The novel tricyclic adenosine analog triciribine (TCN), as the prodrug triciribine phosphate (TCN-5'-phosphate), is currently in clinical trials as an antineoplastic agent, but the biochemical mechanism of TCN cytotoxicity has not been fully elucidated. Preliminary results from the principal investigator's laboratory show that treating L1210 cells with TCN causes accumulation in the G1 phase of the cell cycle, and a slowing of progression through S phase. Therefore, it is proposed to investigate whether cell killing by TCN is cell cycle phase specific. The cell cycle effects reflect a dual effect of TCN on DNA synthesis: inhibition of chain initiation and of elongation. To elucidate the molecular mechanisms, the effects of TCN on formation and ligation of Okazaki fragments and of the 10 kb DNA replication intermediates will be investigated using gel electrophoresis. Positive identification of the nucleotide metabolites of TCN separated in the principal investigator's laboratory, is being undertaken by a collaborator who is an expert in mass spectroscopy. In the principal investigator's laboratory, incorporation of 3H from 3H-TCN into DNA and RNA in L1210 cells will be further investigated to determine whether it is still in the form of TCN, and whether it is incorporated in internucleotide linkage or as an alkylation adduct. This latter possibility would represent a novel mechanism of action. The metabolism of TCN in H.Ep.2 cells will be investigated to elucidate the basis for their high constitutive resistance to TCN. Two new analogs of adenosine, 3'-deoxysangivamycin and 4-amino-3- carboxamido-1-(beta-D-ribofuranosyl)pyrazolo[3,4-d]pyrimidine, will also be included in these studies. The cell cycle perturbations induced during growth inhibition by these analogs will be investigated using flow cytometry, and the cell cycle phase specificity of cell killing will be studied. The cellular metabolism of these analogs also will be investigated. The biochemical mechanisms of cytotoxicity will then be studied further, as suggested by these initial investigations.
