A 1 6

B 09 MODULATION OF HUMAN AROMATASE GENE EXPRESSION BY SPECIFIC ANTISENSE OLIGODEOX'YNUCLEOTIDES

K. Ackermann, J. Fauss and W. Pyerin

The presence of estrogen is considered to play a crucial role for the development of hormone-dependent tumors. Estrogens are generated by an endomembrane-bound cytochrome P450 termed aromatase. This rate- limiting enzyme converts androgens into estrogens by a series of three sequential hydroxylations that results in loss of the angular C-19 methyl group and concomitantly in aromatization of ring A of the steroid ring system. To improve specificity of clinically applied aromatase inhibitors which exclusively are designed to act on enzyme protein, a novel strategy of inhibition was examined: inhibition at the nucleic acid level. An antisense oligodeoxynucleotide complementary to the translation start region of human aromatase transcripts (antisense-arom) was synthesized and employed to inhibit cyclic AMP-triggered aromatase gene expression in cultured human choriocarcinoma cells (JEG-3). Appearance of transcripts following induction by hCG or the membrane permeating dibutyryl cyclic AMP was significantly inhibited (reaching 70% and 60% inhibition, respectively) accompanied by accelerated mRNA degradation. The inhibition at nucleic acid level was paralleled by a decrease of both the aromatase protein and the microsomal aromatase activity. The data appear to indicate the antisense strategy to be a most promising approach for the development of a novel type of specific aromatase inhibitor.

Reference: Ackermann, K., Fauss, J. and Pyerin, W. (1994) Cancer Research 54, 4940- 4946

Biochemische Zellphysiologie, Deutsches Krebsforschtmgszentrum, Im Neuenheimer Feld 280, D-69120 Heidelberg

B 11 PHYSICAL AND BIOCHEMICAL PROPERTIES OF THE HUMAN DNA REPAIR PROTEIN O6-ALKYLGUANINE-DNA ALKYL- TRANSFERASE (AT) K. Bender, M. Federwisch, U. Loggen, J. Thomale, and M.F. Rajewsky Unrepaired OS-alkylguanines in cellular DNA may result in mutations or cell death. The major repair pathway for these DNA lesions involves the DNA repair protein Oa-alkylguanine-DNA alkyltransferase (AT). We have analyzed the DNA sequence specificity of AT-mediated repair in synthetic ds-deoxynucleotides (34-mers) in vitro. The DNA binding constants to oligomers with or without 0 e- ethylguanine (OS-EtGua) located between different flanking bases were determined by gel-retardation assays. The same oligomers were employed for kinetic analyses using a monoclonal antibody- based filter binding assay. All experiments were carried out with recombinant human AT and AT mutants (e. g., truncated at the N- or C-terminus) purified by Ni-chelate chromatography. Milligram quantities of apparently homogeneous protein (as tested by SDS-PAGE) with a high specific activity were produced. No major differences in DNA binding properties were observed with the different substrate molecules. The apparent association constants K~pp for the native AT were of the order of 3 x 105 M 1 for all oligomers tested. Repair rate constants were of the order of 107 M-is -1. Only slightly different values were observed as a function of the base context of the central OS-EtGua. Conclusions: (i) Neither the binding of AT to the substrate DNA nor the repair rate constants show significant sequence specificity; (ii) the postreplicative (OS-EtGua)'T mispair is a good substrate for human AT; (iii) AT binding is not dependent on the presence of the ethyl group on the OS-atom of guanine; and (iv) the N-terminus (first 56 amino acids) of AT neither influenced binding to the substrate DNA nor the repair rate constants.

Institute of Cell Biology (Cancer Research), Research Group I, West German Cancer Center Essen, University of Essen Medical School, Virchow-Strasse 173, D-45122 Essen, Germany.

B 10 PURIFIED DNA POLYMERASES iX, t~, AND S FROM THE HIGHLY MALIGNANT NOVIKOFF HEPATOMA CELLS HAVE LOWERED COPYING FIDELITY. G. Fox, O. Popanda, and H. W. Thielmann Objective of study. We checked whether or not DNA polymerases from tumor cells have mutator properties which might be responsible for the majority of mutations found in tumor progression. Methods. DNA polymerases ct, 8, and ~ were purifed from both normal rat liver and Novikoff hepatoma6cells and compared with regard to a) insertion of nucleotides opposite m Gua (control: Gua) in the template and b) extension of primers with mismatched 3'-OH ends, using steady-state kinetics and the following primer-template:

5' - -3 ' AGA GGA AAG TGA 12-mer 3' - -5' TCT CCT TTC ACT GAT CAC 18-mer

I s me

[for extension studies, the primer was a 13-mer with mismatched 3'-OH end] 6

Results. a) Insertion of nucleotides opposite m Gua in the template. DNA polymerases ct, 5, ~nd z from normal rat liver preferably catalyzed incorporation of dAMP opposite m Gua. When dNTP concentrations were increased in the assays, dCMP (polymerases 8 and e) and dTMP (polymerase ct) were also incorporated. The same insertion pattern was found for the enzymes from malignant cells, however, insertion frequencies were significantly higher for polymerases 8 and E (i. e., misincorporations were more abundant). b) Extension of the DNA primers with mismatched 3'-OH ends. On principle, G:T and G:A mismatches were easily extended by DNA polymerases cq 8 and e from both normal rat liver and Novikoff hepatoma cells. Only the G:G mismatch represented an extension problem. Surprisingly, DNA polymerase c~ from Novikoff bepatoma cells was capable of extending G:A and G:G mismatches much more readily than the enzyme from normal cells. Conclusion. DNA polymerases 0% 8, and e from malignant cells can have altered kinetics of a) base misincorporation and b) mismatch extension, and may thus give rise to mutations in tumor progression.

This work was supported by the Deutsche Krebshilfe, Dr. Mildred Scheel Stiftung, W 28/93/Th 2. DKFZ Heidelberg, Division 340, Im Neuenheimer Feld 280, D-69120 Heidelberg

B 12 THE NUCLEAR FORM OF LACTATE DEHYDROGENASE MODULATES UV =

INDUCED DNA REPAIR SYNTHESIS IN HUMAN FIBROBLASTS. H. W. Thielmann, O. Popanda, and G. Fox Objective 9/" study. Does the nuclear, phosphorylated form of lactate dehydrogenase play a role in UV-induced DNA repair in human fibroblasts? Backaround. Several glycolytic enzymes occur in mammalian cell nuclei, in additi-on to their known presence in the cytoplasm, among them lactate dehydrogenase and phosphoglycerate kinase. The nuclear forms of these enzymes are closely associated with chromatin, bind to unfolded DNA, and may serve as stabilizing factors in DNA synthesis and/or transcription. Methods. a) In vitro DNA synthesis catalyzed by purified DNA polymerases c~, 6, and e from normal and malignant rat liver cells was measured in the presence of the glycolytic enzymes, b) The effects of lactate dehydrogenase and phosphoglycerate kinase upon UV-induced DNA repair synthesis were studied in permeabilized normal human fibroblasts. Results. a) DNA polymerase ct was found to be stimulated, DNA polymerase e was inhibited by lactate dehydrogenase in a concentration-dependent fashion. In contrast, when lactate dehydrogenase was preincubated with phosphoglycerate kinase and ATP, prior to being applied-/n vitro, DNA polymerase ct was inhibited, DNA polymerases 6 a~d_ e were strongly stimulated. Control experiments using both enzymes and [y- P]ATP for preincubation revealed that phosphate groups became attached to lactate dehydrogenase. b) Addition of lactate dehydrogenase and phosphoglycerate kinase inhibited replicative DNA synthesis and UV-induced DNA repair synthesis, again in a dose- dependent manner. Most probably, this effect was mediated through DNA polymerase e which is believed to catalyze i) replicative DNA synthesis on the lagging strand and ii) DNA repair synthesis following UV damage. When lactate dehydrogenase was preincubated with phosphoglycerate kinase and ATP before being added to UV-irradiated permeabilized cells, stimulation of repair synthesis occurred. Phosphoglycerate kinase per se had no effect. Conclusion. Lactate dehydrogenase seems to have a dual function in DNA replication and post-UV DNA repair, depending on its state of modification: the unphosphorylated form slows down both replicative and reparative DNA synthesis through inhibition ofDNA polymerase e, whereas the phosphorylated form stimulates.

This work was supported by the Deutsche Krebshilfe, Dr. Mildred Scheel Stiftung, W 28/93/Th 2. DKFZ Heidelberg, Division 340, Im Neuenheimer Feld 280, D-69120 Heidelberg