Klin Onkol 2005; 18(2): 64-68.
Abstract:
Background: Cellular events occurring downstream from the inhibition of DNA synthesis by methotrexate have remained largely unexplored. Here, we show that chemotherapy-naive lymphoblasts isolated from bone marrow of patients with acute lymphoblastic leukemia cultivated ex vivo with methotrexate resulted in changes in expression of the p53 protein and in upregulation of several p53-regulating genes.
Methods: We investigated induction of p53 protein and p53-dependent genes after 24 hour ex vivo incubation with methotrexate in chemotherapy-naive, bone marrow lymphoblasts taken prior to any therapy. To date, we completed immunocytochemical analysis of p53 and determined changes in expression of genes involved in p53-dependent biological pathways in four patients using cellular pathway-oriented GEArray membranes (www.superarray.com) enabling analysis of 112 transcripts.
Results: Ex vivo experiments were performed with folate-free medium to which 5-methyl tetrahydrofolate was added to the concentration 25 nmol/l to parallel serum conditions. GEArray experiments showed overall decrease of mRNA expression in ex vivo cultivated lymphoblasts after methotrexate treatment. In the fourth patient, also with immunocytochemically observable p53 induction, we observed decrease of expression of several cell cycle-regulating genes such as CDC2 (cdk1), SP1, NDRG (n-myc downstream regulated gene) whereas apoptosis-related genes did not change their expression levels. The APEX (Ref-1) and ARF (p16ink4), the p53-upstream signaling genes encoding proteins involved in p53 interactions showed variable expression. Genes encoding transcription regulators ATM (2 patients with B-ALL) and SIRT1 (4 patients) participating in p53 interactions showed significant decrease of their expression. Interindividual differences were also observed in genes involved in p53 pathways such as CSNK1A1, CSNK2, HIPK2 and JNKK2 gene.
Conclusions: We have observed variable induction of p53 and expression of p53controlled genes. This result may imply that a disorder – though classified as Bor Ttype malignancy – may not respond identically to otherwise identical treatment under identical conditions. We suggest that assessment of changes in expression of p53-controlled genes and p53 protein status may be a tool to determine an intraindividual rate of „functional“ response to high-dose methotrexate therapy. These preliminary results may broaden our understanding on the mechanism of methotrexate action at the cellular level going beyond the traditional DHFR inhibition effect. We succeeded in identifying about 11 genes where changes in expression were observed in all patients. Those genes and their products will be subject of additional investigations to find out connections between gene expression, their products, type of disease and mechanism of therapy response.
