Colin K. Pearson

Cellular euthanasia mediated by a nuclear enzyme: a central role for nuclear ADP-ribosylation in cellular metabolism

Abstract A number of roles have been ascribed to the nuclear ADP-ribosyl transferase, including involvement in DNA repair, cellular differentiation, transformation and gene rearrangements and transpositions. In this brief article we concentrate on the unifying concept that the transferase is fundamentally involved in regulating cellular metabolism in response to DNA damage through modulation of cellular NAD + pools. The ultimate contribution of the enzyme under conditions of extreme DNA damage may be to cause cell death.

Stage-specific distribution of P-glycoprotein in first-trimester and full-term human placenta.

SummaryThe distribution of P-glycoprotein in human placenta has been examined by immunohistochemistry using a battery of monoclonal antibodies (MRK-16, C219 and JSB-1). P-glycoprotein was located on the syncytiotrophoblast microvillus border in first-trimester placentas and some of the placental macrophages (Hofbauer cells) showed weak cytoplasmic staining. In term placentas, however, staining was not observed in the trophoblast but most of the Hofbauer cells displayed strong cytoplasmic staining. In situ hybridization with specific gene probes suggested that both human multidrug resistance genes were expressed in the placenta, although only the multidrug resistance-1 gene product would have been detected by the MRK and JSB-1 antibodies. These results point to distinct functions for P-glycoprotein during the different stages of placental development and indicate that its expression may be under developmental control.

Covalent modification of proteins by ADP-ribosylation

Abstract In addition to its well established role as a cofactor in redox reactions, NAD + serves also as a substrate for a ubiquitous group of enzymes called ADP-ribosyl transferases. These enzymes are found in both the nucleus and cytosol of eukaryotic cells, as components of pathogenic bacterial toxins, and as part of a mechanism for inactivating host cell protein biosynthetic machinery in bacteriophage-infected cells. In this article we provide a brief overview of all known ADP-ribosylation reactions.

The activity and properties of poly(adenosine diphosphate ribose) polymerase in vitro during the embryonic development of the South African clawed toad Xenopus laevis

Characterization of poly(ADPribose) polymerase in isolated nuclei of Xenopus laevis embryos shows that maximum activity in vitro occurs at 25°C in 10 mM Tris-HCl buffer, pH 8.0, containing 20 mM MgCl2, 1 mM dithiothreitol, and 3 mM NaF. Under these conditions the apparent Km for NAD+ was 0.125 mM. The activity of the polymerase during embryogenesis was measured using both a whole embryo extract and isolated embryonic nuclei. Both of these sources of enzyme demonstrated an increase of approximately eightfold in the activity per cell, between early cleavage (stages 2–4; Nieuwkoop and Faber, 1956, “Normal Table of Xenopus laevis (Daudin),” North-Holland, Amsterdam) and late neurula (stages 23–24). From late neurula to early tadpole (stages 38–39) the activity of the extracted enzyme, calculated per cell, decreased by 64%. During the same period the activity of the enzyme in isolated nuclei increased by 40% to reach its maximum activity in early tail bud (stages 27–28), and thereafter decreased by 23%. These results indicate a possible involvement of poly(ADPribose) polymerase in these embryos in the cell differentiation processes, rather than in cell proliferation.

Characterization of density gradients prepared by freezing and thawing a sucrose solution.

Density gradients of sucrose can be prepared in large numbers by successive freezing and thawing of sucrose solutions. Gradients of other solute molecules, such as salt and detergents, also form and this could affect subsequent sedimentation behavior of some molecules. However, the sedimentation behavior of native and denatured DNA of bacteriophage lambda was essentially isokinetic under the conditions used thus making these gradients comparable with ones prepared manually, at least for preparative sedimentation work with nucleic acids.

The Effect of Polyamines on Herpes Simplex Virus Type 1 DNA Polymerase Purified from Infected Baby Hamster Kidney Cells (BHK-21/C13)

The DNA polymerase whose synthesis is directed by the herpes simplex virus type 1 (HSV-1) DNA was purified 545-fold from BHK-21/C13 cells 16 h after infection with the virus. Spermidine and spermine stimulated the activity of the polymerase over the concentration range 0.5 mM to 2.5 mM. This effect was enhanced with increased concentrations of polyamine, maximum stimulation being threefold and fourfold for spermidine and spermine respectively. The diamine, putrescine, had little effect on the enzyme at the concentrations used (0.25 to 1.25 mM).

Poly(adenosine diphosphate ribose) synthesis by isolated nuclei of Xenopus, laevis embryos: Invitro elongation of invivo synthesized chains☆

Abstract We have studied the synthesis of poly(ADP-ribose) by nuclei isolated from Xenopus laevis embryos at different stages of development. Determination of the total chain length of poly(ADP-ribose) molecules by hydroxylapatite column chromatography generally gave higher values than when the radioactive portions of these molecules, synthesized in vitro , were measured by poly(ethyleneimine)-cellulose thin layer chromatography, after snake venom phosphodiesterase digestion. The results show that most of the poly(ADP-ribose) synthesized in vitro is a covalent elongation of molecules previously initiated in vivo .

Multidrug resistance during cancer chemotherapy — biotechnological solutions to a clinical problem

Tumour cells can be resistant to a variety of chemotherapeutic drugs of different structure (multidrug resistance) by expressing a transmembrane pump (P-glycoprotein) on their cell surface. This situation can lead to a failure of cancer chemotherapy as the P-glycoprotein acts by actively pumping the drugs out of cells, thus lowering the intracellular concentration of the drug and, hence, its cytotoxic effectiveness. This review summarizes present and proposed approaches to preventing or circumventing the action of this drug-transporting protein.

Is uracil misincorporation into DNA of mammalian cells a consequence of methotrexate treatment

We have carried out an exhaustive analysis to determine if uracil is incorporated into DNA of different mammalian cells exposed to methotrexate. Both HeLa and human lymphoblastoid cells (CCRF-HSB2) were incubated in medium containing [5-3H] deoxyuridine and 10 microM or 100 microM methotrexate. In some experiments non-radioactive 10mM uracil was present to inhibit uracil-DNA-glycosylase and thus facilitate the subsequent detection of uracil in the DNA. This was extracted and freed of RNA, ribonucleotides and protein with the use of phenol, RNAase, pronase, ethanol precipitation and Sephadex chromatography. DNA was enzymically degraded to nucleosides which were analysed directly by HPLC. We did not detect uracil in the DNA in over 12 different experiments under various conditions and times of drug-treatment. In view of this we caution against ready acceptance of the notion that uracil is incorporated to any significant extent, or indeed at all, in all types of cells exposed to methotrexate.

Increased protein ADPribosylation in HeLa cells exposed to the anti-cancer drug methotrexate.

DNA single-strand breaks (about 200-300 per genome) were transiently detected during the first hour when HeLa cells were incubated for up to 24 h with 100 microM methotrexate. There was an expected increase in ADPribosyltransferase activity, which reached a maximum 2-3-fold stimulation at 3 h but which was still greater than in control cells after 24 h. When hypoxanthine (25 microM) was present in the incubations together with the methotrexate the transferase was no longer activated, although basal, control levels of activity were still present. DNA strand breaks were reduced in number but were still just detectable under these conditions. Cellular NAD+ levels were mostly unaffected by the various drug treatments, except for a small transient decrease after 1 h, possibly as a result of the transferase activation. Methotrexate did not cause an increase in the rate of ADPribose degradation. Degradation of ADPribose residues labelled in a preincubation period in permeabilized cells was more extensive at pH 6.0 was a 50% loss of acid-insoluble radioactivity in 30 min at 26 degrees C. At pH 8.0 the loss did not exceed 30-35% even after 90 min incubation. The activation of the transferase is reflected in a general increase in protein ADPribosylation detected by autoradiography of 32P-labelled proteins in 6.25-18.25%T gradient acrylamide gels. There were three major acceptors with molecular masses of 17, 100 and over 100 kDa, which could be respectively a histone, a transferase-derived peptide fragment and the transferase itself. When ADPribosyltransferase was inhibited with 3-amino-benzamide DNA single-strand breaks were no longer detected. However, this had no observably signficant effect on the kinetics of loss of cell viability (from Trypan blue uptake), cell number or colony-forming ability. Similar results are observed in most cases when the activation of the transferase, resulting from the incubation of cells with methotrexate, is inhibited by hypoxanthine. We conclude from such observations that the enhanced protein ADPribosylation seen in the cells exposed to methotrexate is a direct consequence of drug-exposure, but does not have any significant influence over the course of events leading ultimately to cell death.