First evidence of a functional interaction between DNA quadruplexes and poly(ADP-ribose) polymerase-1.

We discovered that the abundant human nuclear protein poly(ADP-ribose) polymerase-1 (hPARP-1) binds to intramolecular DNA quadruplexes in vitro with high affinity and with a stoichiometry of two proteins for one quadruplex. Using an enzymatic assay, we have shown that hPARP-1 gets catalytically activated upon binding to G-quadruplexes localized at the c-kit promoter and human telomere regions. This is the first example of a truly functional quadruplex-protein interaction, which has possible implications in understanding hPARP-1 mediated mechanisms of transcription regulation and telomere end protection.

DNA-binding properties of poly(ADP-ribose) polymerase: a target for anticancer therapy.

Poly(ADP-ribose) polymerization is a unique post-translation protein modification that utilizes an ADP-ribose moiety from NAD+ to form long and branched polymers attached via glutamic acid residues to nuclear acceptor proteins. The corresponding enzyme, poly(ADP-ribose) polymerase (PARP-1), is a zinc finger-containing protein, which allows PARP-1 binding to either double- or single-strand DNA breaks. The catalytic activity of PARP-1 is strictly dependent on the presence of strand breaks in DNA, and is modulated by the level of automodification. PARP-1 is regarded as an intracellular sensor for DNA strand breaks, and its function has been implicated in cellular processes that require DNA cleavage and rejoining reactions, such as DNA replication, recombination and repair. Recent studies have also implicated PARP-1 in the regulation of gene expression through modification of transcription factors by poly(ADP-ribosyl)ation or its direct binding to gene-regulating DNA sequences. The latter is attributable to PARP's ability to recognize and bind to various structural discontinuities in the DNA duplex in the absence of DNA strand breaks, such as three- or four-way junctions, bent DNA, and base unpaired regions. Cumulatively, these findings indicate that PARP-1 plays a pivotal role in the maintenance of the genome integrity during the normal functioning of eukaryotic cells as well as in the cellular responses to DNA damage, and that PARP-DNA interactions are indispensable for PARP function. This review summarizes the data on DNA-binding properties of PARP-1 and relates them to the development of strategies for sensitizing tumor cells to genotoxic treatments.

Poly(ADP-ribose) polymerase in DNA damage-response pathway: implications for radiation oncology.

Poly(ADP-ribose) polymerase (PARP) catalyzes the transfer of successive units of ADP-ribose moiety from NAD(+) covalently to itself and other nuclear acceptor proteins. PARP is a zinc finger-containing protein, allowing the enzyme to bind to either double- or single-strand DNA breaks without any apparent sequence preference. The catalytic activity of PARP is strictly dependent on the presence of strand breaks in DNA and is modulated by the level of automodification. Data from many studies show that PARP is involved in numerous biological functions, all of which are associated with the breaking and rejoining of DNA strands, and plays a pivotal role in DNA damage repair. Recent advances in apoptosis research identified PARP as one of the intracellular "death substrates" and demonstrated the involvement of polymerase in the execution of programmed cell death. This review summarizes the biological effects of PARP function that may have a potential for targeted sensitization of tumor cells to genotoxic agents and radiotherapy. Int. J. Cancer (Radiat. Oncol. Invest.) 90, 59-67 (2000).

Inhibition of homologue of Slimb (HOS) function sensitizes human melanoma cells for apoptosis.

Homologue of Slimb (HOS)/beta-transducin repeats containing proteins up-regulate nuclear factor kappaB activity by targeting its inhibitor (IkappaB) for ubiquitination and subsequent degradation. We investigated whether inhibition of HOS function may modulate apoptosis in human melanoma cells. Forced expression of the dominant negative HOSdeltaF construct inhibited IkappaB degradation and led to sensitization of melanoma cells to apoptosis induced by tumor necrosis factor alpha with cycloheximide, as well as by cisplatin and ionizing and UV irradiation. These data indicate that HOS plays an important role in controlling the IkappaB-dependent apoptotic pathways in human melanoma.

Regulation of the human poly(ADP-ribose) polymerase promoter by the ETS transcription factor.

Ewing's sarcoma (EWS) cells accumulate elevated steady-state levels of poly (ADP-ribose) polymerase (PARP) mRNA and protein. To understand the molecular mechanisms underlying PARP upregulation, we cloned and analysed the 5'-flanking region of the PARP gene from EWS cells. Nucleotide sequence analysis demonstrated no variations in the PARP promoter region in EWS cells. The PARP promoter encompasses multiple binding motifs for the ETS transcription factor. We have also observed that there is a coordinated up-regulation of the expression of both PARP and ETS1, relative to cells of other human tumor types expressing lower levels of PARP. Transient co-expression of ETS1 in EWS cells resulted in a strong enhancement of PARP-promoter activity. The participation of ETS in the regulation of PARP gene expression was further demonstrated in EWS cells stably transfected with Ets1 antisense cDNA constructs. Antisense-mediated down-regulation of endogenous ETS1 resulted in the inhibition of PARP expression in EWS cells, and sensitized these cells to ionizing radiation. These data provide support for ETS regulation of PARP expression levels, and implicate ETS transcription factors in the radiation response of EWS cells.

Protein changes associated with ionizing radiation-induced apoptosis in human prostate epithelial tumor cells.

Ionizing radiation (IR) is an important component in the therapy of localized prostate cancer. Identification of protein alterations during IR-induced apoptosis prostate cancer cells is an important step toward understanding the new metabolic status of the dying cell. In the present study, we report changes in protein profile that define the execution phase of the apoptotic response in the in vitro model of tumorigenic radiation-transformed SV40-immortalized human prostate epithelial cells (267B1-XR), induced to undergo programmed cell death by IR. We employed an approach that involves use of analytical two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) coupled with Western blotting with specific antisera. Our results point out that apoptotic cells experience significant reduction in the levels of the intermediate filament proteins, keratins-18, 19, vimentin and the associated 14-3-3 adapter proteins. At the same time, molecular chaperones such as glucose-regulated protein 94, calreticulin, calnexin, and protein disulfide isomerase exhibit marked accumulation in these dying cells. The present data indicate that apoptosis-associated processes in prostate epithelial cells include solubilization of the rigid intermediate filament network by specific proteolysis as well as increased levels of endoplasmic reticulum (ER) proteins with chaperone functions.

Differential expression of stathmin during neoplastic conversion of human prostate epithelial cells is reversed by hypomethylating agent, 5-azacytidine.

In a variety of human tumor tissues, including those of prostate and breast, CpG hypermethylation represents one of the mechanisms downregulating the expression of specific proteins, including tumor suppressor proteins. Using 267B1-XR cells generated by ionizing radiation-induced transformation of epithelial cells, derived from neonatal human prostate and immortalized by SV40 (267B1), we now report markedly low levels of expression of the cytoplasmic phosphoprotein stathmin, in addition to several proteins of the actin microfilaments and intermediate filaments that characterize the altered phenotype. Stathmin is emerging as a relay protein integrating signals from diverse pathways during differentiation and neoplastic progression. In this in vitro prostate carcinogenesis model system, where loss of specific-protein expression is a major feature of the transformed 267B1-XR cells, we employed 5-azacytidine treatment followed by 2D-PAGE to reveal if experimental genomic hypomethylation reinstated the levels of any of the differentially expressed proteins. Our data suggest that stathmin represents one such example.

Intermediate filament proteins during carcinogenesis and apoptosis (Review).

The intermediate filament network spreading from the cell periphery to the nucleus forms dynamic linkages between nuclear matrix, actin microfilaments, and the extracellular matrix. The six different types (types I-VI) of IF proteins consisting of nearly 50 different proteins form at least nine different kinds of filaments depending on the tissue types: keratins, lamins, vimentin, desmin, neurofilaments, peripherin, alpha-internexin, glial fibrillary acidic protein and nestin. Their tissue specific expression in normal cells and differential expression/assembly in neoplasia has been of immense value in tumor diagnosis. At the same time, recent in vitro studies point out that keratins, lamins and vimentin are subject to caspase-mediated proteolysis in an apoptosis-related manner. We reviewed the experimentally demonstrated P4-P1 motif specificities of caspases in the selection of substrates in the IF protein family. In addition, we provided clues to possible cleavage of additional IF proteins during programmed cell death, based on acceptable cut site motifs indicated by searches using the PIR protein sequence database. The present review concludes with presentation of evidence on the emerging roles of IFs in association with intermediate filament associated proteins in the dynamic remodeling of the cell during development of neoplastic phenotype and execution of apoptosis.

Identification of keratins 18, 19 and heat-shock protein 90 beta as candidate substrates of proteolysis during ionizing radiation-induced apoptosis of estrogen-receptor negative breast tumor cells.

Induction of apoptosis in the estrogen-receptor negative MDA-MB-468 breast tumor cells has been demonstrated by treatments with cytotoxic agents and growth factors. Using these breast tumor cells, we studied ionizing radiation-induced apoptosis. 2D-PAGE of apoptotic cells indicated keratins 18, 19 and heat-shock protein 90 as candidate substrates of apoptosis-associated proteolysis. At the same time, a motif search revealed possible cleavage-sites in keratins 18, 19 (VEVD) and hsp-90 (DEED) that would yield polypeptides of molecular sizes observed experimentally by immunoblotting with specific antisera. This study provides evidence that the insoluble network of intermediate filament proteins of epithelial cells (keratins), and the associated proteins (heat-shock protein 90) constitute targets of caspase-mediated proteolysis during apoptosis triggered by ionizing radiation.

Apoptosis-associated proteolysis of vimentin in human prostate epithelial tumor cells.

Vimentin intermediate filaments (IF) are responsible for regulation of cell attachment and subcellular organization. Using an in vitro model system of human prostate epithelial cells (267B1-XR), we demonstrate that a series of vimentin proteolytic fragments represent some of the differentially expressed proteins in 2D-gel profiles of the apoptotic cells undergoing ionizing radiation-induced cell death. A caspase-sensitive motif search suggests that the type III IF protein (vimentin) is subject to proteolysis to promote the execution phase of apoptosis, in a manner similar to the well-established type V (lamins) and type I (keratins 18, 19) IF proteins. Furthermore, vimentin and a few of its derived polypeptides, reported to be specific to the apoptotic process, correspond to ubiquinated proteins, thus pointing to the complex interrelationships of protein ubiquination in solubilizing the IF network during apoptosis.

Sodium butyrate induces apoptosis and accumulation of ubiquitinated proteins in human breast carcinoma cells.

To investigate the possible relationship between apoptosis and the ubiquitin pathway we examined the patterns of ubiquitinated proteins in the human breast carcinoma MCF-7 cell line following induction of apoptotic death by sodium butyrate. Apoptosis in these cells was associated with internucleosomal DNA fragmentation and proteolytic cleavage of poly(ADP-ribose) polymerase. By dual in situ antiubiquitin immunofluorescence and chromatin DNA staining, we demonstrated that ubiquitin fluorescence was increased specifically in cells that underwent sodium butyrate-mediated apoptosis. The extent of ubiquitin incorporation into protein conjugates was examined in both adherent (not yet apoptotic) and floating (apoptotic) cell populations. We found that apoptotic cells exhibited enhanced intensity of ubiquitin-immunoreactive conjugates, whereas adherent cells did not. In addition, two-dimensional immunoblot analysis of proteins from apoptotic cells identified a set of isomeric ubiquitinated conjugates located at a pI range of 4. 2 - 4.6 and a Mr approximately of 30 kDa. These data indicate that the ubiquitin pathway may play a role in the sodium butyrate-induced apoptotic program in breast carcinoma cells.

Apoptosis of Ewing's sarcoma cells is accompanied by accumulation of ubiquitinated proteins.

Induction of apoptosis in Ewing's sarcoma cells by ionizing radiation is accompanied by accumulation of ubiquitinated proteins preferentially in the form of conjugates with M(r) greater than 75,000. Furthermore, enhanced antiubiquitin immunofluorescence was detected only in cells that underwent radiation-induced apoptosis, suggesting that the observed alterations in protein ubiquitination are specific to the apoptotic process. To determine the role of the proteasome in apoptosis-associated accumulation of ubiquitin-protein conjugates, we used lactacystin, a highly selective inhibitor of proteasome proteolytic activity. Exposure of Ewing's sarcoma cells to lactacystin resulted in accumulation of ubiquitinated proteins and activation of a Bcl-2-sensitive apoptotic pathways. The latter led to proteolytic cleavage of poly(ADP-ribose) polymerase and fragmentation of nuclear DNA. These findings suggest that proteasome function is required for apoptosis-specific accumulation of ubiquitinated proteins and indicate that functional disorder of the ubiquitin-proteasome system may play an important role in the apoptotic cell death pathway.

Inhibition of Raf-1 protein kinase by antisense phosphorothioate oligodeoxyribonucleotide is associated with sensitization of human laryngeal squamous carcinoma cells to gamma radiation.

BACKGROUND: Previous studies suggest a central role for Raf-1 protein kinase in mitogenic and radiation damage responsive signaling pathways. PURPOSE: Here we report that the expression and enzymatic activity of Raf-1 protein are inhibited in SQ-20B cells exposed to raf antisense oligodeoxyribonucleotide (As-ODNs) directed against the translation initiation site of human c-raf-1 cDNA. In contrast, treatment of SQ-20B cells with an equimolar concentration of raf sense oligodeoxy-ribonucleotide (S-ODNs) had no effect on the expression and activity of Raf-1. RESULTS: We have observed radiosensitization of raf As-ODNs-treated SQ-20B cells. The dose modifying factor of As-ODNs treatment was approximately 1.4. CONCLUSIONS: These studies demonstrate that raf As-ODNs is a DNA sequence-specific radiosensitizer that may have potential for use in the radiation therapy of cancers.

Differential induction of programmed cell death in CD8+ and CD4+ T cells by the B subunit of cholera toxin.

We have recently demonstrated that a short-term treatment of parental splenocytes with the B subunit of cholera toxin (CT-B) abrogates the development of acute GVHD in F1 hybrid mice transplanted with these cells. In order to obtain better insight into the mechanism of the action of CT-B, we studied the effect of CT-B on survival of purified murine T cells and their subsets. We show that treatment with B subunit stimulates apoptosis in T cells, detectable following incubation in vitro. Although apoptosis was noticed in both CD8+ and CD4+ T cell subsets, the treatment preferentially stimulates programmed cell death (PCD) in CD8+ population. Thus, immunosuppressive action of CT-B in vivo may be in part due to its ability to eliminate CD8+ T cells.

Radiation-induced apoptosis of Ewing's sarcoma cells: DNA fragmentation and proteolysis of poly(ADP-ribose) polymerase.

Ewing's sarcoma (ES) cells express high levels of poly(ADP-ribose) polymerase (PADPRP) and are responsive to killing by ionizing radiation. We have determined that ionizing radiation induced a pronounced but reversible G2-M phase cell cycle arrest that was maximum by 24 h after exposure. Following the release from this block, floating cells began to appear. These floating cells were shown to be apoptotic by flow cytometric and DNA fragmentation analyses. We found that apoptosis is a significant component of radiation-induced death in ES cells and that this is accomplished in conjunction with proteolytic cleavage of PADPRP. Two fragments of M(r) 25,000 and M(r) 29,000 containing the PADPRP DNA-binding domain were identified in floating (apoptotic) cells, whereas only the full-length M(r) 116,000 native protein was detected in adherent cells that retained DNA intact. These data are consistent with PADPRP cleavage being an early step in the apoptotic cascade of biochemical events in ES cells after ionizing radiation exposure.

Isolation and characterization of SpTRK, a gene from Schizosaccharomyces pombe predicted to encode a K+ transporter protein.

A novel gene, SpTRK, has been isolated from DNA of the fission yeast Schizosaccharomyces pombe (Sp) by hybridization to an oligodeoxyribonucleotide (oligo) probe designed from a sequence fully conserved between the potassium transporter genes TRK1 and TRK2 of Saccharomyces cerevisiae (Sc). SpTRK is a single-copy gene located on Sp chromosome I. Nucleotide sequence analysis of the cloned gene identified an open reading frame (ORF) with coding capacity for a protein of 833 amino acids (aa). The predicted SpTRK aa sequence showed a high level of conservation relative to the potassium transporters of Sc and Saccharomyces uvarum (Su), particularly within their transmembrane (TM) domains and in aa required for their ion transport functions. A single SpTRK transcript of about 2.7 kb is expressed at high levels in exponentially growing Sp cells, but it is downregulated in cells from stationary cultures.

In vitro effect of 5-fluorouracil, verapamil and hyperthermia on the human pancreatic adenocarcinoma cell line ASPC-1.

The purpose of this study was to assess the efficacy of verapamil (20 microM) and hyperthermia (42 degrees C) as modifiers of 5-fluorouracil (5-FU) used at different concentrations, in inhibiting the growth of human pancreatic adenocarcinoma cells. Combined verapamil and hyperthermia treatment significantly decreased cell count by 63.8% when compared to the control. Verapamil drastically enhanced the growth inhibitory effect of 5-FU at all concentrations. At high-concentration 5-FU (50 micrograms/ml), verapamil and hyperthermia had a synergistic growth-inhibitory effect and caused a significant decrease in cell count by 81.4% in comparison to the control. The modalities analyzed in this study require further investigation and have potential for clinical applicability to pancreatic cancer therapy in the future.

Is the NAD-poly (ADP-ribose) polymerase system the trigger in radiation-induced death of mouse thymocytes?

Agents that induce DNA strand breaks evoke a drop in the NAD content in mouse thymocytes. A decrease in the endogenous NAD content that occurs immediately after gamma-irradiation of thymocytes is entirely attributed to the activation of poly(ADP-ribosylation). The addition of 5 mM benzamide before irradiation prevents the postirradiation drop of the NAD level but has no effect on chromatin degradation and cell death. In contrast to liver nuclei, pre-incubation of mouse thymic nuclei with NAD had no effect on the subsequent chromatin endonucleolysis by Ca2+/Mg2+-dependent endonuclease. It is suggested that the NAD-poly(ADP-ribose) polymerase system is probably not the trigger in the radiation-induced programmed death of mouse thymocytes, but may merely be indicative of the radiation response of these cells.

Early postirradiation chromatin degradation in thymocytes.

The decrease in the average DNA size in thymocytes starts soon after in vivo irradiation and at approximately 45 min reaches a plateau, thereafter showing only minor changes up to 3 h. This fall in extent of chromatin cleavage coincides with the accumulation of 1.0-1.5 kb DNA fragments. Double-strand breaks generated by endonucleases are not randomly distributed along DNA but clustered in such a way that they give rise to fragments of 1-5 nucleosomes in size. Cycloheximide treatment partially inhibits nuclease activity in nuclear extracts isolated from thymus of irradiated mice. This suggest that DNA fragmentation is an early event in programmed death of thymocytes mediated by irradiation. The data indicate that it requires protein synthesis and that it precedes release of polydeoxyribonucleotides.