DNA Ladder Formation Research Articles

Anti‐apoptotic role of TWIST and its association with Akt pathway in mediating taxol resistance in nasopharyngeal carcinoma cells

TWIST, a basic helix-loop-helix transcription factor, has been reported to be associated with development and progression of human cancer. Recently, over expression of TWIST is found in cancer patients with shorter survival and poor response to chemotherapy. Previously, we found that upregulation of TWIST was responsible for the development of acquired resistance to taxol in a nasopharyngeal carcinoma (NPC) cell line, HNE1-T3 (Wang et al., Oncogene, 2004;24:274). In this study, we investigated the underlying molecular mechanisms responsible for the TWIST-mediated taxol resistance. By comparison of the parental HNE1 and its derivative HNE1-T3 cell lines, we found that the resistance to taxol in HNE1-T3 cells was associated with suppression of taxol-induced apoptosis evidenced by decreased expression of Bak and Bax and increased Bcl-2, as well as inhibition of PARP and caspase cleavage and DNA ladder formation. However, there was no correlation between taxol sensitivity and alterations on G2/M cell cycle distribution, suggesting that the TWIST-induced taxol resistance is mediated through protection against apoptosis but not mitotic arrest. Analysis of additional 8 NPC cell lines showed that upregulation of TWIST was associated with resistance to microtubule disrupting agents, especially taxol, and inactivation of TWIST through small RNA interference led to increased sensitivity to taxol-induced cell death. Subsequent studies also demonstrated that the TWIST-mediated taxol resistance may be regulated through its positive involvement with the Akt pathway. Our findings suggest an underlying molecular mechanism responsible for the TWIST-mediated chemodrug resistance and suggest a target for overcoming taxol resistance in cancer cells.

Stage-specific expression of DNaseγ during B-cell development and its role in B-cell receptor-mediated apoptosis in WEHI-231 cells

Here, we describe the non-redundant roles of caspase-activated DNase (CAD) and DNasegamma during apoptosis in the immature B-cell line WEHI-231. These cells induce DNA-ladder formation and nuclear fragmentation by activating CAD during cytotoxic drug-induced apoptosis. Moreover, these apoptotic manifestations are accompanied by inhibitor of CAD (ICAD) cleavage and are abrogated by the constitutive expression of a caspase-resistant ICAD mutant. No such nuclear changes occur during oxidative stress-induced necrosis, indicating that neither CAD nor DNasegamma functions under necrotic conditions. Interestingly, the DNA-ladder formation and nuclear fragmentation induced by B-cell receptor ligation occur in the absence of ICAD cleavage and are not significantly affected by the ICAD mutant. Both types of nuclear changes are preceded by the upregulation of DNasegamma expression and are strongly suppressed by 4-(4,6-dichloro-[1, 3, 5]-triazin-2-ylamino)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-benzoic acid (DR396), which is a specific inhibitor of DNasegamma. Our results suggest that DNasegamma provides an alternative mechanism for inducing nuclear changes when the working apoptotic cascade is unsuitable for CAD activation.

Apoptosis and Necrosis are Involved in the Toxicity of Sauropus androgynus in an In Vitro Study

The raw juice of the young sticks and leaves of Sauropus androgynus (SA) has been widely used as a natural food for body weight reduction and vision protection in Taiwan and Southeast Asia. But as has been reported, SA-associated obliterative bronchiolitis can develop after taking SA for more than 3 months. Lung transplantation was carried out in severe cases. To study the toxic effect, we separated the SA extract into three parts, namely CHCl3, EtOAc and n-BuOH fractions, using polarity dissection. NIH3T3 fibroblasts were treated with the SA fractions 300 microg/mL and subjected to a series of cytotoxic assays. The EtOAc fraction exhibited the strongest effect of cell growth inhibition, followed by the CHCl3 and n-BuOH fractions. Features of condensed chromatin and apoptosis were observed in cells exposed to n-BuOH and EtOAc fractions using fluorescence microscopy. Formation of DNA ladders was also observed in the above cells. Instead, the CHCl3 fraction induced DNA smearing. In bivariate dot plots of annexin V and propidium iodide double staining, necrosis and apoptosis appeared in cells treated with CHCl3 and n-BuOH fractions, respectively, and a mixed type of necrosis and apoptosis appeared in EtOAc fraction-treated cells. Our results indicate that necrosis and apoptosis are involved in the toxic effect of SA in NIH3T3 fibroblasts. More evidence is needed to clarify if necrosis and apoptosis are also related to the pathogenesis of SA-associated obliterative bronchiolitis.

Saucernetin-7 Isolated from Saururus chinensis Induces Caspase-Dependent Apoptosis in Human Promyelocytic Leukemia HL-60 Cells

In the present study, we investigated the effect of saucernetin-7 (a biologically active compound isolated from the underground parts of Saururus chinensi) on the induction of apoptosis and the putative pathways of its action in HL-60 human promyelocytic leukemia cells. Saucernetin-7-treated HL-60 cells displayed several features of apoptosis, including DNA fragmentation, DNA laddering by agarose gel electrophoresis, and externalization of annexin-V targeted phosphatidylserine (PS) residues. z-VAD-fmk (a broad-caspase inhibitor) almost completely suppressed saucernetin-7-induced DNA ladder formation, thereby implicating the caspase cascade in the apoptotic process. We also observed that saucernetin-7 caused the activations of caspase-3, -8 and -9, and that it induced Bid cleavage, the mitochondrial translocation of Bax from the cytosol, and cytochrome c release from mitochondria, but it had no effect on Bcl-2 and Bcl-xL levels. Taken together, the present study demonstrates that saucernetin-7 is a potent inducer of apoptosis and that its activity is facilitated by caspase-8 activation, Bid cleavage, Bax translocation to mitochondria, release of cytochrome c into cytoplasm, and subsequently caspase-3 activation, which offers a potential mechanism for the apoptosis-inducing activity of saucernetin-7.

Lantadene A-induced apoptosis in human leukemia HL-60 cells

Objectives : Lantadene A (LA, 22β -angeloyloxy-3-oxoolean-12-en-28-oic acid) a pentacyclic, triterpenoid isolated from the leaves of the obnoxious weed, Lantana camara L. was evaluated for apoptosis induction in the human leukemia HL-60 cell line. Materials and Methods : The effect of LA on cell proliferation of HL-60 cancer cells was determined by using the MTT assay. The morphological effects of LA-treated HL-60 cancer cells were observed under a fluorescence microscope. DNA fragmentation was observed using gel electrophoresis. Flow cytometry was carried out to observe changes in the cell cycle distribution of the cells. The expression of Bcl-2 and Bax proteins in HL-60 cells was visualized by means of an immunohistochemical assay and cell viability was determined upon treatment with DEVD-CHO (inhibitor of caspase-3) and LA. Results : Typical morphological changes including cell shrinkage, chromatin condensation and characteristic DNA ladder formation in agarose gel electrophoresis were observed. LA-induced marked concentration- and time-dependent inhibition of cancer cell proliferation with an IC 50 value of 19.8 ± 0.10 µg/ml following 48 h incubation. Flow cytometric analysis showed suppressed cell proliferation associated with cell cycle arrest in the G 0 /G 1 phase. LA significantly inhibited cell proliferation of HL-60 cells and induced cell apoptosis by downregulating Bcl-2 and upregulating Bax expression. The peptidic caspase-3 inhibitor, DEVD-CHO (NH 2 -Asp-Glu-Val-Asp-CHO, 2 µM), increased the viability of HL-60 cells, which had been previously treated with LA. Conclusions : The results indicated that LA induces efficient cell apoptosis by activating the caspase-3 pathway and through down- and upregulation of Bcl-2 and Bax expression respectively.

Induction of Apoptosis and Anti-Tumor Effects of a Novel Pan Inhibitor of Bcl-2 and Mcl-1 Apogossypolone (ApoG2) in Multiple Myeloma Cells.

Multiple myeloma remains an incurable malignancy and overall survival has not been improved despite responses to conventional and high-dose chemotherapy. Over-expression of both Bcl-2 and Mcl-1 is frequent in multiple myeloma which renders myeloma cells resistant to apoptosis by chemotherapy, and overexpression is associated with relapse and poorer survival. Inhibition of the anti-apoptotic function of Bcl-2 family member proteins such as Bcl-2 and Mcl-1 represents an attractive new strategy for developing anticancer drugs. Apogossypolone (ApoG2) is a novel derivative of the naturally occurring polyphenolic compound gossypol which has aldehyde moieties removed and further modification to make it pharmaceutically more stable. More particularly, ApoG2 is a potent inhibitor of Mcl-1 and Bcl-2, with Ki value of 25 nM for Mcl-1, 35 nM for Bcl-2, respectively. In this study, trypan blue dye exclusion, Hoechst 33258 staining, DNA ladder formation and annexin-V-PI flow cytometric analysis were used to determine the cellular activities of ApoG2 on cell growth inhibition, cell viability, cell cycle and apoptosis. Cleavage of caspase-3 and caspase-9 was analyzed by colorimetirc assay. Xenograft model of Wus1 cells (from Peking Union Medical College Hospital) in nude mice were used to determine the antitumor activity of compounds. We found that ApoG2 resulted in a dose and time-dependent inhibition of multiple myeloma cell proliferation, with IC50 value to both U266 and Wus1 cells at 0.1 to 0.2 uM at 48 hours after treatment. ApoG2 effectively induced apoptosis of multiple myeloma cells as evidenced by typical morphological changes, transmission electron microscopy, DNA ladder formation and increase in the percentage of cells in subdiploid peak. Colorimetric assays further showed activation of both caspase-3 and caspase-9. In a parallel direct comparison study, ApoG2 was more potent than the parental compound gossypol in both growth inhibition and induction of apoptosis. Of interest, cell cycle analysis of both U266 and Wus1 cells treated with ApoG2 produced a slightly G2 arrest, increasing from 9.7% to 19.6% in U266 cells, and from 9.8% to 31.7% in Wus1 cells, respectively. This was different from gossypol which induced mainly G1 arrest. Preliminary in vivo antitumor activity of ApoG2 was examined in xenograft model of Wus1 cells in nude mice, and growth inhibition (T/C) of 32.7% and 33.4% was obtained at 60 mg/kg, and 40 mg/kg, respectively. In addition, there was no body weight loss for both treated groups in comparison with the vehicle treated mice. Our results demonstrated that a potent pan inhibitor of Bcl-2 and Mcl-1 ApoG2 had significant effect of antiproliferation and induction of apoptosis on multiple myeloma cells in vitro and in vivo. ApoG2 may represent a promising new anticancer agent with a novel molecular mechanism and warrant further investigation as a single agent or in combination for human multiple myeloma with Bcl-2/Mcl-1 overexpression.

Induction of G2/M phase arrest and apoptosis by a novel enediyne derivative, THDB, in chronic myeloid leukemia (HL-60) cells

( Z)-2-(6-(Thieanisyl-2-yl)hexa-3-en-1,5-diynyl)benzenamine (THDB), an enediyne compound, was identified in our laboratory as a novel antineoplastic agent with broad spectrum of antitumor activities against many human cancer cells. THDB was found to inhibit the growth of HL-60 cells in a time-and dose-dependent manner. Cell cycle analysis showed G2/M phase arrest in HL-60 cells following 48 h exposure to THDB. Analysis of the cell cycle regulatory proteins demonstrated that THDB did not change the steady-state levels of cyclin B1, cyclin E, Cdk1 and Cdc25C, but decreased the protein levels of Cdk2 and cyclin A. THDB also caused a marked increase in apoptosis, as characterized by DNA fragmentation (DNA ladder and sub G1 formation), and poly (ADP-ribose) polymerase (PARP) cleavage, which was associated with activation of caspase-3, caspase-8 and caspase-9. Moreover, the THDB-induced apoptosis was significantly attenuated in the presence of specific inhibitors of caspase-3, -8 and -9. These molecular alterations provide an insight into THDB-caused growth inhibition, G2/M arrest and apoptotic death of HL-60 cells.

Fundamental study of small interfering RNAs for ganglioside GD3 synthase gene as a therapeutic target of lung cancers

Gangliosides GD3 and GD2 are specifically expressed in neuro-ectoderm-derived tumors, and are considered to play roles in the malignant properties of those cells. We analysed effects of small interfering (si) RNAs against GD3 synthase gene on the expression of ganglioside GD2 and biological phenotypes of human lung cancer cells expressing GD2. An siRNA could suppress the mRNA level of GD3 synthase gene even by single transfection, whereas repeated transfection was required to suppress GD2 expression on the cell surface. Significant reduction in the cell growth and invasion activity was observed in both lung cancer cell lines examined, when repeatedly transfected with the siRNA twice a week. DNA ladder formation was observed after third transfection, indicating the potent induction of apoptosis. Stable transfection of an RNAi expression vector with H1 RNA promoter was also examined. Transfectant cells with the RNAi expression vector showed almost equivalent suppression of GD2 expression and tumor properties in vitro. Furthermore, the stable transfectant cells showed slower cell growth than the control cells in severe combined immunodeficiency mice. These results suggested that siRNAs and/or RNAi expression vectors to generate siRNAs are promising approach to overcome human lung cancers.

Antitumor activity of polyoxomolybdate, [NH 3Pr i] 6[Mo 7O 24]·3H 2O, against, human gastric cancer model

Polyoxometalates are negatively charged inorganic compounds which contain metal ions such as tungsten, molybdenum, vanadium etc. and which make clusters with the surrounding oxygen atoms. [NH 3Pr i] 6[Mo 7O 24]·3H 2O (PM-8) was found to be a significant antitumor polyoxomolybdates. It had already been reported that the PM-8 suppressed the growth of Co-4 human colon cancer, MX-1 human breast cancer and OAT human lung cancer xenografted in nude mice. However, the mechanism of the antitumor activity has not been clarified. In this study, the antitumor activity of one of the metal oxide clusters (polyoxometalates), hexabis(isopropylammonium) heptamolybdate trihydrate, [NH 3Pr i] 6[Mo 7O 24]·3H 2O (PM-8) were shown in an MTS assay. DNA ladder formation and detection of apoptotic bodies in nuclei were revealed that antitumor activity of PM-8 in MKN45 cells was due to apoptosis. It is concluded that the observation of significant tumor growth suppression of PM-8 in MKN45-bearing mice results from the induction of apoptosis. PM-8 shows promise as a novel anti-cancer agent.

Induction of Apoptosis in HeLa Cells by 3β‐Hydroxyurs‐12‐en‐27‐oic Acid

3Beta-hydroxyurs-12-en-27-oic acid (1), a pentacyclic triterpenoid isolated from the rhizomes of Astilbe chinensis, was structurally very similar to ursolic acid, with the only difference being the interchange of the COOH and Me group at C(14) and C(17). Ursane-type triterpene with a COOH group at C(14) is present in a limited number of natural resources. Compound 1 was found to exhibit more distinctive cytotoxicity toward human cervical squamous carcinoma (HeLa) cells than ursolic acid, suggesting that the position of the COOH group significantly affects the cytotoxicity of ursane-type pentacyclic triterpenes with a COOH group. To elucidate the underlying biological mechanism responsible for the cytotoxicity of 1, we investigated its growth-inhibitory and apoptosis-inducing effect on HeLa cells. Compound 1 induced a marked concentration- and time-dependent inhibition of cell proliferation with an IC50 value of 6.80+/-0.88 microg/ml following 48 h incubation. The drug-treated HeLa cells displayed typical morphological apoptotic characteristics and formation of DNA ladders in agarose-gel electrophoresis. Flow cytometric analysis showed that the cell cycle was arrested in G0/G1 phase by 1, and the apoptotic rate of HeLa cells treated for 48 h with 20 microg/ml of 1 was 21.08+/-2.14%. Also, 1 increased and decreased the expression of Bax and Bcl-2 proteins, respectively, and lowered the mitochondrial transmembrane potential (delta psi(m)). The peptidic caspase-3 inhibitor DEVD-CHO (NH2-Asp-Glu-Val-Asp-CHO, at 2 microM) could increase the viability of HeLa cells previously treated with 1. These results indicate that 1 induces efficient cell apoptosis through down-regulating Bcl-2 expression, up-regulating Bax expression, lowering delta psi(m), and by activating the caspase-3 pathway.

Cell Apoptosis: Requirement of H2AX in DNA Ladder Formation, but Not for the Activation of Caspase-3

Immunofluorescence studies have revealed that H2AX is phosphorylated at the sites of DNA double-strand breaks induced by ionizing radiation and is required for recruitment of repair factors into nuclear foci after DNA damage. Therefore, the function of H2AX is believed to be associated primarily with repair of DNA damage. Here, we report a function of H2AX in cellular apoptosis. Our data showed that H2AX is phosphorylated by UVA-activated JNK. We also provided evidence showing that UVA induces caspase-3 and caspase-activated DNase (CAD) activity in both H2AX wild-type and H2AX knockout mouse embryonic fibroblasts (MEFs). However, DNA fragmentation occurred only in H2AX wild-type MEFs. Furthermore, H2AX phosphorylation was critical for DNA degradation triggered by CAD in vitro. Taken together, these data indicated that H2AX phosphorylation is required for DNA ladder formation, but not for the activation of caspase-3; and the JNK/H2AX pathway cooperates with the caspase-3/CAD pathway resulting in cellular apoptosis.

H2AX Is a Target of the JNK Signaling Pathway that Is Required For Apoptotic DNA Fragmentation

The mechanism of apoptotic signaling by JNK is incompletely understood. In the July 7 issue of Molecular Cell, Lu et al. (2006) report that JNK phosphorylation of H2AX at a noncanonical site is required for caspase-induced DNA fragmentation.

Involvement of caspase activation and mitochondrial stress in trichostatin A‐induced apoptosis of Burkitt's lymphoma cell line, Akata

Epstein-Barr virus (EBV) infects more than 90% of the human population and has a potential oncogenic nature. Trichostatin A (TSA) has potent antitumor activity, but its exact mechanism on EBV-infected cells is unclear. This study examined the effects of TSA on proliferation and apoptosis of the Burkitt's lymphoma cell line, Akata. TSA treatment inhibited cell growth and induced cytotoxicity in both the EBV-negative and -positive Akata cells. TSA sensitively induced apoptosis in both cells, as demonstrated by the increased number of positively stained cells in the TUNEL assay, the migration of many cells to sub-G1 phase by flow cytometric analysis, and the formation of DNA ladders. This suggests that EBV has no effect on the sensitivity to TSA. Western blot analysis showed that the cleavage of PARP and Bid and the activation of caspases are closely related to the TSA-induced apoptosis of the cells. The reduction in mitochondrial transition potential and the release of apoptosis-inducing factor from mitochondria to cytosol was also observed after the TSA treatment, but was suppressed by treating the cells with a cathepsin B inhibitor. Overall, these findings suggest that besides the caspase-dependent pathway, mitochondrial events are also associated with the TSA-induced apoptosis of Akata cells.

Anticancer activity of polyoxomolybdate

Anticancer polyoxomolybdates have been investigated for medical application of polyoxometalates as discrete cluster anions of metal oxides. [NH 3Pr i] 6[Mo 7O 24]·3H 2O (PM-8) has been recognized as one of significant antitumoral polyoxomolybdates. PM-8 had shown the growth suppression against several tumors, for examples, Co-4, human colon cancer, MX-1, human breast cancer, and OAT, human lung cancer. PM-8 showed the tumor growth suppression for MKN-45 human gastric cancer in tumor bearing mice. PM-8 inhibited the cell growth of AsPC-1 which depended on the dose with showing DNA ladder formation and DNA fragmentation, and positive Hoechst staining indicating apoptosis. The ratio of apoptotic cells on flow cytometry analysis were 35%, and 57% with treatment of PM-8 after 48, and 72 h, respectively. One of the anti-tumor activity of PM-8 result from the activation of apoptotic pathway. It is thought that polyoxomolybdates will be applied as a novel anti-tumor agent especially against cancers which are difficult to be treated clinically.

3β‐Hydroxyolean‐12‐en‐27‐oic Acid: A Cytotoxic, Apoptosis‐Inducing Natural Drug Against COLO‐205 Cancer Cells.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.

Hypoxic postconditioning enhances the survival and inhibits apoptosis of cardiomyocytes following reoxygenation: role of peroxynitrite formation

Our previous study has shown that slow or "controlled" reperfusion for the ischemic heart reduces cardiomyocyte injury and myocardial infarction, while the mechanisms involved are largely unclear. In this study, we tested the hypothesis that enhancement of survival and prevention of apoptosis in hypoxic/reoxygenated cardiomyocytes by hypoxic postconditioning (HPC) are associated with the reduction in peroxynitrite (ONOO(-)) formation induced by hypoxia/reoxygenation (H/R). Isolated adult rat cardiomyocytes were exposed to 2 h of hypoxia followed by 3 h of reoxygenation. After 2 h of hypoxia the cardiomyocytes were either abruptly reperfused with pre-oxygenized culture medium or postconditioned by two cycles of 5 min of brief reoxygenation and 5 min of re-hypoxia followed by 160 min of abrupt reoxygenation. H/R resulted in severe injury in cardiomyocytes as evidenced by decreased cell viability, increased LDH leakage in the culture medium, increased apoptotic index (P values all less than 0.01 vs. normoxia control group) and DNA ladder formation, which could be significantly attenuated by HPC treatment applied before the abrupt reoxygenation (P < 0.05 vs. H/R group). In addition, H/R induced a significant increase in ONOO(-) formation as determined by nitrotyrosine content in cardiomyocytes (P < 0.01 vs. normoxia control). Treatment with the potent ONOO(-) scavenger uric acid (UA) at reoxygenation significantly decreased ONOO(-) production and protected myocytes against H/R injury, whereas the same treatment with UA could not further enhance myocyte survival in HPC group (P > 0.05 vs. HPC alone). Statistical analysis showed that cell viability closely correlated inversely with myocyte ONOO(-) formation (P < 0.01). These data demonstrate that hypoxic postconditioning protects myocytes against apoptosis following reoxygenation and enhances myocytes survival, which is partly attributable to the reduced ONOO(-) formation following reoxygenation.

Protective effect of labedipinedilol-A, a novel dihydropyridine-type calcium channel blocker, on myocardial apoptosis in ischemia–reperfusion injury

The effects of labedipinedilol-A, a novel dihydropyridine-type calcium channel blocker with α-/β-adrenoceptor blocking activities, on myocardial infarct size, apoptosis and necrosis in the rat after myocardial ischemia/reperfusion (45 min/120 min) were investigated. Ten minutes prior to left coronary artery occlusion, rats were treated with vehicle or labedipinedilol-A (0.25 or 0.5 mg/kg, i.v.). In the vehicle group, myocardial ischemia–reperfusion induced creatine kinase (CK) release and caused cardiomyocyte apoptosis, as evidenced by DNA ladder formation and terminal dUTP deoxynucleotidyltransferase nick end-labeling (TUNEL) staining. Treatment with labedipinedilol-A (0.25 or 0.5 mg/kg) reduced infarct size significantly compared to vehicle group (18.75 ± 0.65% and 8.27 ± 0.29% vs. 41.72 ± 0.73%, P < 0.01). Labedipinedilol-A also reduced the CK, CK-MB, lactate dehydrogenase (LDH) and troponin T levels in blood. In addition, labedipinedilol-A (0.5 mg/kg) significantly decreased TUNEL positive cells from 19.21 ± 0.52% to 9.73 ± 0.81% ( P < 0.01), which is consistent with absence of DNA ladders in the labedipinedilol-A group. Moreover, labedipinedilol-A pretreatment also decreased calcium content in ischemic–reperfused myocardial tissue. In conclusion, these results demonstrate that labedipindielol-A, through reduction of calcium overload and apoptosis, exerts anti-infarct effect during myocardial ischemia–reperfusion and would be useful clinically in the prevention of acute myocardial infarction.

BAM-SiPc, a novel agent for photodynamic therapy, Induces apoptosis in human hepatocarcinoma HepG2 cells by a direct mitochondrial action

Photodynamic therapy is recently developed as an effective treatment for malignant disease. The therapeutic effect depends on the properties of the photosensitizers. Among the novel photosensitizers we have synthesized, the unsymmetrical bisamino phthalocyanine, SiPc[C3H5(NMe2)2O](OMe) [abbreviated as BAM-SiPc] is particularly active in the HepG2 cell culture model. Fluorescence microscopy has also indicated that it targets the mitochondria. In the present investigation, the biochemical mechanisms of BAM-SiPc leading to cell death were investigated. Photodynamic treatment with BAM-SiPc resulted in the generation of reactive oxygen species and a collapse of mitochondrial membrane potential. The pro-apoptotic Bax protein was translocated from the cytosol to mitochondria; while the level of the mitochondrial anti-apoptotic Bcl-2 protein decreased after photodynamic treatment. Cytochrome c, but not apoptosis-inducing factor, was released from the mitochondria into the cytosol, subsequently resulting in the cleavage of poly(ADP-ribose) polymerase. These events were at least partially responsible for the observed BAM-SiPc induced apoptosis, which was clearly demonstrated by (a) the loss of membrane asymmetry, (b) DNA ladder formation, and (c) the presence of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells.

Mechanism of DNA Damage and Apoptosis Induced by Tetrahydropapaveroline, a Metabolite of Dopamine

Tetrahydropapaveroline (THP), a metabolite of dopamine, has been suspected to be associated with dopaminergic neurotoxicity of L-DOPA. THP induced apoptosis in human leukemia cell line HL-60 cells, but did not in its hydrogen peroxide (H(2)O(2))-resistant clone HP100. THP-induced DNA ladder formation in HL-60 cells was inhibited by a metal chelator. THP induced damage to (32)P-labeled DNA fragments in the presence of metals. In the presence of Fe(III)EDTA, THP caused DNA damage at every nucleotide. The DNA damage was inhibited by free hydroxy radical ((.)OH) scavengers and catalase, suggesting that the Fe(III)EDTA-mediated DNA damage is mainly due to (.)OH generation. In the presence of Cu(II), THP caused DNA damage mainly at T and G of 5'-TG-3' sequence. The inhibitive effect of catalase and bathocuproine on Cu(II)-mediated DNA damage suggested that H(2)O(2) and Cu(I) participate in the DNA damage. This study demonstrated that THP-induced apoptosis via reactive oxygen species generated from reaction of H(2)O(2) and metals plays an important role in cytotoxicity of L-DOPA.

CDNA cloning and functional characterization of Xenopus laevis DNase γ

We report here the cDNA cloning and functional analysis of Xenopus DNase gamma (xDNase gamma). Two forms of cDNAs are isolated from adult spleen: one composing a 933 bp open reading frame for the enzymatically active xDNase gamma protein, and the other encoding an inactive short alternative form. Northern blot analysis revealed that the xDNase gamma mRNA is expressed in spleen, liver, testis, and ovary. xDNase gamma expression is scarcely detected in the tail muscle of tadpoles; however, it increases during metamorphosis and reaches a maximum during the late metamorphic climax. The ectopic expression of xDNase gamma results in the appearance of extensive DNA fragmentation in C2C12 myoblasts after the induction of apoptosis. In contrast, Xenopus DNase I fails to induce apoptotic DNA ladder formation under the same conditions. Our results suggest a possible involvement of xDNase gamma in apoptosis during amphibian metamorphosis.