MODIFICATION OF GROWTH-RELATED ENZYMATIC PATHWAYS AND APPARENT LOSS OF TUMORIGENICITY OF A RAS-TRANSFORMED BOVINE ENDOTHELIAL-CELL LINE BY TREATMENT WITH 5-IODO-6-AMINO-1,2-BENZOPYRONE (INH2BP)

Bovine aortic endothelial cells were converted to a highly tumorigenic cell line by transfection with Ha-ras and stimulation with thrombin. Sustained pretreatment with a non-cytotoxic concentration (600 mu M) o f 5-iodo-6-amino-1,2-benzopyrone (INH2BP), a lipophilic ligand of poly (ADP-ribose) polymerase, abrogated in vivo tumorigenicity, of 10(5) ce lls per inoculum an effect which developed progressively during 2 to 6 weeks of drug treatment. The initial action of the drug was cytostasi s, consisting of an arrest in prophase, extreme cell enlargement consi stent with cytoplasmic hypertrophy, as seen by EM, and dramatic morpho logic changes. Although neither DNA, RNA or protein syntheses are dire ctly affected by INH2BP, apparently newly synthesized cellular DNA is degraded by endonucleases, which are upregulated by the inhibition of their poly-ADP-ribosylation. The drug treated cells exhibited greatly increased respiration and aerobic glycolysis, due to an augmentation o f,glycolytic and respiratory enzymes in enlarged cells. These response s to the drug were reversible in cell cultures following drug removal, within 5-10 days drug exposure but the progressive loss of tumorigeni city in nude mice that developed after 3-6 weeks of drug exposure of c ells, prior to inoculation to nude mice, was not reversible in vivo. D rug treatment produced a sustained 70-80% inhibition of pADPRT in inta ct cells at 600 mu M extracellular concentration of INH2BP. The prereq uisite for the abrogation of tumorigenicity was the maintenance of pAD PRT inhibition. The arrest of cell multiplication and a large decrease of Topo I, especially of Topo II and MAP kinase activities occurred w ithout loss of enzyme protein as assayed in cell extracts of drug-trea ted cells. However INH2BP had no direct effect on these enzymes. Drug treatment down-regulated DNA-methyltransferase, PKC, ODC proteins, dim inished cyclin A protein, but the hypophosphorylated form of Rb protei n was significantly augmented. None of the enzymatic components of sig nal pathways so far studied, were directly affected by INH2BP. The inh ibition of pADPRT by INH2BP coincided with an induction or activation of alkaline phosphatase and leucyl and glutamyl peptidase. The pADPRT content or the expression of pADPRT gene were not influenced by drug t reatment, but the expression of ras gene was completely absent in nont umorigenic drug-treated cells, without a loss of ras gene from genomic DNA. Telomerase activity was not directly influenced by INH2BP treatm ent when assayed in diluted cell extracts, but the addition of homogen eous pADPRT to cell extracts, to approach physiological concentration of this protein in the cell, inhibited telomerase activity by binding of the polymer-free pADPRT to telomer templates. We conclude that inhi bition of pADPRT indirectly down-regulates growth stimulatory signal p athways and sustains growth-arrested cells in culture at a pre-apoptot ic threshold which explains the absence of tumorigenicity in vivo.