Direct role of nucleotide metabolism in C-MYC-dependent proliferation of melanoma cells.

To identify C-MYC targets rate-limiting for proliferation of malignant melanoma, we stably inhibited C-MYC in several human metastatic melanoma lines via lentivirus-based shRNAs approximately to the levels detected in normal melanocytes. C-MYC depletion did not significantly affect levels of E2F1 protein reported to regulate expression of many S-phase specific genes, but resulted in the repression of several genes encoding enzymes rate-limiting for dNTP metabolism. These included thymidylate synthase (TS), inosine monophosphate dehydrogenase 2 (IMPDH2) and phosphoribosyl pyrophosphate synthetase 2 (PRPS2). C-MYC depletion also resulted in reduction in the amounts of deoxyribonucleoside triphosphates (dNTPs) and inhibition of proliferation. shRNA-mediated suppression of TS, IMPDH2 or PRPS2 resulted in the decrease of dNTP pools and retardation of the cell cycle progression of melanoma cells in a manner similar to that of C-MYC-depletion in those cells. Reciprocally, concurrent overexpression of cDNAs for TS, IMPDH2 and PRPS2 delayed proliferative arrest caused by inhibition of C-MYC in melanoma cells. Overexpression of C-MYC in normal melanocytes enhanced expression of the above enzymes and increased individual dNTP pools. Analysis of in vivo C-MYC interactions with TS, IMPDH2 and PRPS2 genes confirmed that they are direct C-MYC targets. Moreover, all three proteins express at higher levels in cells from several metastatic melanoma lines compared to normal melanocytes. Our data establish a novel functional link between C-MYC and dNTP metabolism and identify its role in proliferation of tumor cells.

Effect of Interferon Inducer Neovir on the Sensitivity MDR1(-) and MDR1(+) Cells to Antitumor Drugs.

The ability of synthetic interferon inducer, neovir (sodium 10-methylencarboxylate-9-acridone), was studied dealing with modulation of the sensitivity of HT-29 and K-562 cells, intact and transfected with mdr1 gene, in relation to the cytototoxic action of antitumor drugs. Neovir exerted the direct cytotoxic action on cells of all studied lines. Preliminary incubation of cells with neovir for 24 h efficiently increased the cytotoxic effect of doxorubicin and vincristine. The degree of toxicity enhancement was depended on the drug concentrations, as well as on cell lines. Neovir at the concentration 10 mg/ml had the most pronounced effect. The enhancement of toxic action of doxorubin for HT-29 cells had, as a rule, additive character, while for HT-29 MDR1 cells the interaction was synergistic (CD(50) was decreased by 2.85- and 8.67-fold respectively). The effect of vincristine toxicity enhancement didn't depend on mdr1 gene expression and had synergistic character. Neovir enhanced the cytotoxic effect of neovir in relation to K-562 and K-562 MDR1 cells by 3.18-fold and more than by 100-fold respectively. Also neovir increased the cytotoxic effect of 5-fluorouracil whose accumulation in cell doesn't depend on Pgp expression. Preliminary incubation of HT-29 cells with neovir has resulted in 2000-fold decrease of 5-fluorouracil CD(50) and in 36.6-fold for HT-29 MDR1 cells. Thus, the effect of neovir seems to have no relation to the action on the mechanisms of multiple drug resistance and may be mediated through some other pathways.