TO: <ageing at net.bio.net>
FROM:
Dr. Leonid A.Gavrilov, Ph.D. <aeiveos at glas.apc.org>
A.N.Belozersky Institute
Moscow State University
119899 Moscow, RUSSIA
FAX: 7 (095) 939-0338
7 (095) 939-3181
October 17, 1994
Dear Sirs,
Recently we have received a lot of E-mail messages asking me to
reproduce on the AGEING list the scientific debates between Dr.Leonard
Hayflick and ourselves (Dr.L.A.Gavrilov and Dr.N.S.Gavrilova) that
were published recently by BioEssays (16/8 AUG 1994, p.591-593).
Because of copyright restrictions, we cannot do that for Hayflick's
paper: please see BioEssays. But we can reproduce here our response
to Hayflick's paper in its original form, since BioEssays has eliminated
many important parts of it because of the limited journal space.
Sincerely yours,
Dr.Leonid A.Gavrilov, Ph.D. and
Dr.Natalia S.Gavrilova, Ph.D.
P.S.: Our original paper submitted to BioEssays (and published in
abridged form) is printed below:
--------------------------------------------------------------------------
The Weismann-Swim-Hayflick concept of proliferative limit:
historical and critical comments
by Leonid A.Gavrilov and Natalia S.Gavrilova
Although the Weismann-Swim-Hayflick concept of proliferative limit
was already discussed in great detail in our book (1) which has received a
lot of attention from many scientific journals including BioEssays (2-20),
we are very pleased by invitation of BioEssays to return to this discussion
again and to reply to Dr.Hayflick's comments. Since his comments have a
lot of quite different declarations mixed together, and since not all the
readers of BioEssays have read our book, we shall start our reply from the
very beginning in a chronological manner (in an abridged form since all the
details could be found in our book):
1. The idea that the limited lifespan of organisms is determined by the
limited capacity of somatic cells for division was originally created not by
Dr.Hayflick in 1961, but by the famous German biologist August
Weismann a century ago (21). Moreover, it was Weismann who postulated
that the differences in the longevity of animal species are caused by the
different number of generations that the somatic cells of each species can
produce (the cells of long-lived species are capable for completing more
divisions).
Weismann's idea has received a lot of attention in our century too. For
example, the Nobel Prize winner and the founder of gerontology (and the
father of the term 'gerontology') Ilya Mechnikov devoted a special chapter
for discussing and criticizing the Weismann's theory of cell division limit in
his famous book "Essais optimistes" (22). Since this book was published
for many times (at least in 1907, 1908, 1913, 1964, 1987) any scientist
interested in aging research had an opportunity to read about Weismann's
theory of cell division limit. Certainly, it is very nice that Dr.Hayflick
after some delay of about 20 years has finally received some knowledge about
the Weismann's theory too and there is a complete mutual understanding
now at least in this issue.
2. The author of the first convincing experimental evidence and clear
conclusion that animal cells in culture cannot be propagated indefinitely
was not Dr.Hayflick but another American scientist, Dr. H. Earle Swim
from Western Reserve University School of Medicine in Cleveland, Ohio,
together with his co-authors, Dr. Robert F. Parker and Dr. R.F.Haff (23-25).
In 1959 after analyzing results from 336 publications, including the
results of his own experiments on the serial cultivation of 23 strains of
fibroblasts derived from normal tissues of the rabbit and chick embryo (23),
as well as 51 strains of human fibroblasts derived from foreskin, placenta,
testicle, uterus and embryonic tissues (24), Dr. Swim came to the following
fundamentally important conclusions: "... in most instances where growth
occurs the cells eventually undergo nonspecific degeneration" (25, see
p.145). "The common experience of many investigators indicates that the
early cultivation of cells usually follows a characteristic course which can
be conveniently divided into three phases. In phase I the cells proliferate
rapidly after an initial lag and usually can be transferred serially without
difficulty. Phase II is characterized by a decrease in multiplication to a
point where it usually ceases and the cells are eventually lost as a result of
nonspecific degeneration" (25, p.159). "This was accompanied at first by
an increase in the number of granules in the cytoplasm of the cells; later,
degenerating cells were observed and their numbers increased progressively
until the bottoms of the flasks were covered with a dense layer of cellular
debris..." (23, p.201). The important conclusion was also drawn that when
cells stop multiplying, this is not a methodological artifact caused by such
factors as inoculum size, toxic media, or their inability to proliferate on
glass (24). Finally, Dr. Swim (25) notes that "infrequently a third stage is
recognized by the appearance of actively proliferating cells in phase II
cultures." (p.159). "It should be emphasized that phases I and II represent
the usual pattern, while phase III is a relatively rare event." (25, p.160).
Dr.Swim (25) also noted that in these rare cases of re-appearance of active
proliferation the new proliferating cells often differ from the original cells
both in morphology and growth pattern (cell transformation).
3. In view of the above mentioned quotations from Dr. Swim it is clear
that such Dr.Hayflick's statements as "Swim ... describe no phases" and
"Moorhead and I were the first to ... describe the three phases" are
absolutely wrong. Moreover, the notations used by Dr.Hayflick are
unbelievably similar to notations of three phases introduced earlier by Dr.
Swim (including even the same Roman numbers I, II and III !). Perhaps
Dr.Hayflick might wish to give a rational explanation for such a fantastic
similarity between his papers and the earlier paper by Dr. Swim (25).
4. It is true that Dr.Hayflick was the first to declare that limited
proliferative capacity is the property of all diploid cells and diploid cells
only. Unfortunately, this declaration is wrong in both directions:
First, there are a lot of aneuploid and polyploid cells in 'old' cultures,
thus the lack of diploid karyotype per se is not sufficient for unlimited
proliferation.
Second, virtually unlimited proliferation could be observed for normal
diploid cells too (if these cells do not embark on terminal differentiation).
For example, it turns out that normal diploid mouse embryo cells, which
under standard conditions manifest a growth crisis after 7-10 population
doublings, may be successfully cultivated without any sign of an
approaching growth crisis for at least 200 population doublings. All that is
necessary is to change the composition of the culture medium (excluding
blood serum and adding a number of ingredients, including the epidermal
growth factor). In this case the cells, which are apparently capable of
unlimited multiplication, remain diploid and nontumorigenic (26).
Certain normal diploid cells show a practically inexhaustible capability
for proliferation not only in vitro, but also in vivo. For example, it is well
known that normal cells of drosophila imaginal discs can proliferate
indefinitely if their differentiation inductors are absent (27). It is also
well established that there is no any intrinsic limit to the proliferation
of normal hemopoietic stem cells (27). Thus, the declaration of Dr.Hayflick
that proliferation limit "is an intrinsic property of all normal cells"
is definitely not true.
5. We would agree with Dr.Hayflick that cells "do eventually die". The
only problem is that this declaration means nothing. For example, the
atoms of radioactive elements also "do eventually die", but they do not age
(their 'rate of dying' is constant and does not increase with age). The same
is true for cell cultures: there is no evidence for real aging, i.e., age-
dependent increase in cellular mortality rates. Instead, the cellular cultures
are surprisingly claimed to be senescent and dead simply because they
stopped active proliferation. This is definitely unacceptable definition of
death since according to it all of us have dead brains ! It is clear that
decrease in proliferation rates is not necessarily a manifestation of cell
deterioration and aging; instead, it might be a consequence of 'healthy' cell
differentiation (see our book for details). For this reason the so-called
'aging' in cell cultures may have no any relation to the problems of real
cellular aging.
6. Since Dr.Hayflick decided to "raise serious questions about the
judgement and competence" of Dr.Downes, we would like to mention that
Dr. Downes was very careful in the review of our book and in discussion of
Dr.Hayflick's scientific contribution. Some other book reviewers, for
example, Dr.Brian Merry, made much more definite conclusions: "the
authors justifiably devote a large proportion of this important chapter to a
careful study of the phenomenon of limited in vitro cell division, often
referred to as the Hayflick limit. They present a most thorough and timely
re-appraisal of this data and they question the relevance of this popular
model to the ageing process" (5). Similar comments could be found in
other book reviews published by Free Radical Biology & Medicine (6),
Human Biology (7), British Medical Journal (8), Population and
Development Review (9). Thus, the waiting list for receiving Dr.Hayflick's
penalties is too long to "raise serious questions about the judgement and
competence" of Dr.Downes and any other person who has his own opinion
on Dr.Hayflick's work.
7. Finally, we would like to emphasize that Dr.Hayflick has made a
significant contribution into promotion of the Weismann's ideas,
reproducing Dr. Swim's experimental results as well as their further
development. For this reason in our book we called this scientific approach
the Weismann-Swim-Hayflick concept. The fact that the name of
Dr.Hayflick is mentioned not on the first place was due to historical reason
only and should not be considered as an attack on him personally (perhaps
he might wish to consult his psychologist for this purpose). Instead
Dr.Hayflick might wish to organize a scientific meeting in 1997 to celebrate
the 40th anniversary of the Dr. Swim's discovery at the School of Medicine
at Cleveland where Dr. Swim worked. This meeting might be sponsored by
American Federation for Aging Research where Dr.Hayflick is a key
person, and we would be happy to take part in such a meeting together
with Dr. Hayflick and Dr.Downes to discuss the issues of mutual interest.
Such meeting might be interesting to many readers of BioEssays too.
References
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