An accurate assay of diadenosine 5',5'''- P1,P4-tetraphosphate [A(5') pppp(5')A], which was shown to be formed in vitro in the backreaction of the amino acid activation step, has been developed in various cell lines in culture and in normal mouse liver or hepatoma in vivo. Use of radioactive labeling of acid-soluble nucleotides to high specific activity followed by chromatographic separation techniques yielded levels of Ap4A varying from 5 to 0.05 muM (from 30 pmol/mg of protein to 0.15 pmol), depending on the doubling time of the cell line or the proliferative state of the cells. The levels of Ap4A incells is inversely related to their doubling time, varying from 0.1 X 10(-4) of the cellular ATP levels in slowly growing cells to 20 X 10(-4) of the ATP levels of cells with rapid doubling times. The steady-state levels of ATP of different cell lines, although showing some fluctuations, are not related to the doubling time of the cells. Arrest of cellular proliferation by serum deprivation or amino acid starvation, which does not alter the cellular ATP levels more than 2-fold, does nevertheless cause a decrease of 30 to 50-fold in the Ap4A levels. Inhibition of protein synthesis by pactamycin or puromycin, or inhibition of DNA synthesis by hydroxyurea, leads to a more dramatic decrease of 50 to 100-fold in intracellular Ap4A levels. The metabolic lability of Ap4A is also demonstrated by its rapid depletion after decreases in the ATP/ADP ratio. The possibility of Ap4A being a metabolic "signal nucleotide" that is formed at the onset of protein synthesis and is active in positive growth regulation (positive pleiotypic activation) is discussed.
Treatment of a variety of cultured human tumor cells with low levels (40-80 pM) of adenosine 5' -diphosphate (ADP) or adenosine 5' -triphosphate (ATP) has produced arrest of these cells in the S phase of their cycle followed by cellular death. ADP and ATP are demonstrated to be incorporated into the cellular acid-soluble nucleotide pools, presumably by permeation through the plasma membrane of these cells. Since AMP, adenosine, 3 ' , 5' cyclic AMP, or a variety of diadenosine polyphosphates do not produce similar cytostatic effects or similar alterations of the cellular acid-soluble nucleotide pools, we suggest that the effects of ADP and ATP are not due to their prior breakdown or modification. Cultured animal cells have been widely acknowledged not to incorporate acid-soluble adenine nucleotides although incorporation of exogenous intact nucleoside monophosphate into cellular pools has been demonstrated in a few cases. A 48-hour treatment of logarithmically growing h u m a n tumor cells with 40 pM of ADP or ATP produced substantial arrest of cell populations in the S phase of their cycle and a dramatic reduction in total cellular uridine 5' -triphosphate (UTP) pools. AMP, adenosine, 3',5' cyclic AMP, Ap3A, Ap4A or Ap5A produced small and dissimilar effects. The experiments reported here suggest that the plasma membranes of several lines of human tumor cells are permeable to low levels of intact ADP and ATP. The suggestion of en bloc incorporation of low ADP and ATP levels into human tumor cells is supported by the almost identical effects of ADP and ATP on total cellular acid-soluble nucleotide pools and cellular growth. The rapid conversion of intracellular ADP pools into ATP in tumor cells has been reported.
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