Read The Epigenetics Revolution Online
Authors: Nessa Carey
Tags: #Science/Life Sciences/Genetics and Genomics
The Epigenetics Revolution (31 page)
The type of cells that Peter Jones and his colleagues were testing are usually grown in a flat plastic flask. This looks a little like a see-through version of a hip flask for whisky or brandy, lying on its side. The mammalian cells grow on the flat inside surface of the flask. They form a single layer of cells, tightly packed side by side, but never growing on top of one another.
One morning, after the cells had been cultured with 5-azacytidine for several weeks, the researchers found that there was a strange lumpy bit in one of the culture flasks. To the naked eye, this initially looked like a mould infection. Most people would just discard the flask and make a silent promise to be a bit more careful when culturing their cells in future, to stop this happening again. But Peter Jones did something else. He looked at the lump more closely and discovered it wasn’t a stray bit of mould at all. It was a big mass of cells, which had fused to form giant cells containing lots of nuclei. These were little muscle fibres, the syncytial tissue we met in the discussion of X inactivation. Sometimes the little muscle fibres would even twitch
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This was very odd indeed. Although the cell line had originally been derived from a mouse embryo, it never usually formed anything like a muscle cell. It tended instead to form epithelial cells – the cell type that lines the surfaces of most of our organs. Peter Jones’ work showed that 5-azacytidine could change the potential of these embryonic cells, and force them to become muscle cells, instead of epithelial cells. But why would a compound that killed cancer cells, presumably by disrupting production of DNA and mRNA, have an effect like this?
Peter Jones carried on working on this when he moved from South Africa to the University of Southern California. Two years later, he and his PhD student Shirley Taylor showed that cell lines treated with 5-azacytidine didn’t only form muscle. They could also form other cell types. These included fat cells (adipocytes) and cells called chondrocytes. These produce cartilage proteins, such as those that line the surfaces of joints so that the two planes can glide smoothly over each other.
These data showed that 5-azacytidine wasn’t a special muscle-specifying factor. Very presciently, Professor Jones made the suggestion in his paper reporting this work that, ‘5-azacytidine … causes a reversion to a more pluripotent state’
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. In other words, this compound was pushing the ball a little way back up Waddington’s epigenetic landscape. The ball was then rolling back down the valleys between the hills, into a different final resting place.
3
. In other words, this compound was pushing the ball a little way back up Waddington’s epigenetic landscape. The ball was then rolling back down the valleys between the hills, into a different final resting place.
But there was still no theory as to why 5-azacytidine had this unusual effect. Peter Jones himself tells a lovely self-deprecating story about the turning point in our understanding. His original appointment at the University of Southern California was in the Department of Paediatrics, but he wanted a joint appointment with the Department of Biochemistry. Part of the procedure for obtaining this joint appointment included an extra interview, which he considered quite pointless. Peter Jones described his work with 5-azacytidine in this interview and explained that no-one knew why the compound affected cell pluripotency. Robert Stellwagen, another scientist at the same university who was taking part in the interview asked, ‘Have you thought of DNA methylation?’. Our candidate admitted he not only hadn’t thought of it, he hadn’t even heard of it
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Peter Jones and Shirley Taylor immediately began to focus on DNA methylation and in a very short time showed that this was indeed key to the effects of 5-azacytidine. 5-azacytidine inhibited DNA methylation. Peter Jones and Shirley Taylor created a number of related compounds and tested them for their effects in cell culture. The ones that inhibited DNA methylation also caused the changes in phenotype originally observed for 5-azacytidine. Compounds that didn’t inhibit DNA methylation had no effect on phenotype
5
.
5
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The methylation cul-de-sac
Cytidine (base C) and 5-azacytidine are very similar in chemical structure. They are shown in
Figure 11.1
, which for simplicity only shows the most relevant parts of the structure (called cytosine and 5-azacytosine, respectively).
Figure 11.1
, which for simplicity only shows the most relevant parts of the structure (called cytosine and 5-azacytosine, respectively).
The top half of the diagram is very similar to
Figure 4.1
, showing that cytosine can be methylated by a DNA methyltransferase (DNMT1, DNMT3A or DNMT3B) to create 5-methylcytosine. In 5-azacytosine, a nitrogen atom (N) replaces the key carbon atom (C) that normally gets methylated. The DNA methyltransferases can’t add a methyl group to this nitrogen atom.
Figure 4.1
, showing that cytosine can be methylated by a DNA methyltransferase (DNMT1, DNMT3A or DNMT3B) to create 5-methylcytosine. In 5-azacytosine, a nitrogen atom (N) replaces the key carbon atom (C) that normally gets methylated. The DNA methyltransferases can’t add a methyl group to this nitrogen atom.
Thinking back to
Chapter 4
, imagine a methylated region of DNA. When a cell divides, it separates the two strands of the DNA double helix and copies each one. But the enzymes that copy the DNA can’t themselves copy DNA methylation. As a consequence each new double helix had one methylated strand and one unmethylated one. The DNA methyltransferase called DNMT1 can recognise DNA which has only got DNA methylation on one strand and can replace it on the other strand. This restores the original DNA methylation pattern.
Chapter 4
, imagine a methylated region of DNA. When a cell divides, it separates the two strands of the DNA double helix and copies each one. But the enzymes that copy the DNA can’t themselves copy DNA methylation. As a consequence each new double helix had one methylated strand and one unmethylated one. The DNA methyltransferase called DNMT1 can recognise DNA which has only got DNA methylation on one strand and can replace it on the other strand. This restores the original DNA methylation pattern.
Figure 11.1
5-azacytosine can be incorporated into DNA during the DNA replication which takes place prior to cell division. 5-azacytosine takes the place of a C base, but because it contains a nitrogen atom where there is usually a carbon atom, the foreign base cannot be methylated by DNMT1 in the way that was described in
Figure 4.2
.
5-azacytosine can be incorporated into DNA during the DNA replication which takes place prior to cell division. 5-azacytosine takes the place of a C base, but because it contains a nitrogen atom where there is usually a carbon atom, the foreign base cannot be methylated by DNMT1 in the way that was described in
Figure 4.2
.
But if dividing cells are treated with 5-azacytidine, this abnormal cytidine base is added into the new strand of DNA as the genome gets copied. Because the abnormal base contains a nitrogen atom instead of a carbon atom, the DNMT1 enzyme can’t replace the missing methyl group. If this continues as the cells keep dividing, the DNA methylation begins to get diluted out.
Something else also happens when dividing cells are treated with 5-azacytidine. We now know that when DNMT1 binds at a region where the DNA contains 5-azacytidine instead of the normal cytidine, the DNMT1 becomes stuck there
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. This marooned enzyme is then sent to a different part of the cell and is broken down. Because of this, the total levels of DNMT1 enzyme in the cell fall
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,
8
. The combination of this decrease in the amount of DNMT1, and the fact that 5-azacytidine can’t be methylated, means that the amount of DNA methylation in the cell keeps dropping. We’ll come back in a little while to why this drop in DNA methylation has an anti-cancer effect.
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. This marooned enzyme is then sent to a different part of the cell and is broken down. Because of this, the total levels of DNMT1 enzyme in the cell fall
7
,
8
. The combination of this decrease in the amount of DNMT1, and the fact that 5-azacytidine can’t be methylated, means that the amount of DNA methylation in the cell keeps dropping. We’ll come back in a little while to why this drop in DNA methylation has an anti-cancer effect.
So, 5-azacytidine is an example of where an anti-cancer agent was unexpectedly shown to work epigenetically. Bizarrely, a rather similar thing happened with our second example of a compound which is now licensed to treat cancer
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Another happy accident
In 1971 the scientist Charlotte Friend showed that a very simple compound called DMSO (its full name is
d
i
m
ethyl
s
ulf
o
xide) had an odd effect on the cancer cells from a mouse model of leukaemia.
d
i
m
ethyl
s
ulf
o
xide) had an odd effect on the cancer cells from a mouse model of leukaemia.
When these cells were treated with DMSO, they turned red. This was because they had switched on the gene for haemoglobin, the pigment that gives red blood cells their colour
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. Leukaemia cells normally never switch on this gene and the mechanism behind this effect of DMSO was completely unknown.
10
. Leukaemia cells normally never switch on this gene and the mechanism behind this effect of DMSO was completely unknown.
Ronald Breslow at Columbia University and Paul Marks and Richard Rifkind at Memorial Sloan-Kettering Cancer Center were intrigued by Charlotte Friend’s research. Ronald Breslow began to design and create a new set of chemicals, using the structure of DMSO as his starting point, and then adding or changing bits, a little like making new combinations of Lego bricks. Paul Marks and Richard Rifkind began to test these chemicals in various cell models. Some of the compounds had a different effect from DMSO. They stopped cells from growing.
After many iterations, learning from each new and more complicated set of structures, the scientists created a molecule called SAHA (
s
uberoyl
a
nilide
h
ydroxamic
a
cid). This compound was really effective at stopping growth and/or causing cell death in cancer cell lines
11
. However, it was another two years before the team were able to identify what SAHA was doing in cells. The key moment happened more than 25 years after Charlotte Friend’s breakthrough publication, when Victoria Richon in Paul Marks’ team, read a 1990 paper from a group at the University of Tokyo.
s
uberoyl
a
nilide
h
ydroxamic
a
cid). This compound was really effective at stopping growth and/or causing cell death in cancer cell lines
11
. However, it was another two years before the team were able to identify what SAHA was doing in cells. The key moment happened more than 25 years after Charlotte Friend’s breakthrough publication, when Victoria Richon in Paul Marks’ team, read a 1990 paper from a group at the University of Tokyo.
The Japanese group had been working on a compound called Trichostatin A or TSA. TSA was known to be able to stop cells proliferating. The Japanese group showed that treatment with TSA altered the extent to which histone proteins are decorated with the acetyl chemical group in cancer cell lines. Histone acetylation is another epigenetic modification that we first met in
Chapter 4
. When cells were treated with TSA, the levels of histone acetylation went up. This wasn’t because the compound was activating the enzymes that put the acetyl groups on histones. It was because TSA was inhibiting the enzymes that remove acetyl groups from these chromatin proteins. These proteins are called histone deacetylases, or HDACs for short
12
.
Chapter 4
. When cells were treated with TSA, the levels of histone acetylation went up. This wasn’t because the compound was activating the enzymes that put the acetyl groups on histones. It was because TSA was inhibiting the enzymes that remove acetyl groups from these chromatin proteins. These proteins are called histone deacetylases, or HDACs for short
12
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Victoria Richon compared the structure of TSA with the structure of SAHA, and the two are shown in Figure
11.2
.
11.2
.
Figure 11.2
The structures of TSA and SAHA, with the areas of greatest similarity circled. C: carbon; H: hydrogen; N: nitrogen; O: oxygen. For simplicity, some carbon atoms have not been explicitly shown, but are present where there is a junction of two lines.
The structures of TSA and SAHA, with the areas of greatest similarity circled. C: carbon; H: hydrogen; N: nitrogen; O: oxygen. For simplicity, some carbon atoms have not been explicitly shown, but are present where there is a junction of two lines.
You don’t need a chemistry degree to see that TSA and SAHA look fairly similar, especially at the right hand side of each molecule. Victoria Richon hypothesised that, just like TSA, SAHA was also an HDAC inhibitor. In 1998, she and her colleagues published a paper that showed this was indeed the case
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. SAHA prevents HDAC enzymes from removing acetyl groups from histone proteins, and as a result, the histones carry lots of acetyl groups.
13
. SAHA prevents HDAC enzymes from removing acetyl groups from histone proteins, and as a result, the histones carry lots of acetyl groups.
Beyond coincidence
So, 5-azacytidine and SAHA both decrease cancer cell proliferation, and both inhibit the activity of epigenetic enzymes. Although we could take this as promising support for the theory that epigenetic proteins are important in cancer, perhaps we could just be leaping to conclusions? It might just be a coincidence that both drugs affect epigenetic proteins. After all, the enzymes targeted by the two compounds are very different. 5-azacytidine inhibits the DNMT enzymes, which add methyl groups to DNA. SAHA, on the other hand, inhibits the HDAC family of enzymes, which remove acetyl groups from histone proteins. Superficially, these seem like very different processes. Maybe it’s just coincidence that both 5-azacytidine and SAHA inhibit epigenetic enzymes?
Epigeneticists believe that it is far from being a coincidence. DNA methyltransferase enzymes add a methyl group to the cytidine base. High concentrations of this base are found in the long CG-rich stretches of DNA known as CpG islands. These islands are found upstream of genes, in the promoter regions that control gene expression. When the DNA of a CpG island is heavily methylated, the gene controlled by that promoter is switched off. In other words, DNA methylation is a repressive modification. DNMT activity increases DNA methylation and therefore represses gene expression. By inhibiting these enzymes with 5-azacytidine, we can drive gene expression up.
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