Hayflick Limit - Debates in BioEssays

Leonid Gavrilov gavrilov at ilr.uucp.free.net
Mon Oct 17 12:30:31 EST 1994


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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