA, results in a prolonged binding to chromatin and an increased association of p300 with PCNA, in UV-irradiated cells. Concomitantly, HAT activity of p300 is reduced after DNA damage. In vitro experiments show that inhibition of p300 HAT activity induced by PCNA is relieved by p21, which disrupts the association between recombinant p300 and PCNA. These results indicate that p21 is required during DNA repair to regulate p300 HAT activity by disrupting its interaction with PCNA.

Poly(ADP-ribosylation) is a post-translational modification of proteins with a multifunctional role in basic processes, such as DNA repair, transcription, cell proliferation and death. For decades, this reaction was studied by a small community of researchers, devoted to the identification of signals promoting the conversion of the coenzyme NAD into poly(ADP-ribose) and the further degradation of the polymers. The development of specific inhibitors and the discovery of a superfamily of poly(ADP-ribosylating) enzymes have recently conferred a higher importance to this process. The main features of poly(ADP-ribosylation) together with new research perspectives will be discussed.

The new arylthioindole (ATI) derivatives 10, 14-18, and 21-24, which bear a halogen atom or a small size ether group at position 5 of the indole moiety, were compared with the reference compounds colchicine and combretastatin A-4 for biological activity. Derivatives 10, 11, 16, and 21-24 inhibited MCF-7 cell growth with IC50 values <50 nM. A halogen atom (14-17) at position 5 caused a significant reduction in the free energy of binding of compound to tubulin, with a concomitant reduction in cytotoxicity. In contrast, methyl (21) and methoxy (22) substituents at position 5 caused an increase in cytotoxicity. Compound 16, the most potent antitubulin agent, led to a large increase (56%) in HeLa cells in the G2/M phase at 24 h, and at 48 h, 26% of the cells were hyperploid. Molecular modeling studies showed that, despite the absence of the ester moiety present in the previously examined analogues, most of the compounds bind in the colchicine site in the same orientation as the previously studied ATIs. Binding to beta-tubulin involved formation of a hydrogen bond between the indole and Thr179 and positioning of the trimethoxy phenyl group in a hydrophobic pocket near Cys241.

The proto-oncogene c-myc is involved in multiple cell pathways with opposite effects on cell outcome of death or proliferation. It has been proposed that these different roles depend on the sequestration of c-Myc protein in cellular compartments and/or its phosphorylation. We speculated that subcellular localization of c-Myc protein and of its phosphorylated form (P-c-Myc) could have a role in the different response to paclitaxel (PTX) in two prostate carcinoma cell lines, PC3 and DU145, which undergo either multinucleation or c-myc-dependent apoptosis, respectively. c-myc is amplified only in PC3, but a similar extent of c-Myc phosphorylation was observed in both cell lines after PTX treatment. We found that PTX-induced upregulation of c-myc in DU145 cells, not occurring in PC3 cells, cannot be ascribed to a different protein localization, and that a comparable c-Myc and P-c-Myc nuclear translocation occurs in both cell lines after drug treatment. Thus, subcellular localization of c-Myc and P-c-Myc is not crucial in determining the mode of cell death in these prostate carcinoma cell lines.

It has been reported that cells with ectopic expression of telomerase are more resistant to apoptotic cell death than their normal counterpart. However, controversial results were obtained when the cellular response to oxidative stress was analyzed. The present research was therefore aimed at defining the effect of the oxidative stress induced by tert-butylhydroperoxide (tBOOH) and 2-deoxy-D-ribose (D-ribose) in human fibroblasts from a centenarian (cen3) and, in parallel, on the same cells after telomerase immortalization (cen3tel cells). By studying different parameters of apoptosis in situ (i.e., chromatin condensation, phosphatidylserine externalization, and DNA fragmentation), we found that both tBOOH and D-ribose induce apoptosis to a greater extent in cen3 than in cen3tel cells, suggesting a protective role of telomerase toward apoptotic death. However, monitoring the cell number during treatment with the drugs, we found a decrease in cell number; since this reduction was lower in cen3 fibroblasts compared to cen3tel cells, it is likely that telomerase does not fully protect cells from drug toxicity.

Background: A C-->A polymorphism within the CDH1 (E-cadherin) promoter seems to be associated with a reduced efficiency of gene transcription in vitro. Due to the crucial role of E-cadherin in epithelia, tissue-specific effect of C-->A change on CDH1 transcription was tested and a case-control study was performed on patients affected with epithelial tumors., Patients and Methods: The -178/+93 CDH1 region containing either C or A nucleotide was inserted upstream of the Luciferase reporter gene in the pGL-2 vector, and the construct activity was assessed by transient transfection assay in HeLa and HCT116 cells., Results: A significantly lower activity for pGL-2A was found compared to pGL-2C, both in HeLa (54% decrease) and in HCT116 (67% decrease) cells. Genotyping of 246 controls and 505 patients affected with breast, gastric, colorectal, cervical and endometrial cancers demonstrated an association between the A allele and an increased risk of colorectal, gastric and endometrial tumors (1.66-, 1.81- and 2.35-fold, respectively)., Conclusion: Our data support the notion that the A allele may act as a low-penetrance cancer susceptibility gene.

The nucleolus may undergo disassembly either reversibly during mitosis, or irreversibly in apoptosis, thus allowing the redistribution of the nucleolar proteins. We investigated here by immunocytochemistry the fate of three representative proteins, namely phosphorylated c-Myc, fibrillarin and Ki-67, and found that they behave independently in both processes: they relocate in distinct compartments during mitosis, whereas during apoptosis they may either be cleaved (Ki-67) or be extruded into the cytoplasm with a different kinetics and following an ordered, non chaotic program. The separation of these nucleolar proteins which occurs in early apoptotic nuclei continues also in the cytoplasm, and culminates in the final formation of apoptotic blebs containing different nucleolar proteins: this evidence confirms that the apoptotic bodies may be variable in size, content and surface reactivity, and include heterogeneous aggregates of nuclear proteins and/or nucleic acids.

Proliferating cell nuclear antigen (PCNA) is a homotrimeric protein adopting a ring structure that may encircle DNA. In this form, PCNA functions as a sliding platform to which different types of catalytic and regulatory proteins are tethered to perform DNA transactions such as replication and repair synthesis, methylation, chromatin assembly and remodeling, as well as sister chromatid cohesion. In addition, PCNA coordinates DNA metabolism with cell cycle progression by interacting with cyclins, cyclin-dependent kinases (CDK), and CDK inhibitors. PCNA participates in different pathways of DNA repair, including nucleotide and base excision repair, as well as mismatch repair, by interacting with proteins involved in these processes. A fundamental step in DNA repair involves the transition of PCNA from a freely soluble nucleoplasmic form, to a chromatin-bound form associated with repair sites. This chapter describes biochemical and immunofluorescence methods for detection of the chromatin-bound form of PCNA involved in DNA repair. Cellular fractionation and nuclear extraction procedures are provided for Western blot analysis, as well as for protein-protein interaction studies. An in situ extraction protocol is described for immunostaining, fluorescence microscopy and flow cytometric analyses of nuclear localization, and cell cycle distribution of PCNA associated with DNA repair sites.

Apoptosis occurring in atherosclerotic lesions has been suggested to be involved in the evolution and the structural stability of the plaques. It is still a matter of debate whether apoptosis mainly involves vascular smooth muscle cells (vSMCs) in the fibrous tissue or inflammatory (namely foam) cells, thus preferentially affecting the cell-poor lipid core of the atherosclerotic plaques. The aim of the present investigation was to detect the presence of apoptotic cells and to estimate their percentage in a series of atherosclerotic plaques obtained either by autopsy or during surgical atherectomy. Apoptotic cells were identified on paraffin-embedded sections on the basis of cell nuclear morphology after DNA staining and/or by cytochemical reactions (TUNEL assay, immunodetection of the proteolytic poly (ADP-ribose) polymerase-1 [PARP-1] fragment); biochemical procedures (identifying DNA fragmentation or PARP-1 proteolysis) were also used. Indirect immunofluorescence techniques were performed to label specific antigens for either vSMCs or macrophages (i.e., the cells which are most likely prone to apoptosis in atherosclerotic lesions): the proper selection of fluorochrome labeling allowed the simultaneous detection of the cell phenotype and the apoptotic characteristics, by multicolor fluorescence techniques. Apoptotic cells proved to be less than 5% of the whole cell population, in atherosclerotic plaque sections: this is, in fact, a too low cell fraction to be detected by widely used biochemical methods, such as agarose gel electrophoresis of low-molecular-weight DNA or Western-blot analysis of PARP-1 degradation. Most apoptotic cells were of macrophage origin, and clustered in the tunica media, near or within the lipid-rich core; only a few TUNEL-positive cells were labeled for antigens specific for vSMCs. These results confirm that, among the cell populations in atherosclerotic plaques, macrophage foam-cells are preferentially involved in apoptosis. Their death may decrease the cell number in the lipid core and generate a possibly defective apoptotic clearance: the resulting release of matrix-degrading enzymes could contribute to weakening the fibrous cap and promote the plaque rupture with the risk of acute ischemic events, while increasing the thrombogenic pultaceous pool of the plaque core.

Actinomycin D (AMD) inhibits DNA-dependent RNA polymerases and its selectivity depends on the concentration used; at very high concentrations it may also induce apoptosis. This study investigates the effects of different concentrations (0.01 to 1 microg/ml) of AMD on RNA transcription and maturation and on the organization of nuclear ribonucleoproteins (RNPs), and their relationship with apoptosis induction. Human HeLa cells were used as a model system. At the lowest concentration used, AMD induced the segregation of the nucleolar components and impaired r-RNA synthesis, as revealed by the decreased immunopositivity for bromo-uridine incorporation and for DNA/RNA hybrid molecules. The synthesis of pre-mRNAs, on the contrary, was active, while the immunolabeling of snRNP proteins and of the SC-35 splicing factor strongly decreased on perichromatin fibrils (where they are involved in co-transcriptional splicing). This suggests that the post-transcriptional maturation of extranucleolar RNAs was also affected. Moreover, still in the absence of typical late morphological or biochemical signs of apoptosis (i.e. chromatin condensation), these cells displayed the early apoptotic features, i.e. the externalization of phosphatidylserine residues on the plasma membrane and propidium iodide exclusion in vivo. At the highest concentrations of AMD used, apoptosis massively occurred, with the typical morphological events (progressive chromatin condensation, clustering of snRNPs and SC-35 splicing factor, cell blebbing). However, transcription of hnRNAs was maintained in the residual areas of diffuse chromatin up to advanced apoptotic stages. The inhibition of rRNA synthesis and the defective pre-mRNA maturation seem to be part of the apoptotic process induced by AMD.

The aim of this paper is to review the data in the literature concerning ribonucleoprotein components during apoptosis, where a major rearrangement of RNPs takes place. In parallel with chromatin changes, the nucleoplasmic constituents (perichromatin fibrils; perichromatin granules; interchromatin granules and nuclear bodies) as well as the nucleoli aggregate into heterogeneous clusters called HERDS, in the interchromatin space. Later, these RNP-containing structures are extruded from the nucleus and leave the cell within cytoplasmic blebs. We propose also a role for HERDS as markers of irreversible transcriptional arrest.

Poly(ADP-ribosylation) is involved in DNA repair and replication, transcription, and cell death. For a long time, only one poly(ADP-ribosylating) enzyme was known, named ADPRT/PARP (EC 2.4.2.30). The recent discovery of a family of PARPs has provided a high degree of complexity in the field. Moreover, the finding that poly(ADP-ribosylation) is not confined to the nucleus but is also carried out by cytoplasmic enzymes supports the idea that it could regulate proteins localized in different cellular compartments. In this respect, a reappraisal of the literature on mitochondrial poly(ADP-ribosylation) could be useful, as well as a discussion of its relevance regarding the current "hot" view of poly(ADP-ribosylation) as a mediator of cell death.

Amplification of the c-MYC proto-oncogene is a frequent alteration in hormone refractory prostate carcinomas (HRPC). In an attempt to investigate the role of c-myc in the cellular response to paclitaxel (PTX), we used two HRPC cell lines, DU145 and PC3, characterised by different levels of the protein and by different behaviour in response to taxane. In both cell lines, PTX-induced cell death was a caspase-mediated apoptosis. In DU145 cells, PTX induced an early apoptotic response associated with upregulation of c-myc restricted to the G2/M cell population. This event appeared delayed in the presence of c-myc antisense (AS-c-myc), suggesting an upstream regulation of the protein expression. In addition, the antisense approach provided evidence of an involvement of c-myc in the apoptotic response to the taxane. In contrast, in PC3 cells, the overexpressed c-myc was not modulated by drug-treatment and the addition of AS-c-myc did not affect the cell growth inhibition of PTX. In both cell lines, PTX-induced c-myc phosphorylation was concomitant with the mitotic arrest and not related to the modulation of the activation state of AKT and MAPK kinases. Our data indicate that the cellular response to PTX of HRPC cells can involve c-myc and suggest that its pro-apoptotic role is affected by the genetic background, thus supporting a complex and differentiated HRPC cell response to taxanes.

To investigate the mechanistic basis for the biological properties of anthocyanins, two aglycone anthocyanins [delphinidin (DY) and cyanidin (CY)] were used to examine their effects on cell cycle progression and on induction of apoptosis in human cancer cells (uterine carcinoma and colon adenocarcinoma cells) and in normal human fibroblasts. These compounds differ in the number and position of hydroxyl groups on the beta ring in the molecular structure. Cellular uptake of anthocyanins was confirmed by HPLC analysis and no metabolites were detected. The clonogenic assay showed that CY induces a dose-dependent growth inhibitory effect only in fibroblasts. This effect was confirmed by flow cytometric analysis, showing a significant reduction of cells in S phase. In contrast, DP inhibited cell growth in normal and tumour cell lines. This event is accompanied in fibroblasts by an accumulation of cells in the S phase suggesting a block in the transition from S to G2 phase. On the other hand, in tumour cell lines we observed a reduction of cells in G1 phase, paralleled by the appearance of a fraction of cells with a hypodiploid DNA content, thus demonstrating an apoptotic effect by DP. The occurrence of apoptosis induced by DP was confirmed by morphological and biochemical features, including nuclear condensation and fragmentation, annexin V staining, DNA laddering and poly(ADP-ribose) polymerase-1-proteolysis. Furthermore, the mitochondrial membrane potential of apoptotic cells after treatment with DP was significantly lost. The different effects exerted by DP as compared with CY suggest that the presence of the three hydroxyl groups on the beta ring in the molecular structure of DP may be important for its greater biological activity.

Telomeres are specialized high-order chromatin structures that cap the ends of eukaryotic chromosomes. In vertebrates, telomeric DNA is composed of repetitions of the TTAGGG hexanucleotide, is bound to a set of specific proteins, and is elongated by the reverse transcriptase enzyme telomerase. Telomerase activity is promptly detected in cells with an indefinite replicative potential, such as cancer cells, while is almost undetectable in normal cells, which are characterized by a limited life span. Mounting evidence indicates that the maintenance of telomere integrity and telomerase protect cells from apoptosis. Disruption of the telomere capping function and (or) telomerase inhibition elicit an apoptotic response in cancer cells, while restoration of telomerase activity in somatic cells confers resistance to apoptosis. The possible mechanisms linking telomeres, telomerase and apoptosis are discussed in this review, together with the impact of this field in anticancer research.

Paclitaxel affects microtubule stability by binding to beta-tubulin, thus leading to cell accumulation in the G(2)/M phase, polyploidization, and apoptosis. Because both cell proliferation and apoptosis could be somehow regulated by the protooncogene c-myc, in this work we have investigated whether the c-myc amplification level could modulate the multiple effects of paclitaxel. To this aim, paclitaxel was administered to SW613-12A1 and -B3 human colon carcinoma cell lines (which are characterized by a high and low c-myc endogenous amplification level, respectively), and to the B3mycC5 cell line, with an enforced exogenous expression of c-myc copies. In this experimental system, we previously demonstrated that a high endogenous/exogenous level of amplification of c-myc enhances serum deprivation- and DNA damage-induced apoptosis. Accordingly, the present results indicate that a high c-myc amplification level potentiates paclitaxel cytotoxicity, confers a multinucleated phenotype, and promotes apoptosis to a great extent, thus suggesting that c-myc expression level is relevant in modulating the cellular responses to paclitaxel. We have recently shown in HeLa cells that the phosphorylated form of c-Myc accumulates in the nucleus, as distinct nucleolar and extranucleolar spots; here, we demonstrated that, after the treatment with paclitaxel, phosphorylated c-Myc undergoes redistribution, becoming diffused in the nucleoplasm.

We addressed the analysis of the physical and functional association of proliferating cell nuclear antigen (PCNA), a protein involved in many DNA transactions, with poly(ADP-ribose) polymerase (PARP-1), an enzyme that plays a crucial role in DNA repair and interacts with many DNA replication/repair factors. We demonstrated that PARP-1 and PCNA co-immunoprecipitated both from the soluble and the DNA-bound fraction isolated from S-phase-synchronized HeLa cells. Immunoprecipitation experiments with purified proteins further confirmed a physical association between PARP-1 and PCNA. To investigate the effect of this association on PARP-1 activity, an assay based on the incorporation of radioactive NAD was performed. Conversely, the effect of PARP-1 on PCNA-dependent DNA synthesis was assessed by a DNA polymerase delta assay. A marked inhibition of both reactions was found. Unexpectedly, PARP-1 activity also decreased in the presence of p21waf1/cip1. By pull-down experiments, we provided the first evidence for an association between PARP-1 and p21, which involves the C-terminal part of p21 protein. This association was further demonstrated to occur also in vivo in MNNG (N-methyl-N'-nitro-N-nitrosoguanidine)-treated human fibroblasts. These observations suggest that PARP-1 and p21 could cooperate in regulating the functions of PCNA during DNA replication/repair.

Poly(ADP-ribose)polymerase (PARP-1) and poly(ADP-ribose)glycohydrolase (PARG) are responsible for the transient poly(ADP-ribosyl)ation of proteins in eukaryotic cells. This biochemical reaction plays an active role in DNA replication and repair, transcription, cell differentiation and death. The aim of this study was to investigate the levels and the sub-cellular distribution of such enzymes in rat germinal cells at different stages of differentiation, i.e. in primary spermatocytes and round spermatids, representing meiotic and post-meiotic cells, respectively. The determination of the level of PARP-1 mRNA and protein revealed its higher expression in primary spermatocytes, thus implying that PARP-1 is one of the meiotic genes whose expression is requested at the pachytene phase of the meiosis. We also demonstrated that rat germinal cells contain both the forms of PARG (i.e. of 110 and 60 kDa) so far described in somatic cells. In our experimental system, the large PARG was present and active mainly in the nuclear fraction of primary spermatocytes, whereas round spermatids showed a higher level of the 60 kDa PARG in the post-nuclear fraction. Collectively, our data show a different expression level of PARP-1 and a different endocellular distribution of PARG and suggest a role for the poly(ADP-ribose) turnover in distinct pathways in meiotic and post-meiotic germinal cells.

We have recently demonstrated that a high c-myc endogenous amplification level confers an apoptosis-prone phenotype to serum-deprived colon carcinoma SW613-S cells. The aim of this study was to gain new insights into the features of c-myc-dependent apoptosis, by extending our analysis to different apoptogenic stimuli. The study was carried out on clones, derived from the human colon carcinoma SW613-S cell line, which harbor different levels of endogenous c-myc amplification, and on isogenic cell lines with an enforced c-myc overexpression. Our results indicate that cells with endogenous or transfected exogenous c-myc overexpression (SW613-12A1 and -2G1mycP2Tu1 cell lines, respectively), activate the apoptotic machinery in response to the treatment with etoposide, doxorubicin and vitamin D3, which induce apoptosis through the death receptor Fas. The low levels of c-myc expression present in SW613-B3 and -B3mycC5, seem to be unable to activate Fas-mediated apoptosis, thus suggesting that only a high c-myc expression can bypass the lack of Fas receptor. Apoptosis induction mediated by DNA damage and long-term culture was independent of c-myc expression. A pathway of apoptosis characterized by the activation of the enzyme L-DNase II, was observed in both 12A1 and B3 cell lines.

Cell death by apoptosis requires a precise plan of destruction of DNA and proteins. In this paper, we review the current knowledge on the different DNA-degrading enzymes which are activated in apoptotic cells. The activation of DNases by upstream proteases is also discussed.

Poly(ADP-ribosylation) is a post-translational modification of proteins playing a crucial role in many processes, including DNA repair and cell death. The best known poly(ADP-ribosylating) enzyme, PARP-1, is a DNA nick sensor and uses betaNAD(+) to form polymers of ADP-ribose which are further bound to nuclear protein acceptors. To strictly regulate poly(ADP-ribose) turnover, its degradation is assured by the enzyme poly(ADP-ribose) glycohydrolase (PARG). During apoptosis, PARP-1 plays two opposite roles: its stimulation leads to poly(ADP-ribose) synthesis, whereas caspases cause PARP-1 cleavage and inactivation. PARP-1 proteolysis produces an 89 kDa C-terminal fragment, with a reduced catalytic activity, and a 24 kDa N-terminal peptide, which retains the DNA binding domains. The fate and the possible role of these fragments during apoptosis will be discussed.

The short-chain lipid hydroperoxide analogue tert-butylhydroperoxide induces peroxynitrite-dependent and -independent DNA single strand breakage in PC12 cells. U937 cells that do not express constitutive nitric oxide synthase respond to tert-butylhydroperoxide treatment with peroxynitrite-independent DNA cleavage. Under experimental conditions leading to equivalent strand break frequencies, the analysis of poly(ADP-ribose) polymerase activity showed an increase in PC12 cells but not in U937 cells. The enhanced poly(ADP-ribose) polymerase activity observed in PC12 cells was paralleled by a significant decline in NAD+ content and both events were prevented by treatments suppressing formation of peroxynitrite. Although DNA breaks were rejoined at similar rates in the two cell lines, an inhibitor of poly(ADP-ribose) polymerase delayed DNA repair in PC12 cells but had hardly any effect in U937 cells. The results obtained using the latter cell type were confirmed with an additional cell line (Chinese hamster ovary cells) that does not express nitric oxide synthase. Collectively, our data suggest that tert-butylhydroperoxide-induced peroxynitrite-independent DNA strand scission is far less effective than the DNA cleavage generated by endogenous peroxynitrite in stimulating the activity of poly(ADP-ribose) polymerase.