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Depletion of Nuclear Poly(ADP-Ribose) Polymerase by Antisense RNA Expression: Influences on Genomic Stability, Chromatin Organization, and Carcinogen Cytotoxicity¹

Department of Biochemistry and Molecular Biology, Georgetown University School of Medicine, Washington, DC 20007

Poly(ADP-ribose) polymerase (PADPRP) catalyzes the transfer of multiple ADP-ribose units from NAD to nuclear histone and nonhistone proteins, a reaction that appears to be important in the rejoining of DNA strand breaks during DNA repair and replication. We previously established and characterized a HeLa cell line that was stably transfected with a recombinant expression plasmid containing the mouse mammary tumor virus promoter upstream of a construct encoding PADPRP antisense RNA. We now show that after depletion of PADPRP mRNA as a result of antisense RNA expression, normal PADPRP mRNA concentrations are restored between 8 and 16 h after removal of dexamethasone (which activated the mouse mammary tumor virus promoter). By depleting antisense cells of PADPRP, we demonstrated the contribution of this enzyme to various aspects of nuclear structure and function: (a) amplification of a selectable gene encoding three early enzymes in the pyrimidine biosynthetic pathway was greatly increased in cells depleted of PADPRP; (b) chromatin structure was significantly altered in PADPRP-depleted cells, as indicated by reduced initiation and elongation of poly(ADP-ribose) chains attached to various nuclear protein acceptors, lower levels of poly(ADP-ribosyl)ation of histone H1, and an increased susceptibility of DNA to micrococcal nuclease digestion; and (c) the survival of PADPRP-depleted antisense cells exposed to the DNA alkylating and carcinogenic agent methyl methanesulfonate or nitrogen mustard was significantly reduced relative to that of control cells.

¹ This work was supported in part by Grant CA25344 from the National Cancer Institute and by funding from the United States Air Force Office of Scientific Research through Grant AFOSR-89-0053 and the United States Army Medical Research and Development Command through Contract DAMD17-90-C-0053.

² To whom requests for reprints should be addressed, at Department of Biochemistry and Molecular Biology, Georgetown University, Basic Science Building, Room 349, 3900 Reservoir Road, N.W., Washington, DC 20007.

Received 11/18/93. Accepted 6/27/94.

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