The potential diadenosine polyphosphate analogue precursor, bis(adenosin-W-yl)dodecane (A[CH,],2A) (Chen, H. & McLennan, A. G. (1993) Eul: J. Biochem. 213, 935-944.) is equally toxic to both wild-type and adenosine-kinase-deficient BHK cells at concentrations up to 100 pM ; at higher concentrations, wild-type cells are more sensitive, as are cells over-expressing adenosine kinase. Thus both the nucleoside and its nucleotide products are toxic. In contrast to adenosine toxicity, the toxicity of A[CH,],,A to S-49 T-lymphoma cells could not be reversed by uridine or by L-homocysteine thiolactone. A [CH,] ,,A and all its shorter chain bis(adenosin-W-yl)alkane homologues could relieve the toxicity of low adenosine concentrations (< 20 pM) to $49 cells, mainly through inhibition of adenosine kinase, while relief of the toxicity of high adenosine concentrations (>20 pM) required the longer chain homologues. A [CH,],,A at 10 pM completely eliminated adenosine toxicity. Deoxyadenosine toxicity could also be relieved, but only that due to low concentrations (< 4 pM). A[CH,] ,,A had only a slight stimulatory effect on S-adenosylhomocysteinehydrolase activity.
The bis(adenosin-W-y1)alkancs (A[CH2].A) comprise a series of compounds in which two adenosine residues are linked through their W-amino groups by alkyl chains containing up to 14 methylene units [l, 21. They were synthesized with the intention that they would behave as cell-permeable precursors to analogues of nucleoside(5')polyphospho(5')nucleosides (NpnN), such as adenosine(5')tetraphospho(5')adenosine (ApJA), and therefore be useful as tools for determining the functions of these unusual nucleotides [3].We have previously described some of the toxicological, receptor binding and metabolic properties of these compounds [4]. They are moderately toxic towards a variety of mammalian cell lines; they bind preferentially to A, Ado receptors, although they are also weak agonists at A, receptors, causing an increase in intracellular CAMP ; they are phosphorylated by Ado kinase and adenylate kinase in vitro and in vivo to yield a number of products which have the potential to act as Ap,A analogues.In this study, we show that the biochemical basis for their cytotoxicity is quite distinct from that of Ado and that they are able to relieve completely Ado toxicity and partially relieve dAdo toxicity towards lymphoid cells, a finding that may be relevant to the treatment of hereditary Ado-deaminase deficiency. Some of these results have previously been reported in brief [5].
MATERIALS AND METHODS
MaterialsCell lines and reagents were as previously described [4]. In addition, the BHK ara-Sl0d Ado-kinase mutant [6] was kindly provided by V.-L. Chan, University of Toronto and Bmix AK' Rous-sarcoma-virus-transformed rat embryo fibrohlnsts (A&J clrarninase deficicnt) and B=mix A K ~ cclL (additionally Ado-kinase deficient) 171 were the generous gift of J. Drach, University of Michigan. L-Homocysteine thiolactone (Hcy thiolactone) was from Sigma. Neplanocin A was kindly provided b...
