Transcription of the MAGE-1 gene and the methylation status of its Ets binding promoter elements: a quantitative analysis in melanoma cell lines using a real-time polymerase chain reaction technique

The human MAGE gene family comprises at least 12 highly homologous genes. T his makes it very difficult to assess expression of a single member quantit atively by means of Northern blotting. In order to investigate expression o f the MAGE-1 gene quantitatively we therefore used the recently developed r eal-time polymerase chain reaction (PCR), a novel fluorescence-based quanti tative PCR technique. This powerful technique enables detection of expressi on levels which differ by as much as a factor of 10(5) in magnitude. MAGE-1 expression is known to correlate with demethylated status of the Ets bindi ng sites of its promoter. In a panel of 19 melanoma and nine non-melanoma c ell lines we were able to confirm the relationship between MAGE-1 expressio n and demethylation of the Ets binding promoter region. Five cell lines, ho wever, showed only very slight expression, while the two essential Ets prom oter elements were largely demethylated. Earlier studies have shown that tr eatment of MAGE-1-negative cell lines with the demethylating agent 5-aza-2' -deoxycytidine (DAC) is sufficient to induce MAGE-1 expression. We were abl e to induce clear MAGE-1 expression in two of the non-expressing cell lines by incubation with DAC, although this expression did not reach very high l evels. Consistent with this low level of induction is the observation that the Ets binding sites of the MAGE-1 promoter were not completely demethylat ed in the DAC-treated cell populations. In conclusion, we show in this stud y that the real-time PCR technique is a very useful tool for the quantifica tion of expression of highly homologous genes. (C) 1999 Lippincott Williams & Wilkins.