Sunday, June 03, 2007


DAPK1 stands for Death Associated Protein Kinase 1. Technically, DAPK1 is an actin-filament-associated, calcium calmodulin-dependent, serine/threonine kinase that promotes apoptosis in response to various stimuli including Fas, interferon gamma and TNF alpha. No, I don’t understand all that either. In layman’s terms it is one of the normal proteins in a cell that plays a part in killing cells at the end of their natural lifespans. There are very many of these and ‘apoptosis’ or ‘programmed cell death’ is a very complex process. In fact there are whole scientific journals that are devoted to it. It has been known for a very long time that in CLL there is something wrong with the apoptosis mechanism. Cells don’t die when they are supposed to.

This is not the only thing wrong with CLL cells; they also seem to divide more rapidly than they ought to – at least in those with unmutated IgVH genes. The cause of the failure to die when they should is one of the most investigated puzzles in the whole of CLL science. Most attention has been paid to BCL-2. This is a protein that opposes apoptosis and is present in increased amounts in CLL cells. We also know that in the related disease, follicular lymphoma, they increased amounts of BCL-2 are caused by the chromosomal translocation that is characteristic of the disease: t(14;18) puts the immunoglobulin promoter region next to the bcl-2 gene. (A promoter is a bit of DNA that carries the message ‘make a lot of’. Instead of saying, “make a lot of immunoglobulin” it says, “make a lot of BCL-2”). But that is not the reason for the increased BCL-2 in CLL.

There are other apoptosis related proteins that are abnormal in CLL. Other members of the BCL-2 family such as anti-apoptotic proteins BCL-XL, BAG-1 and MCL-1 are overexpressed, while pro-apoptotic proteins like BAX and BCL-XS are underexpressed. DAPK1 is a pro-apoptotic protein that previously been little studied, but a paper by Nagy and colleagues in the British Journal of Haematology in 2003, mentioned that was underexpressed in CLL patients with del 11q23.

Yesterday, an important paper appeared in Cell (all papers in Cell are important) concerning the importance of DAPK1 in CLL. The paper comes mainly from OSU in Columbus Ohio, with contributions from several other institutions in America, Germany, Sweden and England. To explain what it says I must explain about methylation.

DNA methylation is a type of chemical modification of DNA that can be inherited without changing the DNA sequence; it is one of the epigentic mechanisms that controls how DNA functions. It involves the addition of a methyl (CH3) group to DNA, usually to the carbon atom at position 5 of the Cytosine pyrimidine ring. Characteristically it occurs at a cytosine-phosphate-guanine (CpG) sequence.

The importance of DNA methylation is that it silences the gene. In Man about 1% of DNA bases are methylated, but about 60%-70% of CpG sequences are methylated. Unmethylated CpGs are grouped in clusters called "CpG islands" that are present in the 5' regulatory regions of many genes. These are the gene promoters and if they can be switched off the gene is not translated. In many cancers some of these CpG islands get hypermethylated, silencing the promoter regions for ‘tumor suppressor genes’

Previously studies have uncovered almost 200 abnormally methylated genes that are silenced in this way in CLL. In this study the DAPK1 was silenced in this way in 60 out of 62 cases of sporadic CLL.

Even more interesting was their study of a family in which multiple members had CLL. They were able to study DNA from 3 members of the third generation and three members of the fourth generation. First they identified a region on chromosome 9 which had the greatest linkage to the appearance of CLL.. This area of the genome contains 3 known genes and 11 predicted genes. Among the known genes is DAPK1, and based on what was known of DAPK1 it was decided to concentrate the study on this gene. They started by sequencing this area of the DNA from fibroblasts taken from affected and non-affected family members. Note these were fibroblasts, not CLL cells, because they wanted to look for DNA differences that they were born with rather than ones acquired in the leukemia cells.

By a series of very intricate experiments they were able to compare in an affected individual, the DAPK1 gene on the normal chromosome 9 and the DAPK1 gene on the chromosome 9 that carried the inherited CLL in this family. They found a lot of differences – 281 in fact. These differences were single nucleotide polymorphisms or SNPs. This means that one nucleotide base – an adenine (A), cytosine (C), thymine (T) or guanine (G) – is substituted for another. However, polymorphisms are extremely common. Whether you have blue or brown eyes, fair or dark hair, blood group O or A, ear lobes or not and many other physical characteristics are cause by SNPs. Generally, they are harmless and they are well known. In this case, almost all the 281 SNPs could be eliminated as well known ones that had no malign affect, but there were 4 possibles that could be the reason for inheriting CLL. One of these, and A to G switch at position c.1-6531, was not found in 383 control samples from the US and Northern Europe, but among 263 cases of CLL from the US and Northern Europe one patient was found with the same polymorphism. This patient from Scandinavia had the SNP in both CLL cells and T cells, so presumably this was something he or she was born with, but there is no suggestion that thispatient is related to the American kindred nor any other familial cases. However, no-one knows whether they have long lost cousins back in Europe.

An important control was to look for this SNP among patients with familial CLL. They looked at 75 patients with CLL who had at least one other family member with the disease, but they didn’t find it. This means that although in this particular family this particular polmotphism predisposes to the development of CLL, and although the gene is aberrantly switched off in CLL cells in sporadic cases, it is not implicated in the majority of cases of familial CLL.

They next investigated how the A to G switch manages to reduce DAPK1 expression and it turns out that this switch increases the affinity of DAPK1 for HOXB7, a transcription factor that normally opposes the expression of DAPK1.

To summarize: John Byrd and his colleagues at OSU have identified a gene that is mutated in one family with CLL. The mutation inactivates this gene. But this is not the abnormality in most families with CLL. However, most cases of sporadic CLL also have this gene inactivated, but by an epigenetic mechanism rather than by a hereditary mutation. There is a plausible hypothesis how inactivating this gene could lead to CLL, but it is only one of 200 epigenetically silenced genes in CLL. Several other genes involved in apoptosis are also switched on or off in CLL, sothe full picture has yet to emerge.

1 comment:

Anonymous said...

Thanks for the lucid explanation of the concepts you mention. We've all read of hypermethylation, CpG islands, apoptotic promoters and suppressors, but it is difficult to put all of these isolated concepts into an integrated whole.

I agree that one should not overhype this paper, as it is but one piece to the very complex puzzle that is CLL.

On the other hand, to reach the final solution to the puzzle, one has to start with the pieces. This seems to be one good-sized piece.