Internucleosomal DNA Cleavage Research Articles

Andrew David Hamilton Wyllie. 24 January 1944—26 May 2022

Andrew Wyllie graduated from the University of Aberdeen, becoming an academic pathologist in Aberdeen, Edinburgh and Cambridge. He was the co-discoverer of apoptotic cell death, having observed single cells dying following carcinogen exposure. Together with Alastair Currie and John Kerr, he realized the profound importance of this novel mode of cell death that showed a distinctive series of morphological changes, which he first described as a new cell death process. Wyllie and Currie introduced the term ‘apoptosis’ for this cell death process in a seminal paper in 1972. Another landmark discovery was of chromatin fragmentation in apoptosis, due to activation of an endogenous endonuclease that caused internucleosomal DNA cleavage (‘chromatin laddering’), which was the first biochemical mechanism of apoptosis described. He further characterized chromatin fragmentation in the 1980s, followed by investigations of cell surface changes to produce ‘eat-me’ signals to trigger rapid phagocytosis of the apoptotic cells and bodies, intracellular calcium ion signalling, caspase activation and other mechanisms of apoptosis. His cancer research helped identify the location of APC and generated his demonstration that apoptosis was regulated by oncogenes MYC and RAS and by tumour suppressor genes, such as TP53 . He showed how apoptosis occurred in response to DNA damage and was a key process influencing both carcinogenesis and tumour growth. Andrew made a major scientific observation that changed the understanding of how cells die in health and disease, although it took time for the scientific establishment to understand its fundamental importance. Andrew Wyllie is widely known as the ‘Father of Apoptosis’.

Cell-Free DNA Fragmentation Patterns in a Cancer Cell Line.

Unique bits of genetic, biological and pathological information occur in differently sized cell-free DNA (cfDNA) populations. This is a significant discovery, but much of the phenomenon remains to be explored. We investigated cfDNA fragmentation patterns in cultured human bone cancer (143B) cells using increasingly sensitive electrophoresis assays, including four automated microfluidic capillary electrophoresis assays from Agilent, i.e., DNA 1000, High Sensitivity DNA, dsDNA 915 and dsDNA 930, and an optimized manual agarose gel electrophoresis protocol. This comparison showed that (i) as the sensitivity and resolution of the sizing methods increase incrementally, additional nucleosomal multiples are revealed (hepta-nucleosomes were detectable with manual agarose gel electrophoresis), while the estimated size range of high molecular weight (HMW) cfDNA fragments narrow correspondingly; (ii) the cfDNA laddering pattern extends well beyond the 1–3 nucleosomal multiples detected by commonly used methods; and (iii) the modal size of HMW cfDNA populations is exaggerated due to the limited resolving power of electrophoresis, and instead consists of several poly-nucleosomal subpopulations that continue the series of DNA laddering. Furthermore, the most sensitive automated assay used in this study (Agilent dsDNA 930) revealed an exponential decay in the relative contribution of increasingly longer cfDNA populations. This power-law distribution suggests the involvement of a stochastic inter-nucleosomal DNA cleavage process, wherein shorter populations accumulate rapidly as they are fed by the degradation of all larger populations. This may explain why similar size profiles have historically been reported for cfDNA populations originating from different processes, such as apoptosis, necrosis, accidental cell lysis and purported active release. These results not only demonstrate the diversity of size profiles generated by different methods, but also highlight the importance of caution when drawing conclusions on the mechanisms that generate different cfDNA size populations, especially when only a single method is used for sizing.

Apoptosis Inducing Effects of Thymus Linearis Methanolic Extract in HCT-116 Cells and LC-MS Chemical Profiling of Its Active Constituents

Background:: Cancer is one of the major problems at present, to which vast research is being dedicated to find an effective remedy. Medicinal plants are endowed with numerous molecules that could be effective in multiple diseases including cancer. Thymus linearis, being rich in phenols, terpenoid, and flavonoids have the potential to provide anti-cancer entities. Methods:: The extracts of Thymus linearis were investigated for in vitro anticancer activity using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay on a panel of cancer cell lines. The cellular and nuclear morphology was studied using microscopic techniques. Agarose gel electrophoresis was used for DNA fragmentation analysis. Protein expression was determined by western blotting. LC-MS was used for phytochemical identification. Results:: Among all the extracts, Thymus linearis methanolic (TLM) extract was found to exhibit antiproliferative activity on cell lines to varying degrees. TLM was found to be most potent against HCT-116 with an IC50 of 158μg/ml after 48hrs treatment while being nontoxic to HEK-293 and FR-2 cells under similar concentrations. TLM decreased clonogenic potential of HCT-116 cells. It induced cell shrinkage, membrane blebbing and nuclear fragmentation characteristic of apoptotic in a dose dependent manner in HCT-116 cells. Prominent internucleosomal DNA cleavage was observed in HCT-116 cells after 48hrs TLM treatment. Western blot analysis revealed the up regulation of expression of Bax, caspases 9 and caspases 3 and downregulation of Bcl-2 proteins. The LC-MS data revealed the presence of Salvianolic acid H, Synparvolide C, Thymuside A and Jasmonic acid; 12-Hydroxy, O-β-D-glucopyranoside and polyphenolic flavonoids to which antiproliferative activity can be attributed. Conclusion:: The results suggest that Thymus linearis methanolic extract could be valuable source of anti-cancer agents.

Anticancer Potential of Damnacanthal and Nordamnacanthal from Morinda elliptica Roots on T-lymphoblastic Leukemia Cells

Background: This study reports on the cytotoxic properties of nordamnacanthal and damnacanthal, isolated from roots of Morinda elliptica on T-lymphoblastic leukaemia (CEM-SS) cell lines. Methods: MTT assay, DNA fragmentation, ELISA and cell cycle analysis were carried out. Results: Nordamnacanthal and damnacanthal at IC50 values of 1.7 μg/mL and10 μg/mL, respectively. At the molecular level, these compounds caused internucleosomal DNA cleavage producing multiple 180–200 bp fragments that are visible as a “ladder” on the agarose gel. This was due to the activation of the Mg2+/Ca2+-dependent endonuclease. The induction of apoptosis by nordamnacanthal was different from the one induced by damnacanthal, in a way that it occurs independently of ongoing transcription process. Nevertheless, in both cases, the process of dephosphorylation of protein phosphates 1 and 2A, the ongoing protein synthesis and the elevations of the cytosolic Ca2+ concentration were not needed for apoptosis to take place. Nordamnacanthal was found to have a cytotoxic effect by inducing apoptosis, while damnacanthal caused arrest at the G0/G1 phase of the cell cycle. Conclusion: Damnacanthal and nordamnacanthal have anticancer properties, and could act as potential treatment for T-lymphoblastic leukemia.

Apoptosis during folliculogenesis in pigs

The number of female germ cells in pig fetuses decreases by 70% between day 50 after mating and day 300 after birth. Approximately 55% of antral follicles undergo degeneration (atresia) except during the 3 days before oestrus, when only 15% of the follicles survive to ovulate. Apoptosis, a form of programmed cell death, is recognized as the mechanism of germ cell death and follicle atresia at all stages of folliculogenesis. The internucleosomal cleavage of genomic DNA caused by caspase-induced deoxyribonuclease activity was measured in pig granulosa cells by DNA fluorescence flow cytometry, densitometry of fluorescently labelled internucleosomal DNA fragments and immunohistochemical analysis of the 3' end labelling of deoxyribonuclease- nicked DNA on frozen tissue sections. Follicular atresia during the 3 days before oestrus is associated with a 60-70% decrease in the secretion of FSH. In granulosa cells, apoptosis is associated with decreased cell proliferation and reduced production of oestradiol and inhibin. In cultured pig granulosa cells, FSH and IGF-I are anti-apoptotic and a caspase inhibitor blocked apoptosis, thereby providing evidence of caspase activity. Oocytes in most follicles have resumed meiotic maturation; therefore, one role for apoptosis and follicle atresia may be to act as a barrier to ovulation of oocytes that have not remained in meiotic arrest.

An overview of apoptosis assays detecting DNA fragmentation.

Apoptosis has been recognized as a type of programmed cell death connected with characteristic morphological and biochemical changes in cells. This programmed cell death plays an important role in the genesis of a number of physiological and pathological processes. Thus, it can be very important to detect the signs of apoptosis in a study of cellular metabolism. The present paper provides an overview of methods often being used for detecting DNA fragmentation as one of the most specific findings in apoptosis. To date, three routine assays have been developed for detecting DNA fragmentation: DNA ladder assay, TUNEL assay, and comet assay. All these methods differ in their principles for detecting DNA fragmentation. DNA ladder assay detects the characteristic "DNA ladder" pattern formed during internucleosomal cleavage of DNA. Terminal deoxynUcleotidyl transferase Nick-End Labeling (TUNEL) assay detects DNA strand breaks using terminal deoxynucleotidyl transferase catalyzing attachment of modified deoxynucleotides on the DNA strand breaks. Comet assay can be used for detecting nucleus breakdown producing single/double-strand DNA breaks. The aim of this review is to describe the present knowledge on these three methods, including optimized approaches, techniques, and limitations.

Cell cycle arrest and apoptosis induced by enteroaggregative Escherichia coli in cultured human intestinal epithelial cells.

Enteroaggregative Escherichia coli (EAEC) is an emerging enteric pathogen causing diarrhoeal diseases in multiple epidemiological and clinical settings. However, understanding of the pathogenesis of the disease caused by this organism is still suboptimal. Studies have indicated that enteric bacteria induced cell cycle arrest and apoptosis in host intestinal epithelial cells might play a vital role in the pathogenesis caused by these organisms. In this study an attempt was made to assess EAEC-induced apoptosis and cell cycle modulation in human intestinal epithelial cell lines. INT-407 and HCT-15 cells were infected with EAEC-T8 (clinical isolate) as well as plasmid cured variant of EAEC-T8 (EAEC-pT8). Propidium iodide staining was done to select the time of infection and the incubation period of the infected culture. Apoptosis was further assessed in EAEC infected both the cell lines by annexin-V-FLUOS & propidium iodide, cell death detection ELISA, DNA strand breaks and microscopic analysis. Further, the DNA content of the EAEC-infected cells at different phases of cell cycle was also monitored. We have found that EAEC could induce apoptosis in human small intestinal as well as colonic epithelial cell lines, which was assessed by the expression of phosphatidylserine on host cell surface, internucleosomal cleavage of host cell DNA and microscopic analysis of the characteristic apoptotic features of these cells. EAEC was also found to arrest cells at S phase and G2-M phase of the cell cycle. EAEC-T8 could induce maximum apoptosis and cell cycle modulation in both small intestinal and colonic epithelial cells. Further, we have observed that the plasmid of this organism had maximum contribution to these processes. The outcome of this study has undoubtedly led to a better understanding of the basic mechanism of pathogenesis caused by EAEC.

Cell cycle arrest and mechanism of apoptosis induction in H400 oral cancer cells in response to Damnacanthal and Nordamnacanthal isolated from Morinda citrifolia.

Oral cancer is the eleventh most prevalent cancer worldwide. The most prevalent oral cancer is oral squamous cell carcinoma (OSCC). Damnacanthal (DAM) and nordamnacanthal (NDAM), the anthraquinone compounds, are isolated from the root of Morinda citrifolia L. (Noni), which has been used for the treatment of several chronic diseases including cancer. The objectives of this study were to evaluate the cytotoxicity, cell death mode, cell cycle, and the molecular mechanism of apoptosis induced by DAM and NDAM on OSCC. The cytotoxic effects of these compounds against OSCC cell lines were determined by MTT assay. The cell death mode was analysed by DNA laddering and FITC-annexin V/PI flow cytometric assays. In addition, the mechanism of apoptosis induced by DAM and NDAM was detected using mitochondrial membrane potential, Cytochrome c, and caspases assays. Finally, the effect of DAM and NDAM on cell cycle phase distribution of OSCC cells was detected by flow cytometry. In the present study, DAM and NDAM showed cytotoxicity towards OSCC cell lines and the maximum growth inhibition for both compounds was observed in H400 cells with IC50 value of 1.9 and 6.8μg/ml, respectively, after 72h treatment. The results also demonstrated the inhibition of H400 OSCC cells proliferation, internucleosomal cleavage of DNA, activation of intrinsic apoptosis pathway, and cell cycle arrest caused by DAM and NDAM. Therefore, these findings suggest that DAM and NDAM can be potentially used as antitumor agents for oral cancer therapy.

SOME RECENT BACTERIOLOGICAL AND BIOCHEMICAL STUDIES ON DIARRHEA IN NEWLY BORN CALVES WITH SPECIAL REFERENCE TO DNA FRAGMENTATION IN BLOOD

Rectal swabs and blood samples were collected from (100) diarrheic calves and (20) apparently healthy contact calves using sterile cotton swabs, at different farms in Sharkia Governorate. For hematological and biochemical investigations, a total of 20 blood samples with and without EDTA from healthy animals showed negative bacterial isolation (group 1) and 50blood samples were selected from collected blood samples (100) from animals showed positive bacterial isolation (group 2). Bacteriological examination of fecal samples revealed that E coli was the most prominent cause of calf diarrhea at an incidence of 54.1 %,followed by Salmonella species at an incidence of 17.5 % then Campylobacter at an incidence of 10.8 %. Proteus valgaris was isolated at an incidence of 10 % then Pseudomonas at an incidence of 6.7 %. Serological identification of 65 E. coli isolates, revealed that the E. coli were typed as O157 (38.5%), O119 (23.1%), O25 (7.7), O111 (7.7%), O186 (15.4%), and untypable E. coli (7.7%). Bacteriological examination revealed the isolation of Salmonella from diarrheic calves as well as apparently healthy contact calves. S. typhimurium was the most predominant serovars (52.4%) which indicates that S. typhimurium is the most predominant serovars causing enteritis in calves. S. enteritidis (33.3%) and 3 strains (14.3%) were untypable. Results of antibiogram revealed that, ciprofloxacin, cephalexin and ofloxacin( were the most effective antibiotics for treatment of diarrheic calves. Most isolated bacteria showed different degrees of resistance to Ampicillin, Cefotaxime, Gentamycine. PCR results revealed that 16 (88.9%) out of 18 examined E coli strains encoding blaTEM gene while only two (11.1%) strains were negative. The hematological changes revealed that there was a significant decrease in total red blood cells count, platelets and hemoglobin concentration (p < 0.05) accompanied by higher values of Total leukocyte count (TLC), neutrophilia with lymphopenia recorded in diarrheic calves in comparison with control.Biochemical analysis of positive cases revealed significant increase (P<0.05) in serum globulin content and decrease in total protein, albumin content, resulting in altered A/G ratio, also there was significant increase in urea, creatinine and potassium accompanied by significant decrease in glucose and sodium. In comparison with control amino acid analysis demonstrated significant (P<0.05) decrease in diseased calves. Inter nucleosomal cleavage of host cell DNA, indicated evidence of apoptosis, there was a direct relationship between DNA fragmentation and malondialdehyde level (MDA) while inverse relation with antioxidants.

Cell cycle, proliferation and apoptosis in erythroblasts cultured from patients with β-thalassaemia major.

Cell cycle, proliferation and apoptosis in erythroblasts cultured from patients with β-thalassaemia major.

Gui-ling-gao, a traditional Chinese functional food, prevents oxidative stress-induced apoptosis in H9c2 cardiomyocytes

Functional foods have become an increasingly popular alternative to prevent diseases and maintain body health status. Gui-ling-gao (GLG, also known as turtle jelly) is a well-known traditional functional food popular in Southern China and Hong Kong. This study aimed to investigate the antioxidative and anti-apoptotic effects of GLG, a traditional Chinese functional food, on preventing oxidative stress-induced injury in H9c2 cardiomyocytes. In this study, the antioxidative capacities of GLG were measured by using both a cell-free assay [2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl assay] and biological methods [2,2'-azobis(2-amidinopropane)-induced haemolysis assay and H(2)O(2)-induced cell damage on H9c2 cardiomyocytes]. Additionally, the total phenolic content was measured using the Folin-Ciocalteu method. Furthermore, the anti-apoptotic effect of GLG was evaluated by nuclear staining and a DNA fragmentation assay. GLG was found to have good antioxidant activities and high total phenolic content. In H(2)O(2)-induced cell damage on H9c2 cells, GLG was demonstrated to ameliorate the apoptotic effects, such as nuclear condensations, increased intracellular caspase-3 activity and inter-nucleosomal DNA cleavage, induced by H(2)O(2). The present study demonstrated for the first time that GLG possesses anti-apoptotic potential in vitro and this effect may be mediated, in part, by its antioxidative function. Additionally, the antioxidative capacities of GLG were proved both chemically and biologically. This study provides scientific evidence to prove the anecdotal health-beneficial claim that the consumption of GLG could help the body to handle endogenous toxicants such as free radicals.

Positioning Effects of KillerRed inside of Cells correlate with DNA Strand Breaks after Activation with Visible Light

Fluorescent proteins (FPs) are established tools for new applications, not-restricted to the cell biological research. They could also be ideal in surgery enhancing the precision to differentiate between the target tissue and the surrounding healthy tissue. FPs like the KillerRed (KRED), used here, can be activated by excitation with visible day-light for emitting active electrons which produce reactive oxygen species (ROS) resulting in photokilling processes. It is a given that the extent of the KRED's cell toxicity depends on its subcellular localization. Evidences are documented that the nuclear lamina as well as especially the chromatin are critical targets for KRED-mediated ROS-based DNA damaging. Here we investigated the damaging effects of the KRED protein fused to the nuclear lamina and to the histone H2A DNA-binding protein. We detected a frequency of DNA strand breaks, dependent first on the illumination time, and second on the spatial distance between the localization at the chromatin and the site of ROS production. As a consequence we could identify defined DNA bands with 200, 400 and (600) bps as most prominent degradation products, presumably representing an internucleosomal DNA cleavage induced by KRED. These findings are not restricted to the detection of programmed cell death processes in the therapeutic field like PDT, but they can also contribute to a better understanding of the structure-function relations in the epigenomic world.

DR396, an apoptotic DNase γ inhibitor, attenuates high mobility group box 1 release from apoptotic cells

High mobility group box1 (HMGB1) is a non-histone chromatin chromosomal protein playing an important role in chromatin architecture and transcriptional regulation. Recently, HMGB1 has been shown to be secreted into extracellular milieu in necrosis and apoptosis, and involved in inflammatory responses. However, the mechanism by which apoptotic cells release HMGB1 is unclear. In this study, to investigate the mechanism of HMGB1 release, we searched inhibitors of HMGB1 release from apoptotic cells. As a result, three compounds, 4-(4,6-dichloro-[1,3,5]-triazin-2-ylamino)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-benzoic acid (DR396), Pontacyl Violet 6R (PV6R), and Fmoc-D-Cha-OH (FDCO) in our in-house chemical library were found to inhibit HMGB1 release from staurosporine (STS)-induced apoptotic HeLa S3 cells. Interestingly, these three compounds have been previously categorized into apoptotic DNase γ inhibitors. Therefore, we examined whether apoptotic nucleosomal DNA fragmentation is involved in the release of HMGB1 during apoptosis. Expectedly, DR396, which is the most potent and specific inhibitor of DNase γ, was found to almost completely inhibit both HMGB1 release and internucleosomal DNA cleavage in HeLa S3 cells transfected with DNase γ expression vector and stably expressing DNase γ (HeLa S3/γ cells). These results clearly suggest that nucleosomal DNA fragmentation catalyzed by DNase γ is critical in the release of HMGB1 from apoptotic cells.

Ca2+, Mg2+-dependent DNase Involvement in Apoptotic Effects in Spermatozoa of Sea Urchin Strongylocentrotus intermedius Induced by Two-Headed Sphingolipid Rhizochalin

Previously, we have purified three distinct DNases from spermatozoa of sea urchin Strongylocentrotus intermedius and we suppose the role of Ca(2+), Mg(2+)-dependent DNase (Ca, Mg-DNase) in apoptosis of spermatozoa. Two-headed sphingolipid rhizochalin (Rhz) induced characteristic apoptotic nuclear chromatin changes, internucleosomal DNA cleavage, and activation of caspase-9, caspase-8, and caspase-3 in spermatozoa as was shown by fluorescence Hoechst 33342/PI/FDA analysis, DNA fragmentation assay, and fluorescence caspase inhibitors FAM-LEHD-fmk, FAM-IETD-fmk, and FAM-DEVD-fmk, respectively. Inhibitor of caspase-3 z-DEVD-fmk subdued Rhz-induced internucleosomal ladder formation, which confirmed the major role of caspase-3 in apoptotic DNA cleavage probably through Ca, Mg-DNase activation. Participation of sea urchin Ca, Mg-DNase in apoptosis of spermatozoa was demonstrated by ions Zn(2+) blocking of Rhz-induced DNA fragmentation due to direct inhibition of the Ca, Mg-DNase and internucleosomal cleavage of HeLa S and Vero E6 cell nuclei chromatin by highly purified Ca, Mg-DNase.

A Highly Toxic Cellular Prion Protein Induces a Novel, Nonapoptotic Form of Neuronal Death

Several different deletions within the N-terminal tail of the prion protein (PrP) induce massive neuronal death when expressed in transgenic mice. This toxicity is dose-dependently suppressed by coexpression of full-length PrP, suggesting that it results from subversion of a normal physiological activity of cellular PrP. We performed a combined biochemical and morphological analysis of Tg(DeltaCR) mice, which express PrP carrying a 21-aa deletion (residues 105-125) within a highly conserved region of the protein. Death of cerebellar granule neurons in Tg(DeltaCR) mice is not accompanied by activation of either caspase-3 or caspase-8 or by increased levels of the autophagy marker, LC3-II. In electron micrographs, degenerating granule neurons displayed a unique morphology characterized by heterogeneous condensation of the nuclear matrix without formation of discrete chromatin masses typical of neuronal apoptosis. Our data demonstrate that perturbations in PrP functional activity induce a novel, nonapoptotic, nonautophagic form of neuronal death whose morphological features are reminiscent of those associated with excitotoxic stress.

The antiapoptotic protein AAC-11 interacts with and regulates Acinus-mediated DNA fragmentation

The nuclear factor Acinus has been suggested to mediate apoptotic chromatin condensation after caspase cleavage. However, this role has been challenged by recent observations suggesting a contribution of Acinus in apoptotic internucleosomal DNA cleavage. We report here that AAC-11, a survival protein whose expression prevents apoptosis that occurs on deprivation of growth factors, physiologically binds to Acinus and prevents Acinus-mediated DNA fragmentation. AAC-11 was able to protect Acinus from caspase-3 cleavage in vivo and in vitro, thus interfering with its biological function. Interestingly, AAC-11 depletion markedly increased cellular sensitivity to anticancer drugs, whereas its expression interfered with drug-induced cell death. AAC-11 possesses a leucine-zipper domain that dictates, upon oligomerization, its interaction with Acinus as well as the antiapoptotic effect of AAC-11 on drug-induced cell death. A cell permeable peptide that mimics the leucine-zipper subdomain of AAC-11, thus preventing its oligomerization, inhibited the AAC-11-Acinus complex formation and potentiated drug-mediated apoptosis in cancer cells. Our results, therefore, show that targeting AAC-11 might be a potent strategy for cancer treatment by sensitization of tumour cells to chemotherapeutic drugs.

Investigation on cell death in the megagametophyte of Araucaria bidwillii Hook. post-germinated seeds

The megagametophyte of the Araucaria bidwillii seed is a storage tissue that surrounds and feeds the embryo. When all its reserves are mobilized, the megagametophyte degenerates as a no longer needed tissue. In this work we present a biochemical and a cytological characterization of the megagametophyte cell death. The TUNEL assay showed progressive DNA fragmentation throughout the post-germinative stages, while DNA electrophoretic analysis highlighted a smear as the predominant pattern of DNA degradation and internucleosomal DNA cleavage only for a minority of cells at late post-germinative stages. Cytological investigations at these stages detected profound changes in the size and morphology of the megagametophyte nuclei. By using in vitro assays, we were able to show a substantial increase in proteolytic activities, including caspase-like protease activities during the megagametophyte degeneration. Among the caspase-like enzymes, caspase 6- and 1-like proteases appeared to be the most active in the megagametophyte with a preference for acidic pH. On the basis of our results, we propose that the major pathway of cell death in the Araucaria bidwillii megagametophyte is necrosis; however, we do not exclude that some cells undergo developmental programmed cell death.

The major apoptotic endonuclease DFF40/CAD is a deoxyribose-specific and double-strand-specific enzyme

DFF40/CAD endonuclease is primarily responsible for internucleosomal DNA cleavage during the terminal stages of apoptosis. The nuclease specifically introduces DNA double strand breaks into chromatin substrates. Here we performed a detailed study on the specificity of the nuclease using synthetic single-stranded and double-stranded ribo- and deoxyribo-oligonucleotides as substrates. We have found that neither single-stranded DNA, single-stranded RNA, double-stranded RNA nor RNA-DNA heteroduplexes are cleaved by the DFF40/CAD nuclease. Noteworthy, all types of oligonucleotides that are not cleaved by the nuclease inhibit cleavage of double-stranded DNA. We have also observed that in cells undergoing apoptosis in vivo neither the activation of DFF40/CAD nor oligonucleosomal chromatin fragmentation was temporally correlated with either total cellular or nuclear RNA degradation. We conclude that DFF40/CAD is exclusively specific for double-stranded DNA.

High mobility group proteins stimulate DNA cleavage by apoptotic endonuclease DFF40/CAD due to HMG-box interactions with DNA.

The DFF40/CAD endonuclease is primarily responsible for internucleosomal DNA cleavage during the terminal stages of apoptosis. It has been previously demonstrated that the major HMG-box-containing chromatin proteins HMGB1 and HMGB2 stimulate naked DNA cleavage by DFF40/CAD. Here we investigate the mechanism of this stimulation and show that HMGB1 neither binds to DFF40/CAD nor enhances its ability for stable binding to DNA. Comparison of the stimulatory activities of different truncated forms of HMGB1 protein indicates that a structural array of two HMG-boxes is required for such stimulation. HMG-boxes are known to confer specific local distortions of DNA structure upon binding. Interestingly, the presence of DNA strand cross-links formed by cisplatin or transplatin, which may somehow mimic distortions induced by HMG-boxes, also affects DNA cleavage by the nuclease. The data presented suggest that changes induced in DNA conformation upon HMG-box binding makes the substrate more accessible to cleavage by DFF40/CAD nuclease and thus may contribute to preferential linker DNA cleavage during apoptosis.

Apoptotic Induction with Bifunctional E.coli Cytosine Deaminase-Uracil Phosphoribosyltransferase Mediated Suicide Gene Therapy is Synergized by Curcumin Treatment In vitro

Development of novel suicide gene therapy vector with potential application in cancer treatment has a great impact on human health. Investigation to understand molecular mechanism of cell death is necessary to evaluate the therapeutic application of suicide vectors. For example, the bifunctional E.coli cytosine deaminase & uracil phosphoribosyltransferase fusion (CD-UPRT) gene expression is known to sensitize a wide range of cells toward nontoxic prodrug 5-flurocytosine (5-FC) by converting it to toxic compounds, but the exact pathway of cell death is yet to be defined. Herein, we investigated the mechanism of cell death by 5-FC/CD-UPRT suicide system in both cancer and non-cancer cells and found that the optimum 5-FC concentration led to programmed cell death in vitro. The CD-UPRT expression of transfected cells was measured by the RT-PCR analysis. Biochemical assays, such as mitochondrial activity (MTS) and lactate dehydrogenase (LDH) measurements exhibited cell death. Microscopic experiments showed characteristic onset of apoptosis which was further supported by internucleosomal DNA cleavage of BrdU labeled cellular DNA, appearance of characteristic laddering of chromosomal DNA and involvement of caspase pathway. Furthermore, the 5-FC/CD-UPRT-mediated apoptosis was potentiated with addition of a known anticancer agent curcumin. Our in vitro studies confirmed synergistic induction of apoptotic pathway in the combination treatment. Therefore, combination of 5-FC/CD-UPRT with curcumin could be a potential chemosensitization strategy for cancer treatment.