Medical Hypotheses 4: 1, 58-77, 1978.

GENERAL THEORY ON THE CONTROL OF CELL CYCLE

Semih ERHAN, 2101 Chestnut Street, Philadelphia, PA. 19103, USA.

SUMMARY

A. Cell cycle control in normal cells: The theory developed is based on the premise of the antagonistic

effects of replication trigger ( RT ) and mitotic inhibitor ( MI ), both of which are, in all likelihood, glyco-

proteins. The former can act either on the chromosomes or on the membrane receptors while the latter

acts only on receptors found inside the cellular membranes. The model proposes that: a. These membrane

receptors are glycoproteins; b. RT receptors have molecular symmetry and can bind each other and they

can move freely within the membranes; c. Primary control of proliferation is due to contact inhibition of

movement, whereby RT receptors bind each other and prevent RT from binding them; d. MI acts as a fine

control element and particularly during regeneration of tissues; e. Both RT and Ml act through cyclic nuc-

leotides. B. Differentiation and aging: This is an extension of the model and further suggests that: a. Dif-

ferentiation is a result of the asymmetry found in all cells; b. Differentiation is triggered by a decrease of

a maternal macro-molecule - a particular mRNA - below a threshold level; c. The initial event is the induc-

tion of histone methylases and this event is stimulated by CAMP; d. Aging is an inevitable consequence

of differentiation and is due to secondary interactions developing between histones and non-histone chro-

mosomal proteins (crosslinking, ionic-, -S-S- bonds etc.) of the genes that are not needed for daily activi-

ties of the cells. C. Neoplustic changes: The flawless formation of these receptors in sufficient quantity,

becomes critically important against unscheduled DNA replication and subsequent cell division. Hence

any agent - physical or chemical - that interferes with the formation of these glyco-protein receptor mo-

lecules as well as that leads to the destruction of the already existing membranes or membrane receptors

is a potential carcinogen.

CELL CYCLE CONTROL IN NORMAL CELLS

INTRODUCTION

The finely tuned and extremely well coupled series of reactions which gives rise to the phenomenon called

“ life ” , appears to be directed for self perpetuation. This tendency which expresses itself by the ever pre-

sent urgency of living cells to devide, is tempered in higher organisms by a concern over the control of un-

scheduled cell division. This we observe as early as the appearance of differentiation in the developing em-

bryo. The potential for division, however, is not lost but only masked in the higher organism that emerges

at the end of the differentiation. A cell’s life starts with cell division, followed by an apparently quiescent

period leading to the replication of the genetic material of the cell, which has to occur before the next

round of cell division can take place. There is, again, a seemingly quiescent period even though this one

is usually much shorter than the previous one. Cell division, which is also called mitosis, terminates this

process giving rise to two daughter cells. It is obvious that this phenomenon is cyclical, hence the concept

of cell cycle. The cell cycle is customarily divided into four unequal parts, all of which are experimentally

determinable: Gl , S , G2 , D . Both cell division ( D ) and DNA replication ( S ) could be seen and identi-

fied, under the microscope. For decades, however, the quiescent periods posed a mystery. That is why

they were called gap one ( G1 ) and gap two ( G2 ), for want of an explanation. Now we know that the

precursors necessary for the replication, be they low molecular weight intermediates or enzymes, are pro-

duced during G1, which can be looked upon as growth period. G2 is also a very busy period, where the

activities culminate with the formation of the mitotic apparatus. Many factors have been isolated that

stimulate DNA replication (1,2,3) or inhibit cell division (4,5). Chemically most of them appear to be

glycoproteins.

Thus the events that control the cell cycle assume a dominant role for the well being of the organism.

58

This section will describe a model for the control of cell cycle of higher organisms. The next section will

deal with the problems of differentiation and aging and the last part will try to analyze what causes neoplas-

tic changes.

The model

1. All cells are capable of producing a replication trigger (RT) and a mitotic inhibitor (MI); both of these

molecules are glycoproteins. RT is universal while MI is tissue specific. Both RT and Ml effects are thre-

shold prenomena, i.e., they can only be effective when their concentrations exceed a minimum threshold

level, enabling them to tie up a certain fraction of the membrane receptors.

2. TR can function in two different ways: a. on the cell chromatin, b. on specific membrane receptors

found on the outside of cell membranes.

3. Once synthesized inside the cell RT binds to replication initiation region of the chromosome(s). At

the end of DNA synthesis replication machinery dissociates and RT leaks out of the cell and becomes the

“ humoral factor ” which can act by binding to the membrane receptors.

4. In higher organisms RT is produced continously by certain tissues such as bone marrow, intestinal epi-

thelial, mucosa cells etc., which after being released as described above provide the humoral factor that

circulates in the blood stream and acts as the initiation factor needed to replace cells, in various tissues,

due to normal turnover.

5. RT receptors are found outside the cell membranes and under certain circumstances are free to move

throughout the membranes. They have three dimensional complementarity so that receptors on adjacent

cells can bind each other and make the receptors inaccessible to RT.

6. Genes coding for TR are turned off at the beginning of differentiation while MI is produced continously

throughout the entire life of the organism. When produced simultaneously in a cell, RT prevails over Ml

because it can act directly on chromatin while MI has to act through its own specific receptors found in-

side the cell membrane. However, RT effect lasts only for one cell division.

7. In higher organisms the cell cycle is primarily controlled by contact inhibition of movement ( CI ),

which, by tying up the membrane receptors limits the accessibility of these receptors to RT.

8. Superimposed on this is the control exerted by MI which acts as a fine control of cell division and is

particularly effective toward the end of wound healing and tissue regeneration. This inhibition is depen-

dent upon the total concentration of MI produced by each cell and collected in the tissue, through inter-

cellular bridges. MI synthesis as well as methylation of histones are triggered by CAMP.

Discussion

The cell cycle of all cells appears to be controlled at two points by two different factors:

1. An RT probably acting at Gl/S juncture of the cell cycle (1,2,3,6,7,8)

2. An MI acting at G2/D juncture (4,5,9,10).

All living cells are capable of producing both RT and MI at a constant level. RT is a glycoprotein synthesi-

59

zed under the control of a specific gene. Once synthesized in the cytoplasm it penetrates the nuclear mem-

brane and binds to the initiation region(s) of replication on the chromosome(s). The rest of the replica-

tion related proteins then bind to this RT-DNA complex in a unique three dimentional conformation (11).

At the end of replication the complex dissociates, the RT leaks out of the nucleus and the cell membrane

and becomes the humoral factor. Under these circumstances it can bind to the cellular receptors found

outside the cell membranes. In higher organisms RT is produced continously by some tissues, such as bone marrow etc., and provides a steady supply of humoral factor. Some glycoproteins other than RT, which

may share a short amino acid sequence as well as a few sugars with RT, may also be capable of binding

and thus stimulating the membrane receptors. This may explain why so many seemingly unrelated proteins

have been reported to stimulate replication in various systems. This factor helps to initiate replication

of DNA in those cells which have to divide in order to replace the cells dying due to normal turnover

in various tissues. I propose that these receptors to which RT binds (12,13,14) as well as the MI recep-

tors, which are found inside the cell membrane (15) are also glycoproteins. The receptors for RT are

synthesized in the GI period, with the maximum rate of synthesis toward the end of GI (16). They are

inserted into the membrane after their synthesis is completed. The cell membrane is also very fluid and

mitogen receptors, for instance, have been shown to move within the membrane (17,18). I propose that

the receptors for RT have their binding site outside the cell membrane together with two enzymes: gly-

cosyl- and sialyl-transferases (19,20). Two other enzymes are found inside the membrane within the re-

ceptor complex: guanyl cyclase (G-cyc) and CAMP phosphodiesterase (A-PDE). All of these enzymes are

stimulated when RT binds to the receptors.

The receptors for MIS, which are tissue specific glycoproteins, have their binding site inside the membrane,

together with two enzymes: adenyl cyclase (A-cyc) and cGMP phosphodiesterase (G-PDE). Binding of

MI stimulates these enzymes.

Proximal to G-cyc there is also a Ca++ pump which normally extrudes Ca++ from the cell, but under

the influence of RT the pump promotes Ca++ uptake into the cell (21) (Figure 1).

When RT is bound to a cell receptor, G-cyc as well as A-PDE is stimulated. This leads to an intracellular

increase of cGMP and a decrease of CAMP concentration. Ca++ taken up, which follows cGMP increase

by 10 minutes in lymphocytes (22), reinforces the RT effect by stimulating A-PDE and inhibiting A-

cycl; cGMP formed also stimulates A-PDE (23). Thus the humoral factor creates conditions conducive to

cell proliferation: increased cGMP and decreased CAMP, which stimulates histone methylases as well as

MI production.

This is followed by stimulation of RNA polymerase I, then *RNA polymerase II and III (21) as well as

by phosphorylation of certain acidic chromosomal proteins (24) and transport of a particular cytoplasmic

protein into the nucleus (25) and binding to cellular DNA (21, 26). Ca++, furthermore, appears to be essen-

tial for the induction of furrow formation as it was shown to be needed for deposition of filament in the

wall of incipient and advancing furrow (27). I further propose that the glycoprotein molecules have three-

dimentional complementarity in such a way that receptors on neighboring cells can complement each

other in a lock and key fashion, Because these receptors can move freely within the membrane, when a

few cells meet, a situation which occurs quite regularly in cell cultures, they bind and neutralize each

other. This situation is called “ contact inhibition of movement ” or simply contact inhibition (Cl). Re-

ceptors engaged in CI are no longer available for humoral factor. All of the membrane receptors cannot

and need not to be engaged in Cl, as the effect of RT is a threshold phenomenon. The actual fraction of

the available receptors that need to be engaged in CI, to prevent untimely stimulation by RT, may depend

upon the source of the cells.

MI effect, too, is a threshold phenomenon, hence a fraction of the receptors need to be bound by MI

before its effect can be seen fully. I propose that each receptor of vital organs, which binds an MI mole-

cule, releases a chemical messenger to the outside so that the optimum total concentration of the tissue

60

c;a - +------

5’-AtiP

/

Fig. 1. FIGURE LEGEND

Portion of cell membrane showing elements which participate in the control of cell cycle. MI: mitotic

inhibitor; RT: replication trigger; G-PDE: cGMP phosphodiesterase; Gcyc: guanylcyclase; A-PDE: CAMP

phosphodiesterase; Acyc: adenyl cyclase; GT: glycosyl transferase; ST: sialyl transferase; Cap: calcium

pump. b+> : inhibition; F> : stimulation.

61

MI can be monitored. The optimal tissue concentration of MI is maintained through cell-cell bridges

(28), the MI molecules which are lost being promptly replaced by de nozw synthesis.

When CI occurs the binding sites of the receptors become inaccessible to RT, which immediately leads to

a drop in cGMP concentration and together with Ca’+ extrusion from the cells results in a reversal of the

events described above and sets the stage for the quiescent state of cells. Since CI would occur early in the G I, this point corresponds to the R point for the control of cell proliferation described by Pardee

(29) The effect of RT is good for only a single cell division (30,31). Unless RT is present continously,

cell division stops after one cycle.

The presence of tubulin in various tissues has been. demonstrated (32,33). Treatment of these cells with

cytochalasin B, colcemid, vinblastin (agents known to disrupt microtubule/microfilament systems) was

shown to cause paracrystalline aggregates of tubulin. Supernatant fractions of the homogenates of purified

mouse lymphocytes (corresponding to a membrane enriched preparation) were also shown to have col-

chicine binding activity (34). The presence of microfilament-microtubule systems within the membranes of

CHO-Kl cells have also been proposed, based on the reversible changes elicited by colcemid and cytocha-

lasin, on the one hand, and dibutyryl CAMP, on the other: addition of the CAMP derivative was shown

first to eliminate the appearance of violently extending and retracting knobbed structures found on epithe-

lial-like appearing cell membranes, leading finally to fibroblast-like appearing cells showing CI. Addition

of colcemid to these cells, then, converted them to epithelial-like cells with throbbing knobbed structures

(35.) Polymerizing effect of CAMP on microtubules, however, has not yet been demonstrated conclusively.

During proliferation the cells are most likely to be in an epithelial-like form, where microfilament-microtu-

bule systems of the membranes are not organized. During this period the membrane RT receptors are free

to move within the membrane and bind receptors in adjacent cells. Once this occurs and the receptors

are no longer accessible for the humoral factor then the quiescent stage is reached whereby CAMP concen-

tration increases. I propose that microfilament- microtubule systems are assembled under the influence

of CAMP. During this organization a maximization of the cell-cell contacts is achieved and the tendency

of cells to be aggregated by Con A is reduced.

The receptors that have not been engaged in CI could get involved in Con A or PHA-mediated agglutina-

tion, were it not for a nondialyzable heat-, trypsin- and cycloheximide- sensitive molecule which can also

be removed from the cell surfaces by dilute urea treatment (36) which decreased the chances of unsche-

duled replication. I also suggest that an increase in CAMP concentration initiates the methylation of histo-

nes which increases the affinity of histone binding to DNA.

Application of the model to various in vitro and in vivo conditions

1. Cell culture: Each cell produces a certain amount of RT which leaks out of the cells into the medium

at the end of replication. In order for a sufficient level of RT to be present in the medium, to sustain

growth of all cells, a minimum number of cells have to be used as the inoculum, otherwise certain supple-

ments have to be added into the medium (37) or a feeder layer has to be furnished to supply the necessary

factor(s). Failure to do so results in a dramatic drop in the viability of cells as measured by plating effi-

ciency. As confluence is reached, the surface receptors meet and bind each other, reducing the number

of receptors available to RT below a minimum threshold. Cells that respond to the addition of fresh enri-

ched medium, such as 3T3 cells, may be lacking in the protein removable by dilute urea treatment and

cells may undergo a limited cell division for one cycle.

When confluence is distrubed, say, by a policeman, the number of exposed receptors increases and the RT,

which is present in the medium, initiates replication and cell division. This continues until new cells produ-

62

cd come into contact with each other.

2. IUI ~KSUP\: When some cells die, due to normal turnover, and are autolyzed, their receptors will also be

degraded leaving certain cells with exposed receptors, which will then be accessible to humoral factor

and a limited cell division will follow until replacement of dead cells is completed.

Wound healing represents a similar situation. First cell-cell contact is destroyed by the wound and then

the cells at the site of loss of Cl are exposed to humoral factor, which stimulates cell division. When new11

formed tissue cells reach each other, cell division stops. Here MI may also play a role by blocking any cells

that may not be exposed to other cells, a situation that occurs at the surface of a tissue which does not

touch another tissue, from dividing, ( a detailed discussion will be given below for tissue regeneration).

3. Kr~~c~ten~ticin: The major difference between wound healing and tissue regeneration is the need to stop

cell division when the original, preoperative, size of the organ is reached. This, of course, is also the major

difference between normal cells vs. the neoplastic cells and the third section of this manuscript will deal

with this issue.

The stop signal comes from MI or more precisely, the total concentration of MI of a normal tissue IS what

controls the size of that tissue. In other words there are means by which the organism senses the total MI

concentration of its tissues. The sensor may be a gland, where the concentrations of the molecules relea-

sed by intracellular MI receptors are measured by their binding to other specific receptors.

Of course the cells that are found inside a tissue are still primarily prevented from cell division by Cl,

and MI concentration acts on the peripheral cells. When a portion of liver is removed the following events

are likely to take place: a. Cell-cell contact is eliminated from the cells at the regions releasing the inhibi-

tion, and, b. Cells become accessible to humoral factor which triggers replication, leading to exponential

growth, c. During the 72 hours after hepatectomy, about 75% of total DNA synthesized occurs in hepa-

tocytes, and hepatocytes in zones 1 and 2 account for about 80% of this replication (38). The newly

formed cells are inhibited from further division by CI, d. As the number of cells increases, the total amount

of MI procluced increases and gradually slows down cell division, c. When total MI concentration reaches

the level produced by liver before hepatectomy, cell division stops.

Ligation of a portion of liver limits the total MI available to the remaining tissue thus releasing the inhibi-

tion to cell division under the influence of humoral factor, which readily reaches there through the blood-

stream When the remaining liver reaches normal size, total Ml concentration is restored and further growth

is prevented.

.4fter single-nephrectomy a similar situation prevails. The body of the organism is tuned to function with

a certain level of kidney chalone. When one kidney is removed the inhibition of cell devision is released,

until regeneration taking place in the remaining kidney restores the original Ml concentration.

For all these processes to take place, there is no need for the RI genes of the differentiated cells to bc

derepressed; they take place because of the cascade of events that leads to gene activation. If the R-1‘

genes were activated, and there are circumstances under which this takes place, then one is faced with

a situation known as neoplasia.

63

DIFFERENTIATION AND AGING

Introduction

As mentioned above, the tendency to divide, which is the overwhelming driving force of all living cells

that is seen so forcefully among microorganisms, has been tempered in differentiated higher organisms.

Evolution had selected the path whereby this tendency to divide is checked constantly. Hence in higher

organisms controls against cell division prevail in all but a few tissues. This suppression occurs primarily

by CI and secondarily by repression of genes, which are not needed for day to day activities of differentia-

ted cells through the specific interaction between basic and acidic nuclear proteins and cellular DNA.

Since most of the DNA of higher organisms is protected by nuclear proteins, there must be a difference be-

tween nuclear proteins found on repressed and unrepressed genes. This section will present a model which

attempts to describe the events during differentiation, starting with this difference and its relation to aging.

The model

1. Differentiation is a direct consequence of the polarity which is found in all cells, but specifically in the

egg cells;

2. The asymmetry produced by the first cleavage is expressed by the components of the cell surface. The

temporal order of the synthesis of various membrane components controls the progress of differentiation;

3. The decrease in the concentration of a maternal macromolecule to a critical threshold level triggers

the initial event in differentiation: the formation of histone methylases;

4. Differentiated cells have negative control on cGMP synthesis and positive control on CAMP synthesis

(Figure 2) ;

5. CAMP stimulates alkylation of histones;

6. Phosphorylation of histones is involved in cellular events leading to cell division while acetylations are

related to transcription;

7. Fine tuning of transcription is controlled by methylation of DNA;

8. MI gene is turned on.

Discussion

Any discussion of differentiation becomes very quickly unmanageable unless one agrees upon certain

ground rules. This is so because the methods used to analyze differentiation can to a great extent influence

the interpretation of the results obtained. For instance, there is a dogma which places differentiation and

proliferation into an antagonistic relationship (39,40,41). One can, of course, find many arguments in

favor of (42,43,44) and against (4.5,46,47) this dogma. Another problem arises from the conflict between

the generally accepted view that differentiation is a restriction of expression of most of the available

genes and that the differentiation is the expression of new genes which were repressed earlier (48). Dif-

ferentiation of systems which differentiate as separate elements, such as blood cells or pigment cells, may

follow different rules with respect to division and differentiation than do tissue systems. If on the other

64

(a)

Fi,y 2, FIGURE LEGEND

a) Control of cGMP concentration during cell cycle. Solid line:guanyl cyclase; broken 1ine:cGMP phospho-

diesterase. Activity in arbitrary units.

b) Control of CAMP concentration during cell cycle. Solid 1ine:cAMP phosphodiesterase; broken line

adenyl cyclase. Activity in arbitrary units.

65

hand the ability to synthesize cell or tissue characteristic products is the criterion of differentiation then

few if any cells or tissues differentiate after proliferation, i.e., when in mitotic arrest (49). Of course, with

the availability of increasingly sensitive methods which enable one to follow syntheses of many molecules -

called “ luxury molecules ” by some (48) - very early during differentiation that were believed to be spe-

cific to differentiated cells, the idea of “ quanta1 division ” loses its meaning (505152). In one study

it was found that during the development of X. laevis collagen synthesis starts at gastrulation and increa-

ses rapidly up to the larval stage (53). Based on the observation that in Drosephila embryos, earlier chro-

matin ( blastula ) supports transcription 2,2 times better than later stage chromatin, one has to conclude

that differentiation is a process of decreasing template activity and hence increasing repression even though

the later stages of differentiation may lead to the synthesis of specialty molecules, such as actin, myosin,

haemoglobin, etc. (54).

Thus I favor the following view in my model:

1. Differentiation follows proliferation during embryogenesis. However, this does not rule out further

proliferation of the differentiated cells. These can still undergo differentiation.

2. Even though the synthesis of some special molecules may not be observable during earlier stages of

embryogenesis, more and more genes are turned off as differentiation progresses.

Coming back to the discussion of the model, one may ask: “ is there any justification for the idea of po-

larity in the egg cells?‘. My answer to this question is an unqualified “yes”, for a myriad of reasons.

To begin with one may consider the internal structure of a cell. The contents of a cell are not like a dilute

solution but a semisolid gel (55) where endoplasmic reticulum, mitochondria, ribosomes etc., are distri-

buted unevenly throughout the cytoplasm. The only way one can conceive of a cell as a symmetrical body

is if the contents of a cell were distributed in a spherically or more generally speaking ellipsoidally symme-

trical fashion. It is impossible to consider any real cell, including an ovum, as an ellipsoidally symmetrical

object. Thus division of such a cell will invariably yield two unequal hence asymmetric cells.

In invertebrates, the cytoplasm of egg cells is definitely polar and one sees a localization of the cytoplasm

of an uncleaved egg within a few minutes after fertilization (56). Even the unfertilized egg already poses-

ses polarity in one axis so that sperm is able to enter through certain particular region(s). This polarity

expresses itself in the uneven distribution of the cytoplasm as cleavage occurs. This phenomenon which

is known as “ localization ” has been exploited in developing the complete cell linage of various organisms

(56). Isolated blastomeres were shown to develop into their final shape and size, independent of the input

from adjacent cells (57). This kind of polarity has also been observed in such vertebrates as birds and te-

least fist (58) in the form of yolk and active cytoplasm. Since cleavage planes are always established in the

plane previously occupied by the metaphase plate of the mitotic apparatus, a polarity, once established,

can be expected to continue throughout embryogenesis. What I am suggesting is to consider the ovum

as the stage where asymmetry expresses itself. That orderly shifts of individual cells and cell complexes

during embryogenesis, especially during gastrulation, may be due to differentials of the cell surface pro-

teins has been proposed (59). It is conceivable that during evolutions, synchrony of cells found close to

each other, such as the ones found in Volvox, preceeded differentiation; cell contact points functioning

as primitive signal carriers - channels through which RT might be transported - between cells.

Recently the clustering of Con A receptor sites on certain cell types in early embryos has been demons-

trated (60). The mobility of specific migratory cells was found to be very similar to the mobility of the

invasive malignant cells (61).

Secondly, a glance at a series of photomicrographs showing the progress of cell division in a mouse egg

66

also confirms this feeling of polarity (62). At the four cell stage the cells appear to form a tetrahedral

structure where each cell touches three other cells. As one of the cells divides, one cell makes contact

with four cells while the other four cells make contact with only three other cells. AS another cell di-

vides, the one that was in contact with four others now has contacts with five cells while five others still

touch only three cells. Is this a coincidental observation of an accidental contacts or does it perhaps suggest

that one cell, because of preceeding events, was destined to have this unique property? If egg membrane

were to have certain sites which were destined to become the poles, for instance, then the cleavage planes

could constrain the areas whereby newly divided cells will touch each other. Computer simulation studies

of embryogenesis of the L,yr~neu egg supports the view that differentiation pattern can entirely be caused

by internal cell factors while cell interactions are not involved during the earlier stages (63). If one also

assumes the presence of a gradient field with the maximum near the animal pole, the simulation becomes

much closer to the experimentally obtained results (64). Finally that two interacting gradient fields, with

induction centers on two opposite sides with respect to the main axis of the egg, might be a model appli-

cable to morphogenesis in general (65,66). Today when the idea of all organisms stemming from the first

primordial cell is nearly universally accepted (67,68), one has to conceed that certain basic biochemical

and biological principles will have to apply to all living cells as inviolable laws, even though evolution later

may have introduced differences among them. Furthermore it is more likely that these principles, that

are common to all living organisms, will be closer to the lower organisms, in their expressions, than to the

more highly evolved ones. Thus, I propose that all eggs are polar and sperm can only penetrate the egg

through a particular region of the egg membrane. The fact that most bacteriophages do invade their hosts

only through certain areas on the cell wall (69,70) make this argument more valid because, if an attack

by a parasite has to be so precisely controlled, how can an important event, such as fertilization, be left

to chance penetration of the sperm at any point on the egg membrane?

In summary, I am suggesting that cell surface, inclusive of egg cells, is the major controlling factor in dif-

ferentiation. This conclusion should not be too surprising as the previous section also had concluded that

the membranes were critically important for the precise control of cell cycle.

Locke discovered an axial gradient of positional information which determines both polarity and the

developmental fate of the epidermal cell (71). It was later shown that this gradient behaved like a concen-

tration gradient of a diffusible substance (72). A recent computer simulation of this gradient phenomenon

suggests that the ceils may be come “ set ” at some stage in the cell cycle to the ambient concentration

and that in addition to a concentration gradient of a diffusible substance these set values are necessary

to have complete positional information (73). Differentiation of certain protozoa into a flagellated form

is preceded by starvation (74). It is quite likely that during cell division, following the fertilization of an

egg, a point may be reached where the diffusion of certain nutrients from the outside is not sufficient to

support the optimum rate of cell division. This situation may be considered “ starvation ” of those cells

and it may trigger differentiation. On the other hand, the concentration of many maternal macromolecu-

les such as mRNAs (75) may fall below a critical threshold and thus act as a trigger of differentiation. I propose that the mRNA which codes for fI(m) histone ( fI histone found during earlier stages - morula )

which is different from the fI (g) histone ( fI histone found during gastrulation ) is the key molecule here

and when, due to cell division, its concentration in each cell falls below a preset level, this need for fI

(nl) mRNA triggers the Lie nova transcription of various genes and particularly of histone methylases.

Histone methyiases, initiate differentiation by increasing the binding affinity, of those histones affected

toward the DNA of various genes.

This model is definetely based on the premise that histones are the molecules responsible for repression

of unused genes and hence, for the initiation and progression of differentiation. The argument that his-

tones alone cannot supply the necessary specificity does not take into account the nearly infinite number

of modification possibilities that exist for these proteins. Acetylation, phosphorylation, methylation,

interactions with other peptides and proteins can more than adequately provide for all the variability

67

in structural as well as interactive terms that may be necessary for this purpose. Actually even without

these modifications, histones may be capable of furnishing a very finely tuned control needed during

differentiation. Goodwin, in an elegant analysis, demonstrates conclusively that low repressor specificity

may actually be necessary to achieve two complementary goals simultaneously: a reduction in the number

of accessible cell states from the astronomical number of possibilities to a value commensurate with the

number of cell types in a higher organism and a stabilization of the embryonic decision processes required

for arriving at these states. The well known interactions among histone and nonhistone proteins may very

well correspond to the interactions described by Goodwin (76).

Since histone synthesis occurs contemporaneously with DNA synthesis (77) and since the synthesis of his-

tones takes place even during the earliest stages of embryogenesis (78) there must be a difference between

the histones found in the repressed ( hetero- ) and unrepressed ( eu- ) chromatin. The difference, I propose,

is furnished by the methylation of histones which increase the basicity and thus the binding affinity of

histones toward DNA, and that this methylation is stimulated by CAMP. I also propose that in differentia-

ted cells cGMP production is under positive and CAMP production is under negative control ( Figure 2).

In other words, cGMP phosphodiesterase ( G-PDE ) is active at a low level and the intracellular cGMP

concentration is controlled by turning guanyl cyclase ( G-cyc) on, when needed. Adenyl cyclase ( A-cyc )

on the other hand, is always on in these cells, and furnishes CAMP continously. The intracellular level is

controlled by switching CAMP phosphodiesterase ( A-PDE ) on or off, as needed.

Thus when the quantity of mRNA, which codes for fl(m) histone falls below a critical threshold level,

at the end of morula stage, de nova RNA synthesis begins together with the transcription of histone me-

thylases, for the first time. The synthesis of a different fl(m) up to morula stage, than the fl(g) which is

seen during gastrula stage, has already been established (79). RNA isolated from morula polysomes directs

the synthesis of a high f2b/f2a/ratio of histones, while RNA isolated from gastrula polysomes directs a

low f2b/ f2a ratio of histone synthesis. This observation lends support to the view that f2a is involved in

the heterochromatization of the genes as well as the view that as the embryo moves from morula to gas-

trula there will be a decrease in the template activity of the chromatin. It was also found that fI(m) was not

converted to fl(g) and that their amino acid sequences were different and most importantly, that the

amounts and kinds of histones that bind to DNA in chromatin do differ characteristically from one stage

of development to the other. As mentioned also before, another study has found a quantitative deficiency

of fl in Drosophila blastule chromatin as well as the presence of a non-histone protein not found in older

embryos (54). Blastula chromatin was also shown to have 2,2 times higher template activity than gastrula

chromatin. There is sufficient evidence to suggest that histone fl is involved with the initiation of repli-

cation. The phosphorylation of fl was found not to occur during GI phase of cell cycle but immediately

preceding S phase (8081). Of special interest is the observation that in avian erythrocytes fl is replaced

by histone V (82). Since the nuclei of these erythrocytes are apparently inactive, in both DNA and RNA

synthesis, histone V seems to function by suppressing both activities. This way replacement by histone

V would eliminate any chance of accidental activation of these processes through phosphorylation of

fI. Histone fl was shown to be the most phosphorylated of all histones (83) while it was not methylated

throughout the cell cycle (84). f3 on the other hand was not phosphorylated to any appreciable degree

and it was found that phosphorylation occured in a slow moving minor fraction of f3 (80) and only in

synchronized cultures which were rich in cells undergoind mitosis. At this stage of the cell cycle, phos-

phorylation may very well serve a different function, such as removal f3 histone for realignment, etc.

Phosphorylation of histones was also found to be independent of RNA synthesis.

Acetylation of histones, on the other hand, was found to occur primarily in cells actively engaged in RNA

synthesis. In regenerating rat liver the maximum arginine-rich histone acetylation (3-4 hours post-hepatec-

tomy) was found to precede maximum RNA synthesis ( 6 hours after operation) (85).

In calf thymus cells, incubated with l4 C- acetate, only arginine-rich fractions f2al and f3 were found to

68

be appreciably labelled (86). fI and f2b were not labelled and did not contain n-acetyllysine. However,

they as well as f2a2, were extensively acetylated at their terminal amino position. Rat liver nuclei, too,

incorporate labelled acetate from acetyl-CoA into histones and nonhistone acidic nuclear proteins (87).

The order of acetylation was f3) f2aI> f2b) fI. Histones f3 (70%) and fZal (25%) comprised 95%of all the

label. Pigeon liver acetylase also acetylates histones, where f3 is the one species with the highest phospho-

rylation (88). In cell culture fI was not acetylated (89).

Thus two different phenomena related to gene activation become apparent:

1. Phosphorylation of histones occur if the gene activation is going to lead to DNA replication and cell

division.

2. Acetylation of histones occur if the genes are activated for transcription and translation needed for

various cellular activities but not involved in the replication of DNA.

Of course this is the predominant mode of operation, since there are many sites that can be phosphoryla-

ted as well as acetylated: fI does also get phosphorylated to a very small extent ( ca. lo/,), at a different

site, during hormone action (90). Furthermore it is suggested that acetylation may be involved in fitting

histones into their proper position on DNA (91).

So, referring to the model 1 proposed previously (11) I suggest that the replication initiation site(s) on

chromosome(s) is rich in adenine and thymine and the region(s) is covered by very lysine rich histone fl.

Thus fI of blastula cells of Drosophiliu embryo was found to be quantitatively less than in the adult ani-

mals, supporting the view of its involvement in replication initiation. Phosphorylation of histones was

also found to be independent of RNA synthesis.

I also propose that histones f3 and f2aI are involved with the repression of genes in differentiated organ-

isms. Both of these histones appear to be bound to DNA through divalent cations (92). And fully differen-

tiated old leaves and pith tissues were found to contain more FII and FIII histones (93); however, the lysine/arginine ratio of FIII was found to be different than calf thymus and pea histone FIII, while the

same ratio for FII was similar to that of pea histone.

f2aI is believed to be involved in the superstructure of chromatin, found in heterochromatin. Its synthesis

was found to be increased during transition from morula to gastrula (79), which suggests that there is a

decrease in the amount of DNA able to support RNA synthesis as embryogenesis progresses (94).

Estradiol given in vivo to rats increases template capacity of uterine chromatin within 15 minutes after

injection. This was found to be paralleled by a 50%decrease of histone methylase 1 activity, which methy-

lates some arginine residues in histones, and a 58% decrease in the amount of f3 histone. This decrease

was shown to be due to de uovo synthesis of an acidic, sulphydryl-containing protein (95). This means

that methylation, which normally is expected to increase the binding of histone, was reduced together

with the synthesis of acidic protein prior to transcription. Histone f 3

was also shown to be the most cf-

ficient in repressing RNA synthesis when added to DNA (96,97).

There is also another possibility for modifying DNA-histone interactions: the methylation of DNA bases,

which has been demonstrated to occur, even in the isolated nuclei (98,99). Certain cytosines of mouse

L and Krebs-2 mouse ascites tumor cell DNA were found to be methylated to S-methyl cytosine about

30 minutes after DNA synthesis begins (98). In this system there was an interesting interplay between

protein and DNA methylations, the greater the histones were methylated the lower was the methylation

of DNA. In HeLa cells, there was also a deaminase which converted l/1000 of these Smethyl cytosines to

thymine (99). However, this reaction was found not to occur in isolated nuclei, suggesting that it was

under the control of some cytoplasmic regulator. Methylation occured preferentially in cytosine isostichs

69

and CpG dinucleotides (100). Methylation was found to be increased after trypsin treatment of chromatin,

which dissociates arginine-rich histones (101). These observations appear to support Georgiev’s hypothe-

sis on the control of mammalian genome (102). Since S-methyl cytosine is still complementary to guanine,

this modification does not change the base sequence of the genes and at the next cell division normal cy-

tosine concentration of the DNA will be restored. The effect of this methylation can be expected to re-

duce the affinity of histones f3 and fZal to DNA (98).

In summary, there are adenine and thymine-rich regions on chromosomes which are the sites for initiation

of DNA replication and fl binds to them. There are also cytosine and guanine-rich regions on chromo-

somes which may be the control sites for the transcription of individual genes. These are likely to occur

near the 5’ end of the genes, their cytosines can be methylated and f3 histones bind to them.

The interaction between histones and DNA can also be modified by nonhistone chromatin proteins. As

already mentioned above, the decrease of f3 affinity toward rat uterine DNA was found to correlate with

iie nova synthesis of sylphydryl-containing acidic proteins (95). Drosophila embryos, also, were found

to contain a nonhistone protein at the blastula stage, which disappears later in development (54).

Thus there are many possibilities for a very finely tuned control of the repression and derepression of

genes which can be utilized during differentiation as well as during derepression following hepatectomy

or wound healing.

Aging

What happens to those genes, in differentiated cells, which are not needed for the day to day activities

of the cells in which they are found? I would suggest that secondary interactions take place between adja-

cent histone molecules (103,104) and nonhistone proteins (.54,94) of those genes which are not needed

for transcription as well as replication. These interactions can be ionic bonds, disulfide bonds and hydro-

phobic interactions as well as crosslinks. When such a cell is then induced to undergo cell division, after

hepatectomy or isoproterenol injection, etc., then, before any replication or even transcription can occur,

these secondary interactions have to be dealt with. The enzymes which work on these bonds have to be

induced first and this of course takes time. Indeed, during studies on aging, a delay has repeatedly been

observed which correlates the age of an animal linearly to the delay observed in the onset of the reaction

being studied (105,106). In many protozoa, the rejuvenating effect of conjugation can be attributed to the

elimination of the old macro- and micro-nuclei and the renewed formation of new structures (107,108).

Ebert, too, had proposed that the act of chromosome replication may actually clean inactive genome

regions (109). Thus according to this model aging appears to be inevitable because it is the result of dif-

ferentiation, since in the higher organisms control of unscheduled cell division is of utmost importance.

However, once an understanding is reached of the controls of these phenomena, one might be able to slow

down the process or even to reverse it.

NEOPLASTIC CHANGES

Introduction

There appears to exist a difference of opinion between the members of medical profession and biochemists

as well as molecular biologists, on the nature of neoplastic diseases. The former tend to consider “ cancer ”

as a whole spectrum of diseases, whereas the latter are more inclined to think that all neoplastic diseases

have much in common and that the differences observed are due to the variations of expression dependent

70

upon the site of the

being able to explain

dent that this model

describe how certain

neoplasia.

disease. Hence, according to the proponents of this latter group, there is hope in

the events leading to neoplastic state in one model and one can be reasonably confi-

may be applicable to all forms of the disease. In this part of the manuscript I shall

conditions can alter this control scheme and lead to the changes of hyperplasia and

According to the model developed at the beginning of this paper the primary control of cell division occurs

through CI, which is elicited when glycoprotein receptors on cell surfaces bind each other and prevent

the RT or humoral factor from initiating replication by binding to the same receptor sites. Fine control

of cell division is then initiated by the binding of the MI or chalone to its own specific receptors, found

inside the cell membrane.

Thus the flawless formation of cell membrane receptors in necessary quantity iacquire utmost importance.

In this way the organism insures itself against unscheduled replication and consequent cell division.

It shoulJ then ~0110~ that any event or agent that leads to an interfbrence with thefbmation or destruc-

tion ofgl_ycoprotein receptors, particularly RT receptors, is potentially a carconigcnic agent!

There are several possibilities than can be conceived which may lead to this interference: a. A chemical

or physical agent which can affect membranes directly will lead to inactivatron and to eventual loss of

receptors through its physical destructive effect on membranes, b. A metabolic defect which can interfere

with the synthesis of glycoprotein precursors, sialyl- or glycosyl-transferases leading to incomplete forma-

tion of surface receptors, c. A defect in the formation of A-cyc, which results in an unusually low CAMP

concentration in the cells, d. A continued stimulation of cell division by agents that either act on mem-

brane receptors or on the chromatin, e. Any condition which may lead to a shortening of Cl phase of the

cell cycle.

The model

1. Cell membrane receptors are affected adversely by anyone of the above causes a,b, or e.

2. CI is thus effectively interfered with, a consequence of which is seen as a piling of cells one on top of

another in cell culture.

3. Humoral factor can initiate DNA replication and cell division in accordance with the model described

in the first section of this manuscript, however, since RT receptors are not present in sufficient quality

or quantity, the effect of RT is not limited to only one cell cycle but continues cycle after cycle.

4. CAMP concentration inside the cell falls to very low levels.

5. Histone methylase induction will no longer be possible.

6. MI synthesis will stop.

7. RT genes cannot be turned off and the cells become autonomous in their ability to grow,

8. Up to the point where autonomy for growth is induced, the changes which occur are reversible and

lead only to hyperplasias. After autonomy is reached, the changes which have taken place are practically

irreversible and the state of the tissue can best be described as neoplastic.

9. Conditions leading to a defect in the formation of CAMP can also affect the control of cell cycle by their

71

interference with histone methylation and with MI formation. These conditions may be due to an inhibi-

tion of A-cyc or due to a defect in its synthesis or to an irreversible activation of A-PDE.

10. Viral infection and perhaps even certain bacterial and nematodal infections can also affect cell cycle

by producing RT needed for its own replication. Because of the universal nature of RT, host replication

is also stimulated. Since these RTs are synthesized inside the cell, they act directly on cellular chromatin

and hence are beyond the control of CL So chronic infection may lead to irreversible changes by helping

the dreepression of cellular RT genes,

Discussion

Needless to say, the model for carcinogenesis developed here puts utmost importance on the normal state

of cellular membranes, because the well being of the organism depends upon the balance between Cl,

which inhibits cell division, enhanced by the effect of MI, on the one hand, and the ever present tendency

for cell division, stimulated by RT, on the other. Thus events which effect both of these forces adversely

can initiate the move toward the path of no return.

Membranes can be affected adversely either while being synthesized or after being formed normally. The

maximum rate of synthesis for glycoproteins was found toward the end of the GI phase of cell cycle (110).

Any events that can shorten GI can result at least in less than the required quantity of glycoprotein recep-

tors. Qualitative changes can also conceivably occur if some of the amino sugars or other components as

well as the enzymes involved are not made in sufficient quantity by the time GI is brought to an untimely

end. This phenomenon would then manifest itself as the presence of new cancer specific antigens. Since Gl

is the only variable part of the cell cycle, rapidly growing tumors most probably have a shorter GI. Thus

the model refers to those possibilities without attempting to be specific, in a situation where there are too

many variables.

The most important event in the “ transformation ” of a normal cell into a neoplastic cell is the one which

commits the cell to remain transformed. Up to that point elimination of the cause will allow the cell,

perhaps after some time needed for adjustment, to resume its normal quiescent state; beyond it, the cell

is committed to continue to divide. The model furnishes a precise mechanism about the events which oc-

cur at this point which can be tested experimentally. According to the model, developed in the first sec-

tion of this paper, the critical event during cell division of a normal cell is the turning off of RT genes at

the right time, when no more proliferation is needed. This was postulated to occur when histone methylases

methylate arginine rich f3 histones. Methylases were proposed to be formed under the influence of CAMP.

If, however, due to defective formation of membrane RT receptors, CI cannot take place and some of the

receptors become available for RT or humoral factor to bind to, then cGMP will be produced continously.

This will inhibit the formation of CAMP and will result in histone methylases to remain uninduced. Another

molecule which could not be synthesized under these conditions is MI, which, if produced, would help

degrade the remaining cGMP and increase A-cyc activity.

Membranes can also be destroyed by physical agents, such as irradiation and chemical agents such as hydro-

carbons, which are excellent fat and lipid solvents. There is a well documented example of myeloid meta-

plasia (111) as well as of leukemogenesis (112) in people who are exposed to benzene over long periods.

As early as 1965, the induction of host DNA synthesis after SV 40 and polyoma infection has been de-

monstrated (113). Because RT are universal molecules (1 ,114,115) and also because they are synthesized

inside the cell, they act directly on the cellular chromatin as well as on viral DNA, and their effect is thus

beyond cellular controls through CI.

72

Bacterial as well as nematodal infections have also been implicated prior to the appearance of certain neo-

plastic diseases. A very widespread plant tumor, crown gall, has been demonstrated to be caused by Agro-

bacterium tumafaciens (116). In the animal field Schistosoma haematobium has been shown to cause

hematuria, anemia and in chronic cases calculus formation which may become malignant, after its eggs

are deposited in the veins of the bladder (117). Formation of true stomach tumors was achieved by feeding

rats a nematode found in the muscles of certain species of cockroaches (118). A high incidence of esteo-

sarcoma in the upper airway of the dog was shown to follow infection by Spirocerca lupi (119). Even tho-

ugh the mechanisms which lead to neoplastic changes in these cases are not known, the suggestion made

here that it may be due to RT produced by the infectious agent cannot be dismissed.

The attractiveness of this model lies in its ability to describe neoplastic events which are seen in cells gene-

ration after generation without involving genetic changes. It also does not presume the involvement of

any factor which was not utilized for the description of the control of cell cycle under normal circum-

stances. The ideas promoted can be tested experimentally and the model is capable of improvement as new

insights are gained on the control of metabolic processes.

Abbreviations used:

RT: replication trigger. MI: Mitotic inhibitor; CAMP: adenosine 3’-5’ cyclic monophosphate; cGMP: gua-

nosine 3’-5’ cyclic monophosphate; A cyc: adenyl cyclase; G cyc: guanyl cyclase; A-PDE: CAMP phopho-

diesterase; G-PDE: cGMP phosphodiesterase; GT: glycosyl transferase; ST: sialyl transferase; GI: gap one

phase of cell cycle; G2: gap two phase of cell cycle; S: replication phase of cell cycle; M: mitosis (cell

division) phase of cell cycle; Cap: calcium pump; Cl: contact inhibition of movement; CHO-KI: Chines

hamster ovary cells KI line.

Acknowledgments

This work was presented in part at the X Turkish Haematology Society Meeting in Ankara, 1975

REFERENCES

1.

2.

3.

4.

5.

6.

7.

8.

9. Jablonski JR, Skoog F. Cell enlargement and cell division in excised tobacco pith tissue. Physiol

Erhan S, Reisher S, Frank0 EA, Kamath SA, Rutman RJ. Evidence for the wedge, initiator of DNA

replication. Nature 225, 340, 1970

Dulak NC, Temin HM. Multiplication stimulating activity for chicken ernbryo fibroblasts from rat

liver cell conditioned medium. J Cell Physiol. 81, 161, 1973.

Kohiyama M, Lamfrom H, Brenner S, Jacob F. Multiplication des fonctions indispensables chez des

mutants thermosensibles E. co/i. C.R. Acad Sci. 257, 1979, 1963.

Bullough WS. In Cellular Control Mechanisms and Cancer p. 124, Ed. Emmelot P, Muhlbock 0. Else-

vier Publishing Co. New York, 1964.

Gelfant S. Initiation of mitosis in relation to the cell division cycle. Exptl Cell Res. 26, 395, 1962.

Szent-Gyorgyi A. On retine. Proc Nat1 Acad Sci USA. 57, 1642, 1967.

Puck TT, Waldren CA, Jones C. Mammalian cell growth proteins. I. Growth stimulation by fetuin.

Proc Nat1 Acad Sci USA 59, 192, 1968.

Cohen S. Isolation and biological effects of an epidermal growth stimulating protein. Nat1 Cancer

Inst Monograph 13, 13, 1964.

73

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

Plant. 7, 16, 1954.

Adler HI, Fisher WD, Stapleton GE. Genetic control of cell division in bacteria. Science 154, 417,

1966.

Erhan S. DNA polymerase model. Nature 219, 160, 1968.

Bossman HB, Hagopian A, Eyler EH. Glycoprotein Biosynthesis: Characterization of two glyco-

protein fucosyl transferases in HeLa cells. Arch Biochem Biophys. 128, 470, 1968.

Roth S, McGuire EJ, Roseman S. Evidence for cell surface glysosyl transferases. Their potential

role in cellular recognition. J Cell Biol. 51, 536, 1971.

Aronson NN, Tan LY, Peters BR. Galactosyl transferase, the liver plasma membrane binding site

for asialoglycoproteins. Biochem Biophys Res Communs. 53, 112, 1973.

Thornley AL, Laurence EB. The present state of biochemical research on chalones. Int J Biochem.

6, 313, 1973.

Pasternak CA, Sumner MCB, Collin RCLS. in Cell Cycle Controls p.ll7,Ed. Padilla GM, Cameron

IL, Zimmerman A. Academic Press, New York 1974.

Frye LD, Edidin M. Rapid intermixing of cell surface antigens after formation of mouse-human

heterokaryons. J Cell Sci. 7, 3 19, 1970.

Singer SJ, NIcholson GL. The fluid mosaic model for the structure of cell membranes. Science

175, 720, 1972.

Sudo T, Onodera K. Response of cell surface glycosyl transferases to dibutyryl CAMP in virus

tranformed and normal cells. Exptl cell Res. 91, 191, 1975.

Bossman HB, Winston RA. Synthesis of glycoprotein, glycolipid, protein and lipid in synchronized

L5178 Y cells. J Cell Biol. 45, 23, 1970.

Hadden JW, Johnson EM, Hadden EM, Coffey, RG, Johnson LD. in Immune Recognition p.359.

Ed. Rosenthal AS. Academic Press, New York, 1975.

Kakuichi S, Yamazaki R. Ca++ dependent phosphodiesterase activity and its activating factor

from brain. Biochem Biophys Res Communs. 41, 1104, 1970.

Beavo JA, Hardman JG, Sutherland EW. Stimulation of CAMP hydrolysis by cGMP. J Biol Chem.

246, 3841, 1971.

Hadden JW, Hadden EM, Haddox MK, Goldberg NO. cGMP, a possible intracellular mediator of

mitogenic influence in lymphocytes. Proc Nat1 Acad Sci USA. 69, 3024, 1972.

Teng CS, Teng CT, Allfrey VG. Studies on nuclear acidic proteins. J Biol Chem. 246, 3597, 1971.

Johnson EM, Karn J, Allfrey VG. Early nuclear events in the induction of lymphocyte prolifera-

tion by mitogens. J Biol Chem. 249, 4990, 1974.

Tilney LG, Marsland D. A fine structural analysis of cleavage induction and the furrowing mech-

anism in the egg of A. punctuluta. J. Cell Biol. 42, 170, 1969.

Johnson RG, Sheridan JD. Junctions between cancer cells in culture: ultrastructure and perme-

ability. Science 174, 717, 1971.

Pardee AB. A restriction point for control for normal animal cell proliferation. Proc. Nat1 Acad

Sci USA. 71,1286, 1974.

Prescott DM, Goldstein L. Nuclear-cytoplasmic interactions in DNA synthesis. Science 155, 469,

1967. Jacoby F, Trowel1 OA, Willmer EN. Further observations on the manner in which cell division

of chick fibroblasts is affected by embryo juice. J Exptl Biol. 14, 255, 1937.

Bryan J. Definition of three classes of binding sites in isolated microtubule crystals. Biochem.

11,2611,1972.

Weltman JK, Dowben RM. Relatedness among contractile and membrane proteins: Evidence for

evolution from common ancestral genes. Proc Nat1 Acad Sci USA. 70, 3230, 1973.

Yahara I, Edelman GM. Modulation of lymphocyte receptor mobility by Con A and Colchicine.

Ann NY Acad Sci. 253,455, 1975.

Puck TT, Waldren CA, Hsie AW. Membrane dynamics in the action of dibutryl CAMP and testo-

sterone on mammalian cells. Proc Nat1 Acad Sci USA. 69, 1943, 1972.

74

36.

37.

38.

39.

40.

41.

42

43

44

45.

46.

47.

48.

49

50.

51.

52;

53.

54.

5s.

56.

57

58.

59

60

61

62.

63.

64

Weston JA, Hendricks KL. Reversible transformation by urea of contact inhibited fibroblasts.

Proc Nat1 Acad Sci USA. 69, 3727, 1972.

Eagle J, Washington CL, Levy M, Cohen L. Population dependent requirement by cultured mam-

malian cells for metabolites which they can synthesize. J Bio. Chem. 241, 4994, 1966.

Grisham JW. A morphologic study of DNA synthesis and cell proliferation in regenerating rat

liver. Cancer Res. 22, 842, 1962.

Hertwig 0. Alfgenzeine Biologie, Gustav Fischer Verlag, Jena, Germany 1920.

Weiss 1’. Pviulciples of‘Developme!~t, Holt. New York, 1939.

Peter K. Die Beziehungen zwischen Zellteilung und Zelltatigkeit. Darstellung und Versuch einer

kausalen Betrachtung. Protoplasma 10, 613, 1930.

Weiss P. l)ynamics of Development: Experiments and Ir?ferences. Academic Press, New York,

1968.

Lockwood DH, Stockdale FE, Topper YJ. Hormone dependent differentiation of mammary gland.

Science 156, 945, 1967.

Wessels NK, Cohen JH. Early pancreas organogenesis, morphogenesis, tissue interactions and mass

effects. Devel Biol. 15, 237, 1967.

Cameron IL, Jeter JR. in Developmental Aspects of the Cell Cyck. p.315, Ed. Cameron IL, Padilla

GM, Zimmerman AM. Academic Press, New York, 1971.

Konegsberg I. Diffusion mediated control of cell fusion. Devel. Biol. 26, 13 3, 197 1.

Stockdale FE, O’Neill MC. DNA synthesis, mitosis and skeletal muscle differentiation. In vitro

8, 212, 1972.

Holtzer H. Proliferative and quanta1 cell cycles in the differentiation of muscle, cartilage and red

blood cells. Symp Int Sot Cell Biol. 9, 69, 1970.

Lash JW. in Colzcepts of‘ Development p.197, Ed. Lash JW, Whittaker JR. Sinauer Assoc. Stamford,

Corm. 1974.

Klose J, Flickinger RA. Collagen synthesis in frog embryo endoderm cells. Biochim Biopys Acta

232, 207, 1971.

Marzullo G, Lash JW. Acquisition of the chondrocytic phenotype. Exp Biol Med 1, 213. 1967.

Kosher RA, Searls RL. Sulfated mucopolysaccharide synthesis during the development of RLzt20

pipifTrx Devel Biol. 32, 56, 1973.

Green H, Goldberg B, Schwartz M. Brown DD. The synthesis of collagen during the development

of X. laevis. Devel Biol. 18, 391, 1968.

Elgin SCR, Hood LE. Chromosomal proteins of Dvosopbila embryos. Biochem. 12, 4984, 1973.

Srere I’. Fnzymc concentration in tissues. Science 158, 936, 1967.

Conklin EG. The organization of cell linage of the ascidian egg. J Acad Nat Sci (Philadelphia)

13, 1, 1905.

Wilson EB. Experimental studies in germinal localization. II Experiments on the cleavage mosaic in

PLrtelh and Dcrztdium. J. Exptl Zool. 1, 197, 1904.

Rappaport R. in Coftcepts of Developwzent p.76, Ed. Lash JW, Whittaker JR. Sinauer Assoc, Stam-

ford Conn. 1974.

Gustafson 7‘. Wolpert I,. Cellular movement and contact in sea urchin morphogenesis. Kiol Rev.

42, 422, 1967.

Smith SK. Revel JP. Mapping of Con A binding sites on surface of several cell types. Devcl Biol.

27, -134, 1972.

Moscona AA. Embryonic and plastic cell surfaces: Availability of receptors for Con A anti. wheat

germ agglutinin. Science 171, 905, 1971.

Rex GG. Ciilt,~?l‘?tOgRlpby in Cell Biology p.268, Academic Press, New York, 1963.

Raven C’, Bezem JJ. Computer simulation of embryonic development. 1. Radialized development

of .!-_v~~z~~ca egg. Konink Neder Akad Wetensch. Ser. C 74, 209, 1971.

R:*WJJ Cl’. Bezem JJ. Computer simulation of embryonic development Il. Radialized development

of L I’Y?!IIC.I egg. Konink Neder Adad Wetensch. Ser. C 74, 223, 1971.

75

65.

66.

67.

68.

69.

70.

71.

72.

73.

74.

75.

76.

77.

78.

79.

80.

81.

82.

83.

84.

85.

86.

87.

88.

89.

90.

91.

92.

Raven CP, Bezem JJ. Computer simulation of embryonic development III. Differentiation in the

radialized embryo. Konink Neder Akad Wetensch. Ser. C 75, 20, 1972.

Raven GP, Bezem JJ. Computer Simulation of embryonic development IV. Normal development of

Lymnea egg. Konink Neder Akad Wetensch. Ser. C 76,23, 1973.

Lehninger AL. Biochemistry PP.772 and 781, Worth, New York, 1970.

Black S. A theory on the origin of life. Adv. Enzymol. 38, 193, 1973.

Bayer ME. Ultrastructure and organization of the bacterial envelope. Ann. NY Acad Sci. 235,

6, 1974.

Johnson KE. in Concepts of Development. p.128, Sinauer Assoc. Stamford, Conn. 1974.

Locke M. The cuticular pattern in an insect Rhodinus Prolixus. J Exptl Biol. 36, 459, 1959.

Stupf H. Die Deutung der Riefenmuster bei Rbodinus Pro&us auf Grund eines Konzentration-

gefalles. Naturwiss. 52, 500, 1965.

Lawrence PA, Crick FHC, Munro M. A gardient of positional information in an insect, Rbodinus.

J Cell Sci. 11, 815, 1972.

Fulton C, Kowit JD. Programmed synthesis of flagellar tubulin during cell differentiation in Nae-

gleria. Ann NY Acad Sci. 253, 318, 1975.

Gross PR, Malkin LL, Hubbard M. Synthesis of RNA during oogenesis in the sea urchin. J Mol

Biol. 13, 463, 1965.

Goodwin BC. Histones and reliable control of protein synthesis. Ciba Fdn Study Group 24, p.68,

Little, Brown Co. Boston, Mass, 1966.

Butler WB, Mueller GC. Control of histone synthesis in HeLa cells. Biochim Biophys Acta 294,

481, 1973.

Ruderman JV, Gross PR. Histones and histone synthesis in sea urchin development, Devel Biol.

36, 286, 1974.

Ruderman JV, Baglioni C, Gross PR. Histone mRNA and histone synthesis during embryogenesis.

Nature 247, 36, 1974.

Gurley LR, Walters RA, Tobey RA. Differences in the phosphorylation of histone fractions during

the cell cycle. Arach Biochem Biophys. 154, 212, 1973.

Shepherd GR, Hardin JM, Noland BJ. Dephosphorylation of histone fractions of cultured mam-

malian cells. Arch Biochem Biophys. 153, 599, 1972.

Dick C, Johns EW. A quantitative comparison of histones from immature and mature erythroid

cells of the duck. Biochim Biophys Acta 175, 414, 1969.

Balhorn R, Chalkley R, Granner D. Lysine-rich histone phosphorylation. A positive correlation

with replication. Biochem. 11, 1094, 1972.

Paik WK, Lee HW, Morris HP. Protein methylases in hepatomas. Cancer Res. 32, 37, 1972.

Pogo BGT, Pogo AD, Allfrey VG, Mirsky AE. Changing patterns of histone acetylation and RNA

synthesis in regenerating liver. Proc Natl Acad Sci USA. 59, 13 37, 1968.

Vidali G, Gershey EL, Allfrey VG. Chemical studies of histone acetylation. J Bio. Chem. 243,

6361, 1968.

Gallwitz D, Sekeris CE. Acetylation of histones of rat liver nuclei in vitro by acetyl-CoA. Zeit phys-

iol Chem. 350,150,1969.

Nohara H, Takahashi T, Ogata K. Acetylation of histones by pigeon liver enzymes. Biochim Biophys

Acta 127, 282, 1966.

Wilhelm JA, McCarthy KS. Partial characterization of histone synthesis and histone acetylation

in cell cultures. Cancer Res. 30, 409, 1970.

Elgin SC, Weintraub H. Chromosomal proteins and chromosome structure. Ann Rev Biochem.

44,749, 1975.

Lovie AJ, Dixon GH. Synthesis, acetylation and phosphorylation of histone IV and its binding

to DNA during spermatogenesis in trout. Proc Nat1 Acad Sci USA. 69, 1975, 1972.

von Hahn HP, Heim JM, Eichorn GL. The effect of divalent cations on the isolation of proteins

from rat liver nucleoprotein. Biochim Biophys Acta 214, 509, 1970.

76

93. Srivastava BIS. Relationship of histones to RNA synthesis in plant tissues. Physiol Plant. 24, 27,

1971.

94. Skidmore C, Walker ID, Pardon JF, Richards BM. The structure of partially histone depleted nucleo-

histone, FEBS Letters 32, 175, 1973.

95. Barker KL. Estrogen induced synthesis of histones and a specific nonhistone protein in the uterus.

Biochem. 10, 284, 1971.

96. Izawa M, Allfrey VG, Mirsky AE. The relationship between RNA synthesis and loop structure in

lampbtush chromosomes. Proc Nat1 Acad Sci USA. 49, 544, 1963.

97. Spelsberg TC, Tankerskey S, Hnilica LS. Inhibition of RNA polymerase activity by arginine rich

histones. Experientia 25, 129, 1969.

98. Burdon RH, DNA and protein methylation in mouse tumor cell chromatin. Biochim Blophys

Acta 232, 359, 1971.

99. Vople P, Eremenko T. Preferential methylation of regulatory genes in HeLa cells. FEBS letters

44, 121,1974.

100. Scarano E:, laccarino M, Grippo P, Winckelmans D. On methylation of DNA during development

of sea urchin embryo. J Mol Biol. 14, 603, 1965.

101. Tosi L, Granieri A, Scarano E. Enzymatic DNA modifications in isolated nuclei from developing

sea urchin embryos. Exptl Cell Res. 72, 257, 1972.

102. Georgiev GP. Regulatory Mechanisms for Protein Synthesis in Mammalian Cells. p.25. Ed. San

Pietro A, Lamborg MR, Kenney FT. Academic Press, New York, 1968.

103. D’Anna JA, Isenberg I. A histone cross complexing pattern. Biochem. 13, 4992, 1974.

104. Smerdon MJ, Isenberg I. Conformationai changes in histone GRK (f2al)_ Biochem. 13,4046. 1974.

105. Adelman RC, Stein G, Roth GS, Englander D. Age dependent regulation of mammalian DNA

synthesis and cell proliferation in vivo. Mech. Age. Dev. 1, 49, 1972. 106. Cameron IL. Cell proliferation and renewal in aging mice. J Geront. 27, 162, 1972.

107. Kidder GW. Studies on Conchophthi~us mytili de Morgan I. Arch Protist. 79, 1, 1933.

108. Colkins GN. Uroleptus mobilis II. J Exptl 2001. 29, 121, 1919.

109. Ebert JD. Levels of control: a useful frame of perception. Curr. Top Devel Biol. 3, 15, 1968.

110. Graham JM, Sumner MCB, Curtis DH, Pasternak CA. Sequence of events in plasma membrane

assembly during the cell cycle. Nature 246, 291, 1973.

111. Aksop M, Erdem S, Dincol G. Two rare complications of chronic benzene poisoning: myeloid

metaplasia and paroxymal nocturnal haemoglobinuria. Blut 30, 255, 1975.

112. Aksoy M, Erdem S. Dincol G. Leukemia in shoe workers exposed chronically to benzene. Blood

44, 837, 1974.

113. Weil R, Michel MR, Ruschman GK. Induction of cellular DNA synthesis by polyoma virus. Proc

Nat1 Acad Sci USA. 53, 1468, 1965.

114. Gurdon JB. On the origin and persistence of a cytoplasmic state inducing nuclear DNA synthesis

in frogs’ eggs. Proc Nat1 Acad Sci USA. 58, 545, 1967.

115. Braun AC. Cuncer Problem p.39, Columbia Univ. Press, New York, 1969.

116. Braun AC. Thermal studies on the factors responsible for tumor induction in crown gall. Am J

Bot. 34, 234, 1947.

117. Gillman J, Pratas MD. Histological types and histogenesis of bladder cancer in the Portugese east

African with special reference to bilharzial cystitis. Acta Un Int Contra Cancrum 18,

560, 1962.

118. Fibiger J. Untersuchungen uber eine Nematode (Spiroptera sp.n.) und deren Fahigkeit, papilla-

matose und carcinomatose Geschwulstbildung im Magen der Ratte Hervorzurufen. Zeit f

Krebsforsch. 13, 217, 1913.

119. Brodey RS, O’Brien J, Berg P, Roszel JF. Osteosarcoma of the upper airway in the dog. J Am

Vet Med Assoc. 155, 1460, 1969.

77