Fig. 1. Effect of DOPET on M14 cell viability evaluated using the trypan blue exclusion test. M14 cells were incubated overnight (18 h) in RPMI medium containing increasing amounts of DOPET. Cell viability was evaluated with the trypan blue exclusion test as described in Materials and methods. Results are expressed as percentage of viability related to control. Data are expressed as mean ± SE (n = 3).

Fig. 2. Effect of DOPET on M14 cells viability evaluated using the MTT assay. (A) M14 cells were incubated as described in the figure in the absence or in the presence of increasing amounts of DOPET. (B) M14 cells were incubated as in A. After 24 h, DOPET was removed, cells were suspended in fresh RPMI medium, and the incubation was prolonged up to 72 h. Cell viability was measured using the MTT assay describer in Materials and methods. Values are mean ± SE (n = 3).

Fig. 3. Metabolic fate of DOPET in M14 cells. HPLC profile of [¹⁴C] molecular species detectable in M14 cells incubated overnight (18 h) in presence of [¹⁴C]DOPET. Samples were treated as reported in Materials and methods. Labeled compounds were identified on the basis of their retention time. Time 0 (full circle); overnight incubation (open circle).

Fig. 4. Effect of DOPET on UVA-induced intracellular ROS production in M14 cells. At the end of an overnight incubation in the presence of increasing amounts of DOPET M14 cells were irradiated with UVA (7.5 J/cm²-dose). ROS production was detected with the fluorescent indicator 2,7-DCFH as indicated in Materials and methods. Data are expressed as means ± SE (n = 3). Asterisk indicates value significantly different from UVA-irrradiated sample; P < 0.05.

Fig. 5. Effect of DOPET on lipid peroxidation in M14 cells exposed to UVA radiation. Cells were treated as indicated in Fig. 4. TBARS were assayed as reported in Materials and methods. Data are expressed as means ± SE (n = 5). Asterisks indicate values significantly different from UVA-irrradiated sample; P < 0.05.

Fig. 6. Measurement of z-DEVD-AFC proteolysis catalyzed by M14 cell extract. M14 cells were treated as reported in Fig. 4. After irradiation, cell lysates were prepared and caspase-3 activity was measured as reported in Materials and methods. Cleavage of z-DEVD-AFC was evaluated fluorometrically (excitation at 375 nm, emission at 530 nm). Data are expressed as means ± SE (n = 3). Irradiated cells, ; nonirradiated cells, □.

Fig. 7. Effect of DOPET on protein methyl esterification in UVA-irradiated M14 cells. M14 cells were treated as reported in Fig. 4. (A) Analysis in intact cells: cells were incubated in presence of l-[methyl-³H]methionine, as described in Materials and methods. Protein methyl esterification was evaluated as the difference between the data of samples incubated with and without protein methylation inhibitors. Protein methylation is expressed as ³H-methyl groups incorporated/mg protein. The specific activity of intracellular Ado-[methyl-³H]Met was evaluated by HPLC analysis of cell extract. (B) In vitro analysis: after treatment, cell lysates were prepared and incubated with recombinant human PIMT and the methyl donor S-adenosyl-l-[methyl-¹⁴C]methionine. All results are expressed as the mean ± SE (n = 5);*P < 0.05.

Fig. 8. Overall mechanistic model of action of DOPET in UVA-treated cells. UVA treatment by eliciting oxidative stress is bound to increase protein damage consisting in the formation of l-isoAsp residues, which are recognized and repaired by PIMT. DOPET, in turn, is quantitatively converted into MOPET by COMT, which also consumes AdoMet and produces AdoHcy, just like PIMT. Therefore COMT activity might contribute to the alteration of the AdoMet/AdoHcy ratio (transmethylation potential) and of the balance between oxidative (damaging) events and antioxidant as well as protein repair systems.

PIMT specific activity and AdoMet/AdoHcy ratio in M14 cells

PIMT specific activity was measured in vitro by a radiochemical enzyme assay in the presence of saturating concentrations of both the methyl donor [methyl-¹⁴C]AdoMet and a methyl-accepting protein substrate. 1 U enzyme activity is defined as 1 pmol methyl group incorporated/min. AdoMet and AdoHcy concentrations were determined by HPLC analysis of acid-soluble extracts of M14 cells (see Materials and methods).