Suresh Rattan rattan at imsb.au.dk
Tue Aug 5 04:43:02 EST 1997

Considering that there has been tremendous response (both privately and on
the internet) to my previous posting of my article on the possibilities of
gene therapy for aging, I hereby post some excerts from one of my
forthcoming articles (in press).

=46rom vitagenes to gerontogenes and the other way around
by: Dr. Suresh Rattan/University of Aarhus/Denmark

Our survival and the physical quality of life depends upon an efficient
functioning of various maintenance and repair processes. This complex
network of the so-called longevity assurance processes is comprised of
several genes, which may be called vitagenes. The homeostatic property of
living systems is a function of such a vitagene network. These processes
have to work in the presence of extrinsic and intrinsic sources of damage,
such as environmental and nutritional agents, spontaneous errors of
macromolecular synthesis, post-synthetic modifications of macromolecules
making them inactive or abnormal, and other defects occurring during the
course of normal metabolism. Apparently, this homeostatic network of
vitagenes appears to work optimally during the major part of life allowing
growth, development, differentiation and maturation to occur. There are no
obvious reasons why these maintenance, defense and repair mechanisms could
not operate for ever and make an organism immortal. Yet, a progressive
failure of maintenance underlines and typifies the process of aging.
	Aging has many facets and almost all the experimental data suggest
that aging is an emergent, epigenetic and a meta-phenomenon, which is not
controlled by a single mechanism. Individually no tissue, organ or system
becomes functionally exhausted, even in very old organisms, yet it is their
combined interaction and interdependence that determines the survival of
the whole. Since it is necessary to look for genes as the ultimate
controllers of all biological processes, the term gerontogenes has been
suggested to refer to any genetic elements that are involved in aging.
However, there is still no agreement as regards the nature of the
gerontogene network and the number of genes involved in it.

Evolutionary theories of aging and longevity discount the notion of
adaptive nature of aging. This is because Darwinian evolution, which works
primarily through the process of natural selection for reproductive
success, does not leave any margin for the selection of aging and death as
an advantageous trait for the individual. Evolutionary forces work only on
life processes and do not select for death. Natural selection of, what is
recently termed as a "selecton" is in the form of an efficient vitagene
network to assure its longevity for fulfilling its purpose of life, that
is, reproduction and continuation of generations. The so-called programmed
death or apoptotic mechanisms are essential parts of the developmental
processes (or may even be a component of the homeostatic vitagene network)
necessary for the making of a reproductively successful individual.
	Once the Darwinian purpose of life is fulfilled, there is no reason
(or the selection pressure) left either for the maintenance of the body or
for its destruction. Thus aging, as a failure of maintenance, occurs
without a cause or a purpose. It just happens. Any search for genes which
were selected specifically to cause aging is misdirected and ill-informed.
=46urthermore, the diversity of the forms and variations in which age-relate=
alterations are manifested indicate that the progression of aging is not
deterministic but stochastic in nature. An age-related increase in
variability among individuals, in terms of any physiological, cellular or
biochemical parameter studied, is a reflection of the stochastic nature of
aging. In the words of an anonymous poet, we are born as copies, but we die
as originals.
	Yet, aging appears to have a genetic component of some kind. The
role of genes in aging is evident from an apparent practical limit to
maximum lifespan within a species along with the evidence from studies on
twins, from the human genetic mutants of pre-mature aging, and from genetic
linkage studies for the inheritance of lifespan and for genetic markers of
exceptional longevity. Thus, aging appears to be genetically regulated
without involving any genes which may be held responsible as its cause.
This paradoxical situation of the genetic aspects of aging and longevity on
the one hand and the stochastic nature of the progression of the aging
phenotype can be resolved by developing radically novel views about the
nature of gerontogenes.

	The following lines of research can form the basis of a promising
strategy to understand and modulate the aging process:
1. Studying the extent of maintenance and repair of the genes involved in
maintaining the stability of the nuclear and mitochondrial genome.
2. Studying the efficiency of transcription of various vitagenes and
post-transcriptional processing of their transcripts.
3. Studying the accuracy and efficiency of translation of vitagenes and
analyzing the specificity, stability and turnover of vitagene products
(vitaprins?), including their post-translational modifications.
4. Searching for natural or induced mutants (including transgenic and
knockout organisms) with altered levels of maintenance and repair of the
crucial vitagenes.
5. Searching for age-specific and age-related disease-specific biomarkers
for diagnostic purposes and for monitoring the effects of potential
therapeutic agents.
6. Experimental modulation of various types of maintenance mechanisms and
studying its effects on other levels, such as gene stability and gene
product synthesis and turnover. Some of the main defense processes that may
provide a relatively easy access to experimentation include responsiveness
to stress, efficiency of signal transduction pathways, and regulators of
cell cycle progression.

An organism's ability to respond to stress is a major component of its
homeostatic vitagene network, and altered responsiveness is one of the most
significant features of aging. The so-called heat shock response as an
important mechanism of cellular defense is very well established. It is
also known that the extent of heat shock response decreases during aging.
Therefore, it has been hypothesized that if organisms are exposed to brief
thermal treatment so that their stress response-induced gene expression is
upregulated and this particular pathway of maintenance and repair is
stimulated, one should observe anti-aging and longevity-promoting effects.
Such a phenomenon in which stimulatory responses to low doses of otherwise
toxic substances improve health and enhance lifespan is known as hormesis.
Recently, anti-aging and life-prolonging effects of heat shock have been
reported for Drosophila and the nematodes.
	I have tested the effects of mild but repetitive heat shock on
various cellular and biochemical characteristics of human skin fibroblasts
undergoing aging in vitro. I undertook a series of pilot experiments to
determine suitable temperature conditions which fulfilled the following
criteria: (i) the thermal treatment had no effects on immediate survival of
the cells, as checked by trypan blue exclusion test; (ii) the cells
responded to the thermal treatment by inducing the synthesis of major heat
shock proteins (hsp), as detected by metabolic labeling of cells with
radioactive amino acids followed by SDS-polyacrylamide gel electrophoresis
(PAGE); and (iii) thermally treated cells could be subcultured normally
without any effect on their attachment frequency.
	All experiments were performed on a normal human adult female skin
fibroblast line designated ASS, which has been used previously to test for
the anti-aging effects of the cytokinin hormone kinetin. The present series
of experiments have demonstrated that repetitive and mild heat shock has
several positive and anti-aging effects on human cells in culture
(manuscript in preparation). Briefly the results are summarized as follows.
Human fibroblasts could be exposed to mild heat shock at 41=B0 C repeatedly
during their limited proliferative lifespan in vitro without any apparent
negative effects on survival, attachment frequency, population doubling
rates and cummulative population doubling level potential. Although the
temperatures higher than this (up to 43=B0 C) could stimulate a more intense
heat shock response in terms of hsp synthesis, cells could not survive more
than 7 repeated thermal treatments. Continuous survival of human
fibroblasts for 140 days during which time they underwent about 30 PDs and
received 35 repeated heat shocks at 41=B0 C is a novel effect not observed
	Although there was no prolongation of the proliferative lifespan of
human fibroblasts after repeated heat shock treatment, several other
anti-aging effects were observed. Most dramatically, age-related alteration
in the morphology of cells, which is one of the most obvious change during
cellular aging, was significantly slowed down in heat shocked cells. The
control cultures showed the typical age-related increase in cell size,
flattened appearance, increased morphological heterogeneity, loss of
arrayed arrangement, increased number of lysosomal residual bodies,
increased number of actin filaments and increased proportion of
multinuclear cells during serial passaging. However, the heat-shocked
cultures showed a highly reduced rate of these age-related alterations and
maintained a relatively young morphology even at the end of their
proliferative lifespan. These cells did not undergo significant
enlargement, maintained to a large extent their spindle-shape and arrayed
arrangement, did not accumulate much residual bodies, did not show many
rod-like actin filaments, and had an almost complete absence of
multinucleate cells. Maintenance of young morphology and reduced cell size
is a strong indication of anti-aging effects of heat shock, as also
observed for other anti-aging treatments such as carnosine and kinetin.
	 We are now investigating what other cellular, physiological,
biochemical and molecular effects of repeated heat shock occur in human
cells. Some of the characteristics to be tested are the rates and extent of
transcription and translation of various genes, the extent of gene-specific
and total DNA repair including telomere length, the extent and rates of
protein synthesis and degradation, and the accumulation of molecular
damage, such as oxidative damage in DNA, lipid-peroxidation products and
abnormal proteins.
	Most importantly, the above experiments show that it is possible to
re-tune the vitagene network in such a way that its transmutation to a
virtual gerontogene network is slowed down. Due to the highly complex
nature of interactions within the vitagene network, it is most unlikely
that a complete transmutation of vitagenes into gerontogenes can ever be
prevented. Furthermore, retuning one or more vitagenes may or may not have
significant effects on the final outcome in terms of reducing the rates of
aging and increasing the lifespan. However, what can definitely be achieved
by this approach is that increased levels and/or efficiency of one or more
maintenance and repair pathways will have positive effects in terms of
improving the physical quality of life and increasing its chances of
survival, and ultimately, achieving a healthy old age.

Dr. Suresh I. S. Rattan, PhD; DSc
Laboratory of Cellular Ageing
University of Aarhus
DK-8000 Aarhus - C

Tlf: +45 89 42 50 34 	Fax: +45 86 20 20 11

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