azaserine and 5--methylthioadenosine

5'-Deoxy-5'-methylthioadenosine phosphorylase (MTAP) is involved in the salvage of adenine and methylthio moieties of 5'-deoxy-5'-methylthioadenosine, a byproduct of polyamine synthesis, to adenine nucleotides and methionine, respectively. The gene encoding MTAP, MTAP, is frequently codeleted along with the tumor suppressor gene p16 in malignant cells bearing homozygous deletions in the chromosome 9p21 region. p16-, MTAP- malignant cells have been shown to be more susceptible to the purine de novo inhibitory actions of antifolates such as methotrexate than are p16+, MTAP+ cells. To understand the underlying mechanism, we reintroduced MTAP activity into two p16-, MTAP- cell model systems, the MiaPaCa-2 and PANC-1 human pancreatic carcinoma cell lines, by transfection with MTAP cDNA. It was found that transfection with MTAP cDNA (i) restored both the MTAP-dependent adenine and methionine salvage pathways, (ii) decreased the rates of purine de novo synthesis (18-47% lower than the wild-type or sham-transfected counterparts), and (iii) decreased cellular sensitivity to the antipurine-related growth-inhibitory actions of methotrexate and azaserine. These data support the hypothesis that operation of the MTAP-dependent adenine salvage pathway renders MTAP+ cells less dependent on de novo purine synthesis and hence less susceptible than MTAP- malignant cells to the growth-inhibitory actions of agents (e.g. antifolates) whose mechanism of action in part involves the de novo purine pathway. These findings provide a theoretical basis for the relatively selective action certain antifolates may have against MTAP-deficient malignancies.

Topics: Adenosine; Antimalarials; Antimetabolites, Antineoplastic; Azaserine; Biomarkers, Tumor; Cell Count; Deoxyadenosines; DNA, Complementary; Dose-Response Relationship, Drug; Humans; Methionine; Methotrexate; Pancreatic Neoplasms; Purine-Nucleoside Phosphorylase; Purines; RNA, Messenger; Thionucleosides; Tumor Cells, Cultured

5'-Deoxy-5'-methylthioadenosine, a by-product of polyamine metabolism, is a potent inhibitor of cell proliferation. MTA phosphorylase cleaves MTA into adenine and 5'-methylthioribose-1-P. We studied MTA inhibition and salvage into purine compounds and methionine in concanavalin A-stimulated rat T lymphocytes and in Raji cells. When de novo purine synthesis was inhibited by azaserine (20 microM), low concentrations of MTA, (less than or equal to 20 microM), were able to completely restore cell proliferation in both types of cells. When cells were cultured in a methionine-free medium, MTA (15 microM) completely fulfilled the methionine requirement of Raji cells but only 50% of that of rat T lymphocytes. MTA displayed a dose-dependent inhibition of the proliferation of both types of cells, but in the case of MTA salvage into purines or methionine, the curves were shifted to higher MTA concentrations. In vitro studies by Backlund et al. (Backlund, P.S., Chang, C.P. and Smith, R.A. (1982) J. Biol. Chem. 257, 4196-4202) on rat liver homogenates, suggested that the last step of MTA salvage into methionine may be the transamination of 2-keto-4-methylthiobutyrate to methionine. We present evidence that this is a step physiologically efficient in intact cells.

Topics: Adenosine; Azaserine; Cell Cycle; Cell Line; Concanavalin A; Deoxyadenosines; Humans; Lymphocytes; Methionine; Purine-Nucleoside Phosphorylase; Purines; Thionucleosides