Low Molecular Weight DNA Fragments Research Articles

Anti Cancer Potential of some Indole based Quinoxaline Derivatives against Dalton’s Lymphoma (DL) cells

Indole based quinoxaline derivatives (2a-f) were synthesized and the therapeutic potential of the synthesized compounds (2a-f) was investigated against Dalton’s Lymphoma (DL) cells. It was observed that treatment of DL cells with quinoxaline derivatives (2a-f) showed significant decrease in cell viability, altered morphology, nuclear disintegration and DNA fragmentation into low molecular weight DNA fragments and thus characteristic of apoptosis. In this view, the synthesized compounds may be used as potential therapeutic regimen to treat this type of cancer.

Potential of Seaweed Extract on Growth, Physiological, Cytological and Biochemical Parameters of Wheat (Triticum aestivum L.) Seedlings

Synthetic fertilizers are widely employed in agriculture relative to biofertilizers. Long-term use affects soil fertility, plant vigor, human and livestock health, and the soil microbial community. Seaweeds were employed as biofertilizers in sustainable agriculture to overcome this issue and boost the agricultural productivity. Therefore, the current study investigates the effect of wheat seeds priming with the aqueous extracts of Ulva linza or Corallina officinalis seaweeds at various concentrations (5, 10, 15, 20, and 30%). Wheat grains were primed with seaweed extracts and grown in petri dishes, and the germination rate, growth attributes, physiological parameters, cytological traits, and molecular analyses were evaluated. The physico-chemical characterization revealed that U. linza possessed higher levels of ash, soluble proteins, and sugars, as well as essential macronutrients and amino acids, than C. officinalis. Seedling growth parameters, physiological characteristics, and mitotic index all showed a substantial increase in low concentrations of both extracts when compared to the control. Higher concentrations, on the other hand, exhibited an inhibitory effect as well as certain chromosomal abnormalities. In the treated seedlings, new proteins were expressed, which could be owing to the activity of bioactive components in the extracts. As compared to untreated seedlings, PCR-RAPD analysis utilizing five markers revealed that both seaweed extracts triggered low and high molecular weight DNA fragments, as well as fragments specific to each extract. Seaweed extracts could be employed as an environmentally friendly and soil-safe alternative to typical chemical fertilizers.

Anti cancer potential of 4H- chromene siprooxindole hybrid molecules against Dalton’s Lymphoma (DL) cells

The anticancer efficacy of 4H- Chromene Spirooxindole hybrid molecules 4i-m1 (Samples 1- 5) was investigated on Dalton’s lymphoma (non-Hodgkin lymphoma) cells. Dalton’s lymphoma (DL) is a spontaneous and highly invasive T-cell lymphoma that develops as an ascitic tumor in murines. It was observed that treatment of DL cells with compounds showed significant decrease in cell viability, altered morphology, nuclear disintegration and DNA fragmentation into low molecular weight DNA fragments and thus characteristic of apoptosis. In this view, these compounds may be used as potential therapeutic regimen to treat this type of cancer in experimental mice.

Whole human blood DNA degradation associated with artificial ultraviolet and solar radiations as a function of exposure time

Laboratory investigations were conducted to evaluate the effect of ultraviolet radiation components and solar radiation exposure as a function of time on the degradation of whole human blood DNA from the standpoint of forensic analysis. Ten μL of whole human male blood samples were exposed to UV-A, UV-B, UV-C, and solar radiation at 20 min intervals up to 120 min. Allele frequencies of 16 short tandem repeat (STR) markers were monitored by employing current forensic typing DNA techniques. The STR markers were grouped into high, medium, and low molecular weight categories. Results revealed that even 20 min exposure to 4.89 eV UV-C photons (ʎ = 254 nm) with radiation intensity of 1200 μW/cm2 would degrade whole human male blood DNA samples significantly, making them unfit for human identification due to the breakdown of high molecular weight STRs. Exposure of blood samples to 4.11 eV UV-B photons (ʎ = 302 nm) with radiation intensity of 900 μW/cm2 resulted in complete degradation of high molecular weight STRs after 60 min. Partial breakdown of medium and low molecular weight STRs started after 80 min exposure. The degradation index (DI) values appear to show that the degradation of the DNA template molecule was relatively less in the low molecular weight DNA fragments as compared with high molecular weight DNA fragments. This finding indicates that genetic profiles obtained from whole human male blood exposed to this radiation for 60 min will give inconclusive results. Samples exposed up to 120 min to 3.40 eV UV-A photons (ʎ = 365 nm) and 3.10–3.94 eV photons of solar radiation did not appear to produce appreciable degradation in any of three molecular weight STRs in the whole human blood DNA samples.

Differential Dynamics of the Levels of Low Molecular Weight DNA Fragments in the Plasma of Patients With Ischemic and Hemorrhagic Strokes.

Introduction:To evaluate Low-Molecular-weight (LMW) DNA as a possible prognostic biomarker in acute ischemic and hemorrhagic stroke.Methods:LMW DNA samples were isolated from plasma and cerebrospinal fluid by phenol deproteinization, analyzed by gradient polyacrylamide electrophoresis and quantified by spectrophotometry.Results:Two common types of stroke, i.e. ischemic and hemorrhagic, differ by the temporal dynamics of cell-free DNA (cfDNA) accumulation. In hemorrhagic stroke, an initial increase in LMW DNA levels, most likely reflects an extent of the tissue damage, while in ischemic patients, the LMW DNA levels increase in parallel with the damage caused by hypoxia and subsequent compensatory reperfusion.Conclusion:These time-course data specify optimal assessment windows with maximum differentiating power for stroke outcomes: 24–48 hours post-event for ischemic stroke, and as close as possible to the moment of hospital admission for hemorrhagic stroke. These data also indicate the role of apoptosis in the formation of ischemic focus.

Differential dynamics of the levels of low molecular weight DNA fragments in plasma of patients with ischemic and hemorrhaging strokes

Differential Dynamics of the Levels of Low Molecular Weight DNA Fragments in the Plasma of Patients With Ischemic and Hemorrhagic Strokes

Whole exome sequencing analysis of urine trans-renal tumour DNA in metastatic colorectal cancer patients

BackgroundThe analysis of circulating free tumour DNA (ctDNA) in blood, commonly referred as liquid biopsy, is being used to characterise patients with solid cancers. Tumour-specific genetic variants can also be present in DNA isolated from other body fluids, such as urine. Unlike blood, urine sampling is non-invasive, can be self-performed, and allows recurrent longitudinal monitoring. The features of tumour DNA that clears from the glomerular filtration barrier, named trans-renal tumour DNA (trtDNA), are largely unexplored.Patients and methodsSpecimens were collected from 24 patients with KRAS or BRAF mutant metastatic colorectal cancer (mCRC). Driver mutations were assessed by droplet digital PCR (ddPCR) in ctDNA from plasma and trtDNA from urine. Whole exome sequencing (WES) was performed in DNA isolated from tissue, plasma and urine.ResultsOut of the 24 CRC cases, only four had sufficient DNA to allow WES analyses in urine and plasma. We found that tumour alterations primarily reside in low molecular weight fragments (less than 112 bp). In patients whose trtDNA was more than 2.69% of the urine derived DNA, cancer-specific molecular alterations, mutational signatures and copy number profiles identified in urine DNA are comparable with those detected in plasma ctDNA.ConclusionsWith current technologies, WES analysis of trtDNA is feasible in a small fraction of mCRC patients. Tumour-related genetic information is mainly present in low molecular weight DNA fragments. Although the limited amounts of trtDNA poses analytical challenges, enrichment of low molecular weight DNAs and optimised computational tools can improve the detection of tumour-specific genetic information in urine.

Radioprotective combination of α-tocopherol and ascorbic acid promotes apoptosis that is evident by release of low-molecular weight DNA fragments into circulation

Purpose: Genotoxic stresses, including irradiation, lead to the apoptosis of damaged cells and the release of DNA fragments into circulation. Both α-tocopherol acetate and ascorbic acid possess antioxidant and radioprotective properties. Interestingly, depending on a particular experimental system, the treatment with vitamins may demonstrate either apoptosis-promoting or apoptosis-suppressing effects.Materials and methods: Adult Wistar male rats received total body irradiation with 2–100 Gy doses, while non-irradiated rats served as controls. Oral gavages with vitamins were administered either 10 min or 1 h before irradiation. Control groups were similarly treated with water. Blood samples were collected at 5 h post irradiation. The levels and the composition of circulating DNA were profiled. Chromosomal aberrations were assessed 24 h after irradiation.Results: A substantial dose-dependent increase in circulating low-molecular weight (LMW) DNA levels was observed after whole body irradiation. An order-of-magnitude increase in the proportion of bone marrow cells with chromosomal abnormalities was observed after irradiation at 2 Gy. Single vitamin preparations were not protective, while the combination of α-tocopherol (10 mg/kg) and ascorbic acid (20 mg/kg) displayed a protective effect evident from marked decrease in chromosomal aberrations. In animals treated with a combination of the vitamins only, substantial increases in the release of LMW DNA were observed.Conclusions: Radioprotective combination of α-tocopherol and ascorbic acid promotes apoptosis that is evident by release of low-molecular weight DNA into circulation. We hypothesize that the pretreatment with vitamins provides radioprotection, at least in part, by aiding non-inflammatory, apoptotic elimination of most damaged cells. The microevolutionary nature of observed adaptive response provides mechanistic foundation for the phenomenon of hormesis.

Вариабельность простых нуклеотидных повторов в ДНК клеток периферической крови мужчин, подвергшихся пролонгированному воздействию ионизирующей радиации

Long-term post-radiation changes in the level of microsatellite DNA polymorphism in peripheral blood of the male "Mayak" employees (Ozyorsk, Russia), who had been exposed to prolonged gamma-irradiation during professional activities, were studied. DNA samples were obtained from the Radiobiology Repository of Human Tissue (Southern-Urals Biophysics Institute FMBA) and used as templates for arbitrarily primed PCR. Comparative analysis of the obtained samples of DNA fragments showed a significant increase in the number of high-molecular fragments and reduction in the number of amplified low molecular weight DNA fragments in comparison with the control. However, a direct correlation of the level of DNA polymorphism with the accumulated total dose of radiation was not found. The study of the polymorphism of microsatellite DNA repeats can be used for qualitative assessment of the levels of genetic variability.

Blood and Tissue Leukocyte Apoptosis inTrypanosoma bruceiInfected Rats

Severity of disease in human African try- panosomiasis may be linked, as in other human protozoan infections, to apoptosis of host inflammatory cells during infection. The present study investigates the involvement of leukocyte apoptosis in Trypanosoma brucei infection of rodents. Twenty-seven Wistar rats were infected with 1 × 10 3 T. brucei and leukocyte apoptosis was evaluated by several methods. Apoptotic cell count was performed in blood, spleen, thymus, lymph node and liver during infections, using Light Microscopy (LM), agarose gel electrophoresis and Transmission Electron Microscopy (TEM). Blood and tissues leukocyte apoptosis in infected animals were confirmed by LM, TEM, and agarose gel electrophoresis, which showed low molecular weight DNA fragments. Infected rats sacrificed after day 8 PI showed sig- nificant increase in apoptotic cells in both blood ( p< 0.001) and spleen ( p< 0.05). Peak blood leukocyte apoptosis corresponded with peak parasitemia and the lowest total leukocyte count in all infected rats. Our data provide the first documentation of increased levels of blood leukocyte apoptosis during T. brucei infection. Apoptosis of blood cells during trypanosome infection may represent a putative mechanism for the severity of leukopenia and disease.

Leishmania mexicana: Inhibition of camptothecin-induced apoptosis of monocyte-derived dendritic cells

Macrophages (Mϕ) and dendritic cells (DC) are the major target cell populations of the obligate intracellular parasite Leishmania. Inhibition of host cell apoptosis is a method employed by multiple pathogens to ensure their survival in the infected cell. Leishmania has been shown to protect Mϕ and neutrophils from both natural and induced apoptosis. As shown in this study, apoptosis in monocyte-derived dendritic cells (moDC) induced by treatment with camptothecin was downregulated by coincubation with L. mexicana, as detected by morphological analysis of cell nuclei, TUNEL assay, gel electrophoresis of low molecular weight DNA fragments, and annexin V binding to phosphatidylserine. The observed antiapoptotic effect was found to be associated with a significant reduction of caspase-3 activity in moDC. The capacity of L. mexicana to delay apoptosis induction in the infected moDC may have implications for Leishmania pathogenesis by favoring the invasion of its host and the persistence of the parasite in the infected cells.

Significance of large nuclear vacuoles in human spermatozoa: implications for ICSI

The aim of this study was to determine the extent of DNA fragmentation and the presence of denatured single-stranded or normal double-stranded DNA in spermatozoa with large nuclear vacuoles (LNV) selected by high magnification. Fresh semen samples from 30 patients were prepared by discontinuous isolate concentration gradient. Spermatozoa with normal nucleus (NN) and LNV were selected at ×8400 magnification and placed on different slides. DNA fragmentation was determined by TUNEL assay. Denatured and double-stranded DNA was identified by the acridine orange fluorescence method. DNA fragmentation in spermatozoa with LNV (29.1%) was significantly higher ( P < 0.001) than in spermatozoa with NN (15.9%). Therefore, cleavage of genomic DNA in low molecular weight DNA fragments (mono- and oligonucleosomes), and single-strand breaks (nicks) in high molecular weight DNA occur more frequently in spermatozoa with LNV. Similarly, the percentage of denatured-stranded DNA in spermatozoa with LNV (67.9%) was significantly higher ( P < 0.0001) than in spermatozoa with NN (33.1%). The high level of denatured DNA in spermatozoa with LNV suggests precocious decondensation and disaggregation of sperm chromatin fibres. The results show an association between LNV and DNA damage in spermatozoa, and support the routine morphological selection and injection of motile spermatozoa at high magnification for ICSI.

Discontinuous fragmentation of nuclear DNA during apoptosis revealed by discrete “sub‐G1” peaks on DNA content histograms

Internucleosomal DNA fragmentation is one of the hallmarks of apoptosis. Because the low molecular weight DNA fragments are extracted during cell staining in aqueous solutions, apoptotic cells can be identified on DNA content frequency histograms as cells with fractional ("sub-G(1)") DNA content. The aim of the present study was to explore whether in situ DNA fragmentation during apoptosis is discontinuous or progresses incessantly and if it is discontinuous, to define the resistant to cleavage fraction of DNA that remains stainable with the fluorochrome. The model of activation-induced apoptosis of human lymphocytes was chosen as it provides uniform cell population with identical DNA content (DI = 1.00) that undergo apoptosis. Their apoptosis was induced by multivalent mitogen phytohemagglutinin (PHA) in the absence and presence of geldanamycin (GA), the benzoquinone ansamycin antibiotic which binds to Hsp90 (Heat Shock Protein 90) and alters its function. The cells were stained with acridine orange, the metachromatic fluorochrome that differentially stains cellular DNA and RNA. A sharp, discrete peak representing the subpopulation of "sub-G(1)" cells with highly reproducible DI = 0.42 +/- 0.02 (CV = 5.5 +/- 1.2) was observed on DNA content histograms of lymphocytes whose apoptosis was induced by PHA alone. Two distinct peaks, one representing cell subpopulations with DI = 0.42 (as above) and another, with DI = 0.79 +/- 0.04 (CV = 5.8 +/- 0.4), respectively, were seen in apoptotic cells from cultures stimulated with PHA in the presence of GA. The frequency of cells represented by the sub-G(1) peaks varied depending on time of induction of apoptosis and GA concentration. Apoptosis-induced DNA fragmentation is discontinuous; approximately 42% of DNA is relatively stable and remains within the cell. The data suggest that the stable DNA is associated with nuclear matrix while the degradable fraction represents DNA in loop domains. A transient DNA stabilization is apparent in the presence of GA as evidenced by the presence of cell subpopulations with 79% of DNA retained in the cell. The observed discontinuity of DNA fragmentation appears to reflect sequential involvement of different nucleases and may also be modulated by chromatin structure.

Impairment of the mitochondrial respiratory enzyme activity triggers sequential activation of apoptosis‐inducing factor‐dependent and caspase‐dependent signaling pathways to induce apoptosis after spinal cord injury

The mitochondrion participates in caspase-independent or caspase-dependent apoptotic pathways through the release of apoptosis-inducing factor or cytochrome c. Whether both mitochondrial apoptotic cascades are triggered in the injured spinal cord remains unknown. Here, we demonstrated that neurons, astrocytes and microglia in spinal segments proximal to a complete spinal cord transection underwent two phases of apoptotic cell death. The early phase of high-molecular weight (HMW) DNA fragmentation was associated with nuclear translocation of apoptosis-inducing factor, reduction in mitochondrial respiratory chain enzyme activity and decrease in cellular ATP concentration. The delayed phase of low-molecular weight (LMW) DNA fragmentation was accompanied by cytosolic release of cytochrome c, activation of caspases 9 and 3, and resumption of mitochondrial respiratory functions and ATP contents. Microinfusion of coenzyme Q(10), an electron carrier in mitochondrial respiratory chain, into the epicenter of the transected spinal cord attenuated both phases of induced apoptosis, and reversed the elicited mitochondrial dysfunction, bioenergetic failure, and activation of apoptosis-inducing factor, cytochrome c, or caspases 9 and 3. We conclude that mitochondrial dysfunction after spinal cord transection represents the initiating cellular events that trigger the sequential activation of apoptosis-inducing factor-dependent and caspase-dependent signaling cascades, leading to apoptotic cell death in the injured spinal cord.

Programmed cell death of follicular epithelium during the late developmental stages of oogenesis in the fruit flies Bactrocera oleae and Ceratitis capitata (Diptera, Tephritidae) is mediated by autophagy

In the present study, we describe the features of programmed cell death of ovarian follicle cells, occurring during the late developmental stages of oogenesis in the olive fruit fly, Bactrocera oleae and the medfly, Ceratitis capitata. During stage 14, the follicle cells contain autophagic vacuoles, and they do not exhibit caspase activity in all parts of the egg chamber. Their nuclei are characterized by condensed chromatin, accompanied with high- but not low-molecular weight DNA fragmentation events exclusively detected in distinct cells of the anterior pole. These data argue for the presence of an autophagy-mediated cell death program in the ovarian follicle cell layer in both species. The above results are likely associated with the abundant phagocytosis observed at the entry of the lateral oviducts, where numerous cell bodies are massively engulfed by epithelial cells. We strongly believe that during the termination of the above Dipteran oogenesis, an efficient mechanism of absorption of the degenerated follicle cells is selectively activated, in order to prevent the blockage of the ovarioles and thus robustly support the physiological completion of the ovulation process.

Hypoxia-induced Bax and Bcl-2 protein expression, caspase-9 activation, DNA fragmentation, and lipid peroxidation in mitochondria of the cerebral cortex of newborn piglets: The role of nitric oxide

The present study tests the hypothesis that cerebral hypoxia results in increased ratio of Bax/Bcl-2, activation of caspase-9, lipid peroxidation, and DNA fragmentation in mitochondria of the cerebral cortex of newborn piglets and that the inhibition of nitric oxide synthase by N-nitro- l-arginine during hypoxia will prevent the events leading to mitochondrial DNA fragmentation. To test this hypothesis, six piglets, 3–5 days old, were divided into three groups: normoxic ( n=5), hypoxic ( n=5), and hypoxic–nitric oxide synthase ( n=4). Hypoxic animals were exposed to a FiO 2 of 0.6 for 60 min. Nitric oxide synthase (40 mg/kg) was infused over 60 min prior to hypoxia. Tissue hypoxia was confirmed by measuring levels of ATP and phosphocreatine. Cerebral cortical tissue mitochondria were isolated and purified using a discontinuous ficoll gradient. Mitochondrial Bax and Bcl-2 proteins were determined by Western blot. Caspase-9 activity in mitochondria was determined spectro-fluorometrically using fluorogenic substrate for caspase-9. Fluorescent compounds, an index of mitochondrial membrane lipid peroxidation, were determined spectrofluorometrically. Mitochondrial DNA was isolated and separated by electrophoresis on 1% agarose gel and stained with ethidium bromide. ATP levels (μmol/g brain) were 4.52±0.34 in normoxic, 1.18±0.29 in hypoxic ( P<0.05) and 1.00±0.26 in hypoxic–nitric oxide synthase animals ( P<0.05 vs. normoxic). Phosphocreatine levels (μmol/g brain) were 3.61±0.33 in normoxic, 0.70±0.20 in hypoxic ( P<0.05 vs. normoxic) and 0.57±0.14 in hypoxic–nitric oxide synthase animals ( P<0.05 vs. normoxic, P=NS vs. hypoxic). Bax density in mitochondrial membranes was 160±28 in normoxic and 324±65 in hypoxic ( P<0.001 vs. normoxic). Bcl-2 density mitochondria was 96±18 in normoxic and 98±20 in hypoxic ( P=NS vs. normoxic). Mitochondrial caspase-9 activity (nmol/mg protein/h) was 1.32±0.23 in normoxic and 2.25±0.24 in hypoxic ( P<0.01 vs. normoxic). Levels of fluorescent compounds (μg of quinine sulfate/g protein) were 12.48±4.13 in normoxic and 37.92±7.62 in hypoxic ( P=0.003 vs. normoxic). Densities (OD×mm 2) of low molecular weight DNA fragments were 143±38 in normoxic, 365±152 in hypoxic, ( P<0.05 vs. normoxic) and 163±25 in hypoxic–nitric oxide synthase animals ( P<0.05 vs. hypoxic, P=NS vs. normoxic). The data demonstrate that hypoxia results in increased mitochondrial proapoptotic protein Bax, increased mitochondrial caspase-9 activity, increased mitochondrial lipid peroxidation, and increased fragmentation of DNA in mitochondria of the cerebral cortex of newborn piglets. The administration of a nitric oxide synthase inhibitor, nitric oxide synthase, prior to hypoxia prevented fragmentation of mitochondrial DNA, indicating that the hypoxia-induced mitochondrial DNA fragmentation is NO-mediated. We propose that NO free radicals generated during hypoxia lead to NO-mediated altered expression of Bax leading to increased ratio of pro-apoptotic/anti-apoptotic protein resulting in modification of mitochondrial membrane, and subsequently Ca 2+-influx and fragmentation of mitochondrial DNA.

The Contribution of Apoptosis-inducing Factor, Caspase-activated DNase, and Inhibitor of Caspase-activated DNase to the Nuclear Phenotype and DNA Degradation during Apoptosis

We have assessed the contribution of apoptosis-inducing factor (AIF) and inhibitor of caspase-activated DNase (ICAD) to the nuclear morphology and DNA degradation pattern in staurosporine-induced apoptosis. Expression of D117E ICAD, a mutant that is resistant to caspase cleavage at residue 117, prevented low molecular weight (LMW) DNA fragmentation, stage II nuclear morphology, and detection of terminal deoxynucleotidyl transferase staining. However, high molecular weight (HMW) DNA fragmentation and stage I nuclear morphology remained unaffected. On the other hand, expression of either D224E or wild type ICAD had no effect on DNA fragmentation or nuclear morphology. In addition, both HMW and LMW DNA degradation required functional executor caspases. Interestingly, silencing of endogenous AIF abolished type I nuclear morphology without any effect on HMW or LMW DNA fragmentation. Together, these results demonstrate that AIF is responsible for stage I nuclear morphology and suggest that HMW DNA degradation is a caspase-activated DNase and AIF-independent process.

Evaluation of the nuclear DNA Diffusion Assay to detect apoptosis and necrosis

We applied the nuclear DNA Diffusion Assay, described as an accurate tool to estimate apoptotic and necrotic cells [N.P. Singh, A simple method for accurate estimation of apoptotic cells, Exp. Cell Res. 256 (2000) 328–337] to tobacco root and leaf cells. In this assay, isolated nuclei are embedded in an agarose microgel on a microscope slide and low molecular-weight DNA fragments diffuse into the microgel. Exposure of the roots to hydrogen peroxide significantly increased the average nuclear area of isolated nuclei. After 4 and 24 h of recovery, all DNA damage was repaired. The data clearly demonstrate that the manifestation of diffused nuclei upon exposure to hydrogen peroxide is not the result of non-repairable apoptotic or necrotic DNA fragmentation, but represents repairable genotoxin-induced DNA damage. In contrast, treatment with the alkylating agent ethyl methanesulphonate (EMS) followed by 24 h of recovery produced a significant increase in the average nuclear area. The contribution of apoptosis to this increase cannot be excluded. Heat treatment of leaves at 50 °C for 1–15 min leading to necrosis, and treatment of isolated nuclei with DNase-I, which digests DNA to nucleosome-sized fragments as during apoptosis, also led to a dose-dependent increase in the nuclear area. The use of different fluorochromes (ethidium bromide, DAPI or YOYO-1) for DNA staining yielded similar results in the DNA Diffusion Assay. As all types and sizes of diffused nuclei were observed after EMS and hydrogen peroxide treatments, we were unable to differentiate, on the basis of the structure of the nuclei, between apoptotic or necrotic DNA fragmentation and other types of genotoxin-induced DNA damage in plants.

Function of Polo-like Kinase 3 in NF-κB-mediated Proapoptotic Response

RelA, the p65 subunit of NF-kappaB transcription factors, plays a key role in regulation of antiapoptotic and proapoptotic responses. However, the downstream target genes regulated by RelA-NF-kappaB in the initiation of proapoptotic signaling were not identified. We previously showed that RelA-NF-kappaB functioned as a proapoptotic factor by activating the p53-signaling pathway in response to doxycycline-induced superoxide. In the present study, we demonstrate that the ability of doxycycline/superoxide to induce expression of polo-like kinase 3 (Plk3) depends on NF-kappaB activity. We identified a kappaB binding site in the promoter of Plk3, and this kappaB site is directly involved in its induction by the RelA-NF-kappaB complex. Plk3 formed a complex with p53 and was involved in the phosphorylation of p53 on Ser-20 in response to superoxide. Inhibition of Plk3 expression by Plk3 small interfering RNA suppressed the doxycycline/superoxide-mediated apoptosis. Overexpression of wild-type Plk3 in HCT116 p53+/+ cells induced rapid apoptosis, whereas overexpression of wild-type Plk3 in HCT116 p53-/- cells and the kinase-defective mutant Plk3(K91R) in p53+/+ cells induced delayed onset of apoptosis. Furthermore, mutagenesis of Plk3 showed that the N-terminal domain (amino acids 1-26) is essential for the induction of delay onset of apoptosis. These data show that Plk3 is a RelA-NF-kappaB-regulated gene that induces apoptosis in both p53-dependent and -independent signaling pathways, suggesting a possible mechanism for RelA-NF-kappaB-regulated proapoptotic responses.

Caspase-3 activation is required for corticosterone-induced rat Leydig cell apoptosis

Objective: Study on caspase-3 expression and its activation in CORT-mediated rat Leydig cell apoptosis. Methods: Caspase-3 protein activity, content and the timing of the onset of caspase-3 cleavage in relationship to CORT administration in rats was studied by Western blot. The timing of the onset of caspase-3 activity in Leydig cell was measured by the fluorescence. Results: Low molecular weight DNA fragments that are characteristic of apoptosis were evident in Leydig cells by 12 h of exposure to 100 nmol/L CORT in vitro and it was more pronounced at 24 h. Western blot analysis revealed that procaspase-3 was low in untreated Leydig cells and increased by 6 h of CORT administration. By 12 h, however, procaspase-3 was significantly reduced and the cleaved, active caspase-3 forms appeared and increased through 24 h. In the presence of a specific caspase inhibitor, Ac-DEVD-CHO, Leydig cell apoptosis was suppressed, corroborating the hypothesis that caspase-3 is involved in CORT-mediated cell death. Conclusion: Caspase-3 was implicated in CORT-mediated rat Leydig cell apoptosis.