Repair of DNA lesions induced by polycyclic aromatic hydrocarbons in human cell-free extracts: involvement of two excision repair mechanisms in vitro

Isotretinoin (ITR), a teratogen in many species, is associated with increased oxidative stress. Metallothionein (MT) is an important tissue antioxidant whose concentrations are induced by zinc. To study the role of supplemental Zn as an inducer of embryonic MT, we injected pregnant CD-1 mice subcutaneously with saline vehicle, or 20 or 40 mg/kg Zn on gestational day (GD) 6.5. After 48 h, embryonic MT concentrations increased in a dose-related manner (r = 0.64, P < 0.05) with Zn treatment. The possible protective role of Zn pretreatment against ITR teratogenicity was investigated in vivo and in vitro. CD-1 mice were pretreated with saline or Zn (20 and 40 mg/kg) on GD 8.5 and 9.5. ITR was administered to both groups of mice via three intragastric intubations of 100 mg ITR/kg at 4 h intervals on GD 10.5. On GD 18.5, Zn pre-treated mice demonstrated decreased ITR-mediated growth retardation, cleft palates and postpartum mortality. A reduction in embryonic MT concentrations was observed in mice exposed to ITR. Mouse embryos cultured on GD 8.5 with an addition of 15 micromol/L Zn for 48 h had a sixfold greater MT concentration (688 microg/g protein) than controls. The Zn pretreatment of cultured embryos prevented malformations and lessened growth retardation caused by 24 h exposure to 17 micromol/L ITR. These results suggest that Zn-mediated induction of MT in mouse embryos could protect against ITR teratogenicity.     

DNA-PK-independent rejoining of DNA double-strand breaks in human cell extracts in vitro

PURPOSE: To investigate the role of DNA-dependent protein kinase (DNA-PK) in the rejoining of ionizing radiation-induced DNA double-strand breaks (dsb). MATERIALS AND METHODS: This study employed previously described in vitro assays that utilize nuclei or 'naked' DNA prepared from agarose-embedded cells as a substrate and S-HeLa cell extracts as a source of enzymes. Rejoining of dsb in these assays is absolutely dependent on cell extract and it proceeds, under optimal reaction conditions, to an extent similar to that observed in intact cells. Results were confirmed in a plasmid-based assay for in vitro rejoining of dsb. RESULTS: It is shown that concentrations of wortmannin completely inhibiting DNA-PK activity profoundly affect the rejoining of dsb in vivo, but have no effect on dsb rejoining in vitro. Furthermore, fractionation of cell extracts using ammonium sulphate precipitation, generates protein fractions that are able to support dsb rejoining, despite the fact that they do not contain detectable amounts of either DNA-PKcs or Ku80. Efficient rejoining of dsb in vitro is also observed with extracts of MO59J cells that lack DNA-PK activity. Finally, rejoining of dsb remains unaffected by wortmannin in a plasmid-based assay, and is also detectable with extracts of MO59J cells. CONCLUSIONS: These findings are in contrast with genetic studies demonstrating a requirement for DNA-PK activity for efficient rejoining of dsb in vivo. The difference between in vitro and in vivo results may not be attributed to chromatin structure since wortmannin was without an effect when using nuclei as a substrate. It is speculated that the differences between in vivo and in vitro results can be explained either by assuming the operation of multiple pathways in dsb rejoining, some of which do not require DNA-PK, or by postulating a purely regulatory/damage-sensing role for DNA-PK in intact cells but no direct involvement in dsb rejoining.     

Optimal erythropoietin expression in human hepatoma cell lines requires activation of multiple signalling pathways

The Drosophila wing is divided into anterior and posterior compartments, the latter characterized by the expression of the engrailed gene. A comparative analysis is presented here, and suggests that a primary conserved role of engrailed is to drive growth of limbs along the proximo-distal axis. The Apical Ectodermal Ridge in vertebrate limbs resembles the Antero/ Posterior compartment boundary in fly wings, particularly in molecular aspects. Multiple evidence suggests that the fly wing Antero/Posterior boundary is not the result of differential cell affinities between all anterior and posterior cells, but responds to the area of cell communication between anterior and posterior compartments. Arguments are presented here to support the notion that the compartment boundary is a consequence of decapentaplegic function in the control of growth. Patterning, on the other hand, requires the participation of several genes, among which are engrailed, invected and hedgehog. Finally, regulatory interactions between en/En-1 and hh/Shh may be significant in the context of morphogenetic regulation during normal development.     

In vitro repair of gaps in bacteriophage T7 DNA

An in vitro system based upon extracts of Escherichia coli infected with bacteriophage T7 was used to study the mechanism of double-strand break repair. Double-strand breaks were placed in T7 genomes by cutting with a restriction endonuclease which recognizes a unique site in the T7 genome. These molecules were allowed to repair under conditions where the double-strand break could be healed by (i) direct joining of the two partial genomes resulting from the break, (ii) annealing of complementary versions of 17-bp sequences repeated on either side of the break, or (iii) recombination with intact T7 DNA molecules. The data show that while direct joining and single-strand annealing contributed to repair of double-strand breaks, these mechanisms made only minor contributions. The efficiency of repair was greatly enhanced when DNA molecules that bridge the region of the double-strand break (referred to as donor DNA) were provided in the reaction mixtures. Moreover, in the presence of the donor DNA most of the repaired molecules acquired genetic markers from the donor DNA, implying that recombination between the DNA molecules was instrumental in repairing the break. Double-strand break repair in this system is highly efficient, with more than 50% of the broken molecules being repaired within 30 min under some experimental conditions. Gaps of 1,600 nucleotides were repaired nearly as well as simple double-strand breaks. Perfect homology between the DNA sequence near the break site and the donor DNA resulted in minor (twofold) improvement in the efficiency of repair. However, double-strand break repair was still highly efficient when there were inhomogeneities between the ends created by the double-strand break and the T7 genome or between the ends of the donor DNA molecules and the genome. The distance between the double-strand break and the ends of the donor DNA molecule was critical to the repair efficiency. The data argue that ends of DNA molecules formed by double-strand breaks are typically digested by between 150 and 500 nucleotides to form a gap that is subsequently repaired by recombination with other DNA molecules present in the same reaction mixture or infected cell.     

In vitro wound repair by human gastric fibroblasts: implications for ulcer healing

Fibroblasts modulate epithelial biological activities and play a key role in the ulcer healing process. There is no information regarding the biological response of human gastric fibroblasts to regulatory compounds. The aim of this study was to assess the effects of growth factors and prostaglandins on an in vitro model of human gastric fibroblast wound repair. Subconfluent fibroblast cultures were used to study proliferative responses, determined by [3H]thymidine incorporation into DNA. In vitro wound repair was determined in confluent fibroblast monolayers after mechanical denudation. The presence of putative growth factors secreted by fibroblasts was studied in conditioned medium by heparin-affinity chromatography and immunodetection with specific antibodies. Serum and platelet-derived growth factor (PDGF) -BB induced a dramatic increase in both gastric fibroblast proliferation and closure of wounded cell monolayers, whereas these activities were inhibited by both transforming growth factor (TGF) -beta1 and prostaglandin E1. Basal activities in unstimulated gastric fibroblasts were lower than those obtained in skin fibroblasts. Conditioned medium stimulated fibroblast proliferation and wound repair activity, which was inhibited by the addition of suramin, and was partially dependent on the presence of PDGF-like factor. PDGF is a major, autocrine promotor of human gastric fibroblast-dependent wound repair activities, which are inhibited by prostaglandins and TGF-beta. These findings might be important for future therapeutic ulcer healing approaches.     

Arthroscopic transglenoid multiple suture repair: 2 to 8 year results in 150 shoulders

Although treatment for hypertension is readily available, poor control of hypertension is a major health problem frequently manifested in late life. Researchers believe that one of the major causes of uncontrolled hypertension is failure to take medication as directed. In this preliminary study, the medication-taking behaviors of 48 adults diagnosed with hypertension, ranging in age from 35 to 87, were recorded for 2 months with credit card-sized bar-code scanners. The social-cognitive model (Park, 1992) for understanding medication adherence, which proposes that medication adherence is governed by both beliefs and cognitive factors, was used as a basis for this research. Therefore, measures of health behaviors, attitudes about health and medication taking, and cognitive function were recorded, as well as blood pressure readings. The main findings were that (a) the oldest-old and groups of middle-aged adults were the most nonadherent, whereas the young-old were more likely to adhere than the other age groups; (b) high blood pressure readings predicted adherence to antihypertensive medications; and (c) medication beliefs influenced adherence in some situations.     

Influence of ethanol on in vitro growth of human mammary carcinoma cell line MCF-7

Death is a commonly used concept but is surrounded by much mystery. The concept of death is examined using the Walker and Avant (1995) framework for concept analysis. The use of the concept death is considered in the intensive care unit. In the intensive care unit a conflict often exists between the curing culture and the inevitability of death.     

Delivery of multiple CD8 cytotoxic T cell epitopes by DNA vaccination

Development of CD8 alphabeta CTL epitope-based vaccines requires an effective strategy capable of co-delivering large numbers of CTL epitopes. Here we describe a DNA plasmid encoding a polyepitope or "polytope" protein, which contained multiple contiguous minimal murine CTL epitopes. Mice vaccinated with this plasmid made MHC-restricted CTL responses to each of the epitopes, and protective CTL were demonstrated in recombinant vaccinia virus, influenza virus, and tumor challenge models. CTL responses generated by polytope DNA plasmid vaccination lasted for 1 yr, could be enhanced by co-delivering a gene for granulocyte-macrophage CSF, and appeared to be induced in the absence of CD4 T cell-mediated help. The ability to deliver large numbers of CTL epitopes using relatively small polytope constructs and DNA vaccination technology should find application in the design of human epitope-based CTL vaccines, in particular in vaccines against EBV, HIV, and certain cancers.     

Methyl-directed repair of mismatched small heterologous sequences in cell extracts from Escherichia coli

The methyl-directed DNA repair efficiency of a set of M13mp18 heteroduplexes containing 1-8 or 22 unpaired bases was determined by using an in vitro DNA mismatch repair assay. The unpaired bases of each heteroduplex residing at overlapping recognition sites of two restriction endonucleases allow independent assay of repair on either DNA strand. Our results showed that the repair of small nucleotide heterologies in Escherichia coli extracts was very similar to base-base mismatch repair, being strand-specific and highly biased to the unmethylated strand. The in vitro activity was also dependent on products of mutH, mutL, mutS, and uvrD loci and was equally efficient on nucleotide insertions and deletions. The repair levels of small heterologies were affected by base composition of the heterologies. However, the extent of repair of heteroduplexes containing small heterologous sequences was found to decrease with an increase in the number of unpaired bases. Heteroduplexes containing an extra nucleotide of 22 bases provoked very low level of methyl-directed repair.     

Angiogenic factors expressed by human prostatic cell lines: effect on endothelial cell growth in vitro

Using anterograde transport of WGA-HRP and the experimental degeneration method for identification of corticocuneate (CCT) and primary afferent (PAT) terminals in conjunction with gamma-amino butyric acid (GABA) and glutamate immunocytochemistry, this study has demonstrated that the GABA-immunoreactive (GABA-IR) neurons in the rat cuneate nucleus were post-synaptic to PATs (some of them being glutamate-IR), GABA-IR and GABA-negative terminals. The HRP-labelled CCTs did not make any synaptic contacts with GABA-IR neurons but with some GABA-negative dendrites. PATs labelled by HRP or showing degenerating features made direct synaptic contacts with the dendrites of GABA-IR neurons. Beside the above GABA-IR boutons also showed axosomatic and axodendritic synapses with the GABA-IR neurons. In 'triple labeling' method for GABA, PAT and glutamate, it was found that the PATs which were usually glutamate-positive were presynaptic to the dendrites of GABA-IR neurons. Furthermore, some glutamate-IR terminals which were of non-PAT's origin also synapsed with the dendrites and somata of GABA-IR neurons. It is concluded from this study that the major inputs of GABA-IR neurons were from glutamate immunopositive PATs and glutamate terminals of non-PATs origin; other GABA-IR terminals either intrinsic or extrinsic also contributed to the afferent sources of GABA-IR neurons. The CCTs contributed very little, if any, to this input. It is suggested that the PATs and glutamate-IR terminals on GABA-IR neurons may be involved in lateral inhibition for increase of spatial precision. The synaptic contacts between GABA-IR boutons and dendrites or somata of GABA-IR neurons may provide a possible means for disinhibition.     

Multiple signaling pathways of human interleukin-8 receptor A. Independent regulation by phosphorylation

Interleukin-8 (IL-8) receptor A (CXCR1) couples to a pertussis toxin-sensitive G protein to mediate phospholipase Cbeta (PLCbeta) activation and cellular responses. Responses to CXCR1 are attenuated by prior exposure of neutrophils to either IL-8, a cleavage product of the fifth component of complement (C5a) or n-formylated peptides (formylmethionylleucylphenylalanine, fMLP). To characterize the role of receptor phosphorylation in the regulation of the CXCR1, a phosphorylation-deficient mutant, M2CXCR1, was constructed. This receptor, stably expressed in RBL-2H3 cells, coupled more efficiently to G protein and stimulated enhanced phosphoinositide hydrolysis, cAMP production, exocytosis, and phospholipase D activation, and was resistant to IL-8-induced receptor internalization. The rate and total amount of ligand stimulated actin polymerization remained unchanged, but interestingly, chemotaxis was decreased by approximately 30% compared with the wild type receptor. To study the role of receptor phosphorylation in cross-desensitization of chemoattractant receptors, M2CXCR1 was coexpressed with cDNAs encoding receptors for either fMLP (FR), C5a (C5aR), or platelet-activating factor (PAFR). Both C5aR and PAFR were cross-phosphorylated upon M2CXCR1 activation, resulting in attenuated guanosine 5'-3'-O-(thio)triphosphate (GTPgammaS) binding in membranes. In contrast, FR and M2CXCR1 were resistant to cross-phosphorylation and cross-inhibition of GTPgammaS binding by other receptors. Despite the resistance of M2CXCR1 to cross-phosphorylation and receptor/G protein uncoupling, its susceptibility to cross-desensitization of its Ca2+ response by fMLP and C5a, was equivalent to CXCR1. Regardless of the enhancement in certain receptor functions in M2CXCR1 compared with the wild type CXCR1, the mutated receptors mediated equivalent PLCbeta3 phosphorylation and cross-desensitization of Ca2+ mobilization by FR, C5aR, and PAFR. The results herein indicate that phosphorylation of CXCR1 regulates some, but not all of the receptors functions. While receptor phosphorylation inhibits G protein turnover, PLC activation, Ca2+ mobilization and secretion, it is required for normal chemotaxis and receptor internalization. Since phosphorylation of CXCR1 had no effect on its ability to induce phosphorylation of PLCbeta3 or to mediate class-desensitization, these activities may be mediated by independently regulated pathways.     

Evidence for involvement of yeast proliferating cell nuclear antigen in DNA mismatch repair

DNA mismatch repair plays a key role in the maintenance of genetic fidelity. Mutations in the human mismatch repair genes hMSH2, hMLH1, hPMS1, and hPMS2 are associated with hereditary nonpolyposis colorectal cancer. The proliferating cell nuclear antigen (PCNA) is essential for DNA replication, where it acts as a processivity factor. Here, we identify a point mutation, pol30-104, in the Saccharomyces cerevisiae POL30 gene encoding PCNA that increases the rate of instability of simple repetitive DNA sequences and raises the rate of spontaneous forward mutation. Epistasis analyses with mutations in mismatch repair genes MSH2, MLH1, and PMS1 suggest that the pol30-104 mutation impairs MSH2/MLH1/PMS1-dependent mismatch repair, consistent with the hypothesis that PCNA functions in mismatch repair. MSH2 functions in mismatch repair with either MSH3 or MSH6, and the MSH2-MSH3 and MSH2-MSH6 heterodimers have a role in the recognition of DNA mismatches. Consistent with the genetic data, we find specific interaction of PCNA with the MSH2-MSH3 heterodimer.     

Modelling human sperm-egg interactions in vitro: signal transduction pathways regulating the acrosome reaction

Recent advances in characterizing sperm surface receptors and ion channels, when combined with the rapidly expanding knowledge of interactions among second messenger systems in somatic cells, permit formulation of a tentative molecular mechanism for the regulation of the human sperm acrosome reaction. As spermatozoa pass through the cumulus mass, progesterone binds to its sperm surface receptor, alkalinizes the sperm head cytosol and potentiates changes in intracellular ionized calcium. Primary binding of spermatozoa to egg involves receptors for mannosyl, N-acetylglucosaminyl and, possibly, fucosyl residues of the glycosylated zona protein, ZP3. These receptors aggregate on multivalent ligand binding, migrate to the equatorial region along an actin filament network formed between the plasma and acrosomal membranes during capacitation, and activate a G protein/protein kinase A/protein kinase C second messenger system and a secondary proteolysis signal. Binding of a receptor tyrosine kinase to ZP3 amino acid residues simultaneous with the sugar recognition event triggers tyrosine phosphorylation signalling. All signals combine to open a voltage-dependent calcium channel. The resulting elevated calcium signal depolymerizes the inter-membrane actin network and activates phospholipases, leading to an acrosome reaction.     

Development of CD4 and CD8 single positive T cells in human thymus organ culture: IL-7 promotes human T cell production by supporting immature T cells

This paper describes novel model systems to study the development of human T cells. Fragments of neonatal human thymus (HUNT) can be cultured in vitro; the initial majority population of CD4, CD8 double-positive (DP) thymocytes is not maintained in organ culture. These cells are rapidly replaced by populations of CD4 or CD8 single-positive (SP) T cells. In addition, allogeneic thymic chimeras can be established by the addition of human cord blood (HUCB) mononuclear cells as a source of T progenitor cells to the organ cultures. Culture results in the acquisition of a mature SP T cell phenotype by the donor cells similar to that found when HUCB is allowed to develop in xenogeneic murine scid/scid fetal thymus organ culture. The number of immature and mature T cells produced by organ cultures can be differentially increased by the addition of exogenous IL-7, stem cell growth factor, IL-1, or GM-CSF. Anti-IL-7 antibody inhibits T cell production. Taken together, the results suggest that human T cell development occurs in these in vitro systems in a similar manner, regardless of the species origin of the thymic stromal cells in the culture, and that exogenous cytokines can be used to expand subpopulations of developing T cells.     

Replication of UV-irradiated DNA in human cell extracts: evidence for mutagenic bypass of pyrimidine dimers

We have examined the efficiency and fidelity of simian virus 40-origin-dependent replication of UV-irradiated double-stranded DNA in extracts of human cells. Using as a mutational target the alpha-complementation domain of the Escherichia coli lacZ gene in bacteriophage M13mp2 DNA, replication of undamaged DNA in HeLa cell extracts was highly accurate, whereas replication of DNA irradiated with UV light (280-320 nm) was both less efficient and less accurate. Replication was inhibited by irradiation in a dose-dependent manner. Nonetheless, covalently closed, monomer-length circular products were generated that were resistant to digestion by Dpn I, showing that they resulted from semiconservative replication. These products were incised by T4 endonuclease V, whereas the undamaged replication products were not, suggesting that pyrimidine dimers were bypassed during replication. When replicated, UV-irradiated DNA was used to transfect an E. coli alpha-complementation host strain to score mutant M13mp2 plaques, the mutant plaque frequency was substantially higher than that obtained with either unirradiated, replicated DNA, or unreplicated, UV-irradiated DNA. Both the increased mutagenicity and the inhibition of replication associated with UV irradiation were reversed by treatment of the irradiated DNA with photolyase before replication. Sequence analysis of mutants resulting from replication of UV-irradiated DNA demonstrated that most mutants contained C-->T transition errors at dipyrimidine sites. A few mutants contained 1-nt frameshift errors or tandem double CC-->TT substitutions. The data are consistent with the interpretation that pyrimidine dimers are bypassed during replication by the multiprotein replication apparatus in human cell extracts and that this bypass is mutagenic primarily via misincorporation of dAMP opposite a cytosine (or uracil) in the dimer.     

Suppression of human cancer cell proliferation by lipoxygenase inhibitors and gamma-radiation in vitro

The effects of inhibitors of arachidonic acid oxidative metabolism, gamma-radiation and/or their combinations on proliferation and cell cycle were studied in human breast carcinoma HS578T and monoblastoid U937 cell lines. While piroxicam an inhibitor of cyclooxygenase pathway, had no significant effects on cell proliferation, inhibitors of lipoxygenase pathway, nordihydroguaiaretic acid and esculetin, suppressed [3H]-thymidine incorporation and cell growth. The latter agents also differed in their modulation of cell cycle parameters depending on the cell line and the time of treatment. When the cells were preirradiated with gamma radiation (5 Gy) and treated with the drugs (at concentrations 50 mumol/l and higher) the effects on cell proliferation were mostly additive. On the other hand, the results suggest that antiproliferative effects could be significantly strengthened when lower doses (25 mumol/l) of lipoxygenase inhibitors were combined with a low dose (1 Gy) of gamma-radiation. Experiments monitoring the reversibility of the effects after single or combined treatment with the agents showed that irradiation suppressed the ability of U937 cells to restore cell proliferation, and that these effects may be strenghtened by esculetin. In conclusion, our results (1) suggest that the lipoxygenase pathway plays a significant role in proliferation of cancer HS578T and U937 cells in vitro, and (2) implicate the possibility of more effective antiproliferative effects after combined treatment of cells with gamma-radiation and lipoxygenase inhibitors.     

In vitro stability of polymerase chain reaction-generated DNA fragments in serum and cell extracts

1. The identification of cytokine genes expressed in the central nervous system is critical to understanding the immune network in various diseases of brain, such as infection, degeneration, and malignancy. 2. Expression of cytokine genes in human astrocytoma cell lines and in fresh brain specimens was studied by the reverse-transcribed/polymerase chain reaction method. 3. The correlation between clinical malignancy and cytokine gene expression within malignant glioma was examined, especially regarding the relevancy of inhibitory cytokines, such as transforming growth factor-beta and interleukin-10.     

Growth control of a human glioma cell line by multiple autocrine loop blockade

The crystal structures of the ligand binding domain of a bacterial aspartate receptor suggest a simple mechanism for transmembrane signaling by the dimer of the receptor. On ligand binding, one domain rotates with respect to the other, and this rotational motion is proposed to be transmitted through the membrane to the cytoplasmic domains of the receptor.