adenosine-kinase and 8-chloroadenosine

Conflicting reports have attributed 8-chloro-cAMP (Cl-cAMP)-mediated inhibition of tumor cell growth to either a toxic 8-chloro-adenosine (Cl-AdR) breakdown product or a Cl-cAMP-mediated decrease in ratio of Type I to Type II regulatory (R) subunits of protein kinase A (PKA). Using the MCF-7 human breast cancer and S49 mouse lymphoma cell lines as models, we show that the effects of Cl-cAMP and other cAMP analogs on growth and R subunit expression are unrelated. MCF-7 cell growth was insensitive to most analogs and inducers of cAMP, but was potently inhibited by Cl-cAMP acting through uptake and phosphorylation of its Cl-AdR breakdown product. Possible roles of adenosine receptors or P(2) purinoceptors in these Cl-cAMP-mediated growth effects were ruled out by studies with agonists and antagonists. Cholera toxin markedly decreased the ratio of Type I to Type II R subunits in MCF-7 cells without affecting growth, while growth inhibitory concentrations of Cl-cAMP or Cl-AdR had insignificant effects on this ratio. In S49 cells, where PKA activation is known to inhibit cell growth, PKA-deficient mutants retained sensitivity to both Cl-cAMP and the related 8-bromo-cAMP. Adenosine kinase (AK)-deficient S49 cells were inhibited only by higher concentrations of these 8-halogenated cAMP analogs. Of the commonly used cAMP analogs, only 8-(4-chlorophenylthio)-cAMP acted purely as a cyclic nucleotide-having no effect on PKA-deficient cells, but strongly inhibiting both wild-type and AK-deficient cells. Where growth inhibitory concentrations of most cAMP analogs reduced RI expression in the AK-deficient mutant, a functionally equivalent concentration of (N(6), O(2'))dibutyryl-cAMP maintained or increased this expression.

Topics: 2-Chloroadenosine; 8-Bromo Cyclic Adenosine Monophosphate; Adenosine Kinase; Animals; Antineoplastic Agents; Cell Division; Cyclic AMP-Dependent Protein Kinase Type II; Cyclic AMP-Dependent Protein Kinases; Gene Expression; Humans; Mice; Mutation; Protein Subunits; Tumor Cells, Cultured

The nucleoside 8-chloro-adenosine (8-Cl-Ado) is currently being developed for treatment of multiple myeloma and leukemias. Although accumulation of the phosphorylated drug product is known to occur within cell lines, its metabolic fate in plasma or circulating cells in animals is unclear. The purpose of the present study was to determine the pharmacology of 8-Cl-Ado in rodents through examination of plasma and cellular levels of parent drug and metabolites. In addition, we sought to determine whether an inhibitor of adenosine deaminase, 2'-deoxycoformycin (dCF), could enhance intracellular formation of 8-Cl-ATP by preventing degradation of 8-Cl-Ado to 8-Cl-inosine (8-Cl-Ino).. A validated HPLC assay permitted simultaneous determination of 8-Cl-Ado, 8-Cl-adenine (8-Cl-Ade), dCF, and 8-Cl-Ino. Radiolabeled cellular nucleotides were obtained from peripheral blood mononuclear cells (PBMC) of both mice and rats using a perchloric acid extraction procedure and were separated by HPLC.. Stability of 8-Cl-Ado in the presence or absence of dCF was examined in fresh plasma from mice, rats and humans. Conversion of 8-Cl-Ado to 8-Cl-Ino was only marginally affected by coincubation with dCF. In CD(2)F(1) mice given 8-Cl-Ado i.p. at 100 mg/kg, there was rapid appearance in plasma of both 8-Cl-Ade and 8-Cl-Ino. The identities of the metabolites were confirmed by mass spectrometry. The plasma [(3)H]8-Cl-Ado concentration 1 h after drug injection was 1.3 micro M in mice while the intracellular levels of [(3)H]8-Cl-AMP and [(3)H]8-Cl-ATP were 1 m M and 350 micro M, respectively. Mice that had received dCF (2 mg/ml) 30 min prior to [(3)H]8-Cl-Ado had 27% less intracellular [(3)H]8-Cl-ATP in PBMC compared to mice without dCF pretreatment. The pharmacokinetics of 8-Cl-Ado were examined in greater detail in Sprague-Dawley rats. Animals were given [(3)H]8-Cl-Ado (42.5 mg/kg, i.v.) by itself or 30 min following injection of dCF (4 mg/kg). Mononuclear cells in mice accumulated 350 or 1200 micro M [(3)H]8-Cl-ATP 1 h after injection of either 50 or 100 mg [(3)H]8-Cl-Ado, respectively. The major metabolite in these cells was the monophosphate, which was four- to sevenfold higher in concentration than the triphosphate metabolite. In rats, [(3)H]8-Cl-AMP concentrations in PBMC were similar to those of the triphosphate metabolite which achieved a peak of 90 micro M 2 h after a bolus injection of 8-Cl-Ado (40 mg/kg). Cellular clearance of 8-Cl-ATP appeared to be slow: 24 h after injection of 8-Cl-Ado the cellular concentration of 8-Cl-ATP was still 40 micro M.. The use of dCF did not significantly alter 8-Cl-ATP levels in PBMC and is not considered to be a useful therapeutic strategy. Even though a portion of 8-Cl-Ado is metabolically inactivated in plasma, high levels of cytotoxic 8-Cl-ATP accumulated intracellularly in these animals and were retained for a considerable length of time. Further development of 8-Cl-Ado is recommended.

Topics: 2-Chloroadenosine; Adenosine Deaminase; Adenosine Kinase; Adenosine Triphosphate; Animals; Antimetabolites, Antineoplastic; Biotransformation; Chromatography, High Pressure Liquid; Female; Inosine; Leukocytes, Mononuclear; Male; Mice; Pentostatin; Phosphorylation; Prodrugs; Rats; Rats, Sprague-Dawley; Species Specificity

Previous work with 8-chloro-cAMP (8-Cl-cAMP) has raised questions as to whether it works as a cAMP analogue or as a nucleoside analogue after its conversion to 8-chloro-adenosine (8-Cl-Ado). Although degradation of 8-Cl-cAMP to 8-Cl-Ado in culture medium or plasma has been shown, cellular pharmacology data are missing. The purpose of the present study was to identify the cellular metabolism of these drugs and their actions in a human multiple myeloma cell line. The cells were incubated with either 8-Cl-Ado or 8-Cl-cAMP to follow the cellular metabolism of these agents. Both 8-Cl-cAMP and 8-Cl-Ado incubation resulted in the accumulation of 8-Cl-Ado mono-, di-, and tri-phosphate (8-Cl-ATP), however, the triphosphate was the major cytotoxic metabolite. Accumulation of 8-Cl-ATP was dependent on both the exogenous concentration of 8-Cl-Ado and incubation time. At the 10 microM level of 8-Cl-Ado, >400 microM 8-Cl-ATP accumulated in multiple myeloma cells after continuous incubation for 12 h. Similar incubation with 8-Cl-cAMP also resulted in accumulation of 8-Cl-ATP in the cells, albeit at a lower level. The formation of 8-Cl-ATP from 8-Cl-cAMP was inhibited by >80% in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine in the medium, suggesting extracellular conversion of 8-Cl-cAMP to 8-Cl-Ado. Cells lacking Ado kinase did not accumulate 8-Cl-ATP, either from 8-Cl-Ado or 8-Cl-cAMP, and were resistant to these agents. There was also a decline in the endogenous level of the cellular ATP pool parallel to the accumulation of 8-C1-ATP. The elimination of 8-Cl-ATP was biphasic and slow from the cells. The accumulation of 8-Cl-ATP and a decline in the ATP pool inhibited RNA synthesis but did not affect DNA synthesis for up to 12 h of incubation. Taken together, these data demonstrate that the cytotoxic metabolite of 8-Cl-Ado and 8-Cl-cAMP is 8-Cl-ATP. Hence, 8-Cl-cAMP serves as a prodrug and is converted to 8-Cl-Ado in medium with subsequent phosphorylation to accumulate as 8-Cl-ATP in cells. At the cellular level, 8-Cl-ATP is associated with a decrease in the endogenous ATP pool; at the nuclear level, it inhibits RNA synthesis.

Topics: 2-Chloroadenosine; 8-Bromo Cyclic Adenosine Monophosphate; Adenosine Kinase; Adenosine Triphosphate; Antineoplastic Agents; Cell Cycle; Cell Division; Dose-Response Relationship, Drug; Humans; Multiple Myeloma; Time Factors; Tumor Cells, Cultured