Endonuclease activation and chromosomal DNA fragmentation during apoptosis in leukemia cells.

Apoptotic endonuclease is a key enzyme that mediates regulated DNA fragmentation and chromatin condensation in response to apoptotic signals such as the Fas ligand, ionizing radiation, and anticancer agents. An endonuclease that is activated specifically by caspase-3 has been identified in humans and mice. The human gene for this protein has been termed DFF40 (DNA fragmentation factor, 40-kd subunit) or caspase-activated nuclease (CPAN), whereas the mouse homologue has been named caspase-activated deoxyribonuclease (CAD). Although CAD/DFF40 is known as a major apoptotic nuclease, mice lacking inhibitor of CAD (ICAD) (also known as DFF45) are viable and still show DNA fragmentation, suggesting that alternative endonucleases play an important role during apoptosis. Endonuclease G has been reported to possibly be responsible for DNA fragmentation in various cells during apoptosis. Furthermore, we also have found that apurinic/apyrimidinic endonuclease 1 (Ape1) and its N-terminal-truncated form (AN34) are involved in DNA fragmentation during apoptosis in leukemia cells. In this review, we describe the features of several endonucleases that are involved in the apoptosis of human leukemia cells. Apoptotic endonuclease may vary among different leukemia cell types.

Granzyme M directly cleaves inhibitor of caspase-activated DNase (CAD) to unleash CAD leading to DNA fragmentation.

Granzyme (Gzm)M is constitutively highly expressed in NK cells that may play a critical role in NK cell-mediated cytolysis. However, the function of GzmM has been less defined. Just one report showed GzmM induces a caspase-independent death pathway. In this study, we demonstrate a protein transfection reagent Pro-Ject can efficiently transport GzmM into target cells. GzmM initiates caspase-dependent apoptosis with typical apoptotic nuclear morphology. GzmM induces DNA fragmentation, not DNA nicking. GzmM can directly degrade inhibitor of caspase-activated DNase to release the nuclease activity of caspase-activated DNase for damaging DNA. Furthermore, GzmM cleaves the DNA damage sensor enzyme poly(ADP-ribose) polymerase to prevent cellular DNA repair and force apoptosis.

PCR-based generation of shRNA libraries from cDNAs.

BACKGROUND: The use of small interfering RNAs (siRNAs) to silence target gene expression has greatly facilitated mammalian genetic analysis by generating loss-of-function mutants. In recent years, high-throughput, genome-wide screening of siRNA libraries has emerged as a viable approach. Two different methods have been used to generate short hairpin RNA (shRNA) libraries; one is to use chemically synthesized oligonucleotides, and the other is to convert complementary DNAs (cDNAs) into shRNA cassettes enzymatically. The high cost of chemical synthesis and the low efficiency of the enzymatic approach have hampered the widespread use of screening with shRNA libraries. RESULTS: We report here an improved method for constructing genome-wide shRNA libraries enzymatically. The method includes steps of cDNA fragmentation and endonuclease MmeI digestion to generate 19-bp fragments, capping the 19-bp cDNA fragments with a hairpin oligonucleotide, and amplification of the hairpin structures by PCR. The PCR step converts hairpins into double-stranded DNAs that contain head-to-head cDNA fragments that can be cloned into a vector downstream of a Pol III promoter. CONCLUSION: This method can readily be used to generate shRNA libraries from a small amount of mRNA and thus can be used to create cell- or tissue-specific libraries.

Yeast cell death caused by mutation of the OST2 gene encoding the epsilon-subunit of Saccharomyces cerevisiae oligosaccharyltransferase.

An essential epsilon-subunit of oligosaccharyltransferase Ost2 is a yeast homolog of mammalian highly conserved DAD1 (defender against apoptotic death). In hamster cells, the Gly38Arg mutation in DAD1 causes apoptosis at restrictive temperatures due to a defect in N-linked glycosylation. To analyze the function of Ost2 in yeast cell death, we constructed Saccharomyces cerevisiae strains expressing Gly58Arg (corresponding to the Gly38Arg mutation in hamster DAD1), Gly86Arg, and Glu113Val mutant Ost2. At elevated temperatures, ost2 mutants arrested growth by decreasing cell viability. Phosphatidylserine exposure, a phenotypic marker of apoptosis in mammalian cells, was found in ost2 mutant cells at 37 degrees C, although DNA fragmentation was not clearly detected. A high concentration of sorbitol compensates for the temperature sensitivity of the ost2 mutant. These results suggest that apoptosis-like cell death in ost2 mutants is caused by the secondary effect of overall reduced protein N-linked glycosylation.

The activin-follistatin system and in vitro early follicle development in goats.

The aim of the present study was to investigate the effects of activin-A and follistatin on in vitro primordial and primary follicle development in goats. To study primordial follicle development (experiment 1), pieces of ovarian cortex were cultured in vitro for 5 days in minimal essential medium (MEM) supplemented with activin-A (0, 10 or 100 ng/ml), follistatin (0, 10 or 100 ng/ml) or combinations of the two. After culture, the numbers of primordial follicles and more advanced follicle stages were calculated and compared with those in non-cultured tissue. Protein and mRNA expression of activin-A, follistatin, Kit ligand (KL), growth differentiation factor-9 (GDF-9) and bone morphogenetic protein-15 (BMP-15) in non-cultured and cultured follicles were studied by immunohistochemistry and PCR. To evaluate primary follicle growth (experiment 2), freshly isolated follicles were cultured for 6 days in MEM plus 100 ng/ml activin-A, 100 ng/ml follistatin or 100 ng/ml activin-A plus 200 ng/ml follistatin. Morphology, follicle and oocyte diameters in cultured tissue and isolated follicles before and after culture were assessed. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) reactions were performed to study DNA fragmentation in follicles. In experiment 1, it was found that goat primordial follicles were activated to develop into more advanced stages, i.e. intermediate and primary follicles, during in vitro culture, but neither activin-A nor follistatin affected the number of primordial follicles that entered the growth phase. Activin-A treatment enhanced the number of morphologically normal follicles and stimulated their growth during cortical tissue culture. The effects were, however, not counteracted by follistatin. The follicles in cultured goat tissue maintained their expression of proteins and mRNA for activin-A, follistatin, KL, GDF-9 and BMP-15. Fewer than 30% of the atretic follicles in cultured cortical tissue had TUNEL-positive (oocyte or granulosa) cells. Activin-A did not affect the occurrence of TUNEL-positive cells in follicles within cortical tissue. In experiment 2, addition of activin-A to cultured isolated primary follicles significantly stimulated their growth, the effect being counteracted by follistatin. Absence of such a neutralizing effect of follistatin in the cultures with ovarian cortical tissue can be due to lower dose of follistatin used and incomplete blockage of activin in these experiments. In contrast to cortical enclosed atretic follicles, all atretic follicles that had arisen in cultures with isolated primary follicles had TUNEL-positive cells, which points to differences between isolated and ovarian tissue-enclosed follicles with regard to the followed pathways leading to their degeneration. In summary, this in vitro study has demonstrated that cultured goat primordial follicles are activated to grow and develop into intermediate and primary follicles. During in vitro culture, the follicles maintain their ability to express activin-A, follistatin, KL, GDF-9 and BMP-15. The in vitro growth and survival of activated follicles enclosed in cortical tissue and the in vitro growth of isolated primary follicles are stimulated by activin-A.

Molecular cloning of rHAUSP encoding a deubiquitinating enzyme in rat testis.

The tumor suppressor protein p53 is stabilized by the herpes-virus-associated ubiquitin-specific protease (HAUSP), a deubiquitinating enzyme. We previously isolated and characterized a mouse orthologue of HAUSP, mHAUSP. In this study, we have identified a rat orthologue of HAUSP, rHAUSP, from the rat testis by RT-PCR using primers used for cloning mHAUSP. rHAUSP cDNA encodes 3,312 bp and 1,103 amino acids with a molecular weight of approximately 135 kDa containing highly conserved Cys, Asp (I), His, and Asn/Asp (II) domains characteristic of the ubiquitin-specific processing proteases. pI value of rHAUSP is 5.31. In vivo and in vitro deubiquitinating enzyme assays demonstrated that rHAUSP has deubiquitinating enzymatic activity. The over-expression of rHAUSP induced cell death of cervical adenocarcinoma cells.

Anti-inflammatory effects of indomethacin's methyl ester derivative and induction of apoptosis in HL-60 Cells.

Indomethacin is used as an anti-inflammatory drug and a nonselective cyclooxygenase inhibitor. When indomethacin in methanol was photo-irradiated with an Hg lamp, methyl ester, ethyl ester, and gamma-lactone derivatives of indomethacin were produced. In the present study, we found that the methyl ester derivative of indomethacin (M-IN) could more potently inhibit prostaglandin E(2) (PGE(2)) and nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX 2) protein expression from lipopolysaccharide (LPS)-stimulated RAW 264.7 cells than indomethacin, similar to the effect of a non-steroidal anti-inflammatory drugs (NSAID). On the other hand, the results showed that M-IN with an IC(50) value maintained at 36.9 microg/ml for 12 h exhibited stronger cytotoxicity than ethyl ester, gamma-lactone derivatives of indomethacin, and indomethacin in promyelocytic leukemia HL-60 cells. Moreover, a series of biochemical analyses determined that M-IN caused apoptotic bodies, DNA fragmentation, and enhanced PARP and pro-caspase 3 degradation in HL-60 cells. These above results indicate that the photosynthesized product, M-IN, had stronger anti-inflammatory effects in LPS-stimulated RAW 264.7 cells and cytotoxicity effects in HL-60 cells than the parent drug, indomethacin.

Simple determination of human sperm DNA fragmentation with an improved sperm chromatin dispersion test.

OBJECTIVE: To improve the sperm chromatin dispersion (SCD) test and develop it as a simple kit (Halosperm kit) for the accurate determination of sperm DNA fragmentation using conventional bright-field microscopy. DESIGN: Method development, comparison, and validation. SETTING: Medical genetics laboratory, academic biology center, and reproductive medicine centers. PATIENT(S): Male infertility patients attending the Reproductive Medicine Center. A varicocele patient and a group of nine fertile subjects. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): [1] The quality of chromatin staining in relaxed sperm nuclear halos and tail preservation; [2] SCD scoring reproducibility; [3] comparison with the sperm chromatin structure assay in 45 samples; [4] frequency of sperm with DNA fragmentation after incubation with increasing doses of the nitric oxide donor sodium nitroprusside and in sperm samples for 9 fertile men, 46 normozoospermic patients, 23 oligoasthenoteratozoospermic patients, and a subject with varicocele. RESULT(S): The sperm nuclei with DNA fragmentation, either spontaneous or induced, do not produce or show very small halos of DNA loop dispersion after sequential incubation in acid and lysis solution. The improved SCD protocol (Halosperm kit) results in better chromatin preservation, therefore highly contrasted halo images can be accurately assessed using conventional bright-field microscopy after Wright staining. Moreover, unlike in the original SCD procedure, the sperm tails are now preserved, making it possible to unequivocally discriminate sperm from other cell types. The chi2 test did not detect significant differences in the mean number of sperm cells with fragmented DNA as scored by four different observers. The intraobserver coefficient of variation for the estimated percentage of spermatozoa with fragmented DNA ranged from 6% to 12%. There was good correlation between the SCD and the sperm chromatin structure assay DNA fragmentation index (intraclass correlation coefficient R: 0.85; percent DNA fragmentation index mean difference: 2.16 significantly higher for SCD). Using the Halosperm kit, a dose-dependent increase in sperm DNA damage after sodium nitroprusside incubation was detected. The percentage of sperm cells with fragmented DNA in the fertile group was 16.3 +/- 6.0, in the normozoospermic group, 27.3 +/- 11.7, and in the oligoasthenoteratozoospermic group, 47.3 +/- 17.3. In the varicocele sample, an extremely high degree of nuclear disruption was detected in the population of sperm cells with fragmented DNA. CONCLUSION(S): The improved SCD test, developed as the Halosperm kit, is a simple, cost effective, rapid, reliable, and accurate procedure, for routinely assessing human sperm DNA fragmentation in the clinical andrology laboratory.

Critically short telomeres are associated with sperm DNA fragmentation.

The influence of critical telomeric attrition, a well-known trigger of apoptosis and cell arrest, on sperm DNA fragmentation was studied in late-generation knockout mice for Terc, the RNA component of telomerase, as a model of choice. Terc knockout mice had a sixfold mean increase in the percentage of sperm cells with fragmented DNA.

Yet another test of sperm chromatin structure.

Sperm chromatin can affect reproductive performance, and it may be analyzed by measuring susceptibility of DNA to breakage using assays such as the sperm chromatin structure assay, comet assay, TUNEL, and DNA ladders. The newly proposed test, Halosperm, may not provide additional information beyond that obtained with existent evaluations.

Halosperm is an easy, available, and cost-effective alternative for determining sperm DNA fragmentation.

The characteristics of Halosperm make this kit a reasonable alternative to allow basic and clinical research on sperm DNA fragmentation in any basic laboratory around the world.

Effect of sperm DNA damage and sperm protamine deficiency on fertilization and embryo development post-ICSI.

The aim of this study was to evaluate the effect of sperm DNA damage and protamine deficiency on fertilization and embryo development post-intracytoplasmic sperm injection (ICSI), and also to assess the effect of protamine deficiency on DNA damage. Semen samples were collected from 28 patients participating in the ICSI programme. Following sperm preparation and ICSI, the remaining processed semen samples were used to assess protamine deficiency and DNA damage employing chromomycin A3 (CMA3) staining and comet assay, respectively. Comet parameters, CMA3 percentage positivity, fertilization rate, embryo cleavage score and embryo quality score were assessed. Except for CMA3, none of the comet parameters showed significant correlation with fertilization rate. However, among comet parameters, head area and head intensity showed positive correlation with the embryo cleavage score, while comet mean intensity and head mean intensity showed a significant negative correlation with CMA3 positivity. Results of this study demonstrate that DNA fragmentation is more frequent in protamine-deficient spermatozoa. Unlike protamine deficiency, sperm DNA fragmentation does not preclude fertilization. Nonetheless, embryos derived from spermatozoa with high DNA damage have a lower potential to reach blastocyst stage.

Circulation DNA: biological implications for cancer metastasis and immunology.

Circulation cell free DNA (cf-DNA) is of considerable interest to oncology researchers seeking to isolate specific cancer markers. Here, we focus on the origin and biological implications of cf-DNA, exploring its potential roles in cancer biology and medicine. We hypothesize that cf-DNA is primarily released by living cancer cells in addition to apoptotic or necrotic cancer cells for three reasons: (1) following radiotherapy, cf-DNA quantities are significantly reduced in a high percentage of patients although radiation-induced massive apoptosis is expected; (2) cancer cell DNA concentration in cultured supernatants increases with cell proliferation when few apoptotic or necrotic cells are present; and (3) DNA concentration increases in normal lymphocyte cultures following stimulation with phytohemagglutinin, lipopolysaccharide or antigen. Our hypotheses have major biological implications in cancer biology. First, cancer cf-DNA may transform normal cells and form adjacent or remote metastases or second primary cancer. In this context, we also have raised an alarming advice that the cancer may be potentially infectious. Secondly, if a normal cf-DNA contains cytokine sequence, it may behave like an intrinsic DNA vaccine, producing therapeutic cytokine. If normal cf-DNA contains a sequence of a non-mutated oncogene or tumor suppressor gene, homologous recombination with the cancer genome may occur leading to knock out mutated oncogene or tumor suppressor gene that could thus elicit a spontaneous remission of cancer.

Defining the breakpoints of the quaking(viable) mouse mutation reveals a duplication from a Parkin intron.

The quakingviable (qkv) mutant mouse shows a recessive neurological phenotype that includes central nervous system (CNS) dysmyelination, seizures, and tremor associated with voluntary movement. The molecular defect of qkv has been previously reported to be a spontaneous approximately 1 megabase (Mb) deletion in the proximal region of mouse chromosome 17 that occurred in the DBA mouse strain more than four decades ago. The mutation has recently been shown to affect three genes in the region: Quaking (qk), Parkin-coregulated gene (Pacrg), and Parkin. Here we determine the exact deletion breakpoints and demonstrate that the mutation is not just comprised of a approximately 1.1 Mb deletion, but also harbors a small 163 bp duplication fragment between the deletion breakpoints. Although the distal deletion breakpoint is within the fifth intron of the mouse Parkin gene, the duplicated sequence is derived from the sixth Parkin intron and shows positive transcriptional activity on a reporter gene in vitro. This complexity provides insight into a well-studied neurological mutant and may have a role in affecting the phenotype observed.

Developmental program of epileptogenesis in the brain of EL mice.

PURPOSE: We recently observed inducible nitric oxide synthetase (iNOS) expression and decreased Cu, Zn-superoxide dismutase (Cu, Zn-SOD) activities in the hippocampus of epileptic mutant EL mice at the age of 30 weeks. In addition, the immediate early gene (IEG) c-fos is unusually expressed in the interictal period, suggesting activation of protein cascades associated with the epileptogenesis. Furthermore, DNA fragmentation has been detected preferentially in the hippocampus CA1 and the parietal cortex of EL mouse brain. It remains to be seen, however, how these abnormalities are related to the DNA fragmentation, and whether neuronal cell loss is involved. The present study was designed to address these issues. METHODS: NOS isoenzymes, pro- (Bax) and antiapoptotic factors (Bcl-2, Bcl-XL), and neurotrophic factors (brain-derived neurotrophic factor, BDNF; neurotrophin-3, NT-3; fibroblast growth factor-2, FGF-2) were determined by immunoblotting in the EL mouse brain at various developmental stages. Hematoxylin-eosin staining was applied to the formalin-fixed brains to examine the cell loss in the tissue. IEG expression in the interictal period was analyzed by in situ hybridization by using the 35S x-ray emulsion method. RESULTS: nNOS was the major component of NOS in the hippocampus of either EL or control DDY mice. In EL mice, however, iNOS was detectable at the age of 10 weeks, at which the animals usually experience the first seizures. eNOS, which appears in DDY brain, could scarcely be identified. Even in the interictal period, EL mice expressed c-fos continuously, preferentially in the parietal cortex and hippocampal CA1. In DDY mice, very low steady-state levels of Bcl-2 and Bax remained constant throughout development. In EL mice, these Bcl-2 and Bax levels were increased even before experiencing frequent seizures. BDNF in EL mice markedly increased temporarily during ictogenesis and epileptogenesis in their early periods. Unexpectedly, no cell loss was found in the hippocampus. CONCLUSIONS: DNA fragmentation without cell loss found in EL mouse brains appears to result from initial activation and later inactivation of the apoptotic process. Neurotrophic factors may play a role in the ictogenesis and the epileptogenesis during the early development. These gene expressions closely related to the periods critical for ictogenesis and epileptogenesis may be of particular importance in the development of antiepileptic drugs (AEDs) with novel mechanisms.

Neutral lipid isolated from endosperm of Job's tears inhibits the growth of pancreatic cancer cells via apoptosis, G2/M arrest, and regulation of gene expression.

BACKGROUND: The neutral lipid isolated from the endosperm of Job's tears (NLEJ) has been known to possess an anticancer activity with relatively low toxicity. The present study was designed to examine its antiproliferative effects in the PaTu-8988 and SW1990 human pancreatic cancer cells and to investigate its potential mechanism(s). METHODS: Pancreatic cancer cells were treated with NLEJ to evaluate cell viability, cell cycle progression, nuclear morphology, DNA fragmentation and annexin V binding analysis. Regulation of gene expression was determined by using cDNA microarrays and western immunoblotting. RESULTS: The NLEJ induced a dose- and time-dependent inhibition of proliferation in both PaTu-8988 and SW1990 cell lines. Further studies were carried out with only the PaTu-8988 cells. Flow cytometry analysis showed that NLEJ blocked cell cycle progression at the G(2)/M phase. There was also an increase in annexin V binding and DNA fragmentation, indicative of apoptosis. The cDNA microarray analysis with cell cycle- and apoptosis-targeted arrays showed that the expression signals of 24 genes were found to be significantly altered at 24 h of NLEJ treatment. These genes are involved in cell cycle control (e.g. p21, p27, CDK2, and cyclins), apoptosis regulation (e.g. bcl-2 and bax), and signal transduction (e.g. ATM, RAD50, and p53). Some of these results were confirmed by western blot analysis. CONCLUSIONS: These data show that NLEJ inhibits pancreatic cancer cell growth through induction of apoptosis and cell cycle arrest as well as regulation of gene expression in vitro. Therefore, NLEJ might be a chemotherapeutic agent against pancreatic cancer.

Telomere length predicts embryo fragmentation after in vitro fertilization in women--toward a telomere theory of reproductive aging in women.

OBJECTIVE: Telomeres are DNA repeats which cap and protect chromosome ends, facilitate homologue pairing and chiasmata formation during early meiosis, and shorten with cell division and exposure to reactive oxygen to mediate aging. Early germ cells contain telomerase, a reverse transcriptase which adds telomeres to 3-prime DNA ends, but telomerase activity declines in oocytes, fixing telomere length earlier during development. Experimentally induced telomere shortening in mice disrupts meiosis, impairs chiasmata formation, halts embryonic cell cycles, and promotes apoptosis in embryos, a phenotype which mimics reproductive senescence in women. Ethical constraints limit study of human embryos to nondestructive assays, such as morphologic evaluation under transmission optics, but cytoplasmic fragmentation is a reliable marker of apoptosis. STUDY DESIGN: Study design consisted of observational study of effect of telomere length in human eggs on cytoplasmic fragmentation, and on other morphologic features of preimplantation embryos. To test the hypothesis that telomere shortening triggers apoptosis in human embryos, we evaluated telomere length as a predictor of cytoplasmic fragmentation in embryos from women undergoing in vitro fertilization. RESULTS: Telomere length negatively predicted fragmentation in day 3 preimplantation embryos, after controlling for patient age and basal follicle stimulating hormone level. Telomere length did not predict other features of preimplantation embryo morphology. CONCLUSION: The finding that telomere length in human eggs predicts cytoplasmic fragmentation in embryos provides evidence that telomere shortening induces apoptosis in human preimplantation embryos, consistent with a telomere theory of reproductive senescence in women.

Growth characteristics of canine distemper virus in a new cell line CCT cells originated from canine malignant histiocytosis.

Canine distemper virus (CDV) growth and the morphological characterization were examined in a cell line established from a canine malignant histiocytosis (CCT cell line). The susceptibility of the CCT cells to 3 CDV strains, FXNO, YSA-TC and MD-77 was shown by detection of the antigen in the indirect fluorescent assay. After passaging 4 and 9 times through the CCT cells, only FXNO strain could produce the syncytia where demonstrated the antigens. Titers of 9 passaged viruses through the CCT cells showed slightly higher in the CCT cells than those in Vero cells. Morphological characterization of karyorrhexis and specific DNA ladder by extracted DNA electrophoresis indicated apoptosis in the CDV infected CCT cells.