alanosine and purine

Methylthioadenosine phosphorylase (MTAP), an enzyme involved in purine and methionine metabolism, is present in all normal tissues but is frequently deficient in a variety of cancers. It has been suggested that this metabolic difference between normal and cancer cells may be exploited to selectively treat MTAP-negative cancers by inhibiting de novo purine synthesis and by depleting L-methionine. However, these therapeutic strategies have only been tested in naturally occurring MTAP-positive and -negative cell lines, which might have additional genetic alterations that affect chemotherapeutic sensitivity. Therefore, it is of importance to examine the feasibility of enzyme-selective treatment using paired cell lines that have an identical genotype except for MTAP status. MTAP-negative A549 lung cancer cells were transfected with eukaryotic expression vectors encoding MTAP cDNA in sense and antisense orientations. The resultant stable transfectomas were treated with inhibitors of de novo purine synthesis such as methotrexate, 5,10-dideazatetrahydrofolate, and L-alanosine and by methionine depletion. The A549 cells transfected with an antisense construct (antisense transfectoma) expressed no MTAP protein and were more sensitive to both purine and methionine depletion than were cells expressing MTAP protein (sense transfectoma). Methylthioadenosine was able to completely rescue the sense transfectoma but not the antisense transfectoma from growth inhibition by depletion of purine and methionine. These results prove that MTAP deficiency contributes directly to the sensitivity of cancer cells to purine or methionine depletion. Inhibition of de novo purine synthesis, combined with methionine depletion in the presence of methylthioadenosine, is a highly selective treatment for MTAP-negative cancers.

Topics: Alanine; Antimetabolites, Antineoplastic; Drug Resistance, Neoplasm; Feasibility Studies; Genetic Vectors; Humans; Lung Neoplasms; Methionine; Methotrexate; Neoplasm Proteins; Purine-Nucleoside Phosphorylase; Purines; Tetrahydrofolates; Transcription, Genetic; Transfection; Tumor Cells, Cultured

Methotrexate pretreatment of L1210 cells had been shown previously by us to cause an enhancement of the intracellular accumulation of 5-fluorouracil and of the formation of 5-fluorouracil nucleotides which was correlated with synergistic cytotoxicity. This effect of methotrexate was associated with increases in 5-phosphoribosyl-1-pyrophosphate, the cofactor required for the conversion of 5-fluorouracil to 5-fluorouridine-5'-monophosphate (FUMP). Because these influences on 5-fluorouracil metabolism were most likely mediated by the activity of methotrexate as an inhibitor of purine synthesis, the effects of other agents that inhibit purine synthesis were examined. An inhibitor of amidophosphoribosyltransferase, 6-methylmercaptopurine ribonucleoside, the glutamine antagonists, azaserine and 6-diazo-5-oxo-L-norleucine (DON), and the L-aspartate analogue inhibitor of adenylsuccinate synthetase, L-alanosine, all reduced the incorporation of [1-14C]glycine into adenine and guanine bases isolated from nucleic acids. Each drug also resulted in intracellular elevations of 5-phosphoribosyl-1-pyrophosphate that were 15- to 25-fold greater than control levels. These alterations in de novo purine nucleotide synthesis were associated with enhanced intracellular 5-fluorouracil accumulation and synergistic cytotoxicity.

Topics: Alanine; Animals; Azaserine; Cell Line, Tumor; Cell Survival; Diazooxonorleucine; Dose-Response Relationship, Drug; Fluorouracil; Methotrexate; Nucleic Acid Synthesis Inhibitors; Purines; Thioinosine; Thionucleotides; Uracil Nucleotides