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Gene Silencing Revisited – Now a Hot
Item (May 2003)
Gene silencing was recently featured in
a Seattle Times story, a reminder to clinicians that we will have to wrap
our minds around these new concepts in order to be well informed.
Developments arising from basic molecular biology are moving to center
stage. However, before reporting the news, let me first offer my
simplistic primer about the basic biology of this area of DNA genetic
infomatics. My brief review begins with a mental cartoon describing an
activated cellular receptor traveling into the cell's nucleus and heading
toward DNA, at which point it finds it has to penetrate a fuzzy molecular
barrier created by the histones, the "spools" around which the DNA is
wrapped. An access "cleft" must be created, and is achieved by the
insertion of small molecular "props" (acetyl groups) that push apart the
tight bonds of the barrier, thus facilitating the receptor's passage.
(Conversely, a open barrier may be closed by removing the acetyl groups, a
process termed "de-acetylation".) Once past the barrier, the business end
of the receptor (with all its helpers: the coactivators, corepressors and
the transcription assembly) must find free access to the DNA target, the
promotor site where a gene's transcription begins. Another possible
obstacle may frustrate successful access to DNA. If a methyl group "sits
on top" and preempts access to the promotor site, the proper "fit" of the
receptor complex and the DNA is prevented. No DNA contact, no
transcription. This "methylation" is termed "epi-genetic", literally
"above the genome", and can also be termed "imprinting", and is a common
method of regulating gene expression. (The Roche pharmaceutical company
must think this area of biology is important since it recently invested
$100,000,000 in the Seattle-Berlin biotect company, "Epigenetics," to
pursue potential therapeutic developments.) If the methyl group is removed
then transcription can begin. What to methylate; what to deacetylate ?! It
remains a fascinating unsolved mystery how the cell "knows" how and when
to do these things... So much for the primer. Now on to the news.
The recent Seattle Times article highlighted a new test that utilizes the
identification of methylated genes as a marker of the risk of developing
colon cancer. The basis for the news was the study published in SCIENCE,
March 14, 2003, "Loss of IGF2 [Insulin-like Growth Factor] Imprinting [methylation]:
A Potential Marker of Colorectal Cancer Risk." Humans inherit two copies
(alleles) of each gene - one maternal, one paternal. The gene of interest
here is IGF2, an important gene for many of our internal systems which
stimulates proliferation and activity of its targets. In the normal state
we receive one of two alleles for this gene methylated, hence silenced.
The test assesses the extent of methylation of these two alleles, and if
both are unmethylated, and therefore both functional, that
individual gets an extra boost of growth factor, thereby raising the risk
of developing colon cancer to 5 times normal ! A converse example can be
found in uterine endometrial cancer where the invasiveness of tumor is
enhanced by unwanted methylation of a "tumor suppressor" gene, E-cadherin,
that normally serves to inhibit migration of tumor cells away from the
primary site (CANCER 2003;97:1002).
Returning the discussion to methylation in prostate cancer, I'll report
the most recent article by Drs. Sidransky and Epstein (J UROL 2003, March)
in which they follow-up on their earlier work [discussed in March PCa
Commentary] utilizing a quantative test for methylation of the gene,
GSTP1, which codes for the protective cellular antioxidant, glutathione.
In the new work they tested a single core from each of 29 prostate biopsy
sets. These cases were considered problematic and non-diagnostic by the
initial pathologists. By using the methylation marker they were able to
diagnose cancer in 11 of 15 cores. Four cores had a single foci of cancer
of only 1 mm. and 7 had 0.05 mm. foci. The Gleason sum for each cancer
case was < 6. On full review, fourteen additional specimens were benign
and none of these showed methylation. Clearly the argument is being made
in support of this technology for diagnosis, especially confirmatory
information in difficult cases.
When it becomes clear that unwanted methylation can cause adverse effects,
it's natural to speculated whether therapy could be developed that would
de-methylate silenced genes whose function is desirable. Perhaps a drug
that demethylates the GTSP1 prostate gene might serve as treatment to
avert progression of very early prostate cancer. An example of such a
medication exists [5-azacytidine] and has been tried in a hematologic
malignancy, but the drug is quite toxic and only is suitable to
intravenous administration. A newly developed agent suitable for oral
administration was report in the JOURNAL of the NATIONAL CANCER INSTITUTE,
March, 2003 - "Inhibition of DNA Methylation and Reactivation of Silenced
Genes by Zebularine". The study was conducted in mice and demonstrated
successful demethylation . Most exciting to the researchers, zebularine
slowed the growth of tumors compared to tumors in the control animals.
Caution is in order, however, when intervention is aimed at this basic
level of the operating systems of cellular function. A brief communication
in the February 19, 2003 issue of the JNCI reported that when tested in
tissue culture using pancreatic cancer cells, the demethylating agent
5-azacytidine reversed the methylted silence of several tumor suppressor
genes thereby promoting aggressive tumor growth. Clearly very careful
studies will be required before drugs of this type are approved for human
use. It makes one recall the old adage - "Be careful what you wish for,
you might just ...."
Bottom Line: Tests of gene methylation are promising for diagnosis
and risk assessment.
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