Gene targeted DNA double-strand break induction by (125)I-labeled triplex-forming oligonucleotides is highly mutagenic following repair in human cells.

A parallel binding motif 16mer triplex-forming oligonucleotide (TFO) complementary to a polypurine-polypyrimidine target region near the 3'-end of the SupF gene of plasmid pSP189 was labeled with [5-(125)I]dCMP at position 15. Following triplex formation and decay accumulation, radiation-induced site-specific double-strand breaks (DSBs) were produced in the pSP189 SupF gene. Bulk damaged DNA and the isolated site-specific DSB-containing DNA were separately transfected into human WI38VA13 cells and allowed to repair prior to recovery and analysis of mutants. Bulk damaged DNA had a relatively low mutation frequency of 2.7 x 10(-3). In contrast, the isolated linear DNA containing site-specific DSBs had an unusually high mutation frequency of 7.9 x 10(-1). This was nearly 300-fold greater than that observed for the bulk damaged DNA mixture, and >1.5 x 10(4)-fold greater than background. The mutation spectra displayed a high proportion of deletion mutants targeted to the(125)I binding position within the SupF gene for both bulk damaged DNA and isolated linear DNA. Both spectra were characterized by complex mutations with mixtures of changes. However, mutations recovered from the linear site-specific DSB-containing DNA presented a much higher proportion of complex deletion mutations.

In vitro repair of complex unligatable oxidatively induced DNA double-strand breaks by human cell extracts.

We describe a new assay for in vitro repair of oxidatively induced DNA double-strand breaks (DSBs) by HeLa cell nuclear extracts. The assay employs linear plasmid DNA containing DNA DSBs produced by the radiomimetic drug bleomycin. The bleomycin-induced DSB possesses a complex structure similar to that produced by oxidative processes and ionizing radiation. Bleomycin DSBs are composed of blunt ends or ends containing a single 5'-base overhang. Regardless of the 5'-end structure, all bleomycin-induced DSBs possess 3'-ends blocked by phosphoglycolate. Cellular extraction and initial end joining conditions for our assay were optimized with restriction enzyme-cleaved DNA to maximize ligation activity. Parameters affecting ligation such as temperature, time, ionic strength, ATP utilization and extract protein concentration were examined. Similar reactions were performed with the bleomycin-linearized substrate. In all cases, end-joined molecules ranging from dimers to higher molecular weight forms were produced and observed directly in agarose gels stained with Vistra Green and imaged with a FluorImager 595. This detection method is at least 50-fold more sensitive than ethidium bromide and permits detection of /=50% conversion of substrate to product is achieved with simple substrates such as restriction enzyme-cleaved DNA. Using our assay we have observed a 6-fold lower repair rate and a lag in reaction initiation for bleomycin-induced DSBs as compared to blunt-ended DNA. Also, end joining reaction conditions are DSB end group dependent. In particular, bleomycin-induced DSB repair is considerably more sensitive to inhibition by increased ionic strength than repair of blunt-ended DNA.

In vitro effects of soy phytoestrogens on rat L6 skeletal muscle cells.

Soy isoflavones display estrogenic activity in humans and animals, and thus are referred to as phytoestrogens. This study was performed to observe the effects of the soy isoflavones genistein, daidzein, and glycitein on cell cultures of rat skeletal muscles. [3H]Thymidine incorporation was used to determine cell proliferation, while protein synthesis and degradation were determined by tracking radiolabeled leucine. For the proliferation studies, insulin, estradiol, genistein, daidzein, or glycitein was supplemented at 0, 0.04, 0.08, 0.16, 0.31, 0.63, 1.25, 2.5, 5, 10, or 20 microM, respectively, or in combinations with final concentrations of 0, 0.1, 1, or 10 microM. Genistein reacted most similarly to estradiol, inhibiting proliferation at > or = 1 microM (P < .001). A combination of phytoestrogens resulted in significant inhibition of cell proliferation, but not to the extent observed with genistein alone. For the protein synthesis and degradation experiments, treatments of 0.1 microM dexamethasone or 1 microM concentrations of insulin, genistein, daidzein, or glycitein were used. Phytoestrogens did not inhibit or stimulate protein degradation or synthesis (P > .05). A one-tailed univariate analysis of variance revealed a trend (P < or = .1) in protein stimulation with genistein and glycitein treatments. These results suggest that the tyrosine kinase inhibiting activity of genistein may be affecting phosphorylation of the mitosis-promoting factor, preventing the advancement of the mitotic cell cycle. In addition, at higher total combined concentrations, daidzein and glycitein may be able to outcompete genistein for receptor sites. These results suggest that soy isoflavones in the diet may potentially modulate normal growth and development in humans and animals that ingest soy-based products.

Nature of endothelin binding in the porcine ovary.

We investigated the nature of endothelin (ET) binding in the porcine ovary. We demonstrated the presence of high affinity (Kd = 0.72; 95% confidence interval = 0.43-1.1 nM) binding sites for ET-1 in the porcine ovary. The binding capacity for this ET-1-specific binding site was 97 pmol/micrograms DNA (95% confidence interval = 90-107). Autoradiographic studies showed that putative ET receptors reside in the granulosa cell layer of the maturing Graafian follicle and in the vascular components of the corpora lutea. The relative abundance of ET receptors was greatest in granulosa cells of large antral follicles, whereas ET binding was absent in granulosa cells of preantral follicles and in luteal cells. ET binding by cultured granulosa cells was further characterized by RRA and shown to exhibit a rank order of binding affinities for different ET isopeptides. The observed rank order indicates that the ET receptors present on granulosa cells are of the ET(A) receptor subtype. The radioreceptor studies also indicated that granulosa cells collected from large antral follicles (9-10 mm in diameter) have a greater binding capacity for [125I]ET-1 in culture than granulosa cells collected from smaller follicles. When cultured granulosa cells were exposed to 100 nM ET-1 or 14 nM 12-O-tetradecanoylphorbol 13-acetate overnight, the percentage of specific [125I]ET-1 binding was reduced (12% and 50%, respectively), indicating a down-regulation of the ET receptor by these treatments. In summary, we have characterized the distribution, isopeptide specificity, relative abundance, and down-regulation of putative ovarian endothelin receptors of subtype ET(A) on swine granulosa cells. Such results in conjunction with other available literature strongly suggest that granulosa cells of maturing Graafian follicles are targeted by ET-1. An additional physiological role for ET-1 in the ovary is suggested by the presence of putative ET receptors in the vasculature of the corpus luteum.

Gene targeted agents: new opportunities for rational drug development.

In addition to traditional drug development methods designed to modulate the activity of protein targets, knowledge of disease gene DNA sequences provides an opportunity for the highly rational design of therapeutic agents that act at the DNA level through sequence-specific interactions. Among the ligands capable of binding DNA in a precise, sequence-specific manner are oligonucleotides, peptide nucleic acids and polyamides. Various strategies employing these agents to either transiently or permanently alter gene expression have been investigated over the past decade. During the past two to three years, important steps have been taken to illustrate the therapeutic potential of these ligands. Triple-helix (triplex) forming oligonucleotides have been particularly effective DNA-targeting agents with a wide range of applications, including the positive and negative transcriptional regulation of target genes, as well as the controlled delivery of site-specific mutations. This review will focus upon recent advances involving the use of sequence-specific DNA-binding ligands to modify gene expression and/or structure, with particular emphasis on the use of triplex-forming oligonucleotides in these roles.

Definition of Soybean Genomic Regions That Control Seed Phytoestrogen Amounts.

Soybean seeds contain large amounts of isoflavones or phytoestrogens such as genistein, daidzein, and glycitein that display biological effects when ingested by humans and animals. In seeds, the total amount, and amount of each type, of isoflavone varies by 5 fold between cultivars and locations. Isoflavone content and quality are one key to the biological effects of soy foods, dietary supplements, and nutraceuticals. Previously we had identified 6 loci (QTL) controlling isoflavone content using 150 DNA markers. This study aimed to identify and delimit loci underlying heritable variation in isoflavone content with additional DNA markers. We used a recombinant inbred line (RIL) population ( $n=100$ ) derived from the cross of “Essex” by “Forrest,” two cultivars that contrast for isoflavone content. Seed isoflavone content of each RIL was determined by HPLC and compared against 240 polymorphic microsatellite markers by one-way analysis of variance. Two QTL that underlie seed isoflavone content were newly discovered. The additional markers confirmed and refined the positions of the six QTL already reported. The first new region anchored by the marker BARC-Satt063 was significantly associated with genistein ( $P=0.009$, $Rcirc;2=29.5\%$ ) and daidzein ( $P=0.007$, $Rcirc;2=17.0\%$ ). The region is located on linkage group B2 and derived the beneficial allele from Essex. The second new region defined by the marker BARC-Satt129 was significantly associated with total glycitein ( $P=0.0005$, $Rcirc;2=32.0\%$ ). The region is located on linkage group D1a+Q and also derived the beneficial allele from Essex. Jointly the eight loci can explain the heritable variation in isoflavone content. The loci may be used to stabilize seed isoflavone content by selection and to isolate the underlying genes.

Genomic Regions That Underlie Soybean Seed Isoflavone Content.

Soy products contain isoflavones (genistein, daidzein, and glycitein) that display biological effects when ingested by humans and animals, these effects are species, dose and age dependent. Therefore, the content and quality of isoflavones in soybeans is a key to their biological effect. Our objective was to identify loci that underlie isoflavone content in soybean seeds. The study involved 100 recombinant inbred lines (RIL) from the cross of 'Essex' by 'Forrest,' two cultivars that contrast for isoflavone content. Isoflavone content of seeds from each RIL was determined by high performance liquid chromatography (HPLC). The distribution of isoflavone content was continuous and unimodal. The heritability estimates on a line mean basis were 79% for daidzein, 22% for genistein, and 88% for glycitein. Isoflavone content of soybean seeds was compared against 150 polymorphic DNA markers in a one-way analysis of variance. Four genomic regions were found to be significantly associated with the isoflavone content of soybean seeds across both locations and years. Molecular linkage group B1 contained a major QTL underlying glycitein content (P = 0.0001, R(2) = 50.2%), linkage group N contained a QTL for glycitein (P = 0.0033, R(2) = 11.1%) and a QTL for daidzein (P = 0.0023, R(2) = 10.3%) and linkage group A1 contained a QTL for daidzein (P = 0.0081, R(2) = 9.6%). Selection for these chromosomal regions in a marker assisted selection program will allow for the manipulation of amounts and profiles of isoflavones (genistein, daidzein, and glycitein) content of soybean seeds. In addition, tightly linked markers can be used in map based cloning of genes associated with isoflavone content.

Use of the PBS2 uracil-DNA glycosylase inhibitor to differentiate the uracil-DNA glycosylase activities encoded by herpes simplex virus types 1 and 2.

The bacteriophage PBS2 encoded uracil-DNA glycosylase (UNG) inhibitor was examined for its effect upon the nuclear UNG activities of KB, HeLa, and Vero cells infected with herpes simplex virus (HSV) type 1 or 2 and mock-infected cells. UNG activity from HSV-1 infected cells exhibited the greatest sensitivity to inhibition by the inhibitor, while UNG activity from cells infected with HSV-2 exhibited the greatest resistance. This differential effect was dependent upon the virus, cell line, and buffer system used in the reaction. Furthermore, the PBS2 UNG inhibitor's differential effect, provides a means of distinguishing the herpesvirus UNG activities from one another, and from the cellular UNG activity. Therefore, this method of identification should prove to be useful for the purification and characterization of the viral enzymes from infected cell nuclear extracts.

Absolute structure determination of the highly biologically active bisdehydrodoisynolic acids.

In a project designed to relate the unexpected in vivo and in vitro properties exhibited by (+)- and (-)-bisdehydrodoisynolic acid with their absolute stereochemical structure, an X-ray crystal-structure analysis was undertaken of the highly estrogenic, poorly binding (-) enantiomer. (1) and (13)C NMR spectra are also reported for the first time. The crystal structure shows the cis juxtaposition of the carboxyl and ethyl groups, which are separated by a large torsion angle, and that only the carbon atom holding the carboxyl group is out of the plane in which the remainder of the fused three-ring moiety lies. The crystal structure, which unequivocally characterizes the (-) enantiomer as cis-13(S),14(R) and, implicitly, the (+) enantiomer as cis-13(R),14(S), will be useful in continued studies aimed at explaining the selective estrogen receptor modulation (SERM) of these enantiomers which, in some cases, produces significantly different end-organ effects compared to those of estradiol, in both males and females, affording the promise of a variety of therapeutic and pharmacologic applications.

Identification of defective illegitimate recombinational repair of oxidatively-induced DNA double-strand breaks in ataxia-telangiectasia cells.

Ataxia-telangiectasia (A-T) is an autosomal-recessive lethal human disease. Homozygotes suffer from a number of neurological disorders, as well as very high cancer incidence. Heterozygotes may also have a higher than normal risk of cancer, particularly for the breast. The gene responsible for the disease (ATM) has been cloned, but its role in mechanisms of the disease remain unknown. Cellular A-T phenotypes, such as radiosensitivity and genomic instability, suggest that a deficiency in the repair of DNA double-strand breaks (DSBs) may be the primary defect; however, overall levels of DSB rejoining appear normal. We used the shuttle vector, pZ189, containing an oxidatively-induced DSB, to compare the integrity of DSB rejoining in one normal and two A-T fibroblast cells lines. Mutation frequencies were two-fold higher in A-T cells, and the mutational spectrum was different. The majority of the mutations found in all three cell lines were deletions (44-63%). The DNA sequence analysis indicated that 17 of the 17 plasmids with deletion mutations in normal cells occurred between short direct-repeat sequences (removing one of the repeats plus the intervening sequences), implicating illegitimate recombination in DSB rejoining. The combined data from both A-T cell lines showed that 21 of 24 deletions did not involve direct-repeats sequences, implicating a defect in the illegitimate recombination pathway. These findings suggest that the A-T gene product may either directly participate in illegitimate recombination or modulate the pathway. Regardless, this defect is likely to be important to a mechanistic understanding of this lethal disease.

Mutation of the uracil DNA glycosylase gene detected in glioblastoma.

Despite extensive characterization of genetic changes in gliomas, the underlying etiology of these tumors remains largely unknown. Spontaneous DNA damage due to hydrolysis, methylation, and oxidation is a frequent event in the brain. Failure of DNA repair following this damage may contribute to tumorigenesis of gliomas. Uracil DNA glycosylase (UDG), an enzyme which excises uracil from DNA, is an important component of the base excision repair pathway. The sequence of a human homologue of uracil DNA glycosylase gene (UNG) has been recently identified. We performed PCR-based SSCP mutational analysis of UNG in 11 sporadic gliomas (six glioblastomas, two anaplastic astrocytomas, and three oligodendrogliomas) and eight glioblastoma cell lines. One out of six sporadic glioblastomas had a point mutation in exon 3, which resulted in a missense mutation in codon 143. None of the eight glioblastoma cell lines or the five non-glioblastoma sporadic gliomas showed a mutation. Genetic alterations of UNG may play a role in the development of a subset of primary glioblastomas.

Induction of estrus in ovariectomized cows and heifers: effects of estradiol benzoate and gonadotropin releasing hormone.

Three experiments were conducted with ovariectomized (OVX) cows and heifers to investigate potential neuroendocrine mechanisms controlling estrous behavior. In Exp. 1, 10 OVX cows were treated with either 125 micrograms estradiol benzoate and 10 cc saline (125 micrograms EB + SAL), 125 micrograms EB and 500 micrograms gonadotropin releasing hormone (125 micrograms EB + GnRH), 250 micrograms EB and 10 cc SAL (250 micrograms EB + SAL), 250 micrograms EB and 500 micrograms GnRH (250 micrograms EB + GnRH) or 500 micrograms EB and 10 cc SAL (500 micrograms EB + SAL) in a replicated 5 X 5 Latin-square design. During the 48 h following EB injection, 2-h observation blocks were alternated with 2-h non-observation blocks. During each 2-h observation block, 14 behavioral interactions were monitored. The percentage of cows in estrus was lower for cows receiving 125 micrograms EB as compared with those given the higher doses. However, the cows receiving 125 micrograms EB + SAL did not differ in their estrous response from those receiving 125 micrograms EB + GnRH. The interval from injection to the onset of estrus and the duration of estrus were similar for all treatments. In Exp. 2, 10 OVX heifers were subjected to the same treatments and observation procedures utilized in Exp. 1. The results of Exp. 2 were similar to those of Exp. 1. In Exp. 3, 10 OVX cows were treated with either 300, 600, 1,200, 2,400 or 4,800 micrograms EB in a replicated 5 X 5 Latin-square design.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterization of the herpes simplex virus type 2-encoded uracil-DNA glycosylase.

The herpes simplex virus type 2 (HSV-2) uracil-DNA glycosylase (UNG) was from the nuclei of infected KB cells using ion exchange, affinity, and chromatofocusing chromatography techniques. Chromatography on DNA cellulose revealed two distinct HSV-2-encoded UNGs. One species, designated A, was purified approximately 324-fold, while the second species, designated B, was purified approximately 130-fold. The HSV UNG species B was observed to unidirectionally convert to the A species, suggesting that the B species binding to DNA cellulose may be the result of an association with other DNA binding proteins. SDS-PAGE demonstrated that both species A and B had molecular weights of 32,000. The HSV-2-encoded UNGs could be distinguished from the cellular nuclear UNG based upon differences in their behavior on the chromatography matrixes and by their molecular weights. There were no significant differences in the biochemical properties of the HSV-2-encoded or nuclear UNGs. Furthermore, all of these UNGs reacted with a monoclonal antibody produced against the human placental UNG. These data support recent studies, at both the DNA and the amino acid levels, which have demonstrated that this enzyme is highly conserved between species.

Human HeLa cell enzymes that remove phosphoglycolate 3'-end groups from DNA.

We have purified three chromatographically distinct human enzyme activities from HeLa cells, that are capable of converting bleomycin-treated DNA into a substrate for E. coli DNA polymerase I. The bleomycin-treated DNA substrate used in this study has been characterized via a 32P-postlabeling assay and shown to contain strand breaks with 3'-phosphoglycolate termini as greater than 95% of the detectable dose-dependent lesions. The purified HeLa cell enzymes were shown to be capable of removing 3'-phosphoglycolates from this substrate. Also 3'-phosphoglycolate removal and nucleotide incorporation were enzyme dependent. In addition, all three Hela cell enzymes have been determined to possess Class II AP endonuclease activity. The enzymes lack 3'----5' exonuclease activity and are, therefore, dissimilar to exonuclease III--an E. coli enzyme that can remove 3'-phosphoglycolate.

Ovarian follicle apoptosis in bovine growth hormone transgenic mice.

Growth hormone directly or via insulin like-growth factor-I has been shown to inhibit preovulatory follicle apoptosis, which is the underlying mechanism of follicular atresia. We studied the levels of apoptosis in the ovaries of transgenic mice expressing bovine growth hormone. Female bovine growth hormone transgenic mice (n = 10) and nontransgenic litter mates (n = 8) were killed at early proestrus. Ovaries were collected, sectioned, and processed using a nonradioactive in situ method for apoptosis detection. Follicles were classified and counted on the basis of size and level of apoptosis. Our results demonstrate that the percentage of ovarian follicles containing apoptotic cells was lower in transgenic versus normal mice (30% vs. 46%; P < 0.05). The percentage of follicles undergoing heavy apoptosis was lower (P < 0.05) in transgenic versus control animals in preovulatory and early antral follicles, but it was not different in preantral follicles. The percentage of healthy preovulatory follicles was also higher in transgenic versus normal mice (7.4% vs. 4.3%; P < 0.05). These results indicate that growth hormone overexpression in transgenic mice significantly decreases follicle apoptosis, and thus atresia in the mouse ovary, therefore leading to increased propensity for ovulation in these animals.

In situ amplification of the cytochrome P-450 cholesterol side-chain cleavage enzyme mRNA in single porcine granulosa cells by IGF-1 and FSH acting alone or in concert.

To investigate the cellular mechanisms and cell-cell heterogeneity of the actions of insulin-like growth factor-1 (IGF-1) and follicle-stimulating hormone (FSH) exerted alone and in combination on ovarian cholesterol side-chain cleavage gene expression (P450scc mRNA) in (pig) granulosa cells, we implemented semiquantitative in situ molecular hybridization at the single target-cell level. To this end, a 1-kb cDNA specific to the catalytic region of porcine p450scc gene was subcloned into pGEM-3 and directionally transcribed in vitro in the presence of 35S-dUTP to yield radiolabeled antisense (and sense, negative control) cRNA hybridization probes. Swine granulosa cells harvested nonenzymatically from immature (1-5 mm) Graafian follicles were anchored on eight-chamber multiwell slides and treated with control solvent, human recombinant IGF-1 (10nM), ovine FSH (10nM), or both hormones, for 48 h to stimulate progestin biosynthesis maximally. After appropriate cellular permeabilization, cRNA hybridization, and solvent washes, granulosa cells were exposed to Kodak NTB-2 emulsion for 6 wk. Semiquantitative automated image analysis software (NIH IMAGE 1.5) was used to evaluate the number of silver grains deposited/20,000 square pixels. Specificity controls included labeled sense riboprobe, pretreatment with RNase, and 100-fold molar excess unlabeled cRNA. Grain counts and their distributions were examined by ANOVA and the Wilcoxon nonparametric test. The mean number of silver grains deposited per granulosa cell increased over control (reflecting specific P450scc mRNA expression) in granulosa cells pretreated with IGF-1, FSH, or IGF-1 + FSH (p < 0.05 by ANOVA). The rank order of abundance of expression of P450scc mRNA (grains/ovarian cell) was (IGF-1 + FSH) > FSH > IGF-1 > control treatment. Distributional analysis showed that each treatment introduced skewed distributions toward granulosa cells expressing more P450scc per cell than controls (p < 0.01). The median grain count of granulosa cells treated with FSH was significantly increased over that of IGF-1 treatment (p < 0.05). Treatment with both IGF-1 and FSH further shifted the grain count distribution per cell to favor granulosa cells expressing more P450scc mRNA compared to IGF-1 or FSH treatment alone (p < 0.05). Accordingly, a demonstrable mechanism of IGF-1 and FSH's regulation of specific P450scc gene expression at the single granulosa cell level is amplification in the number of target ovarian cells expressing this enzymatically rate-determining gene transcript. Interestingly, the induction of P450scc mRNA is not sufficient to explain fully the synergistic increases in progesterone accumulation driven by combined treatment with IGF-1 and FSH, thus suggesting that other steroidogenic control points are also targets of IGF-1/FSH action.

A cooperative interaction between soy protein and its isoflavone-enriched fraction lowers hepatic lipids in male obese Zucker rats and reduces blood platelet sensitivity in male Sprague-Dawley rats.

Soy protein diets lower plasma cholesterol in hyperlipoproteinemic human subjects, as well as in animal models. We fed 7-wk-old male obese (fa/fa) and lean Zucker rats a modified AIN-76 diet (20 g protein/kg diet) containing casein (C), low isoflavone soy protein (38 mg isoflavones/kg diet; LI), or high isoflavone soy protein (578 mg isoflavones/kg diet; HI) for 70 d. In obese rats, plasma total cholesterol was 21 and 29% lower in the LI and HI groups, respectively, than in the C group (P: