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Transcription Abnormalities Potentiate Apoptosis of Normal Human Fibroblasts

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Abstract

Background

Apoptosis is a natural process by which damaged and potentially tumorigenic cells are removed. Induction of apoptosis is important in chemotherapy aimed at eliminating cancer cells. We address the mechanisms by which this process can be triggered in cells that are recalcitrant to cell death induced by DNA-damaging agents.

Materials and Methods

Normal human fibroblasts and lymphoblasts, and fibroblasts with defined genetic changes, were treated with DNA-damaging agents and inhibitors of transcription. Western blotting was used to study the expression of some of the key factors involved in the response to DNA damage and the induction of apoptosis, namely, p53, p21WAF1,Cip1, Mdm2, Bax, and CD95 (Fas/APO1). Apoptosis was followed by various criteria, including DNA fragmentation, specific proteolysis, cell morphology, viability, and FACS scan for sub-G1 cells.

Results

Normal human fibroblasts were more resistant than lymphoblasts to DNA damage-induced apoptosis. The DNA-damaging agents mitomycin C and cisplatin induced rapid apoptosis of fibroblasts with defects in the repair of transcribed DNA, compared with wild-type cells or those with defects in overall genome repair. Short-term treatment with inhibitors of RNA polymerase II transcription, actinomycin D, and α-amanitin induced rapid cell death of normal fibroblasts. These results show that there is a link between defective transcription and apoptosis. Treatments and genetic backgrounds that favored apoptosis were associated with efficient and prolonged induction of p53 and often altered or unbalanced expression of its downstream effectors p21WAF1,Cip1 and Mdm2, whereas there were no changes in Bax or CD95 (Fas/APO1).

Conclusion

Transcription inhibitors increase p53 levels and are better inducers of apoptosis than DNA-damaging agents in some cell types. Apoptosis might be triggered by blocked polymerases and/or faulty expression of downstream effectors.

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Acknowledgments

We are grateful to Jan H. Hoeijmakers, Nicholas J. Jaspers, David P. Lane, and Gilbert De Murcia for generously providing us with cells and antibodies and to Sagar Sengupta for kind help. We thank Thierry Léveillard and Sagar Sengupta for critical reading of the manuscript, members of the Gene Medecine Department of Rhone-Poulenc Rohrer (especially Bruno Tocque, Laurent Bracco, Laurent Debussche, and Emmanuel Conseiller) for continual help and encouragement, the staff of the IGBMC facilities for their invaluable help, and various funding agencies, including: the Centre National de la Recherche Scientifique, the Institut National de la Santé et de la Recherche Médicale, the Centre Hospitalier Universitaire Régional, the Association pour la Recherche sur le Cancer, the Fondation pour la Recherche Médicale, the Ligue Nationale Française contre le Cancer, the Ligue Régionale (Haut-Rhin) contre le Cancer, the Ligue Régionale (Bas-Rhin) contre le Cancer (the Legs Meyer), and the Bioavenir Program (Ministère de la Recherche et Ministère de l’Industrie).

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  1. Ladislav Andera

    Present address: Laboratory of Leukocyte Antigens, Institute of Molecular Genetics, Videnska 1083, 14220, Praha 4, Czech Republic

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  1. Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS, INSERM, ULP, Illkirch, France

    Ladislav Andera &  Bohdan Wasylyk

  2. IGBMC, 1 rue Laurent Fries, B.P. 163, 67404, Illkirch, France

    Bohdan Wasylyk

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Andera, L., Wasylyk, B. Transcription Abnormalities Potentiate Apoptosis of Normal Human Fibroblasts. Mol Med 3, 852–863 (1997). https://doi.org/10.1007/BF03401721

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