DNA Agarose Electrophoresis Research Articles

Dry Bio-Decontamination Process in Reduced-Pressure O2 Plasma

The main objective of this work was to fully understand the bio-decontamination process in a reduced-pressure oxygen plasma. Gram-negative Escherichia coli species was chosen as the target microorganism in this test. The comparison of decontamination efficacy between plasma total and UV radiation individually under various treatment parameters and tests of DNA agarose electrophoresis were made to evaluate the inactivation effect of UV radiation. The quantity of protein leakage and the concentration of malondialdehyde (MDA), which are markers of the end products of lipid peroxidation, in bacterial suspension after treatment were determined to estimate the contribution of both charged particles and free radicals for bacterial death. In addition, a scanning electronic microscope was used to visualize the plasma effect on microorganisms. The results showed that the essential action of the oxygen plasma on Escherichia coli is believed to be attributed to the fast and intense etching on cell membrane by electrons and ions. Attacks on polyunsaturation fatty acid (PUFA) in the cell membrane by oxygen free radicals and the destruction of the DNA in the cell by UV radiation are accessorial during an effective decontamination process.

DNA sequence polymorphism within the bovine adenosine monophosphate deaminase 1 (AMPD1) is associated with production traits in Chinese cattle.

The objectives of the present study were to detect an 18-bp deletion mutation in the bovine adenosine monophosphate deaminase 1 (AMPD1) gene and analyze its effect on growth traits in 2 Chinese cattle breeds using DNA sequencing and agarose electrophoresis. The five 19-bp polymerase chain reaction products of the AMPD1 gene exhibited 3 genotypes and 2 alleles: WW: homozygote genotype (wild-type); DD: homozygote genotype (mutant-type); WD: heterozygote genotype. Frequencies of the W allele varied from 66.15-70.35%. The associations between the 18-bp deletion mutation in the AMPD1 gene with production traits in 226 Jia-Xian red cattle was analyzed. The animals with genotype WW showed significantly higher heart girth and body weight than those with genotypes WD and DD at 24 months (P < 0.01). Our results indicate that the deletion mutation in the AMPD1 gene is associated with production traits, and may be used for marker-assisted selection in beef cattle breeding programs.

Alginic Acid-Coated Chitosan Nanoparticles Loaded with Legumain DNA Vaccine: Effect against Breast Cancer in Mice

Legumain-based DNA vaccines have potential to protect against breast cancer. However, the lack of a safe and efficient oral delivery system restricts its clinical application. Here, we constructed alginic acid-coated chitosan nanoparticles (A.C.NPs) as an oral delivery carrier for a legumain DNA vaccine. First, we tested its characteristic in acidic environments in vitro. DNA agarose electrophoresis data show that A.C.NPs protected DNA better from degradation in acidic solution (pH 1.5) than did chitosan nanoparticles (C.NPs). Furthermore, size distribution analysis showed that A.C.NPs tended to aggregate and form micrometer scale complexes in pH<2.7, while dispersing into nanoparticles with an increase in pH. Mice were intragastrically administrated A.C.NPs carrying EGFP plasmids and EGFP expression was detected in the intestinal Peyer’s patches. Full-length legumain plasmids were loaded into different delivery carriers, including C.NPs, attenuated Salmonella typhimurium and A.C.NPs. A.C.NPs loaded with empty plasmids served as a control. Oral vaccination was performed in the murine orthotopic 4T1 breast cancer model. Our data indicate that tumor volume was significantly smaller in groups using A.C.NPs or attenuated Salmonella typhimurium as carriers. Furthermore, splenocytes co-cultured them with 4T1 cells pre-stimulated with CoCl2, which influenced the translocation of legumain from cytoplasm to plasma membrane, showed a 4.7 and 2.3 folds increase in active cytotoxic T lymphocytes (CD3+/CD8+/CD25+) when treated with A.C.NPs carriers compared with PBS C.NPs. Our study suggests that C.NPs coated with alginic acid may be a safe and efficient tool for oral delivery of a DNA vaccine. Moreover, a legumain DNA vaccine delivered orally with A.C.NPs can effectively improve autoimmune response and protect against breast cancer in mice.

Two-dimensional intact mitochondrial DNA agarose electrophoresis demonstrates the complexity of mtDNA structural forms and in vivo supercoiling by TFAM

Two-dimensional intact mitochondrial DNA agarose electrophoresis demonstrates the complexity of mtDNA structural forms and in vivo supercoiling by TFAM

A simple and effective SuperBuffer for DNA agarose electrophoresis

In the paper, we describe a unique effective electrophoresis buffer for DNA agarose electrophoresis, called SuperBuffer. Using this buffer, electrophoresis could be performed within 10 min at voltages as high as 25 V/cm. In addition, DNA fragments of different lengths could be isolated clearly even at lower agarose gel concentrations and the DNA recovery efficiency was higher than that of the TAE/TBE running buffers. The SuperBuffer still retained its electrophoretic effect even after several uses.

A novel 18-bp deletion mutation of the AMPD1 gene affects carcass traits in Qinchuan cattle

The objectives of the present study were focused on detecting deletion mutation in bovine AMPD1 gene, and analyzing its effect on body measurement and carcass traits in Qinchuan cattle by using DNA sequencing and agarose electrophoresis methods. The 198-bp PCR products of AMPD1 gene exhibited three genotypes and two alleles were revealed: A and B. The frequencies of genotype AA/AB/BB in Qinchuan populations was 0.7163, 0.2233 and 0.0605. The χ(2)-test analysis demonstrated that the breed was not in agreement with Hardy-Weinberg equilibrium (P < 0.05). The association of the 18-bp deletion mutation of AMPD1 gene with body measurement and carcass traits of Qinchuan cattle were analyzed. The cattle with AA genotype had slaughter weight and carcass weight than those with genotype AB (P < 0.01 or P < 0.05). These results suggest that the 18-bp deletion mutation may influence the carcass traits in Qinchuan cattle.

Propolis inhibits the proliferation of human leukaemia HL-60 cells by inducing apoptosis through the mitochondrial pathway

Propolis, a natural product derived from plant resins collected by honeybees, has been reported to exert a wide spectrum of biological functions. This research aimed at investigating the effect of propolis on the proliferation of human leukaemia HL-60 cells and whether propolis might induce apoptosis in HL-60 cells. The results showed dose- and time-dependent decreases in the proliferation of HL-60 cells treated with propolis (above 3 µg mL−1 of propolis). Further studies revealed that the anti-proliferative effects of propolis were caused by inducing apoptosis. Agarose electrophoresis of genomic DNA of HL-60 cells treated with propolis showed the ladder pattern typical for apoptotic cells. Propolis induced the activation of caspase-3 and cleavage of poly (ADP-ribose) polymerase in HL-60 cells. Propolis also induced the release of cytochrome c from mitochondria to cytosol. Taken together, these findings demonstrate that the inhibitory effect of propolis on HL-60 cell proliferation is caused by inducing apoptosis through the mitochondrial pathway.

The Apoptosis-Inducing Effects of HIV vpr Recombinant Eukaryotic Expression Vectors with Different Mutation Sites on Transfected Hela Cells

The vpr gene of human immunodeficiency virus type 1 (HIV-1) plays an important role in the pathogenicity of viruses. Previous studies showed that mutation of certain sites in HIV-1 Vpr amino acid sequences might influence the clinical process of infected individuals and attenuate its apoptosis-inducing capacity on infected cells. The present study was designed to transfect hela cells with several HIV-1 vpr fragments carrying specific mutation sites, and to observe the distinction of apoptosis-inducing effects of different HIV-1 vpr variant fragments on host cells and explore the possible mechanisms. According to the previous results, 14 typical vpr variant fragments were chosen from Chinese HIV-1 infected individuals. After PCR amplification of vpr gene, the products were purified and double digested with Hind and BamHI. Then pcDNA3.1 (+) eukaryotic expression plasmids were used for the ligation and transduction experiments. The recombinant plasmids were transiently transfected into Hela cells with liposomes, and meanwhile the blank cell and empty vector cell were established as control. RT-PCR was used to detect the mRNA expression of target genes; fluorescent microscope for observing apoptotic cells with Hoechst staining; DNA agarose electrophoresis for detecting apoptotic gradient band; Annexin assay for detecting cell apoptosis; and Caspase activity test for exploring the pathway of cell apoptosis. After transfected with 14 vpr gene segments, the Hela cells exhibited various apoptosis-inducing capabilities. We found that the HIV-1 Vpr segments with mutation at the 70(th), 85(th), 86(th), or 94(th) site showed lower apoptosis-inducing capabilities than other segments on Hela cells, and the apoptosis-inducing ability of HIV-1 vpr gene on host cells might be related to its subtype. Meanwhile, we found that the Caspase3 activity of transfected cell carrying these mutated fragment sites was decreased as compared to the cell with other fragments. This study firstly found that the HIV-1 Vpr fragments with the 70(th), 85(th), 86(th), or 94(th) site mutation might attenuate their apoptosis-inducing abilities on Hela cells. One of the mechanisms might be the attenuation of Caspase-3 activity.

The Antiproliferation Effect of Berbamine on K562 Resistant Cells by Inhibiting NF‐κB Pathway

Imatinib mesylate is effective against Ph chromosome-positive leukemia; however, resistance has been reported. High expression of bcr-abl in mRNA and protein levels, and other alterations were found in patients who experienced imatinib treatment failures and thus it is important to design alternative treatment strategies. The aim of this study was to evaluate the in vitro effect of berbamine, on imatinib-resistant chronic myelogenous leukemia (CML) K562 (K562-r) cells, and explore the mechanisms. The growth of K562-r cells was examined using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Morphological analysis and DNA agarose electrophoresis were used to detect apoptosis in K562-r cells, and the extent of the cells in the sub-G1 cell cycle phase was measured using flow cytometry. The expression levels of BCR-ABL, phospho-BCR-ABL, and nuclear factor kappaB (NF-kappaB), IkappaBalpha, phospho-IkappaBalpha, IkappaB kinases alpha(IKKalpha), and Survivin were determined by Western blot. bcr-abl mRNA expression was determined by RT-PCR. MTT assays indicated that berbamine significantly inhibited the proliferation of K562-r cells. Cells with characteristics of apoptosis were confirmed by morphology examination and DNA agarose electrophoresis and percentage of apoptosis were increased after treatment with berbamine. The results also showed that berbamine was able to down-regulate BCR-ABL and phospho-BCR-ABL proteins by affecting bcr-abl mRNA expression and decrease expression of nuclear NF-kappaB, phospho-IkappaBalpha, IKKalpha, and Survivin. Collectively, berbamine could inhibit the proliferation of K562-r cells and induce apoptosis. The mechanisms may be related at least in part, to inhibit BCR-ABL and its downstream NF-kappaB signaling. Berbamine may provide an alternative candidate for the treatment of patients with CML resistant to imatinib therapy.

Protocol for the quantitative assessment of DNA concentration and damage (fragmentation and nicks)

An ultrasensitive protocol is presented for the quantitative assessment of fragmented and nicked dsDNA using PicoGreen and consists of four methods. The first quantifies the concentration of DNA, whereas the second (quantitative complement of the Comet assay) quantifies the degree of DNA fragmentation seen in a typical DNA agarose electrophoresis gel. Both methods have sensitivity of 5 pg of DNA. The third method (quantitative counterpart of the electrophoresis-based qualitative apoptotic and necrotic DNA assays) quantifies the polyethylene glycol-fractionated small-size (0-1 kb) fragmented DNA. This method also detects up to 5 pg of damaged DNA and requires a minimum sample of quantity 0.2 ml of 2.5 microg ml(-1). The fourth method measures the percentage of DNA nicks by alkaline DNA unwinding and requires up to 15 pg of DNA sample. The time required for processing 10 DNA samples is 1/2, 1, 13 and 1 h for the first, second, third and fourth method, respectively.

The effects of Hedgehog-Gli 1 signaling pathway on proliferation and apoptosis of hepatic stellate cells

To investigate the effect of Hedgehog-Gli1 signaling pathway on proliferation, apoptosis and activation of hepatic stellate cells (HSCs) in vitro. The expression of Shh, Smo, Ptc and Gli-1 in HSC-T6 cells was analyzed by RT-PCR. HSC-T6 cells were incubated with various concentration of cyclopamine (0, 50, 100, 150, 200, 250 mumol/L) for 24 hours, cell viability was checked by MTT colorimetric assay, cell cycle was analyzed by flow cytometry, apoptosis was assayed by agarose electrophoresis of DNA and PI-Annexin V fluorescent staining, and the mRNA levels of Gli-1, TGF beta 1, PDGF and Bcl-2 were quantified by real-time RT-PCR. RT-PCR indicated that the components of the Hedgehog-Gli1 signaling pathway were expressed in HSC-T6 cells. MTT assay indicated that cyclopamine inhibited cell viability in a concentration dependant manner (F = 636.81, P less than 0.01). Flow cytometry indicated that cells were piled up at G0/G1 phase in cyclopamine treated cells (65.08%+/-1.50%) as compared to control cells (55.41%+/-2.54%, t = -8.05, P less than 0.01). Cyclopamine treatment resulted in apoptosis as indicated by DNA fragmentation and PI-Annexin V staining. The mRNA levels of Gli-1, TGF beta 1, PDGF and Bcl-2 in cyclopamine treated cells were significantly lower than that in control cells (P less than 0.01). Cyclopamine may inhibit the Hedgehog-Gli1 signaling, and hence repress proliferation and promote apoptosis in hepatic stellate cells.

Coelomic fluid of the earthworm Eisenia fetida induces apoptosis of HeLa cells in vitro

Earthworm, Dilong in Chinese, is a traditional Chinese medicine used to treat many diseases including tumors. To investigate whether the coelomic fluid of the earthworm Eisenia fetida inhibits viability and induces apoptosis of HeLa cells, MTT assay, AO/EB fluorescent staining method and DNA agarose electrophoresis were applied. The cytotoxicity of coelomic fluid on HeLa cell showed a concentration dependent manner after 48 h treatment as detected by the MTT assay. Coelomic fluid (1 mg/ml) exhibited toxic effects on HeLa cells with an inhibition rate of 84.22%, leading to cell lysis. The inhibition rates in 0.1 mg/ml and 0.01 mg/ml treatments were 10.24% and 2.99%, respectively. Morphological changes of typical apoptosis were observed by AO/EB staining in 0.1 mg/ml and 0.01 mg/ml concentration with apoptotic rates of 79.1% and 22.2%, while the cells were necrotic in 1 mg/ml concentration as indicated by red stained cells. The agarose gel electrophoresis of DNA revealed a smear pattern at the concentrations of 0.1 mg/ml and 0.01 mg/ml of coelomic fluid. At the concentration of 0.1 mg/ml, the coelomic fluid induced apoptosis in HeLa cells in a time dependent manner. This work suggests that some of the coelomic fluid components might be useful for pharmaceutical applications in the treatment of cancer, but our knowledge is still very limited. Testing this hypothesis will require intense controlled investigations.

Betanin a betacyanin pigment purified from fruits of Opuntia ficus-indica induces apoptosis in human chronic myeloid leukemia Cell line-K562

Betalains are water-soluble nitrogenous vacuolar pigments present in flowers and fruits of many caryophyllales with potent antioxidant properties. In the present study the antiproliferative effects of betanin, a principle betacyanin pigment, isolated from the fruits of Opuntia ficus-indica, was evaluated on human chronic myeloid leukemia cell line (K562). The results show dose and time dependent decrease in the proliferation of K562 cells treated with betanin with an IC(50) of 40 microM. Further studies involving scanning and transmission electron microscopy revealed the apoptotic characteristics such as chromatin condensation, cell shrinkage and membrane blebbing. Agarose electrophoresis of genomic DNA of cells treated with betanin showed fragmentation pattern typical for apoptotic cells. Flow cytometric analysis of cells treated with 40 microM betanin showed 28.4% of cells in sub G0/G1 phase. Betanin treatment to the cells also induced the release of cytochrome c into the cytosol, poly (ADP) ribose polymerase (PARP) cleavage, down regulation Bcl-2, and reduction in the membrane potentials. Confocal microscopic studies on the cells treated with betanin suggest the entry of betanin into the cells. These studies thus demonstrate that betanin induces apoptosis in K562 cells through the intrinsic pathway and is mediated by the release of cytochrome c from mitochondria into the cytosol, and PARP cleavage. The antiproliferative effects of betanin add further value to the nutritional characteristics of the fruits of O. ficus-indica.

Induction of apoptosis in K562 cells by jolkinolide B

Jolkinolide B, a bioactive diterpene isolated from the roots of Euphorbia fischeriana Steud, has various biological and pharmacological properties. In this study, the cytotoxicity of highly purified jolkinolide B was tested in human chronic myeloid leukemia (K562) and 2 other cell lines (human esophageal carcinoma Eca-109 and human hepatoma HepG2). The results indicate a significant decrease in the proliferation of all the 3 cell lines when treated with jolkinolide B for 24 h; the IC50 value of cytotoxicity was 12.1 microg/mL (for K562 cells), >50.0 microg/mL (for HepG2 cells), and 23.7 microg/mL (for Eca-109 cells). Further study of K562 cells involving fluorescence and transmission electron microscopy revealed characteristic apoptotic features, such as cell shrinkage, membrane blebbing, loss of microvilli, and nuclear condensation. Agarose electrophoresis of genomic DNA showed a typical fragmentation pattern for apoptotic cells. A kinetic cell-cycle analysis demonstrated that the cell cycle was arrested in the G1 phase. All these results suggest that the anti-proliferation effect of jolkinolide B on K562 cells is achieved by arresting the cell cycle in the G1 phase and subsequently inducing apoptosis.

Altered Bcl-2 and Bax expression is associated with cultured first trimester human cytotrophoblasts apoptosis induced by hypoxia

目的 探讨缺氧对妊娠早期细胞滋养细胞凋亡以及相关蛋白表达的影响.方法采用原代培养的妊娠早期细胞滋养细胞,用基因组DNA琼脂糖凝胶电泳和流式细胞术以及Western印迹等方法比较缺氧和正常氧体外培养后细胞凋亡以及凋亡相关蛋白Bcl-2和Bax表达的差异.结果妊娠早期的细胞滋养细胞缺氧培养24 h后电泳出现凋亡的典型特征DNAladder,同时细胞凋亡率增加[(4.4±0.8)%和(17.4±3.0)%,P＜0.05],并伴有Bcl-2蛋白表达减少和Bax蛋白表达增加(P＜0.05).结论缺氧促进妊娠早期细胞滋养细胞凋亡,伴有Bcl-2和Bax蛋白表达的改变。

Targeted Disruption of FANCC and FANCG in Human Cancer Provides a Preclinical Model for Specific Therapeutic Options

How specifically to treat pancreatic and other cancers harboring Fanconi anemia gene mutations has raised great interest recently, yet preclinical studies have been hampered by the lack of well-controlled human cancer models. We endogenously disrupted FANCC and FANCG in a human adenocarcinoma cell line and determined the impact of these genes on drug sensitivity, irradiation sensitivity, and genome maintenance. FANCC and FANCG disruption abrogated FANCD2 monoubiquitination, confirming an impaired Fanconi anemia pathway function. On treatment with DNA interstrand-cross-linking agents, FANCC and FANCG disruption caused increased clastogenic damage, G2/M arrest, and decreased proliferation. The extent of hypersensitivity varied among agents, with ratios of inhibitory concentration 50% ranging from 2-fold for oxaliplatin to 14-fold for melphalan, a drug infrequently used in solid tumors. No hypersensitivity was observed on gemcitabine, etoposide, 3-aminobenzamide, NU1025, or hydrogen peroxide. FANCC and FANCG disruption also resulted in increased clastogenic damage on irradiation, but only FANCG disruption caused a subsequent decrease in relative survival. Finally, FANCC and FANCG disruption increased spontaneous chromosomal breakage, supporting the role of these genes in genome maintenance and likely explaining why they are mutated in sporadic cancer. Our human cancer cell model provides optimal controls to elucidate fundamental biologic features of individual Fanconi anemia gene defects and facilitates preclinical studies of therapeutic options. The impact of Fanconi gene defects on drug and irradiation sensitivity renders these genes promising targets for a specific, genotype-based therapy for individual cancer patients, providing a strong rationale for clinical trials.

Somatostatin levels and polymorphonuclear neutrophil life in intestinal ischemia reperfusion of macaque

To investigate the association of somatostatin (SST) levels in plasma or jejunum tissue and polymorphonuclear neutrophil (PMN) life in intestinal ischemia reperfusion of macaque. MODS model in macaque was established by clamping superior mesenteric artery. The levels of SST in plasma and jejunum tissue were measured by radioimmunoassay. After incubated with SST, the apoptotic PMN were determined by the morphological studies, flow cytometer and DNA agarose electrophoresis. The expression of SSTR on PMN was detected by biosensor. SST levels in macaque plasma or jejunum tissue were significantly decreased after intestinal ischemia reperfusion (IR) injury. The apoptotic rate of PMN was obviously reduced (15.4% +/- 1.4% to 3.5% +/- 0.5%, P < 0.05). However, the number of apoptotic macrophage from abdominal cavity was clearly increased. The apoptotic rate of PMN was significantly increased (20.0% +/- 2.2% to 50.2% +/- 1.8%, P < 0.01) after incubated with SST in vitro. Specific DNA ladder bands from PMN were visualized with agarose electrophoresis. Specific bindings between PMN and the antibodies for SSTR1 (548 RU/ml +/- 20 RU/ml) or SSTR2 (28 RU/ml +/- 21 RU/ml) were detected. The physiological apoptosis of PMN in macaque could be induced by SST through SSTRs on PMN. The decreased SST levels in macaque plasma or jejunum tissue in the state of intestinal ischemia reperfusion might extend PMN life and promote the occurrence of MODS.

Effects of berbamine on growth of leukemia cell line NB4 and its mechanism

To determine effects of berbamine on the growth of leukemia cell line NB4 and explore its possible mechanisms. The growth of NB4 cells was examined with MTT assay. Morphological analysis and DNA agarose electrophoresis were used to detect apoptosis in NB4 cells, and the apoptosis rate was measured by flow cytometry. The PML/RAR alpha mRNA was determined by nested-PCR, and the Survivin mRNA was tested by RT-PCR. The expression of caspase 3 protein in NB4 cells was evaluated by flow cytometry. The growth of NB4 cells was inhibited significantly after treated with berbamine at different concentrations for different time points, the IC(50)value was 3.860 microg/ml at 48 hours. Morphology analysis showed the characteristics of apoptosis, and the DNA agarose electrophoresis showed the typical DNA ladder. The apoptosis rate increased from 2.83% to 58.44% after treated with berbamine at 12 microg/ml for 48 hours. The expression of PML/RAR alpha mRNA presented no significant changes, however, Survivin mRNA was decreased dramatically. The protein expression of Caspase 3 increased significantly from 2.06% to 70.89% after treated with berberine at a concentration of 12 mug/ml for 48 hours. Berbamine could inhibit the growth of leukemia cell line NB4. The induction of cell apoptosis may be one of the mechanisms for suppressing the growth of leukemia cell line NB4. Inhibition of Survivin mRNA and upregulation of Caspase 3 protein might be also involved in cell apoptosis.

Molecular immune mechanism of C‐phycocyanin from Spirulina platensis induces apoptosis in HeLa cells in vitro

C-phycocyanin (C-PC), a water-soluble protein pigment, isolated from Spirulina platensis, is of great importance because of its various medical and pharmacological properties. In the present study, we first investigated the effect of highly purified C-PC on growth and proliferation of HeLa cells in vitro. The results indicated that there was a significant decrease in the number of cells that survived for HeLa cells treated with C-PC compared with control cells untreated with C-PC. Further electron-microscopic studies revealed that C-PC could induce characteristic apoptotic features, including cell shrinkage, membrane blebbing, microvilli loss, chromatin margination and condensation into dense granules or blocks. Agarose electrophoresis of genomic DNA of HeLa cells treated with C-PC showed fragmentation pattern (DNA ladder of oligomers of 180-200 bp) typical for apoptotic cells. Flow-cytometric analysis of HeLa cells treated with different concentrations of C-PC demonstrated an increasing percentage of cells in sub-G0/G1 phase. In addition, we found that C-PC could promote the expression of Fas and ICAM-1 (intercellular cell-adhesion molecule 1) protein, while it held back the Bcl-2 (B-cell lymphocytic-leukaemia proto-oncogene 2) protein expression. This suggested that C-PC could induce the activation of pro-apoptotic gene and downregulation of anti-apoptotic gene expression and then facilitate the transduction of tumoural apoptosis signals that resulted in the apoptosis of HeLa cells in vitro. Caspases 2, 3, 4, 6, 8, 9, and 10 were activated in C-PC-treated HeLa cells, which suggested that C-PC-induced apoptosis was caspase-dependent. C-PC treatment of HeLa cells also resulted in release of cytochrome c from the mitochondria into the cytosol that was related to apoptosis of C-PC-treated HeLa cells.

Lipids Deposited in Human Atheromatous Lesions Induce Apoptosis of Human Vascular Smooth Muscle Cells

To clarify whether lipids deposited in human atheromatous lesions induce apoptosis of vascular smooth muscle cells (SMC) and to identify the main component in deposited lipids responsible for inducing apoptosis, we examined the effect of lipids extracted from human atheromatous lesions on apoptosis of cultured SMC and analyzed the content of cholesterol in the lipids. When lipids extracted from atheromatous lesions were added to SMC, agarose electrophoresis of DNA showed a ladder pattern, DNA fragmentation assay detected an increase of fragmented DNA, and flow cytometric analysis demonstrated an increase of apoptotic cells. When the extracted lipids were fractionated by Sep-Pak ODS column, addition of the oxysterol-rich fraction to SMC resulted in a DNA ladder pattern and positive staining of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL). The oxysterol-rich fraction also increased fragmented DNA and apoptotic cells to a greater extent than the other two fractions. HPLC analysis showed that the quantity of 7-ketocholesterol in extracted lipids was large enough to induce SMC apoptosis. These results suggest that lipids deposited in human atheromatous lesions may induce apoptosis of SMC and that oxysterols may be important factor contributing to induce apoptosis among deposited lipids.