From: Robert J. Bradbury (bradbury@aeiveos.com)
Date: Mon May 01 2000 - 07:41:54 MDT
On Mon, 1 May 2000, John Clark wrote:
> > I had commented on the problem that increased numbers of cell divisions
> > would effectively lead to greater malignancy.
>
To which John had replied:
> But after 93 divisions, 50% more than in a normal lifetime, there was still
> no evidence of an increase in malignancy. Ok the divisions were done in
> vitro not in an animal but I would think, perhaps foolishly, that if
> anything that would increase the likelihood of cancer occurring in the
> cells not decrease it.
John, has pointed out the points in my argument that I'm glossing over.
The question is: What are the sources of mutation that lead to malignancy
(and perhaps aspects of aging)?
(a) Radiation
(b) Oxidative stress
(c) Toxins in the cellular "environment" (derived from food).
(d) Mutations by DNA Polymerase in copying the DNA.
Now, look at the difference between replication in the laboratory dish
where you are pushing the cells as fast as possible (about 1 generation
per day) in "perfect" culture media, with much lower exposure to toxins
a very uniform level of oxygen (so you can tune the antioxidant defenses
and/or repair rates appropriately) and a much reduced level of radiation
esposure simply due to a shorter time period.
It is well known that DNA damage "cleanup" is supposed to occur during
both transcription and DNA replication. This is however imperfect and
certain types of damage result in repairs that must cause mutations
(otherwise *where* would they come from?).
So, the cells are certainly exposed to lower levels of radiation and
toxins and perhaps oxidative stress. They are constantly replicating
the DNA (and therefore conducting the repairs that occur during
replication). What we need are good data on the mutation rates
in cell culture vs. in vivo to get any kind of a handle on this.
Such data probably exists but I've never seen any "comments" on
it vis-a-vis impact on aging and/or the development of malignancies.
To transform normal cells into malignant cells in vitro, the most
common methods involve hitting them with a hefty dose of radiation
or chemical mutagen. If you just let them divide, malignant cells
occur rarely if at all. I seem to recall statements to the effect
of -- you can get them if you are very patient with mouse cells
but rarely if at all with human cells. How much of this is due
to differences in the in vitro vs. in vivo environment and how
much is due to the much smaller quantity of cells in culture vs
a living organism, I am unsure.
The simple observation that cells rarely transform in cell culture
would seem to indicate that the majority of mutations must be coming
from (a-c) above. Kornberg and Baker [1] document the E. coli
polymerase "natural" error rate at < 10^-10 per base pair. If
the error rate is similar in humans (a reasonable assumption) that
would imply 1 mutation every 3 cell divisions. Given 90% or
so of the DNA is junk and many mutations are "silent" due to
the redundant code, you would need hundreds of cell divisisions
before an "effective" mutation occured. Most of these would have
no effect on creating malignant cells because there only ~1% of the
genes are involved in replication. Hits in the ~70% of the genes
turned off in a particular cell type will do nothing, hits
in housekeeping genes may make the cell grow less efficiently
(so it eventually disappears from culture due to competition),
and hits in a few genes might result in apoptosis so it also
disappears.
Larry Loeb (who was one of my professors) has argued fairly strongly
that the first step in malignancy is the creation in a single cell
of a "mutator phenotype", i.e. a mutation in a gene like those
involved in DNA repair. The increased cancer rates found in
Xeroderma Pigmentosum are due to mutations in the ~10 genes
that do a specific type kind of DNA repair. I suspect if they
did the same type of cloning operation (creating long telomeres)
in cows or sheep that had defects in the repair genes, that you
would begin to see malignant cells in culture. Or perhaps if they
did the cell culture experiments with 50 kg of cells instead of
milli- or micro-grams.
>
> >that is a *far cry* from keeping alive an organism over many years
> >(where background radiation plays a significant hazard).
>
> Good point but if they increased the normal background radiation by a factor
> of 60 or 70 while the cells divided 93 times in vitro and they still didn't
> get a catastrophic increase in cancer then that would eliminate that worry.
> I think. Anyway, it doesn't seem like a particularly difficult experiment
> but to my knowledge it has not been done.
I agree that it is interesting idea. I suspect you could probably tease
the information out of the existing literature on radiation, oxidative
stress, toxin exposure, etc. I may put this on my list as a low level
project.
>
> > Ever since Fossil's book, everyone has thought that "aging" is
> > associated with telomere shortening and quite simply that is crap.
>
> It sounds like you're saying that there is no possibility telomere shortening
> has anything to do with aging, not in any way shape or form. Skepticism is
> always healthy in science but cynicism is not and I don't think the evidence
> can support such an extreme view.
>
The problem is that Fossil attempted to make telomere shortening responsible
for *all* aging. First, that violates what we know about the declining
force of natural selection with age and the Hamilton/Williams theories
regarding pleiotropic genes (with early benefits but late harms).
Second it makes no sense since there is lots of documentation on aging
that occurs in the tissues of the body where cell division does not
occur (collagen cross-linking or protein glycosylation for example).
Just so my perspective is clear, IMO, telomere shortening *does* play
a role in aging in all dividing tissues. So that would include:
skin, tongue, stomach & intestines, immune system, endothelial cells, and
sometimes liver. The diseases of the aged that result from those
*are* dry & brittle with poor healing, poor tase, poor nutrient absorption,
increased susceptibility to pathogens, e.g. flu, stroke and liver
cirrosis (respectively).
However, heart disease (involving immune system foam cells and/or endothelial
cells in areas of athersclerotic plaques) and cancer (in cells that are
programmed for or biased for division) are the two leading causes of death
in our society. These are diseases of *over* replication, not *under*
replication. It is open to debate whether making human telomeres
"shorter" would have any impact on these. Why? Because if you make
the telomeres shorter in endothelial cells, you might cut down
on heart attacks from occlusions due to excessive replication in regions
where plaques develop, but you might increase strokes due to the senscence
of cells in high-stress arterial regions where the cells need to divide
more frequently. In cancer, the question is whether telomerase is
turned on *before* or after the mutator phenotype develops? I.e.,
is telomerase eventually turned on because it is a required element
of malignancy in cells with the mutator phenotype, or is telomerase
turned on as a precursor for accumulating enough mutations to "exhibit"
a mutator phenotype that eventually leads to malignancy?
So, while I will freely grant that telomeres play an important role
in aging, they are no magic bullet.
As far as the ACT speculations go, there is no evidence I'm aware
of that indicates that the telomeres in human adult muscle stem cells,
neuronal stem cells, etc. are "too short" to grow replacement tissue.
If you can grow a single heart from a single muscle stem cell, it
doesn't matter if the telomeres in those 2nd generation hearts
are too short to grow 3rd generation hearts. Why? Because I
only need to harvest 10 muscle stem cells from your existing
1st generation heart to grow enough hearts for 10 lifetimes.
So if I am "cynical", it is because West is using terms like
"breakthrough", when in fact it is something that may be entirely
unnecessary.
If it does turn out that the telomeres are too short in your stem
cells, doesn't it make much more sense to simply turn on telomerase
in those stem cells (as cancer cells do) *without* resorting to
the messy procedure of cloning your cells into denucleated oocytes
(that at least currently would have to be harvested from human female
volunteers)! Now, if it turned out that you could get the magical
telomere lengthening by putting human nuclei into mouse denucleated
oocytes, that really would be a breakthrough!
Robert
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