Figure 6

Expression of DNMT1 and DNMT3a in MCF-7 cells. The expression of DNMT1 and DNMT3a mRNA is decreased in MCF-7 cells treated with hydralazine. The lower band intensity was confirmed by densitometric (right) analysis, (control is untreated cells). MCF-7 cells were growth arrested for 48 hours by serum deprivation, then treated for 24 hours with hydralazine at 10 μM and then RNA extracted for analysis. Reverse transcription was carried out as previously described [110] using random hexamers, superscript II reverse transcriptase (Life Technologies) and 2.5 μg of total RNA as recommended by the manufacturer in a total volume of 50 μL. One microliter of RT reaction was used for subsequent PCR amplification for each of the desired transcripts with dNTPs and Amplitaq (Applied Biosystems). Primer sequences used were as follows: DNMT1 sense 5-gat cga att cat gcc ggc gcg tac cgc ccc ag-3 and antisense 5-atg gtg gtt tgc ctg gtg c-3. DNMT3a sense 5-ggg gac gtc cgc agc gta cac-3 and antisense 5-cag ggt tgg act cga gaa atc g-3. Amplification conditions were: 94 C° for 5 min 1 cycle; 94 C° 30 sec, (58°C for DNMT1 and 65°C for DNMT3a) annealing temperature for 1 min, and 72 C° for 30 sec, 35 cycles.