Original Paper: Bovenzi, V. and R. L. Momparler, 2001. Antineoplastic action of 5-aza-2′deoxycytidine and histone deacetylase inhibitor and their effect on the expression of the retinoic acid receptor β and estrogen receptor α genes in breast carcinoma cells. Cancer Chemotherapy and Pharmacology 48: 71–76.

Cancer cells do not stop dividing. In some types of cancer, this happens because oncogenes that signal the go-ahead at cell cycle checkpoints are continually expressed, while tumor suppressor genes that inhibit progression through checkpoints are silenced. So when a group of researchers observed a pattern of gene silencing in breast cancer cells, they wondered if they could intervene. If the genes had been silenced as the result of DNA methylation or histone deacetylation, perhaps the researchers could induce expression of the genes by reversing DNA methylation and/or histone deacetylation.

Recall that DNA methyltransferase adds methyl groups onto cytosines in DNA, while a demethylase catalyzes the reverse reaction. Methylation of promoter regions in DNA inhibits gene expression; demethylation activates gene expression.

Recall, too, that chromatin can undergo remodeling as histones undergo reversible acetylation and deacetylation of lysine residues. Acetylation of histones activates gene expression; deacetylation inhibits gene expression.

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With this in mind, the researchers decided to treat the breast cancer cells with two different inhibitors: AZA, a compound that inhibits DNA methyltransferase; and TSA, a compound that inhibits histone deacetylase. The researchers looked at the effects of both inhibitors, first separately and then together (see graph at right), on the growth of the cancer cells.

In another experiment, the researchers measured the expression of two genes that have been shown to be silenced in these cancer cells. At the beginning of the experiment, cancer cells were either incubated with each inhibitor separately or with the two inhibitors combined. The researchers then isolated RNA from the cells and used the polymerase chain reaction (PCR) to amplify mRNA that had been expressed from the genes of interest. They compared the quantities of the amplified products, which were proportional to the quantities of the original mRNA transcripts that had been expressed in the cells during their treatment. The results are shown in the table below.

Questions

Question 1

An inhibitor of DNA methylation restores tumor suppressor activity in the cancer cells. The same effect is observed with an inhibitor of histone deacetylation. These results suggest that both DNA methylation and histone deacetylation operate to shut down expression of genes that suppress tumor growth.

Question 2

The data show that inhibitors of both histone deacetylation and DNA methylation act together with greater effect than either inhibitor alone. This suggests that some gene expression is inhibited by DNA methylation, some by histone deacetylation, and some by both processes. In this last group of genes, both inhibitors must be required to release the genes from being silenced.)

Question 3

Gene 1 can be reactivated by either inhibitor, but Gene 2 can be reactivated only by a combination of the two inhibitors. For Gene 1, the extent of DNA methylation and histone deacetylation may not be great. In this case, either DNA demethylation or histone acetylation may tip the balance just enough to alter the nucleosome to allow access of RNA polymerase to bind to the promoter and express the gene. In the case of Gene 2, RNA polymerase may be prevented from binding to the promoter for two reasons: the promoter site could be heavily methylated and histones could also be deacetylated, thus preventing loosening of the nucleosome to expose the promoter region. Therefore, for Gene 2 both DNA methylation and histone deacetylation must be inhibited for expression to be restored.