Plant polyphenols mobilize nuclear copper in human peripheral lymphocytes leading to oxidatively generated DNA breakage: implications for an anticancer mechanism

It was earlier proposed that an important anti-cancer mechanism of plant polyphenols may involve mobilization of endogenous copper ions, possibly chromatin-bound copper and the consequent pro-oxidant action. This paper shows that plant polyphenols are able to mobilize nuclear copper in human lymphocytes, leading to degradation of cellular DNA. A cellular system of lymphocytes isolated from human peripheral blood and comet assay was used for this purpose. Incubation of lymphocytes with neocuproine (a cell membrane permeable copper chelator) inhibited DNA degradation in intact lymphocytes. Bathocuproine, which is unable to permeate through the cell membrane, did not cause such inhibition. This study has further shown that polyphenols are able to degrade DNA in cell nuclei and that such DNA degradation is inhibited by neocuproine as well as bathocuproine (both of which are able to permeate the nuclear pore complex), suggesting that nuclear copper is mobilized in this reaction. Pre-incubation of lymphocyte nuclei with polyphenols indicates that it is capable of traversing the nuclear membrane. This study has also shown that polyphenols generate oxidative stress in lymphocyte nuclei which is inhibited by scavengers of reactive oxygen species (ROS) and neocuproine. These results indicate that the generation of ROS occurs through mobilization of nuclear copper resulting in oxidatively generated DNA breakage.     

Oral administration of copper to rats leads to increased lymphocyte cellular DNA degradation by dietary polyphenols: implications for a cancer preventive mechanism

To account for the observed anticancer properties of plant polyphenols, we have earlier proposed a mechanism which involves the mobilization of endogenous copper ions by polyphenols leading to the generation of reactive oxygen species (ROS) that serve as proximal DNA cleaving agents and lead to cell death. Over the last decade we have proceeded to validate our hypothesis with considerable success. As a further confirmation of our hypothesis, in this paper we first show that oral administration of copper to rats leads to elevated copper levels in lymphocytes. When such lymphocytes with a copper overload were isolated and treated with polyphenols EGCG, genistein and resveratrol, an increased level of DNA breakage was observed. Further, preincubation of lymphocytes having elevated copper levels with the membrane permeable copper chelator neocuproine, resulted in inhibition of polyphenol induced DNA degradation. However, membrane impermeable chelator of copper bathocuproine, as well as iron and zinc chelators were ineffective in causing such inhibition in DNA breakage, confirming the involvement of endogenous copper in polyphenol induced cellular DNA degradation. It is well established that serum and tissue concentrations of copper are greatly increased in various malignancies. In view of this fact, the present results further confirm our earlier findings and strengthen our hypothesis that an important anticancer mechanism of plant polyphenols could be the mobilization of intracellular copper leading to ROS-mediated cellular DNA breakage. In this context, it may be noted that cancer cells are under considerable oxidative stress and increasing such stress to cytotoxic levels could be a successful anticancer approach.     

Plant polyphenols mobilize endogenous copper in human peripheral lymphocytes leading to oxidative DNA breakage: a putative mechanism for anticancer properties

Plant polyphenols are important components of human diet and a number of them are considered to possess chemopreventive and therapeutic properties against cancer. They are recognized as naturally occurring antioxidants but also act as prooxidants catalyzing DNA degradation in the presence of transition metal ions such as copper. Using human peripheral lymphocytes and Comet assay we have previously confirmed that resveratrol-Cu(II) is indeed capable of causing DNA degradation in cells. In this paper we show that the polyphenols alone (in the absence of added copper) are also capable of causing DNA breakage in cells. Incubation of lymphocytes with neocuproine inhibited the DNA degradation confirming that Cu(I) is an intermediate in the DNA cleavage reaction. Further, we have also shown that polyphenols generate oxidative stress in lymphocytes which is inhibited by scavengers of reactive oxygen species and neocuproine. These results are in further support of our hypothesis that anticancer mechanism of plant polyphenols involves mobilization of endogenous copper, possibly chromatin bound copper, and the consequent prooxidant action.     

Resveratrol-Cu(II) induced DNA breakage in human peripheral lymphocytes: implications for anticancer properties

Resveratrol (3,4',5-trihydroxy stilbene), a plant derived polyphenol found in mulberries, grapes and red wine is considered to possess chemopreventive properties against cancer. It is recognized as a naturally occurring antioxidant but also catalyzes oxidative DNA degradation in vitro in the presence of transition metal ions such as copper. Using a cellular system of lymphocytes isolated from human peripheral blood and Comet assay, we have confirmed that resveratrol-Cu(II) system is indeed capable of causing DNA degradation in cells such as lymphocytes. Also, trans-stilbene, which does not have any hydroxyl groups, is inactive in the lymphocyte system. Pre-incubation of lymphocytes with resveratrol indicates that it is capable of either traversing the cell membrane or binding to it. Our results are in partial support of our hypothesis that anticancer properties of various plant derived polyphenols may involve mobilization of endogenous copper and the consequent prooxidant action.     

Resveratrol-induced apoptosis is enhanced in low pH environments associated with cancer

Many critical factors such as hypoxia, nutrient deficiency, activation of glycolytic pathway/Warburg effect contribute to the observed low pH in tumors compared to normal tissue. Studies suggest that such tumor specific acidic environment can be exploited for the development of therapeutic strategies against cancer. Independent observations show reduction in pH of mammalian cells undergoing internucleosomal DNA fragmentation and apoptosis. As such, our group has extensively demonstrated that anticancer mechanisms of different plant polyphenols involve mobilization of endogenous copper and consequent internucleosomal DNA breakage. Copper is redox active metal, an essential component of chromatin and is sensitive to subtle pH changes in its microenvironment. Here we explored whether, acidic pH promotes growth inhibition, apoptosis, and DNA damaging capacity of chemopreventive agent resveratrol. Our results reveal that growth inhibition and internucleosomal DNA fragmentation induced apoptosis in Capan-2 and Panc-28 pancreatic cancer cell lines (and not in normal HPDE cells) by resveratrol is enhanced at lower pH. Using comet assay, we further demonstrate that DNA breakage by resveratrol is enhanced with acidification. Membrane permeable copper specific chelator neocuproine (and not iron chelator orthophenanthroline) abrogated growth inhibition and apoptosis by resveratrol. Western blot results show enhanced activation of DNA laddering marker H2.aX by resveratrol at acidic pH that was reversed by neocuproine and not by orthophenanthroline. Our findings provide irrevocable proof that low pH environment can be turned into tumor weakness and assist in eradication of cancer cells by resveratrol.     

Redox cycling of endogenous copper by thymoquinone leads to ROS-mediated DNA breakage and consequent cell death: putative anticancer mechanism of antioxidants

Plant-derived dietary antioxidants have attracted considerable interest in recent past for their chemopreventive and cancer therapeutic abilities in animal models. Thymoquinone (TQ) is the major bioactive constituent of volatile oil of Nigella sativa and has been shown to exert various pharmacological properties, such as anti-inflammatory, cardiovascular, analgesic, anti-neoplastic, anticancer and chemopreventive. Although several mechanisms have been suggested for the chemopreventive and anticancer activity of TQ, a clear mechanism of action of TQ has not been elucidated. TQ is a known antioxidant at lower concentrations and most of the studies elucidating the mechanism have centered on the antioxidant property. However, recent publications have shown that TQ may act as a prooxidant at higher concentrations. It is well known that plant-derived antioxidants can switch to prooxidants even at low concentrations in the presence of transition metal ions such as copper. It is well established that tissue, cellular and serum copper levels are considerably elevated in various malignancies. Copper is an important metal ion present in the chromatin and is closely associated with DNA bases, particularly guanine. Using human peripheral lymphocytes and comet assay, we first show that TQ is able to cause oxidative cellular DNA breakage. Such a DNA breakage can be inhibited by copper-chelating agents, neocuproine and bathocuproine, and scavengers of reactive oxygen species. Further, it is seen that TQ targets cellular copper in prostate cancer cell lines leading to a prooxidant cell death. We believe that such a prooxidant cytotoxic mechanism better explains the anticancer activity of plant-derived antioxidants.     

Characterization of the endogenous deoxyribonuclease involved in nuclear DNA degradation during apoptosis (programmed cell death).

Cell death by apoptosis occurs in a wide range of physiological events including repertoire selection of lymphocytes and during immune responses in vivo. A hallmark of apoptosis is the internucleosomal DNA degradation for which a Ca2+,Mg(2+)-dependent endonuclease has been postulated. This nuclease activity was extracted from both rat thymocyte and lymph node cell nuclei. When incubated with nuclei harbouring only limited amounts of endogenous nuclease activity, the ladder pattern of DNA fragments characteristic of apoptosis was induced. This extractable nucleolytic activity was immunoprecipitated with antibodies specific for rat deoxyribonuclease I (DNase I) and was inhibited by actin in complex with gelsolin segment 1, strongly pointing to the presence of a DNase I-type enzyme in the nuclear extracts. COS cells transiently transfected with the cDNA of rat parotid DNase I expressed the enzyme, and their nuclei were able to degrade their DNA into oligosome-sized fragments. PCR analysis of mRNA isolated from thymus, lymph node cells and kidney yielded a product identical in size to that from rat parotid DNase I. Immunohistochemical staining with antibodies to rat DNase I confirmed the presence of DNase I antigen in thymocytes and lymph node cells. The tissue distribution of DNase I is thus extended to tissues with no digestive function and to cells which are known to be susceptible to apoptosis. We propose that during apoptosis, an endonuclease indistinguishable from DNase I gains access to the nucleus due to the breakdown of the ER and the nuclear membrane.     

Oxidation-reduction reactions in Ehrlich cells treated with copper-neocuproine.

The interaction of 2,9-dimethyl-1,10-phenanthroline (neocuproine or NC) and its copper complex with Ehrlich ascites tumor cells was studied. NC is frequently used as a negative control in studies of in vitro DNA degradation by copper phenanthroline and has also found use as a potential inhibitor of damage from oxidative stress in biological systems. NC inhibited Ehrlich cell growth in monolayer culture over 48 h treatment by 50% at 0.05 nmol/10(5) cells. Addition of 5- to 100-fold ratios of CuCl2 to NC (at 0.035 nmol NC/10(5) cells) produced progressively more growth inhibition. Addition of 1:0.5 ratios of NC to CuCl2 over the range of NC concentrations 0.08-0.2 nmol/10(5) cells/mL resulted in DNA single-strand breakage during 1-h treatments as measured by DNA alkaline elution. Concomitant addition of catalase or dimethyl sulfoxide (DMSO) inhibited DNA strand scission, while superoxide dismutase enhanced breakage. Catalase and DMSO also inhibited induction of membrane permeability by the copper complex of NC. These cellular effects apparently result from the intracellular generation of hydroxyl radical from H2O2. NC facilitated the uptake of copper into cells, though it was initially bound as a copper-histidine-like complex. The internalized copper was reduced to Cu(I), bound mostly as (NC)2Cu(I). To explain the (NC)2Cu-dependent generation of hydroxyl radical, it is hypothesized that glutathione successfully competes for Cu(I), converting it to a redox-active form that can catalyze the reduction of molecular oxygen to .OH. Model studies support this view. Radical scavengers did not reverse growth inhibition produced by NC or NC + CuCl2.     

Role of nuclear proteins on [H]actinomycin D binding during lymphocyte mitogenesis

The uptake of [³H]actinomycin D ([³H]AD) by ConA-stimulated lymphocytes was followed during 96 h of incubation and correlated with the level of nuclear proteins in the nucleus, DNA synthesis and the degree of AD-induced inhibition of RNA and DNA synthesis. During the first 48 h there is a parallel increase of drug binding to cells and a rising level of non-histone proteins (NHP) in the nucleus. During the next 48 h, DNA synthesis occurs, drug uptake decreases and the nuclear level of NHP continues to rise. The level of histones remains constant during 96 h. The variations in cellular [³H]AD uptake during 96 h are not due to changes in cell membrane permeability, since similar variations in drug binding are observed in isolated cell nuclei. NHP, obtained as 0.25 M NaCl extracts of cell nuclei, increase binding of [³H]AD to nuclei isolated from non-stimulated lymphocytes, while histones have no such effect. NHP extracted with phenol, after washing the nuclei with salt and acid solutions, or extracted with 0.25 M NaCl from non-stimulated and stimulated lymphocytes and Chang liver cells are equally active to bind [³H]AD to nuclei of non-stimulated lymphocytes. NHP from Chang cells, purified by DNA-cellulose chromatography using calf thymus DNA, stimulated [³H]AD binding to lymphocyte nuclei, indicating that the drug-binding activity is due to proteins binding to DNA. NHP increase binding of [³H]AD to pure DNA in the absence of histones. The degree of [³H]AD binding to ConA-stimulated lymphocytes during 96 h correlated with the degree of inhibition of RNA and DNA synthesis by AD.     

Role of nuclear proteins on [3H]actinomycin D binding during lymphocyte mitogenesis

The uptake of [³H]actinomycin D ([³H]AD) by ConA-stimulated lymphocytes was followed during 96 h of incubation and correlated with the level of nuclear proteins in the nucleus, DNA synthesis and the degree of AD-induced inhibition of RNA and DNA synthesis. During the first 48 h there is a parallel increase of drug binding to cells and a rising level of non-histone proteins (NHP) in the nucleus. During the next 48 h, DNA synthesis occurs, drug uptake decreases and the nuclear level of NHP continues to rise. The level of histones remains constant during 96 h. The variations in cellular [³H]AD uptake during 96 h are not due to changes in cell membrane permeability, since similar variations in drug binding are observed in isolated cell nuclei. NHP, obtained as 0.25 M NaCl extracts of cell nuclei, increase binding of [³H]AD to nuclei isolated from non-stimulated lymphocytes, while histones have no such effect. NHP extracted with phenol, after washing the nuclei with salt and acid solutions, or extracted with 0.25 M NaCl from non-stimulated and stimulated lymphocytes and Chang liver cells are equally active to bind [³H]AD to nuclei of non-stimulated lymphocytes. NHP from Chang cells, purified by DNA-cellulose chromatography using calf thymus DNA, stimulated [³H]AD binding to lymphocyte nuclei, indicating that the drug-binding activity is due to proteins binding to DNA. NHP increase binding of [³H]AD to pure DNA in the absence of histones. The degree of [³H]AD binding to ConA-stimulated lymphocytes during 96 h correlated with the degree of inhibition of RNA and DNA synthesis by AD.     

Non-enzymatic fast repair of DNA oxidative damage might also exist in cells

Many studies have shown that in a chemical system natural polyphenols can rapidly repair DNA oxidative damage. In this paper we report that in a cellular system the non-enzymatic fast repair activities of two natural polyphenols might also exist. The viability of a Chinese hamster ovary cell line (AA8) highly expressing the XRCC1 gene (a DNA repairing protein) treated with H2O2 is significantly higher than that of a normal Chinese hamster ovary cell line (CHO). Following inhibition of the enzymatic repair system by different inhibitors--methoxyamine (MX), 3-aminobenzamide (3AB) or nicotinamide (NIC)--DNA oxidative damage by H2O2 increased 2-5-fold in both cell lines. However, when natural polyphenols--rosmarinic acid (RA) or verbascoside (VER)--were added, DNA oxidative damage was significantly reduced. This decrease of DNA oxidative damage by RA or VER is not due to their scavenging activity for reactive oxygen species (ROS) because cells suffered from heavy ROS throughout the whole experimental process. Therefore, the decrease of DNA damage might be due to their non-enzymatic fast repair mechanisms. Further investigation showed that H2O2 induced a drop in the mitochondrial membrane potential (MMP), and that RA and VER were able to attenuate the drop. Previous studies have shown that H2O2 initiates a chain of events in cells, involving mtDNA damage, a drop in MMP and loss of repair activity. These results, taken together with our present results, suggest that the non-enzymatic fast repair mechanism exists not only in chemical systems but also might exist in cells.     

Experimental studies on ultralow frequency pulsed gradient magnetic field inducing apoptosis of cancer cell and inhibiting growth of cancer cell

The morphology characteristics of cell apoptosis of the malignant tumour cells in magnetic field-treated mouse was observed for the first time. The apoptotic cancer cell contracted, became rounder and divorced from adjacent cells; the heterochromatin condensed and coagulated together along the inner side of the nuclear membrane; the endoplasmic reticulums(ER) expanded and fused with the cellular membrane; many apoptotic bodies which were packed by the cellular membrane appeared and were devoured by some lymphocytes and plasma. Apoptosis of cancer cells was detected by terminal deoxynucleotidyl transferase mediated in situ nick end labeling(TUNEL). It was found that the number of apoptosis cancer cells of the sample treated by the magnetic field is more than that of the control sample. The growth of malignant tumour in mice was inhibited and the ability of immune cell to dissolve cancer cells was improved by ultralow frequency(ULF) pulsed gradient magnetic field; the nuclei DNA contents decreased, indicating that magnetic field can block DNA replication and inhibit mitosis of cancer cells. It was suggested that magnetic field could inhibit the metabolism of cancer cell, lower its malignancy, and restrain its rapid and heteromorphic growth. Since ULF pulsed gradient magnetic field can induce apoptosis of cancer cells and inhibit the growth of malignant tumour, it could be used as a new method to treat cancer.     

Experimental studies on ultralow frequency pulsed gradient magnetic field inducing apoptosis of cancer cell and inhibiting growth of cancer cell

The morphology characteristics of cell apoptosis of the malignant tumour cells in magnetic field-treated mouse was observed for the first time. The apoptotic cancer cell contracted, became rounder and divorced from adjacent cells; the heterochromatin condensed and coagulated together along the inner side of the nuclear membrane; the endoplasmic reticulums (ER) expanded and fused with the cellular membrane; many apoptotic bodies which were packed by the cellular membrane appeared and were devoured by some lymphocytes and plasma. Apoptosis of cancer cells was detected by terminal deoxynucleotidyl transferase mediated in situ nick end labeling (TUNEL). It was found that the number of apoptosis cancer cells of the sample treated by the magnetic field is more than that of the control sample. The growth of malignant tumour in mice was inhibited and the ability of immune cell to dissolve cancer cells was improved by ultralow frequency (ULF) pulsed gradient magnetic field; the nuclei DNA contents decreased, indi     

Intracellular iron, but not copper, plays a critical role in hydrogen peroxide-induced DNA damage

The role of intracellular iron, copper, and calcium in hydrogen peroxide-induced DNA damage was investigated using cultured Jurkat cells. The cells were exposed to low rates of continuously generated hydrogen peroxide by the glucose/glucose oxidase system, and the formation of single strand breaks in cellular DNA was evaluated by the sensitive method, single cell gel electrophoresis or "comet" assay. Pre-incubation with the specific ferric ion chelator desferrioxamine (0.1-5.0 mM) inhibited DNA damage in a time- and dose-dependent manner. On the other hand, diethylenetriaminepentaacetic acid (DTPA), a membrane impermeable iron chelator, was ineffective. The lipophilic ferrous ion chelator 1,10-phenanthroline also protected against DNA damage, while its nonchelating isomer 1,7-phenanthroline provided no protection. None of the above iron chelators produced DNA damage by themselves. In contrast, the specific cuprous ion chelator neocuproine (2,9-dimethyl-1,10-phenanthroline), as well as other copper-chelating agents, did not protect against H(2)O(2)-induced cellular DNA damage. In fact, membrane permeable copper-chelating agents induced DNA damage in the absence of H(2)O(2). These results indicate that, under normal conditions, intracellular redox-active iron, but not copper, participates in H(2)O(2)-induced single strand break formation in cellular DNA. Since BAPTA/AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester), an intracellular Ca(2+)-chelator, also protected against H(2)O(2)-induced DNA damage, it is likely that intracellular Ca(2+) changes are involved in this process as well. The exact role of Ca(2+) and its relation to intracellular transition metal ions, in particular iron, needs to be further investigated.     

Nucleocytoplasmic distribution of the ovalbumin serpin PI-9 requires a nonconventional nuclear import pathway and the export factor Crm1.

Proteinase inhibitor 9 (PI-9) is a human serpin present in the cytoplasm of cytotoxic lymphocytes and epithelial cells. It inhibits the cytotoxic lymphocyte granule proteinase granzyme B (graB) and is thought to protect cytotoxic lymphocytes and bystander cells from graB-mediated apoptosis. Following uptake into cells, graB promotes DNA degradation, rapidly translocating to the nucleus, where it binds a nuclear component. PI-9 should therefore be found in cytotoxic lymphocyte and bystander cell nuclei to ensure complete protection against graB. Here we demonstrate by microscopy and subcellular fractionation experiments that PI-9 is present in the nuclei of human cytotoxic cells, endothelial cells, and epithelial cells. We also show that the related serpins, PI-6, monocyte neutrophil elastase inhibitor (MNEI), PI-8, plasminogen activator inhibitor 2 (PAI-2), and the viral serpin CrmA exhibit similar nucleocytoplasmic distributions. Because these serpins lack classical nuclear localization signals and are small enough to diffuse through nuclear pores, we investigated whether import occurs actively or passively. Large (approximately 70 kDa) chimeric proteins comprising PI-9, PI-6, PI-8, MNEI, or PAI-2 fused to green fluorescent protein (GFP) show similar nucleocytoplasmic distributions to the parent proteins, indicating that nuclear import is active. By contrast, CrmA-GFP is excluded from nuclei, indicating that CrmA is not actively imported. In vitro nuclear transport assays show that PI-9 accumulates at a rate above that of passive diffusion, that it requires cytosolic factors but not ATP, and that it does not bind an intranuclear component. Furthermore, PI-9 is exported from nuclei via a leptomycin B-sensitive pathway, implying involvement of the export factor Crm1p. We conclude that the nucleocytoplasmic distribution of PI-9 and related serpins involves a nonconventional nuclear import pathway and Crm1p.     

Ascorbic acid mobilizes endogenous copper in human peripheral lymphocytes leading to oxidative DNA breakage: a putative mechanism for anticancer properties

Several decades back ascorbic acid was proposed as an effective anticancer agent. However, this idea remained controversial and the mechanism of action unclear. In this paper, we show that ascorbic acid at a concentration reported to be achievable through high doses of oral consumption is capable of cytotoxic action against normal cells. Several antioxidants of both animal as well as plant origin including ascorbic acid also possess prooxidant properties. Copper is an essential component of chromatin and can take part in redox reactions. Previously we have proposed a mechanism for the cytotoxic action of plant antioxidants against cancer cells that involves mobilization of endogenous copper ions and the consequent generation of reactive oxygen species. Using human peripheral lymphocytes and Comet assay we show here that ascorbic acid is able to cause oxidatative DNA breakage in normal cells at a concentration of 100–200 μM. Neocuproine, a Cu(I) specific sequestering agent inhibited DNA breakage in a dose dependent manner indicating that Cu(I) is an intermediate in the DNA cleavage reaction. The results are in support of our above hypothesis that involves events that lead to a prooxidant action by antioxidants. The results would support the idea that even a plasma concentration of around 200 μM would be sufficient to cause pharmacological tumor cell death particularly when copper levels are elevated. This would account for the observation of several decades back by Pauling and co-workers where oral doses of ascorbic acid in gram quantities were found to be effective in treating some cancers.     

Isolation of Ca/Mg-dependent endonuclease of cell nuclei as the basic component in the chromatin nuclease complex of human lymphocytes

Highly purified preparations of Ca/Mg-dependent cell nuclear endonuclease have been obtained from human spleen lymphocytes. The purification was 2000-fold, and the enzyme was a polypeptide with a molecular weight of 57-54 kD. The study of its binding with monoclonal antibody enzyme, produced by various hybridoma strains obtained upon the immunization of mice with non-fractionated nuclear extract has shown that Ca/Mg-dependent endonuclease was the best expressed endonuclease of lymphocyte cellular nuclei. Possible production of some other cell nuclear endonucleases during processing of CH/Mg-dependent endonuclease is suggested.     

Characterization of apoptosis-like endonuclease activity in avian thymocytes.

In the current study the internucleosomal DNA cleavage activity associated with apoptosis was investigated in avian thymocytes. Thymocyte nuclear proteins from glucocorticoid-treated chickens were incubated with chicken red blood cell (cRBC) nuclei, and DNA degradation was analyzed by agarose gel electrophoresis and fluorescence-activated flow cytometry. The thymocyte nuclear extract contained an endonuclease activity that degraded cRBC chromatin at internucleosomal sites as detected by agarose gel electrophoresis. Flow cytometry analysis of cRBC nuclei that were treated with thymocyte nuclear proteins demonstrated a loss of cellular DNA as a function of the amount of added nuclease activity. Furthermore, it was demonstrated that the thymocyte nuclear extract contained a nuclease activity that was capable of degrading radiolabelled naked 32P-DNA into acid soluble DNA fragments. All three assay methods demonstrate that the thymocyte nuclease activity can be inhibited by EDTA, zinc ions and the nuclease inhibitor aurintricarboxylic acid. Based on the analysis of cofactor requirement of this nuclease activity and its susceptibility to inhibitors, the endonuclease activity present in avian apoptotic thymocytes appears to be identical to the mammalian counterpart.     

Protein kinase C and calcium ion in mitogenic response of macrophage-depleted human peripheral lymphocytes

For mitogenic response of macrophage-depleted human peripheral lymphocytes, 12-O-tetradecanoylphorbol-13-acetate (TPA) or 1-oleoyl-2-acetylglycerol (OAG) and Ca2+ ionophore are both needed in addition to a small quantity of plant lectin, phytohemagglutinin (PHA). PHA alone is not sufficient to produce the cellular response. The addition of TPA or OAG to these cells induces the activation of protein kinase C as assayed by the phosphorylation of its endogenous substrates. Apparently, TPA or OAG and A23187 together substitute for macrophages and act synergistically to potentiate the DNA synthesis of this lymphocyte preparation. The results suggest that protein kinase C activation and Ca2+ mobilization are essential and that additional receptor occupation by PHA is necessary for producing cell proliferation.