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Telomeres, telomerase, and aging: Origin of the theory

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Abstract

In 1971 I published a theory in which I first formulated the DNA end replication problem and explained how it could be solved. The solution to this problem also provided an explanation for the Hayflick Limit, which underpins the discovery of in vitro and in vivo cell senescence. I proposed that the length of telomeric DNA, located at the ends of chromosomes consists of repeated sequences, which play a buffer role and should diminish in dividing normal somatic cells at each cell doubling. I also proposed that the loss of sequences containing important information that could occur after buffer loss could cause the onset of cellular senescence. I also suggested that for germline cells and for the cells of vegetatively propagated organisms and immortal cell populations like most cancer cell lines, an enzyme might be activated that would prevent the diminution of DNA termini at each cell division, thus protecting the information containing part of the genome. In the last few years, most of my suggestions have been authenticated by laboratory evidence. the DNA sequences that shorten in dividing normal cells are telomeres and the enzyme that maintains telomere length constant in immortal cell populations is telomerase.
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Experimental Gerontology, Vol. 31, No. 4, pp. 443-448, 1996
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Historical Perspective
TELOMERES, TELOMERASE, AND AGING: ORIGIN OF
THE THEORY
ALEXEY M. OLOVNIKOV 1
Institute of Biochemical Physics of the Russian Academy of Sciences, Chernahovskogo 5, kv. 94,
Moscow 125319 Russia
Abstract--In
1971 I published a theory in which I first formulated the DNA end replication
problem and explained how it could be solved. The solution to this problem also provided an
explanation for the Hayrick Limit, which underpins the discovery of in vitro and in vivo cell
senescence. I proposed that the length of telomeric DNA, located at the ends of chromosomes
consists of repeated sequences, which play a buffer role and should diminish in dividing
normal somatic cells at each cell doubling. I also proposed that the loss of sequences
containing important information that could occur after buffer loss could cause the onset of
cellular senescence. I also suggested that for germline cells and for the cells of vegetatively
propagated organisms and immortal cell populations like most cancer cell lines, an enzyme
might be activated that would prevent the diminution of DNA termini at each cell division,
thus protecting the information containing part of the genome. In the last few years, most of
my suggestions have been authenticated by laboratory evidence. The DNA sequences that
shorten in dividing normal cells are telomeres and the enzyme that maintains telomere length
constant in immortal cell populations is telomerase.
Key Words: marginotomy, telomere, telomerase, DNA end-underreplication problem, Hayflick
Limit, cancer cells, normal ceils
INTRODUCTION
THIS IS the history of a theory about the Hayflick Limit and the DNA end-underreplication
problem, which, when first published in 1971, went almost unnoticed for 20 years until recent
research results dramatically proved its validity.
In the last few years an enormous amount of information has been published on the identi-
fication and behavior of telomeres and telomerase and the role of each in normal cell senescence
and cancer cell immortality both in vitro and in vivo. This essay describes the historical
developments that led to this remarkable series of discoveries.
The importance of chromosome ends, called telomeres, was first recognized by McClintock
(1941) and by Muller (1938) who defined telomeres as the functional ends of chromosomes as
J Fax: (011) (7095) 214-9269; E-mail: am@olovnikov.msk.ru
(Received 9 November
1995;
Accepted
26
January
1996)
443
444 A.M. OLOVNIKOV
distinct from random broken ends. Beginning from about 1966 I had understood that the
replication of linear DNA by DNA polymerase would result in the loss of terminal sequences
unless some mechanism existed that would maintain the ends of chromosomes. The mechanism
by which this terminal sequence loss could be explained was called end-underreplication.
In 1971, I published a paper in which I described the existence of the DNA end-
underreplication problem and proposed a theory for its solution. However, it was not until the
last few years that laboratory studies have provided confirmation of the theory that I proposed
25 years ago.
It has been discovered that the ends of linear chromosomes consist of a repeated sequence of
bases whose length decreases with each cell division. It is thought that when the chromosome
shortening reaches a critical length, further events prevent the cell from dividing. In immortal
cancer cells, an enzyme is produced that adds new sequences onto the ends of chromosomes at
each DNA replication, thus maintaining the chromosome length constant. In this way cancer
cells divide indefinitely and, thus, achieve immortality. It is quite gratifying to see one's theory
ultimately supported by laboratory results, and I would like to explain how my theory arose.
I formulated the theory of telomere shortening when I was a postdoctoral student at the
Gamelaya Institute of the Academy of Sciences of the USSR in 1966. Academician Gamelaya,
a vaccinologist and for whom the Institute was named, was a colleague of the great Russian
biologist, Elie Metchnikov, the discoverer of phagocytosis and the theory that aging results from
inadequate fermentation activities of the intestinal flora.
My chief was Dr. Lev A. Zilber, a man of great intellect and one of the originators of the idea
that viruses may cause cancer. Zilber was a "Renaissance" man who spent several years of
incarceration in the Gulag Archipelago as an alleged Japanese spy. When Zilber was freed he
returned to the Gamelaya under the direction of the same person who was his tormentor in the
Gulag. In a dispute with the director, Zilber hurled a black marble blotter base at the face of the
director, a high ranking KGB officer, but he missed. This occurred during the Khrushchev thaw
and the director was soon replaced. The directors of the Gameleya then changed several times
with some having been appointed more for their achievements as KGB members than as
members of the scientific community.
In Zilber's Department of Immunology and Oncology, I worked in two laboratories. One was
the antibody biosynthesis laboratory of Aaron E. Gurvich who was coinventor of immunosor-
bents and also coined this term instead of immunoadsorbent. I also studied in the laboratory of
Gary I. Abelev who discovered the presence of alpha-fetoprotein in some tumors. Abelev, like
Zilber who died in 1966, struggled with another director, a KGB Colonel, to save Zilber's
Department. The struggle ended when Abelev moved his laboratory to another institute in
Moscow. Both Gurvich and Abelev helped me greatly by providing a letter of recommendation
to Andrey N. Belozersky, the Academician of our Academy of Sciences. In that letter they asked
Belozersky to submit my strange theory on telomeres to Doklady, which is the local Proceedings
of the Academy of Sciences.
My theory looked strange because it stated that a portion of genomic DNA was regularly lost
with each round of DNA replication and that the losses occurred at the ends of chromosomes.
The fact that DNA in eukaryotic chromosomes was linear was not entirely settled at that time.
But, Belozersky gave me a recommendation and my paper appeared in Doklady in 1971. Thus,
my "Theory of Marginotomy" began its official existence.
But, that is getting ahead of the story.
Until the Autumn of 1966, I never thought about the replication of the DNA double helix
termini. This changed when I attended a lecture by Alexander Y. Friedenstein, a cell biologist
TELOMERES, TELOMERASE, AND AGING 445
at Moscow University. His lecture was on the new phenomenon discovered by Leonard Hay-
flick, in which it was reported that normal human cells, unlike immortal, abnormal or cancer
cells, have a limited capacity for replication. This was, in fact, the second time that I heard about
this phenomenon. I first heard about it at the Gameleya Institute of Epidemiology and Micro-
biology of the Academy of Medical Sciences when I was a postdoctoral student but it made no
strong impression. But, when I heard about it for the second time at Moscow University I was
simply thunder-struck by the novelty and beauty of the Hayflick Limit. I thought about this as
I returned home from the University and walked along the quiet Moscow streets that were paved
with gold-colored leaves on that early evening in late Fall as I made my way to the subway
station.
The Theory of Marginotomy came to me in that Moscow subway station. I heard the deep roar
of an approaching train coming out from the tunnel into the station itself. I imagined the DNA
polymerase to be the train moving along the tunnel that I imagined to be the DNA molecule. I
thought that this polymerase cannot begin to copy from the very beginning because there is a
dead zone between the front end of the polymerase molecule and its catalytic center. This is
analogous to the dead zone between the front end of a subway car standing at the beginning of
the subway platform and the nearest entrance door to the first car. After this serendipitous
underground brainstorm, which happened in the Fall of 1966, I wrote to Hayflick to ask some
questions about his discovery and he sent additional unpublished data to me. I then spent several
years thinking about this idea before publishing it in the central journal of our Academy of
Sciences, Doklady Academii Nauk SSSR (Proceedings of the Academy of Sciences of the
USSR) (Olovnikov, 1971).
So, it was the Hayflick Limit that started me thinking about an explanation for his finding and
its possible link with the DNA end replication problem. Hayflick's finding was for me like
Ariadne's thread was for Theseus, who followed it to escape from the labyrinth. During the
subsequent months, as I commuted to my laboratory via that subway station and saw the
approaching train, my mind returned repeatedly to thoughts about how the Hayflick Limit might
be tied to the DNA end-replication problem.
In 1972 I had an opportunity to present my theory at the 9th International Congress of
Gerontology in Kiev in the Ukraine.
The director of the Gamaleya Institute at the time that I was on its staff was rumored to be
a colonel in the KGB. He forbid all of his staff from participating in the Congress. I believe that
this was done in connection with the intent to keep the Russian biochemist and gerontologist,
Zhores Medvedev, from attending the same Congress. I well remember my deep indignation
when the director personally told me that the publication of my abstract in the Congress
Proceedings was sufficient and that he forbid me to attend in person. Immediately after leaving
his office I arranged to take my vacation and left for the Congress in Kiev.
After I presented my short paper, there among my listeners was Hayflick. I asked him for his
opinion and he said that it was very interesting but obviously required support from experi-
mental data. Later, I learned that he did not speak to me at length because he was heavily
involved in attempts to learn the fate of his kidnaped friend, Zhores Medvedev, who like me,
had been forbidden to come to the Kiev Congress and had taken his vacation to circumvent the
official denial. Medvedev was later found to have been interrogated by the local police and then
taken by them to the Kiev railroad station and put on a train back to Moscow. He was protected
from harm because of the international outcry made by many Western scientists at the confer-
ence who were organized by Hayflick to come to the aid of their colleague.
More than two decades have passed since my publication. Although I had no doubts about the
446 A.M. OLOVNIKOV
accuracy of my proposal, nevertheless it has been an uncertain and lonely 20 years for me
because, like most innovative ideas, it was unacceptable to many biologists. There was, how-
ever, at least one notable exception.
At an April, 1974 session of the annual meeting of the Federation of American Societies for
Experimental Biology (FASEB), chaired by Hayrick, Robin Holliday presented a lecture in
which he described my theory and referred to my first paper (Olovnikov, 1971). Holliday's
lecture was subsequently published, and represents the first reference to my original paper in
Russian on Marginotomy by a Western scientist (Holliday, 1975).
It was not until a letter arrived a few years ago that I began to feel encouraged about my old
speculations. The letter came from Calvin Harley, who was then at McMaster University in
Canada. He described his experimental evidence (Harley
et al.,
1990) that telomere shortening
could explain the Hayrick Limit (Hayrick and Moorhead, 1961). It was a very exciting and
inspiring moment for me. My thoughts returned to that Autumn day when I left the lecture on
the Hayflick Limit and descended into the Moscow subway station. So, for me the telomere
shortening story has its origin with the report of Hayrick and Moorhead.
In the intervening years telomerists have supplied a remarkable amount of scientific data in
support of the Theory of Marginotomy (for reviews, see Harley, 1991; Levy
et al.,
1992;
Greider, 1993, 1994; Blackburn, 1994; Harley
et al.,
1993; Rhyu, 1995). However, because
most Western scientists do not read the Russian scientific literature, reference to my theory is
made to my later 1973 paper published in English in the Journal of Theoretical Biology, despite
the fact that I made reference to my original Russian publication (Olovnikov, 1971) on the first
page of my 1973 paper (Olovnikov, 1973). Thus, my original and primary publication was made
in 1971 (Olovnikov, 1971) and before the paper by James Watson in 1972, in which he
independently made a similar proposal about the DNA end-underreplication problem (Watson,
1972). Watson, however, did not come to a conclusion on the significance of the phenomenon
for somatic and germline cells or for cancer and aging.
I also published another paper in 1972 on the same problem in which I discussed incomplete
replication at the termini of chromosomes and DNA shortening due to removal of the RNA
primer from the DNA end and the peculularitfies, of DNA polymerase construction and move-
ment. I used, as an example, lymphocyte proliferation (Olovnikov, 1972).
My 1971 paper in Doklady, written in Russian (Olovnikov, 1971), can be summarized as
follows: I first described the "end-underreplication problem" as incomplete replication of the
ends of the DNA double helix and gave to this phenomenon the name "marginotomy." Now,
I realize that this term is not a good one because it may sound unusual to an English speaker's
ear. The phenomenon is often designated in the current literature as "end-underreplication."
I then described in my 1971 paper (Olovnikov, 1971) that the circle form of bacterial DNA
and of all circular DNA in the prokaryotic world offered a form of protection from "margi-
notomy" or underreplication of the DNA termini because a circle has no ends.
I also described my explanation of the Hayrick Limit, in which I proposed that during each
round of DNA replication that occurs during the doubling of normal somatic cells, a portion of
telomeric DNA is lost because of end-underreplication. In this way the cell can count the
number of cell doublings it has already performed. After the loss of a critical portion of the
telomeric DNA, the cells will change their normal, young phenotype to an old phenotype. This
is the Hayrick Limit that results in the process of cellular senescence (Hayrick and Moorhead,
1961; Hayrick, 1965). My predictions were subsequently confirmed by Harley
et al. (1990)
and
other researchers and called a mitotic clock or the telomere hypothesis.
I also explained (Olovnikov, 1971) that germ line cells and tumor cells are able to protect their
TELOMERES, TELOMERASE, AND AGING
447
telomeric DNA from shortening by the expression of a special form of DNA polymerase that
does not exist in normal somatic cells. This polymerase has been identified as telomerase by
Greider and Blackburn (1985, 1989), Morin (1989), and others (Henderson and Larson, 1991).
I also proposed a variant of protection from marginotomy or from DNA end-underreplication by
proposing that a DNA fragment coming from the outside may attach to the telomere end. This
was experimentally confirmed in
Drosophila
by Biessmann
et al.
(1990).
Finally, in my 1971 paper (Olovnikov, 1971), I proposed that the gene of the DNA poly-
merase, which is specific for a germ line cells' immortality, also is expressed in cancer cells,
thus endowing them with immortality also. The enzyme, of course, is now called telomerase and
is now studied by many groups.
One of my reasons for writing this Historical Commentary is to explain to my English-
speaking colleagues the full content of my 1971 paper. I do this because there does not seem to
be a complete understanding of its content as demonstrated by some of the erroneous references
and historical confusion that has now crept into the enormously expanding scientific literature
on the origin of the ideas that led to the discovery of telomere shortening and telomerase.
In particular, I find that my 1971 paper is frequently overlooked in favor of the 1972 paper
by Watson when references are made to the end-replication problem. In the field of cellular
senescence I find many papers that refer to my 1973 paper rather than to my original paper of
1971. I also find authors who do, indeed, refer to my 1971 paper but do not realize that it was
in this same paper that I also proposed the telomeric mitotic counter in senescing cells that
exhibit the Hayrick Limit. Reference to the 1972 paper of Watson is frequently the only
reference made to the origins of the telomerase hypothesis when, in fact, he independently
arrived at this idea that I first speculated upon in my 1971 paper (Olovnikov, 1971).
I am continuing with my theoretical work on the end replication problem and will soon
publish some thoughts on why telomere shortening might benefit normal cells. I prefer to pursue
theoretical ideas because they fascinate me and someone must propose unifying ideas before any
experiments can be designed.
Theoretical work also has the advantage of not requiring great financial resources. My
Western readers are well aware that we were once separated by an Iron Curtain. Today, we are
separated from the other world by a Golden Curtain. Our libraries are starved for recently
published books and journals, and many of our research laboratories are without modern in-
struments or sufficient supplies to conduct meaningful biological research.
Nonetheless, a misty morning does not signify a cloudy day.
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