book

The existence of such cell lines poses another question: are they really
“immortal” or the cells in them will die after the 50 duplications as Hayflick
has established earlier? In his opinion, “if these cell lines prove to be
“immortal”, then the reasons for the alterations of age in all living things are
outside the cells” (Hayflick, 1989).
In my opinion the biological aging in the multicellular organisms is a
natural process and essentially it cannot be practically avoided. The reason
is the cell differentiation approved in the course of evolution during the
building multicellular organisms from their unicellular ancestors. Nature has
not had an alternative. To create higher multicellular organisms (including
the man) it has formed different cell colonies, tissues and organs with the
necessary heterogeneity of their cell composition and functional diversity,
restricting the number of divisions within certain limits. Cell divisions in the
different types of specialized cells (epithelial, muscle, nervous,
erythrocytes, etc.) vary not only in number, but also regarding the time
necessary for their realization. That is why the 50 duplications of cell
populations, established by Hayflick as a limiting factor, should not be
considered a constant, since they can be less, and possibly more.
Limited cell division is valid only for the differentiated cells of higher
multicellular organisms. By creating cell cultures from them and liberating
the cells from the “mysterious influence” of cell differentiation, it is
reasonable to accept that their behaviour will be similar to that of their
ancient ancestors, i.e. inevitably the cell lines will become “immortal”.
As far as the process of cell differentiation is ultimately reduced to
blocking the DNA-synthesis, many authors suppose that including of
specific proteins will lead to deblocking its synthesis, which is observed in
so-called dedifferentiation. Elucidating this question will contribute to
revealing the reasons for cell differentiation and dedifferentiation. That is
why, at present a serious attention is paid to these two mutually connected
and at the same time opposite processes.
Naturally a question arises: can the very DNA aging? For the present
there is no definite answer to this question. But having in mind that in all
unicellular organisms and in the cell cultures of plant and animal origin
already obtained through a routine procedure the division and development
of cells can continue without limits, infinitely theoretically (in the presence of
the necessary conditions), at least within relative broad limits senescence
and daying off of DNA are not observed. But during the prolonged
cultivation alterations occur in it, which lead to “aging”. In natural state that
is avoided by exchanging genetic material in the lower, or fertilization in the
higher organisms.

The entering into the essence of biological aging begins with the
discovery of methods of cultivation epithelial and fibroblast cells of higher-
organism tissues, mainly of animal origin in the beginning (Carrel,
Burrows, 1911; Carrel, 1912). The obtained experimental data have
enabled A. Carrel to arrive at a conclusion that somatic cells do not
senescence in vitro and are able to unlimited reproducion (Carrel, 1912,
1913 a, b).
The interest in this problem still more increases after establishing the
limit to the number of divisions in normal (diploid) cells taken from human
tissues and developed in cell cultures (Swim, Parker, 1957; Hayflick,
Moorhead, 1961). In this respect great is the contribution of Leonard
Hayflick (1965), who for the first time makes an attempt to connect the
limited dividing ability of the cells from cell cultures (fibroblast) with the
process of aging.
In its review Hayflick (1989) notes that even in sequoias and bristlecone
pines in California and Nevada, with a life-span of several thousand years,
living cells can be found only in peripheral layer of the cambium and in the
needles not older than 30 years. The other cells are dead and continue to
exist due to the cambium, which is renovate constantly.
Of great importance are Hayflick’s findings leading to the conclusion that
cell cultures obtained from higher organisms (normal embryonic human cells)
have a limited replicative ability. They inevitably senescence and die after
approximately 50 doublings of the cell population, which he called
phenomenon of phase III. Besides, cells possess amazing “memory” and
they “remember” at which number of division have been stored even after
being kept for 27 years in liquid nitrogen (minus 180°C).
The above data raise an essential question: is it possible normal cells
of cell cultures, obtained from higher multicellular organisms and placed in
suitable conditions of development and division to avoid the aging and their
inevitable death? Or briefly speaking — can they avoid biological aging?
The answer of this question is closely related to the elucidation of
possibility such type of cells to be transformed into “immortal”, like the
independently existed unicellular organisms (bacteria, blue-green and
green microalgae, yeasts, etc.), which can reproduce without time limits, if
the conditions necessary for their existence are present.
Here, it is necessary to explain the meaning of “immortal”. This does
not mean that they exist several hundred or thousand years. On the
contrary, their life cycle is only several hours or days. The difference is that
they do not die, but from the mother cells through continuous division new
generations of daughter cells are obtained.
According to Hayflick at present there are thousands of “immortal” cell
lines. As well-known he has shown the L-cell line, isolated from mouse
tissues in 1940 and HeLa-line from human cells used in many laboratories
all over the world since 1951 up to now.

exactly the opposite: its regulation by sudden protein destruction, or
proteolysis, triggered by a molecule named uliquitin”.
For obvious reasons cell is an object of numerous investigations. In the
literature there are given still a number of factors having certain influence
over cell development and division. Nevertheless, the problem connected
with the causes for beginning or stopping of cell division remains far from
elucidation. That is why, it is reasonable to be referred to the “white spots”
of cell biology.
Depending on the structural organization of cells and their place in
evolutionary hierarchy, they can divide continuously (non-stop), to
differentiate and again to dedifferentiate. Thorough review on these
problem is given by Hay (1962). All that argues for the presence of complex
genetic mechanisms determining their broad capabilities for adaptation,
acquired in ontogenesis and phylogenesis. Some of these questions are
related with the biological aging and neoplasms, which will be considered in
the next Section 3. 6.

Biological Aging and Neoplasms

Section 3.6. Biological aging and neoplasms are key problems of biology,
which affect man directly. In recent several decades they attract the
attention of the investigators. Numerous investigations are carried out, but
the causes and mechanisms connected with their appearance and
development as biological processes are not elucidated.
There are different opinions, hypotheses and theories on the nature
and causes for the biological aging and the emergence of neoplasms.
Except mistakes in synthesis of macromolecules, damages of the genetic
material, effect of carcinogenic factors of the surroundings and oncoviruses
(Watson, 1976, and others), recently some authors direct the attention to
the cell cycle, the differentiation and proliferation of the cells (see Goldstein,
1990; Cohen, Ellwien, 1990; North, 1991).

Biological Aging

As a phenomenon the biological aging (senescence) is observed in both
the animal and plant world. The time duration preceding biological death
varies for the different species. The age limit of human life-span is
considered to be about 115—120 years, while for some plants —
bristlecone pine (Pinus aristata) and sequoia (Sequoiadendron giganteum)
— it is of the order of 4—5 millennia.
These data, however, refer to the studying of the problem at multicellular
organism level. Firmly believing that its essence is encoded in the cells which
build the organisms, our attention will be mainly focused on that.

Still unclear are the reasons for the transition of the cells from the
restriction point R to G₁ -phase, i.e. to a new cell division. According to one
of the existing hypotheses there is a necessity of accumulating certain
amount of unstable (trigger) proteins. This supposition is based on data
that their highest content is observed only in phase G1 (Pardee et al., 1978;
Rossow et al., 1979).
Cell division can also be controlled by a number of mechanisms
operating on the feedback principle. These mechanisms are of special
importance, since they limit the uncontrolled accumulation of cells in
individual tissues or organs that can lead to lethal consequences for the
given organism, as it is in the tumour formations. This is most clearly
expressed in animal tissues and organs. For instance, in a state of repose
liver cells begin to divide rapidly after separating a part of this organ and
stop the dividing, when the mass reaches the normal status. Epithelial
mammalian cells, developing in a cell culture on a solid nutrient medium,
also stop the dividing when there is no vacant place for them. This
phenomenon has termed contact retention (Folkman, Moskona, 1978).
Cell division can be caused not only when there is a vacant place for
their growth, but also when they are in a suitable encirclement. In the
multilayer skin epidermis division is observed in only one of the basal cell
layers located on the basal membrane, separating them from the derm. If
the basal cells are located deeper into the derm or above it and have lost
the contact with the basal membrane, they stop to divide. That is explained
with the presence of so-called position signals (Rutter et al., 1973; French
et al., 1976; Bryant et al., 1977, etc.) for which a little is known up to now. It
is assumed that they are realized by means of a number of growth factors
like specific proteins, small molecules of peptide or steroid character,
hormones circulating in blood or substances operating within short
distances — local chemical mediators.
The control and realization of cell division is a complex process.
Undoubtedly it includes different mechanisms, the genetic material and a
number of specific proteins. Some of them were already mentioned, and
possibly there are still unknown ones. Recently the attention was attracted
by a protein widely spread in all living cells — so-called ubiquitin (from
Latin ubique — everywhere, all over). According to Bradbury et al. (1981) in
mammal tissues ubiquitin participates with 2—3% of the amount of
conservative histones. This is a very stable globular protein. The discovery
of bifurcate protein A24, consisting of ubiquitin covalently bound with
histone H2A, is the first direct proof of the role of ubiquitin in chromatin
structure and functions.
“Researchers once expected — noted Barinaga (1995) — that the cell
cycle to be regulated mainly by the well-timed production of regulatory
proteins. But today one of the hottest topics in the cell-cycle field deals with

Causes for Starting or Stopping of the Cell Division

Section 3.5. Elucidating the causes for starting or stopping of the cell
division is one of the basic problems of cell biology. The presence of certain
factors is necessary to stimulate the cell to divide. Some authors (Gurwitsch,
1926; Wasserman, 1929; Swann, 1957) divide these factors in two groups: a)
factors determining the readiness of the cell to divide; b) factors realizing the
entry of cell into division, being prepared for that.
To the first group Swann (1957) refers: increasing the mass of cell;
synthesis of precursors necessary for the division (including DNA and
proteins); creating certain level of energetic reserve; accumulation of SH-
containing compounds (mainly glutathione); synthesis of nuclear RNA; the
mechanisms controlling the sensitivity to temperature and irradiation. A
great importance is attached to various other substances — hormones,
active components of embryonic extracts, etc.
Undoubtedly, these factors play an important role in the processes
proceeding during the preparation of cell for division. There are close
connections and interactions among them. The absence of only one of
them leads to retention or stopping the cell division. Besides, these factors
possess certain independence. Cell growth can proceed in absence of
DNA-synthesis and vice versa, blocking the systems responsible for
sensitivity to the temperature do not terminate cell growth and DNAsynthesis, etc.
Much more unclear is the role of the factors realizing the entry of cell
into division. In this respect also various suppositions are expressed:
alterations in nuclear-plasmatic proportions (Hertwig, 1903, 1908; Popoff,
1908); the necrohormones (Harberlandt, 1921); mitogenic irradiations
(Gurwitsch, Gurwitsch, 1948); activation and reactivation of specific genes
(Epifanova 1965), etc.
The complexity of the cell mechanisms is most clearly expressed in the
processes related with the realization of cell division. Every living cell has to
divide and “prolong its existence” through the subsequent generations or to
die. This is its life, i.e. cell cycle. Its duration varies within a great limits.
To a great extent the differences in the duration of cell cycle are
determined by the period, when the cells are in G1-phase. Some cells
divide very slowly, staying in G1 for days or years. After passing this phase
and in the beginning of S-phase up to the completion of mitosis the
processes are realized an equal same speed, not corresponding to the
previous one. At the end of G1-phase there is a moment after which their
retention is not possible any more. This moment has been termed
restriction point (R-point). After R-point the cells inevitably acomplish their
cycle, irrespective of the influence of surroundings, of course if it is not fatal
(Pardee, 1974).

consecutively, i.e. asynchronously. Then, it should not surprise us that in
living nature the asynchronous division has been estabished as a natural
biological process. It is more suitable in view of potential energetic
possibilities of the cells, depending on a number of factors including the
conditions of surroundings.
Often in the literature the terms “synchronous” and “asynchronous”
cell culture are mixed up with those of synchronous and asynchronous
division. Obtaining synchronous cultures is achieved by using definite
methods in order to lead the cell population to the same phase of
development, while synchronous or asynchronous division is the mode of
realizing the reproduction in the time. The synchrony index varies but
never achieves 100%. That is why it is better to use the terms
synchronized and asynchronized cell culture instead of synchronous and
asynchronous. According to Mazia (1963) “nobody has succeeded to
force cells to grow synchronously and to maintain such a synchrony for a
long time”.
The cause for impossibility to obtain completely synchronized cell
cultures is the asynchronous nuclear division and daughter cell formation.
Because of that the nuclei available in mother cells, though having equal
genetic material, are not equalized regarding their mitotic stage. That is
why the extent of synchronization of a given population should be
considered relative and cannot be determined only on the basis of the
number of nuclei and morphological characteristics. It is also necessary
to take into consideration the phases of development of the nuclei in cells
that are synchronized.

factors — nutrient substances, temperature, pH of the medium, potential
genetic and energetic possibilities of the cells, etc. It is difficult to ensure
them simultaneously, especially in natural conditions. For example, in
case of changing only one of the factors necessary for the development
of an optimum synchronized culture of Scenedesmus acutus (temperature
below 20°C or pH below 6) under equal other conditions, the cells begin
to form two or three nuclei, instead of four, eight or more, and divide very
slowly.
2. What is the way of realizing the formation of daughter cells and the
distribution of cytoplasm in the mother cell, i.e. whether it occurs
immediately after each nuclear division or after reaching a “crucial”
number of nuclei, when the mother cell will start releasing the new
generation of daughter cells?
This questions has been attracted the attention already at the time of
Smith (1914), then of Mazia (1963), Pickett-Heaps (1975) and many other
investigators. But even now it is still unclear. According to Pickett-Heaps
“the divisions that occur after the completion of mitosis have proved to be
most difficult regarding ultrastructure studies and they are largely resisted
analyses”.
3. What are the reasons and mechanisms determining the “decision”
of a nucleus to start a subsequent division or not to divide and form a
daughter cell?
This is a real “mystery” of cell biology, and it will be very hard to be
solved. Here is the key to the asynchronous division approved in the
course of evolution. In my opinion it is realized asynchronously,
consecutively on the principle “nucleus — cell”. Violating this principle
leads to multinuclear cells, mentioned in the previous Section 3. 3 —
symplasts, osteoclasts, HeLa-cells, etc., where cell division does not
proceed normally.
Accepting the asynchronous nuclear and cell division as a natural
biological process (Nicolov, 1997 a) gives rise to the necessity to change
the trends and methods of cell research. The reason for such a mode of
division should be looked for mainly in the genetic material, in eventual
differences between the copies of DNA-replication and in the energetic
possibilities of cells, acquired as a result of the metabolic processes in
them.
Let pay attention to the energetic aspect of the question, that could
be the decisive evolutionary factor determining the way of realizing the
reproduction of nuclei and cells in time. It is known that every biological
process needs some energy. Undoubtedly, nuclear and cell division is
connected with such processes. If in one cell of Scenedesmus acutus or
Clorella vulgaris there are four or eight nuclei and they are to reproduce
simultaneously, i.e. synchronously in eight or sixteen, respectively, much
more energy for a given time will be necessary, than if that proceeds

formation of 16 or 32 autospores, and after breaking down the mother-cell
envelope the cycle is repeated.
Similar are the cases with T. Sedova and B. Zakryś. Studying the
sporogenesis in the unicellular green alga Palmellococcus (Sedova,
1972) has observes a cell with three nuclei, which gives grounds to draw
a picture of asynchronous division, but she overlooks this fact without any
explanation. In Euglena cells (Zakryś 1980, 1983) has registered uneven
number of nuclei and a generation with uneven number of daughter “cells,
considered by him as” aberrative divisions with rudimentary character.
A great number of such cases can be given from the literature. That
demonstrates the enormous influence of the conception of synchronous
division on interpreting the results of this kind of studies.
Analyzing the results of the studies performed on unicellular green
algae of Scenedesmus and Chlorella genera (Nicolov, and Nicolov et al.,
1982—1997 a) made it possible to draw the following important
conclusions:
First. Nuclear division occurs asynchronously up to the formation of
“crucial” number of nuclei, and when the mother cells have formed the
corresponding cell coenobia they go on to division. At that always in one of
the nuclei, more advanced in its development than the others, the next
division is realized earlier.
Second. The formation of daughter cells (autospores) also occurs
asynchronously, by analogy with nuclear division.
Third. Observing two dividing nuclei in one cell (see Fig. 3–18 e)
cannot be considered proof of a synchronous process of division.
Reasonably is to suppose that these two nucleus are in different phases,
which cannot be recorded by a cytological picture fixing a given moment
statically. That can be consider time-coincidence caused by the dynamic
processes ensuring the rapid formation of numerous nuclei necessary for
obtaining a great amount of daughter cells (autospores).
Fourth. The presence of even number of nuclei (two, four, eight,
etc.) as well as obtaining such number of daughter cells in one
coenobium, which meets the basic requirement of the conception of
synchronous division, does not mean that nuclear and cellular
reproductions have occured synchronously. These cases can be consider
separate stages of one asynchronous process, where there are both even
and uneven number of nuclei and daughter cells (see Fig. 3–24).
The asynchronous nuclear and cell division poses a lot of questions.
Three of them deserve a special attention.
What kind of process is that realizing the asynchronous nuclear
reproduction, i.e. whether it is uniform, with equal periods of the individual
cycles or it is irregular?
On the basis of the essence of asynchrony as biological process one
can suppose its irregularity. Its realization is a function of combining many

al., 1978; Ghosh, Paweletz, 1984; Stephenson, Gooday, 1984;
Petrashchuk, Onishchenko, 1987, etc.). It should be noted that their
interpretations are not unidirectional and in most cases they are considered
as unusual phenomena or exceptions. Some of the above mentioned
authors (Johnson, Rao, 1971; Ghosh et al., 1978; Ghosh, Paweletz, 1984)
try to find the reasons for these “exceptions” in differences in size, nature
and origin of the nuclei, non-optimal conditions of the nutrative regimen, in
metabolism, compartmentalization of the cytoplasm and their like.
Until recently the observed cytological pictures of asynchronous
division caused a perplexity. In his book “Mitosis and Physiology of Cell
Division” D. Mazia (1963) considers “unusual phenomenon” the
registered by Haque (1953) asynchronous division of nuclei in one of the
two binucleate cells, representing pollen grains of Tradescantia paludosa
(Fig. 3–28). In confirmation of his opinion he cites the words of Holden
and Mota (1956) that “even in unusual cases, when two nuclei in the cell
undergo mitotic divisions with a different speed, they can enter in
prophase simultaneously”.

image

image

Figure 3–28. Synchronous (A) and asynchronous (B) mitosis in 
binucleate pollen grains of Tradescantia paludosa (After Haque, 1953; 
From Mazia, 1963). 

Peshkov and Rodionova (1964) have also registered a cell with two
nuclei, which are in different phases of division. After that Peshkov (1966)
analyzing the peculiarities of cytology and karyology of some unicellular
and multicellular green algae considers a mistake the interpretation of this
figure, given in their earlier work, where they have described it as a single
metaphase plate, and actually these are two separate groups of
chromosomes in different mitotic phases. As a result of this analysis he
has supposed that in Chlorella vulgaris during the transition to prophase
the mitotic division can proceed asynchronously. Nevertheless, after that
this great researcher emphasizes that divisions occur synchronously, one
after another, passing through the individual mitotic phases up to the

Figure 3–27. Histograms of synchronized (1—5) and asynchronized cell 
cultures of Scenedesmus acutus (Nicolov et al.,1987). 
1 — zero point; 2 — at the second hour; 3 — at the fourth hour; 4 — at 
the eight hour; 5 — at the tenth hour; 6 — asynchronized culture 
N — sample 1 ml.

In the literature there are data about single cases of asynchrony
observed during DNA-synthesis, in some phases of the mitosis and the
behaviour of nuclei. Such data are met in the papers of many investigators
(Wimber, 1961; Zeuthen, 1963; Nilova, Sukhanova, 1964; Stubblefield,
1964; Harris, 1965, 1967; Harris, Watkins, 1965; Oftebro, Wolf, 1967; Kato,
Sandberg, 1967, 1968; Johnson, Harris, 1969; Krishan, Ray-Chaudhury,
1969; Rao, Johnson, 1970; Johnson, Rao, 1971; Heneen, 1971; Ghosh et