Ole F. Nielsen

Antagonistic effect of aclarubicin on camptothecin induced cytotoxicity: role of topoisomerase I.

The cellular target of camptothecin and several of its derivatives has been identified as topoisomerase I. Central to the cytotoxic action of camptothecin is the drugs ability to stimulate formation of topoisomerase I mediated DNA cleavages. Here we demonstrate that the intercalating antitumor agent aclarubicin inhibits camptothecin induced DNA single strand breaks in cells as measured by alkaline elution. When purified topoisomerase I was reacted with DNA, aclarubicin inhibited the formation of enzyme mediated DNA breaks induced by camptothecin. High aclarubicin concentrations (10 and 100 microM) caused a slight stimulation of topoisomerase I mediated DNA cleavage at a few distinct DNA sites. The cytotoxicity associated with camptothecin treatment measured in clonogenic assays was antagonized by preincubation with aclarubicin. This inhibitory effect of aclarubicin upon camptothecin action holds implications for the scheduling of aclarubicin in combination therapy with anticancer agents directed against topoisomerase I. Aclarubicin also inhibits the effect of topoisomerase II directed agents [such as etoposide (VP16), amsacrine (mAMSA), etc.] suggesting that aclarubicin acts against the two topoisomerases.

Poly(ADP-ribose) metabolizing enzymes in nuclei and nucleoli of Tetrahymena Pyriformis

Poly(ADP-ribose) is formed in the nuclei of eucaryotic cells from NAD’ by the enzyme poly(ADPribose)polymerase and is degraded at the glycosidic bonds by poly(ADP-ribose)glycohydrolase. The physiological function of poly(ADP-ribose) in intact cells is unknown. Possible functions include a role in cell proliferation, differentiation, DNA replication, repair or transcription. The subject has been reviewed [ 1,2]. We have chosen a specific defined gene system to study the physiology of poly(ADP-ribose). The system chosen is the ribosomal genes of exponentially growing Tetrahymena. It is a well defined, specific gene system in which both replication and transcription may be studied. Indeed the average transcriptional template activity of Tetrahymena nucleoli is at least 25fold greater than that of whole nuclei [3]. We have asked whether poly(ADP-ribose) is made in isolated nucleoli. We show that not only is it synthesized in association with those ribosomal genes, but that the specific activity of both the synthetic and elongating enzymes in nucleoli is about the same as for the bulk of the chromosomal DNA. Thus, the very high level of ribosomal transcriptional activity is not associated with a correspondingly high activity of poly(ADPribose) enzymes, although these enzymes occur in purified nucleoli.

An Electron Microscopic Analysis of Transcription of Nucleolar Chromatin Isolated from Tetrahymena pyriformis

Transcriptionally active nucleoli and solubilized nucleolar chromatin were visualized by electron microscopy. The palindromic structure of the chromatin was demonstrated by spreading the chromatin on glow-discharged grids. In the presence of single-strand binding Eco HDP protein the preribosomal RNA transcripts are seen attached to the RNA-polymerase molecules in the electron micrographs. Each palindrome contains two preribosomal RNA genes. The strict termination properties of the transcription are indicated by the absence of transcriptional complexes in the distal parts of the molecules. — Investigation of the process of transcription in nucleolar chromatin being depleted of a termination protein by ammonium sulfate-treatment showed in agreement with biochemical studies that only some of the RNA polymerases terminate properly while others transcribe into the distal spacer region. The elongation rate is estimated to be slightly lower than in the gene region. The results are discussed in relation to biochemical studies of the transcriptional properties of the chromatin.

Isolation of the ribosomal RNA gene from Tetrahymena in the state of transcriptionally active chromatin.

Summary The chromatin form of the gene coding for ribosomal 17S and 25S RNA can be isolated from exponentially growing Tetrahymena cells in a yiled of 30–40%. The final purity with respect to DNA is more than 95%. The isolated ribosomal chromatin contains endogenously active RNA polymerase and sediments as a 50S particle on sucrose gradients.

Purification and characterization of an inducible mitochondrial DNA polymerase from Tetrahymena thermophila

Treatment of the eukaryotic organism Tetrahymena with various types of DNA-damaging agents has been reported to cause a 35-fold induction of a mitochondrial DNA polymerase. We here report that the enzyme can be induced in large-scale cultures by exposure of the cells to thymine starvation and/or intercalating agents. The induced DNA polymerase has been purified to near homogeneity, with a specific activity of approx. 300,000 units/mg protein. The relative molecular mass of the active form of the enzyme is approx. 100,000, as determined by glycerol gradient sedimentation. The subunit structure has been analysed by SDS polyacrylamide gel electrophoresis of the highly purified preparation and by immunoprecipitation with a monoclonal antibody directed to the DNA polymerase. A polypeptide of Mr 47,000 has been observed to be a subunit of the enzyme. This corresponds to the size of the subunits suggested for mitochondrial DNA polymerase from chicken embryos and mouse myeloma cells.

Studies of Molecular Events in Isolated Nucleolar Chromatin in Response to Chemicals and Ionizing Radiation

Chromatin is an important target for the effect of chemicals and ionizing radiation in living cells. The structural alterations induced lead to disturbance of the genetic expression, i.e. of the biosynthesis of RNA and of protein. One useful approach to explore the molecular background of various observed end-effects is to study the effect of damage on a specific gene which has been isolated in its chromatin form.